NAFLD in normal weight individuals

Nonalcoholic fatty liver disease (NAFLD) can develop in lean individuals. Despite a better metabolic profile, the risk of disease progression to hepatic inflammation, fibrosis, and decompensated cirrhosis in the lean is similar to that in obesity-related NAFLD and lean individuals may experience more severe hepatic consequences and higher mortality relative to those with a higher body mass index (BMI). In the absence of early symptoms and abnormal laboratory findings, lean individuals are not likely to be screened for NAFLD or related comorbidities; however, given the progressive nature of the disease and the increased risk of morbidity and mortality, a clearer understanding of the natural history of NAFLD in lean individuals, as well as efforts to raise awareness of the potential health risks of NAFLD in lean individuals, are warranted. In this review, we summarize available data on NAFLD prevalence, clinical characteristics, outcomes, and mortality in lean individuals and discuss factors that may contribute to the development of NAFLD in this population, including links between dietary and genetic factors, menopausal status, and ethnicity. We also highlight the need for greater representation of lean individuals in NAFLD-related clinical trials, as well as more studies to better characterize lean NAFLD, develop improved screening algorithms, and determine specific treatment strategies based on underlying etiology.

Introduction
Nonalcoholic fatty liver disease (NAFLD) is a chronic, progressive condition that arises from intrahepatic fat accumulation in the absence of monogenic metabolic disorders, infections, steatogenic medications, or significant alcohol consumption [1]. Since it was first described in 1980 [2], NAFLD has become the most common liver disease in Western populations [3,4]. NAFLD is also recognized as a common chronic disease worldwide, affecting ~ 24% of the population [5], with a growing prevalence [6,7]. In the United States, nonalcoholic steatohepatitis (NASH), a severe form of NAFLD characterized by hepatic inflammation and often accompanied by fibrosis, is the major cause of chronic liver disease and is emerging as the most common indication for liver transplantation [8].
Obesity is a risk factor for the development of NAFLD [9,10] and the prevalence of NAFLD increases in parallel with BMI [11]. Nevertheless, NAFLD is not inextricably linked with obesity, as many individuals with obesity maintain normal intrahepatic content, while a significant number of lean individuals develop NAFLD, even in the absence of insulin resistance, type 2 diabetes (T2D), and related metabolic comorbidities [10]. Early observations by Bellentani et al. [12] noted a NAFLD prevalence of entity and defined it as "lean-NAFLD". However, because body weight is not a component of the diagnostic criteria for NAFLD, and describing NAFLD itself as lean is imprecise, "NAFLD in lean individuals" has been suggested as a more accurate description of this condition [17]. In agreement, we utilize this terminology here.
Although NAFLD in lean individuals is not uncommon, the pathophysiology of the disease in lean patients remains poorly characterized. While some characteristics of NAFLD are shared among individuals in obese and lean BMI groups, not all lean individuals with NAFLD have metabolic antecedents that predispose to hepatic dysfunction. In these individuals, factors such as dietary composition, lifestyle factors, and genetic susceptibility may contribute to the development of NAFLD. Despite potentially distinct etiologies, NAFLD in lean individuals appears to follow a disease progression similar to that in patients with overweight or obesity, indicating that the absence of excess corporeal adiposity does not confer protection against hepatic inflammation, fibrosis, or decompensated cirrhosis. As discussed in the following sections, some studies have even reported more severe histological presentation and higher mortality in NAFLD patients with normal BMI compared to higher BMI groups.
Because NAFLD is a clinically silent disease in most cases, the absence of early signs and symptoms, coupled with normal laboratory and anthropometric measures, likely blind clinicians to the presence of NAFLD in lean individuals. However, lean NAFLD patients are at risk for progression to severe liver disease and possibly even increased mortality, warranting efforts to promote awareness of NAFLD in lean individuals. In this review, we summarize the literature on NAFLD prevalence, characteristics, outcomes, and mortality in lean individuals and discuss factors that may contribute to the development of NAFLD in this patient population. We also address clinical strategies for the screening and management of NAFLD in lean individuals based on suspected etiologies.

Prevalence of NAFLD in lean individuals
The prevalence of NAFLD in lean individuals has been estimated almost exclusively using BMI as the sole criterion to describe body habitus. The international definition for normal BMI is defined as < 25 kg/m 2 . However, in Asians and Pacific Islanders, a BMI of < 23 kg/m 2 is used due to the occurrence of visceral adiposity and risk of developing comorbidities, such as T2D and cardiovascular disease, at a lower BMI than that found in Europeans [18]. Using these thresholds to define lean individuals, a comprehensive survey of available studies based on BMI cut-offs of < 25 kg/m 2 (< 23 kg/m 2 for Asians) for lean individuals indicates that the prevalence of NAFLD ranges from 5 to 34% (Table 1). Of note, there is significant heterogeneity among these studies, which vary by geography, method of NAFLD determination, design, sample size, and comparison group. For example, the assignment of NAFLD was based on a number of different methods including liver biopsy, abdominal ultrasonography, computed tomography, liver function tests (i.e., hepatic transaminases), magnetic resonance imaging, controlled attenuation parameter, and several indices. Some studies were population-based, while others were hospital-based or located at a tertiary care clinic. Sample sizes ranged from 39 to more than 10,000, with the majority of studies emanating from South and East Asia. Comparator groups were either lean NAFLD compared to all lean or lean NAFLD compared to all NAFLD.
The highest NAFLD prevalence rates (e.g., > 30%) were observed in India, and were based on estimates obtained from community-based populations, indicating a lower degree of bias compared to studies in which participants were recruited from hospitals or tertiary liver clinics. This increased prevalence, coupled with observations that lean, healthy, sedentary, non-smoking Asian-Indian men have a three-to fourfold increased prevalence of insulin resistance associated with a twofold increase in hepatic fat content compared to Eastern Asian, Black, Caucasian, and Hispanic men [19], suggests that this lean population may be at particular risk for NAFLD due to yet-to-be-identified factors.
In addition to the individual studies in Table 1, a comprehensive meta-analysis using data from 84 studies (n = 10,530,308) found that within the NAFLD population, 19.2% (95% Confidence Interval [CI] 15.9-23.0) of participants were lean [20]. However, in the general population (23 studies; n = 113,394), comprising all individuals regardless of NAFLD status, only 5.1% (95% CI 3.7-7.0) had NAFLD in the presence of normal BMI. Among the lean population (19 studies; n = 49,503), 10.6% (95% CI 7.8-14.1) had NAFLD. The authors noted high heterogeneity among the results, although in general, European countries appeared to have the highest, and Asian countries the lowest, prevalence of NAFLD in nonobese individuals. In a similar analysis of 21 studies (N = 55,936), Shi et al. [21] estimated an overall NAFLD prevalence rate of 10.2% (95% CI 7.6-13.6%) in lean populations, also noting significant heterogeneity. Data from the Global NAFLD/NASH Registry comprising data from 18 countries found that approximately 8% of the patients were lean, based on BMI, and exhibited fewer components of the metabolic syndrome, fewer comorbidities, and less cirrhosis [22]. The year in which data were collected and sample size significantly impacted estimates of NAFLD prevalence, but BMI cut-off, region of study,  population source, and method of diagnosis also exerted nonsignificant effects on the observed heterogeneity. Like obesity-associated NAFLD [6], the prevalence of NAFLD in lean individuals is also increasing. In studies with data collected before 2000, the prevalence was 5.6%, but from 2001 to 2010, and after 2011, rates were estimated at ~ 11.0% and 12.6%, respectively, reflecting the trend of increasing prevalence in the overall population [23]. Several groups have estimated NAFLD prevalence in nonobese populations using combined normal and overweight BMI groups (i.e., < 30 kg/m 2 [< 25 kg/m 2 in Asians]). In China, a population study comprising 911 individuals recruited from the census database of the Hong Kong government reported a NAFLD prevalence of 19.3% in nonobese subjects [24]. In a cross-sectional study of individuals receiving health checkups in Japan, NAFLD prevalence in nonobese individuals was estimated at 15.2% [25]. NAFLD prevalence in nonobese, nondiabetic Belgians undergoing biopsy for chronic liver disease was 2.8% (50/1777) [16]. Estimates in nonobese populations are similar to those found in lean populations (Table 1), revealing potential limitations of BMI cut-off for NAFLD screening in the general population.

Clinical characteristics
A number of studies have compared clinical characteristics between lean and non-lean NAFLD cohorts. An early study by Vos et al. [16] observed the presence of NASH and fibrosis in 61% and 55% of the lean group, respectively. Relative to healthy controls, these individuals were less insulin sensitive and had higher triglyceride levels. However, these observations were based on a BMI cut-off < 30 kg/m 2 for lean individuals and a relatively small sample size (31 "lean" and 48 obese individuals with NAFLD and eight healthy controls), thereby, limiting the conclusions to be drawn.
A survey of available studies indicates that, in general, lean individuals with NAFLD have a more favorable metabolic profile compared to those with a higher BMI ( Table 2). Measures of metabolic markers such as waist circumference, triglyceride levels, fasting plasma glucose, HDL-C (high-density lipoprotein-cholesterol), adiponectin levels, and Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) appear to be intermediate between lean individuals without NAFLD and those with both NAFLD and obesity ( Table 2). Many studies report a male dominance and younger age relative to the non-lean group. Multivariate analysis of data from the National Health and Nutrition Examination Survey III (NHANES III) showed that lean individuals with NAFLD were more commonly Hispanic with T2D and hypertension compared to lean control individuals without liver disease, but relative to individuals with overweight or obesity, NAFLD in lean individuals was independently associated with younger age, female sex, and a lower prevalence of insulin resistance and hypercholesterolemia [26]. Among NAFLD patients living in Olmstead County (Minnesota), female predominance (66.9%) and a higher proportion of Asian and African American individuals (13.2%) were found in lean individuals compared to overweight (47.2%; 6.5%) and obese (56.1%; 7.4%) groups [27]. Feldman et al. [28] reported lower serum levels of lysophosphatidylcholines and phosphatidylcholines and higher levels of glutamate in lean individuals with NAFLD relative to lean healthy controls, but no differences in level of physical activity or frequency of fast-food consumption were observed between the two weight groups. In this study, lean individuals with NAFLD had levels of severely impaired glucose tolerance almost identical to those in found in NAFLD patients with obesity, and ~ 30% were diagnosed with T2D. Glucose homeostasis thus appears to be abnormal in some lean subjects with NAFLD, confirming data suggesting that the accumulation of liver fat may be of particular importance to the development of insulin resistance and diabetes even in the absence of obesity [28].
Despite the better metabolic profile generally observed in lean individuals with NAFLD relative to those with obesity, the risk of disease progression to NASH is comparable to that experienced in NAFLD patients with overweight or obesity. In 56 Greek subjects with liver biopsy-documented NAFLD, the severity of inflammation and fibrosis did not differ between weight groups and NASH prevalence in lean individuals was 50% (compared to 68.8% in the non-lean NAFLD group) [29]. Another study found that 42% of lean Italian NAFLD patients had NASH, of which 42.3% had a fibrosis score of 2 or higher [30]. In lean Austrians with NAFLD, rates of portal inflammation, lobular inflammation, hepatocyte ballooning, perisinusoidal and periportal fibrosis, and NASH were similar between lean and non-lean groups [31,32], although the proportion of lean individuals with cirrhosis was significantly higher compared to non-lean groups (11% vs. 2-3%) [32]. In NHANES III participants, individuals with normal BMI had the same risk of cirrhosis and decompensation, malignancy, and cardiovascular h Recruited from population of apparently healthy individuals or health checkup program    events as those in the overweight and obese categories, indicating that in these individuals, a normal BMI does not confer protection against progression or severity of liver dysfunction in the context of NAFLD [27].
In contrast, a multinational study found that lean subjects with NAFLD had significantly less steatosis, lobular inflammation, ballooning, and advanced liver fibrosis compared to the non-lean group, although 50% and 10% of lean individuals displayed mild/moderate fibrosis and advanced fibrosis, respectively [33]. Findings from metaanalyses studies were in agreement with a more favorable metabolic profile and milder disease progression in lean individuals with NAFLD. Sookoian and Pirola [34] reported that lean NAFLD patients showed less severe histological features than NAFLD patients with overweight or obesity and were less likely to have NASH. However, 33% of lean individuals with NAFLD had NASH, and it should be noted that in this analysis, all but one of the studies in Asian populations failed to use the BMI cut-off point of < 23 kg/m 2 , and therefore, included overweight individuals in the estimates. Shi et al. [21] found that lean and nonobese NAFLD patients were predominantly male and had a significantly lower rate of hypertension, lower waist circumference, lower levels of fasting plasma glucose, triglycerides, and uric acid, and higher levels of HDL-C compared to NAFLD patients with obesity. No significant differences were observed between these two groups with respect to diabetes prevalence, age, and levels of total cholesterol and low-density lipoprotein-cholesterol (LDL-C), suggesting that lean/ nonobese NAFLD patients may have a risk for developing metabolic diseases similar to NAFLD patients with obesity. Alam et al. observed that lean and non-lean NAFLD patients had similar characteristics and shared common risk factors [35].

Outcomes and mortality
Few studies have investigated differences in outcomes and mortality between lean and non-lean individuals with NAFLD (Table 2). Lean Swedish patients with NAFLD, despite a better prognostic profile at baseline, including a lower prevalence of NASH and advanced fibrosis, showed an increased risk for development of severe liver disease during follow-up compared to patients with a higher BMI, even after adjustment for age and fibrosis stage at baseline [36]. This unexpected finding may indicate that the lean individuals in this study experienced a faster rate of fibrosis progression relative to patients with a higher BMI. In a cohort of 1339 NAFLD patients from Australia, Italy, Spain, and the United Kingdom, followed for a median period of 7.6 years, new onset diabetes, cardiovascular events, extrahepatic cancers, liver-related events, and hepatocellular carcinoma (HCC) occurred in 6.2%, 7.3%, 4.7%, 8.9%, and 1.0% of lean individuals, respectively [33]. The incidence of these complications, as well as overall survival, were not significantly different between normal and high BMI groups.
Using data from the NHANES III with a median follow-up period of 17.8 years, Golabi et al. [37] reported that the weighted, unadjusted all-cause mortality was significantly higher in lean individuals with NAFLD compared to lean individuals without NAFLD (40.9% vs. 17.9%, P < 0.001). In lean NAFLD patients, the unadjusted hazard ratio (HR) for all-cause mortality was 2.44 (95% CI 1.77-3.37), which remained significant after adjusting for demographic variables, metabolic components, and primary comorbidities. Likewise, weighted unadjusted cardiovascular mortality was also significantly higher in lean individuals with NAFLD (15.1% vs. 3.7%, P < 0.001), showing a 238% increased risk of cardiovascular mortality, following adjustment. In 299 Austrian NAFLD patients (38 lean, 165 overweight, and 93 obese) over a follow-up period of 8.4 years, lean individuals had a lower overall mortality compared to overweight patients, but a mortality rate similar to NAFLD patients with obesity [32]. Notably, in this population, lean patients had a significantly higher mortality rate from liver-related causes compared to the overweight (11% vs. 4%) and obese groups (11% vs. 4%). In NAFLD patients from Olmstead County, the normal BMI group had a higher risk of death relative to the high BMI group [27]. The most common causes of death in the normal BMI group were malignancy (25.7%), cardiovascular event (21.6%), and infection (13.5%). In contrast to the findings reported by Feldman et al. [32], who reported significantly higher numbers of fatal liver-related events in the lean BMI group compared to higher BMI groups, mortality due to hepatic events was significantly lower in the normal BMI group (1.4%) compared to the obese BMI group (10.4%), but similar to the overweight BMI group (2.0%), and no significant differences in mortality from cardiovascular events or malignancy were observed among the three groups. A recent study by Zou et al. [38] found that lean NAFLD patients had the highest 15-year cumulative allcause mortality (76.3%) compared to nonobese NAFLD patients (51.7%), NAFLD patients with obesity (27.2%), and individuals without NAFLD (20.7%). The analysis revealed similar patterns related to cardiovascular disease (16.9% vs. 5.6% vs. 8.8%, respectively, P = 0.0013).
Combined, these studies indicate that despite lower adiposity, less severe dyslipidemia, and lower levels of hepatic transaminases, lean individuals with NAFLD are at similar or greater risk as those with higher BMI for cardiovascular disease, malignancy, progressive liver disease, and increased all-cause mortality associated with NAFLD. The reason(s) for this relative increase in risk has

Possible causes of NAFLD in lean individuals
Recent epidemiological and clinical studies have identified a number of factors that may contribute to the development of NAFLD in the absence of excess adiposity (Fig. 1). The main classes of these factors include environmental determinants, of which the role of dietary composition has been the best studied, genetic susceptibility, endocrine dysfunction, and metabolic derangement. Some of these factors are known to interact with one another to modulate NAFLD risk, oftentimes in the presence of increasing visceral adiposity, regardless of BMI, suggesting a common metabolic pathway that underlies NAFLD development in all individuals regardless of body habitus.
In particular, regular consumption of sugar-sweetened beverages is strongly associated with NAFLD in adults [39,[45][46][47] and children [48]. In adults, soft drink consumption predicts NAFLD, even following adjustment for dietary composition and physical activity level [47]. Soft drink consumption in NAFLD patients with no risk factors for metabolic syndrome was three times higher than in healthy controls and was significantly associated with the presence of fatty liver [49].
Fructose is a monosaccharide that, together with glucose, forms sucrose (table sugar). Regular dietary fructose consumption induces hepatic de novo lipogenesis and endoplasmic stress, impairs fatty acid oxidation, depletes beneficial microbial species in the gut [50], and promotes hepatic inflammation through the generation of both uric acid and gut-derived endotoxins [51][52][53][54][55]. Due to the constellation of these metabolic effects, dietary fructose may indirectly promulgate hepatic insulin resistance, an important factor in the development of NAFLD [52]. The relationship between dietary fructose and NAFLD is an area of active investigation [56][57][58][59][60][61][62][63]. For example, in adults with NAFLD, daily fructose consumption was associated with higher fibrosis stage [64], while in children, high fructose intake was independently associated with NASH [65].
Thus far, dietary interventions for NAFLD have been limited to non-lean individuals. In a comparison of fatand carbohydrate-restricted diets in adults with obesity and NAFLD, only the latter led to significant reductions in hepatic fat fraction [66]. Interestingly, the carbohydrate-restricted group also exhibited significantly greater reductions in insulin resistance, abdominal fat mass, and body fat mass compared to the fat-restricted group [66]. Eight weeks of sugar restriction in adolescent males with NAFLD yielded significant decreases in hepatic fat fraction, mean body weight, and mean levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transferase (GGT), and total cholesterol [67]. Reduced liver fat was independent of changes in weight or measures of adiposity [67]. Similarly, isocaloric substitution of starch for sugar over a period of 9 days resulted in reduced levels of liver fat, visceral adipose tissue, fractional de novo lipogenesis, and insulin resistance in children with obesity who reported habitually high (> 50 g/day) sugar consumption [68]. The strongest effects of a low fructose, low glycemic index, and low glycemic load diet on metabolic parameters were observed in children with NASH, who also had the highest reported dietary intakes of fructose at baseline, compared to children with NAFLD, and healthy controls [69]. Consistent with these results, current EASL guidelines now recommend a Mediterranean diet with avoidance of processed foods and added fructose for individuals with NAFLD [3].
Some dietary components have been associated with NAFLD in lean individuals. Deprivation of choline, an essential nutrient for human health [70], for 3 weeks in healthy male volunteers resulted in increased ALT activity [71]. Extended depletion of choline for up to 42 days resulted in significant liver dysfunction in men and postmenopausal women [72][73][74]. Postmenopausal women from the NASH Clinical Research Network study with low dietary choline intake had worse fibrosis (OR 3.37; p = 0.002) after adjusting for age, race, obesity, triglycerides, alcohol, and steroid use [75]. In normal-weight Chinese women, higher dietary choline intake was associated with a lower risk of NAFLD [76]. The Adequate Intake (AI) for choline is 550 mg/day for men and 425 mg/day for women, although analyses of dietary patterns reveal that the vast majority of individuals do not achieve these levels [76][77][78]. Because choline is present at more abundant levels in animal-derived foods, vegetarians and vegans may have a greater risk of deficiency, and therefore a higher risk of NAFLD due to inadequate intake [77,78].
In the choline biosynthesis pathway, phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the formation of phosphatidylcholine, which is required for the secretion of VLDL (very low-density lipoproteincholesterol) in the liver [79]. PEMT −/− mice fed a diet high in fat and sucrose rapidly develop hepatic steatosis, inflammation, and fibrosis; dietary choline supplementation ameliorated liver damage in these animals [80][81][82][83][84]. In humans, hepatic PEMT expression was observed to be lower in NASH patients compared to NAFLD patients, and was significantly correlated with platelet counts, which decline in tandem with progression of fibrosis [80]. Lower hepatic PEMT expression was also associated with lower BMI [80], in keeping with the demonstration that PEMT −/− mice are protected from high fat diet-induced obesity and insulin resistance [84,85]. Because the PEMT gene is regulated by estrogen [86,87], the detrimental impact of choline deficiency may be exacerbated following the menopausal transition, consistent with findings from choline depletion [73,88] and observational studies [75]. Together, findings from human and mouse studies suggest that choline deficiency, whether due to low PEMT expression or inadequate dietary intake, may be related to the development of NAFLD and a susceptibility toward progressive disease specifically in lean individuals.
Other environmental factors that may modulate NAFLD include alcohol consumption and cigarette smoking. The diagnostic criteria for NAFLD requires the absence of significant alcohol consumption, although there is not yet a global consensus among professional societies on what level is considered "significant". In the United States, alcohol limits for men and women are 294 g/week and 196 g/week, respectively [89], while in Europe and Asia, limits are 210 g/week (men)/140 g/week (women) [3] and 140 g/week (men)/70 g/week (women) [3,90,91], respectively. However, a retrospective study using prospectively recorded data found that French NAFLD patients who consumed more than 7 units/week (i.e., 56 g) experienced a significantly higher mortality than those consuming less, and conversely, alcohol consumption of less than 1 unit/week (8 g) was significantly associated with improved survival in NAFLD patients [92]. In the NAFLD population, alcohol consumption in excess of 7 units of alcohol/week significantly increased the probability of death from cardiovascular causes and was associated with a significantly higher rate of overall complications, as well as cardiovascular-and cancerrelated complications [92]. Moderate alcohol consumption (30 g/day for men and 20 g/day for women) has also been associated with worse fibrosis in NAFLD [93]. Other large and multiethnic studies have likewise reported a significant relationship between low-to-moderate alcohol consumption and increased mortality and morbidity risk [94][95][96]. Interestingly, alcohol consumption worsens liver disease in individuals with obesity. A BMI > 30 kg/m 2 was found to double the hepatoxicity of alcohol [97] and synergistically increase the risk of HCC [98]. The role of modest alcohol consumption and NAFLD in lean individuals has not yet been investigated.
Cigarette smoking is associated with NAFLD onset [99], progression to fibrosis, and increased risk for severe liver disease [100][101][102]. In Chinese men with a history of heavy smoking and moderate alcohol consumption, NAFLD risk was 85% higher compared to individuals who neither drank nor smoke [103].

Genetic susceptibility
Obesity is the strongest independent risk factor for NAFLD, even after adjusting for age, sex, total cholesterol, triglycerides, HDL, LDL, glucose, uric acid, homocysteine, creatinine, AST, ALT, and hypertension [11]. However, even in the presence of severe obesity and the corresponding chronic caloric excess, some individuals do not develop NAFLD. Conversely, the relationship between risk of NAFLD and BMI is J-shaped, with risk increasing below BMI of 19 from a nadir [11]. These data suggest that there are putative NAFLD susceptibility and/ or protective factors that can modify the effects of BMI, particularly genetic variation [104]. The single nucleotide variant (SNV) resulting in the I148M substitution (rs738409) in the patatin-like phospholipase domaincontaining protein 3 gene (PNPLA3) is the major genetic risk factor for NAFLD known to date [105]. In addition to PNPLA3, variants in MBOAT7 (membrane-bound O-acyltransferase domain-containing 7) and TM6SF2 (trans-membrane 6 superfamily antigen 2) have been associated with NAFLD [106]. Most genetic association studies have been performed in individuals with classical obesity-related NAFLD and few data are available from lean populations. In a study of 904 community dwelling Japanese participants, in whom the prevalence of NAFLD was 12.4%, 41.4% and 59.1% in lean, overweight, and obese groups, the PNPLA3 rs738409 risk genotype (GG) increased NAFLD risk in lean subjects by more than twofold compared with overweight and obese participants [107]. No differences in risk were found for the MBOAT7 or TM6SF2 NAFLD risk alleles after stratifying by BMI. In a study of 187 Austrian participants, Feldman et al. [28] observed a higher rate of PNPLA3 risk alleles in lean individuals with NAFLD compared with the lean control group, with a frequency comparable to NAFLD patients with obesity. In addition, a significantly greater proportion of lean individuals with NAFLD carried the rs58542926 risk allele (4%), relative to non-lean NAFLD patients (0.3%). In Italian patients with lean NAFLD, the only variable associated independently with NASH and a fibrosis score ≥ 2 was the presence of the rs738409 (PNPLA3) risk allele [30]. The rs738409 risk genotype was also associated with NAFLD in lean individuals in a Sri Lankan population [108] and nonobese Japanese subjects [109]. A recent study found the highest NAFLD risk increase among carriers of the rs738409 risk genotype in 529 lean subjects (OR 6.04, 95% CI [2.62, 13.91]), compared with 162 and 213 individuals with overweight (OR 3.43, 95% CI [1.06, 11.14]) and obesity (OR 2.51, 95% CI [0.93, 6.78]), respectively [110]. Other groups have not found evidence for statistically significant differences for NAFLD risk alleles in PNPLA3 and TM6SF2 between weight groups [31,111].
A risk genotype (AA) at the V175M variant (rs7946) in PEMT, which results in a partial loss of PEMT activity, was 1.7-fold higher in individuals with NAFLD compared to normal controls [112]. Additional PEMT variants (rs12325817, rs4646343, and rs3761088) were associated with developing liver dysfunction in response to a choline-depleted diet [88]. Two other PEMT variants, rs1531100 and rs4646365, increased liver damage risk in postmenopausal women [88]. Variants in genes from the choline biosynthesis pathway, namely choline kinase A, moderated the effects of a low choline diet [88], while a genetic signature comprised of variants in the choline and 1-carbon metabolism pathways were associated with severity of hepatic steatosis [113]. Using an unbiased exome sequencing approach in a discovery set of two lean NAFLD patients and six lean controls, only rs7946 (PEMT) and rs2290532 in oxysterol-binding protein-related protein 10 (OSBPL10) were found to be associated with NAFLD [114]. Genotyping in a validation cohort of 191 lean individuals with NAFLD and 105 lean controls revealed a threefold higher risk of NAFLD in carriers of the rs7946 risk genotype, but no significant differences were found for the OSBPL10 variant.
Lipodystrophies are a group of heterogeneous rare genetic disorders characterized by the common phenotype of deficient adipose tissue without nutritional deprivation or increased metabolism [115]. The inability to store lipids as fat leads to several adverse complications including NAFLD and liver fibrosis, which can lead to cirrhosis. Pathogenic variants in several genes can cause familial partial lipodystrophies including peroxisome proliferator-activated receptor gamma (PPARG ), lamin A/C (LMNA), perilipin 1 (PLIN1), hormone-sensitive lipase (LIPE), cell death-inducing DFFA-like effector C (CIDEC), and Akt murine thymoma viral oncogene homolog 2 (AKT2) [116]. Hepatic steatosis is an almost universal finding in these individuals, suggesting that NAFLD in lean individuals may be a type of ectopic fat deposition similar to lipodystrophy. Genetic evidence supports such a mechanism. A polygenic risk score associated with insulin resistance and decreased adiposity in the lower extremities, both of which are features of lipodystrophy, has been reported [117]. Subsequently, Chen et al. [118] determined that the lipodystrophy polygenic risk score was associated with NAFLD, increased liver fibrosis, and decreased lower extremity fat mass.

Other factors
NAFLD can develop against the backdrop of endocrine disturbances, often by exacerbating hormone-related metabolic alterations. For example, women are at high risk of developing NAFLD and NASH following the menopausal transition [119], likely due to the loss of protection conferred by estrogens, in combination with subclinical disturbances in metabolic parameters prior to menopause, such as mild to moderate levels of adiposity, dyslipidemia, and impaired glucose tolerance. Hyperandrogenism is also associated with hepatic steatosis and fibrosis in women, independent of insulin resistance and adiposity [120,121], although increased circulating testosterone levels in middle-aged women are associated with higher visceral adiposity [122]. Hyperthyroidisminduced NAFLD is regarded as a distinct disease entity [123] and thyroid hormone supplementation improves liver dysfunction [124]. To date, no studies have specifically focused on the role of endocrine factors in NAFLD risk in lean individuals, although many changes in hormonal levels are accompanied by increasing visceral adiposity, which yields ramifications for NAFLD susceptibility, even in lean individuals.
There is some evidence suggesting that high dietary fat or fructose intake in animals can synergistically enhance the effects of estrogen deficiency, leading to exaggerated effects on hepatocellular injury [125,126]. Similarly, interactions between choline deficiency and hormonal status may modulate NAFLD risk. For example, postmenopausal women had significantly worse fibrosis compared to premenopausal women, although both groups had similarly low levels of choline intake [75]. As noted above, reduced endogenous production of estrogen results in diminished PEMT expression, which may lead a greater susceptibility to the development of NAFLD in postmenopausal women with chronic states of choline deficiency [86,87].

Screening and clinical management of NAFLD in lean individuals: outstanding questions
Despite the prevalence and adverse outcomes associated with NAFLD in normal weight individuals, there are no global consensus guidelines for NAFLD screening, nor is screening in the general population recommended by any professional societies. As shown in Table 3, practice guidance statements developed by the American Association for the Study of Liver Diseases (AASLD), and intended for use by physicians and other health professionals [89], do not recommend routine screening for NAFLD in high-risk groups (i.e., T2D or obesity) due to the uncertain evidence supporting diagnostic tests, treatment options, and the long-term benefits or costeffectiveness of screening. Some specialists in the United States recommend screening individuals at risk of developing liver disease, such as those older than 50 years and with T2D or metabolic syndrome, using liver function tests and abdominal ultrasound in a primary care setting and imaging or prediction algorithms to assess the presence of fibrosis and subsequent diagnosis of NASH and staging of fibrosis [132].
A review of current international guidelines has recently been published [133]. In contrast to the AASLD, European and Asian guidelines recommend that screening be considered for groups considered at risk for developing NAFLD, including patients with obesity and T2D [3,90,91]. With respect to lean individuals, guidelines for NAFLD screening become less clear. Many guidelines acknowledge the importance of NAFLD in lean individuals, especially those of Asian ancestry or who exhibit features of metabolic syndrome [3,90,134]. The development and distribution of consistent screening and risk assessment guidelines will be critical to ensure optimal clinical management for all Table 3 Summary of AASLD practice guidelines for the screening, evaluation, and treatment of NAFLD [89] Screening Routine Screening for NAFLD in high-risk groups attending primary care, diabetes, or obesity clinics is not advised because of uncertainties surrounding diagnostic tests and treatment options, along with lack of knowledge related to long-term benefits and cost-effectiveness of screening There should be a high index of suspicion for NAFLD and NASH in patients with type 2 diabetes Systematic screening of family members for NAFLD is not recommended Evaluation Patients with unsuspected hepatic steatosis detected on imaging who have symptoms or signs attributable to liver disease or have abnormal liver chemistries should be evaluated as though they have suspected NAFLD and worked up accordingly Patients with incidental hepatic steatosis detected on imaging who lack any liver-related symptoms or signs and have normal liver biochemistries should be assessed for metabolic risk factors (e.g., obesity, diabetes mellitus, or dyslipidemia) and alternate causes for hepatic steatosis, such as significant alcohol consumption or medications When evaluating a patient with suspected NAFLD, it is essential to exclude competing etiologies for steatosis and coexisting common chronic liver disease In patients with suspected NAFLD, persistently high serum ferritin, and increased iron saturation, especially in the context of homozygote or heterozygote C282Y HFE mutation, a liver biopsy should be considered High serum titers of autoantibodies in association with other features suggestive of autoimmune liver disease (> 5 upper limit of normal aminotransferases, high globulins, or high total protein to albumin ratio) should prompt a work-up for autoimmune liver disease Initial evaluation of patients with suspected NAFLD should carefully consider the presence of commonly associated comorbidities such as obesity, dyslipidemia, insulin resistance or diabetes, hypothyroidism, polycystic ovary syndrome, and sleep apnea Treatment Weight loss generally reduces hepatic steatosis, achieved either by hypocaloric diet alone or in conjunction with increased physical activity. A combination of a hypocaloric diet (daily reduction by 500-1000 kcal) and moderate-intensity exercise is likely to provide the best likelihood of sustaining weight loss over time Weight loss of at least 3-5% of body weight appears necessary to improve steatosis, but a greater weight loss (7-10%) is needed to improve the majority of the histopathological features of NASH, including fibrosis Pharmacological treatments, such as pioglitazone and Vitamin E, aimed primarily at improving liver disease should generally be limited to those with biopsy-proven NASH and fibrosis. Bariatric surgery can be considered in otherwise eligible obese individuals with NAFLD or NASH NAFLD patients [133]. Knowledge of disease etiology, screening, detection methods, and consensus guidelines are becoming increasingly important for adequate clinical care of both lean and obese NAFLD patients, especially for primary care physicians, who are the providers in the best position to make an initial diagnosis.
Most of the guidelines do not directly address screening and treatment of NAFLD in lean individuals. There are thus many questions that arise when considering the screening and clinical management of NAFLD in this patient population. For example, is visceral adiposity, rather than the overall amount of body fat, more relevant for NAFLD pathogenesis in lean individuals than it is in those with higher BMI? If so, are there better alternatives to the use of BMI as a marker for adiposity for NAFLD screening? Some investigators have argued that waist circumference is a more accurate representation of body fat distribution and a better method with which to identify individuals at higher risk of developing cardiometabolic disease [135]. However, despite the relative simplicity and low financial cost, implementation of waist circumference measurements as a standard measure of adiposity in primary care faces systemic obstacles, and in many settings, would require problematic process reconfiguration. However, obtaining waist circumference measurements may be clinically significant for lean individuals, who, despite having a normal BMI, may have some degree of visceral adiposity and consequently, an increased risk for NAFLD.
A major question is whether NAFLD in lean individuals represents a distinct clinicopathological entity requiring specific management, as suggested by many researchers [16,27,31,32,36,136], or is it a sub-phenotype of classical obesity-associated NAFLD that will respond to the current approach of weight loss and control of insulin resistance, hypertension, and hyperlipidemia [17]? Certainly, many of the same factors that increase susceptibility to NAFLD are shared between normal weight and overweight individuals. Further, even among normal weight individuals, those with NAFLD appear to have slightly worse metabolic features. For example, Kim et al. [14] observed significant differences in sex, waist circumference, triglyceride level, and logarithm HOMA-IR between normal weight subjects with and without NAFLD. These differences in clinical and laboratory measures between normal weight individuals with or without NAFLD were comparable to those observed between overweight individuals with or without NAFLD, suggesting that in this cohort, NAFLD in lean individuals is a clinical entity similar to obesity-related NAFLD [14]. However, the risk of insulin resistance, hypertriglyceridemia, hyperuricemia, and central obesity in NAFLD patients compared with those without NAFLD was higher in lean individuals than those with overweight.
Lean individuals with NAFLD have also been found to respond to diet and lifestyle modifications typically utilized in the treatment of obesity-related NAFLD. In one study, loss of only 5% of initial body weight was demonstrated to result in remission of NAFLD in 75% of individuals [137]. Likewise, a 5% reduction in weight in 35 NAFLD patients (14 lean and 21 with obesity) yielded significant improvements in ALT and AST levels, hepatic steatosis, and liver stiffness [138]. In this intervention, NAFLD was resolved in 57.1% of lean individuals [138]. Combined, these data suggest that lifestyle modifications and weight loss are appropriate to reduce NAFLD, at least in some lean individuals. More research to determine whether reductions in central obesity, through a nutritional regimen and exercise, are appropriate therapeutic approaches in lean individuals with NAFLD.
It will also be important to determine whether therapeutics under investigation for classical obesity-related NAFLD will also be effective in lean NAFLD patients. Clinical trials addressing the potential effectiveness of drugs such as SGLT2 (sodium-glucose transport protein 2) inhibitors, GLP-1 (glucagon-like peptide 1) receptor agonists, obeticholic acid, pioglitazone, or saroglitazar in lean individuals with NAFLD are urgently needed [139].
Despite the similarities in NAFLD across the BMI spectrum, there may be cases in which NAFLD in lean individuals represents a distinct disease entity, and here, interventions that specifically address the pathophysiological triggers must be developed and tested. In these cases, the etiology and pathogenesis of NAFLD may inform the most appropriate treatment strategy. More studies to identify potential genetic factors that specifically contribute to NAFLD without obesity (or are masked by the presence of obesity) and uncover interactions with lifestyle factors that modulate their impact would provide a deeper understanding of disease risk in lean individuals. The role of various dietary factors or specific macronutrient composition as significant contributors to NAFLD risk in lean individuals remains largely unexplored. Many research studies have consistently demonstrated a link between liver dysfunction and choline deficiency, which has the unusual phenotype of resistance to diet-induced obesity, but are there other micronutrients that contribute to NAFLD, and if so, do they interact with functional genetic variants, as observed between choline and PEMT SNVs? Finally, there may be additional, as-yet unknown environmental factors, including herbal supplements, that contribute to the development of NAFLD in lean individuals.
Recent efforts to apply data-driven cluster analysis identified five distinct subtypes of diabetes, showing distinct patient characteristics and differential risk for diabetic complications [140]. This level of stratification of patients with a notably heterogeneous disease may lead to more focused treatment strategies instead of a onesize-fits-all approach, which represents the current state of diabetes care. We envision the application of similar cluster analysis to identify a spectrum of individuals from those who may have relatively mild NAFLD with little chance of progression to those who are on a rapid trajectory to advanced disease with severe complications. Such stratification may also lead to specific treatment strategies.
Going forward it will be important to assess the variance in NAFLD prevalence in lean populations, according to different ancestries, ethnicities, and geographies, and determine risk factors that may be more important for some groups than for others. Knowledge of the longterm consequences of NAFLD in the lean and the rate and severity of progression to NASH compared to classical obesity-associated NAFLD will also be important for the development of precise treatment strategies.

Conclusions
It is not uncommon for lean individuals to develop NAFLD and NASH, despite a healthier metabolic phenotype than that observed in classical obesity-related NAFLD. We posit that NAFLD develops in lean individuals due to a distinct array of contributing etiologies, including dietary composition, genetic susceptibility, and hormonal status. In the absence of suspicious laboratory findings, lean individuals are not likely to be screened for NAFLD, nor for metabolic diseases associated with NAFLD. Awareness of menopausal status, genetic factors, ethnicity, dietary factors (especially added sugar, refined carbohydrates, and saturated fat/cholesterol), choline deficiency, and alcohol consumption patterns may be of value in assessing NAFLD risk in lean individuals.
Much more work is needed not only to address the questions raised above, but also to promote greater awareness among practitioners about the potential health risks associated with NAFLD in lean individuals. Efforts aimed at the development of screening algorithms that are less dependent on BMI and hepatic transaminase levels, implementation of more precise treatment strategies based on underlying pathoetiology, and inclusion of lean individuals in NAFLD-related clinical trials are necessary to reduce the burden of NAFLD in this patient group. Further, additional studies to characterize the lean NAFLD population and identify factors that modulate NAFLD risk in the absence of clinically significant metabolic dysfunction are urgently needed. Finally, recognition that NAFLD in some lean individuals may resemble classical obesity-related NAFLD, while in others, it may represent a distinct clinical entity, provides a foundation by which different strategies for clinical management can be devised. Early detection, combined with the appropriate steps to mitigate NAFLD through lifestyle modifications and clinical interventions, may effectively prevent the progression to NASH in lean individuals.


s2fieldsofstudy: Biology Chemistry

text:
Preclinical Evaluation of a Novel Small Molecule Inhibitor of LIM Kinases (LIMK) CEL_Amide in Philadelphia-Chromosome Positive (BCR::ABL+) Acute Lymphoblastic Leukemia (ALL)

Ph+ (BCR::ABL+) B-ALL was considered to be high risk, but recent advances in BCR::ABL-targeting TKIs has shown improved outcomes in combination with backbone chemotherapy. Nevertheless, new treatment strategies are needed, including approaches without chemotherapy for elderly patients. LIMK1/2 acts downstream from various signaling pathways, which modifies cytoskeleton dynamics via phosphorylation of cofilin. Upstream of LIMK1/2, ROCK is constitutively activated by BCR::ABL, and upon activation, ROCK leads to the phosphorylation of LIMK1/2, resulting in the inactivation of cofilin by its phosphorylation and subsequently abrogating its apoptosis-promoting activity. Here, we demonstrate the anti-leukemic effects of a novel LIMK1/2 inhibitor (LIMKi) CEL_Amide in vitro and in vivo for BCR::ABL-driven B-ALL. The IC50 value of CEL_Amide was ≤1000 nM in BCR::ABL+ TOM-1 and BV-173 cells and induced dose-dependent apoptosis and cell cycle arrest in these cell lines. LIMK1/2 were expressed in BCR::ABL+ cell lines and patient cells and LIMKi treatment decreased LIMK1 protein expression, whereas LIMK2 expression was unaffected. As expected, CEL_Amide exposure caused specific activating downstream dephosphorylation of cofilin in cell lines and primary cells. Combination experiments with CEL_Amide and BCR::ABL TKIs imatinib, dasatinib, nilotinib, and ponatinib were synergistic for the treatment of both TOM-1 and BV-173 cells. CDKN2Ako/BCR::ABL1+ B-ALL cells were transplanted in mice, which were treated with combinations of CEL_Amide and nilotinib or ponatinib, which significantly prolonged their survival. Altogether, the LIMKi CEL_Amide yields activity in Ph+ ALL models when combined with BCR::ABL-targeting TKIs, showing promising synergy that warrants further investigation.

Introduction
Acute lymphoblastic leukemia (ALL) results from leukemic transformation of lymphoid B-and T-lineage precursors caused by multiple genetic events, including chromosomal aberrations, gene mutations, and rearrangements [1]. In contrast to other subtypes 2 of 11 of ALL, including the more frequent pediatric patients, the incidence of the Philadelphia chromosome (Ph+)-positive B-ALL (BCR::ABL+) increases with age in up to 50% of patients and was considered to signal a poor prognosis before tyrosine kinase inhibitors (TKI) targeting BCR::ABL were combined with classical backbone chemotherapy [2,3]. Even strategies without classical chemotherapy have been tested using combinations of TKI and bispecific and conjugated antibodies [4,5]. Nevertheless, relapsed and refractory disease remains frequent in the elderly and new treatment strategies are needed.
The inhibition of kinases in the signaling pathway acting downstream of BCR::ABL+ would be of particular interest for relapsed and resistant disease caused by mutations of the BCR::ABL kinase domain and subsequent clonal evolution [6,7]. It was demonstrated that Rho kinases (ROCK) were constitutively activated in BCR::ABL-transformed cells in a way that was dependent on the BCR::ABL-induced activation of PI3 kinase and RhoGT-Pase [8]. As a result of pharmacologic inhibition of ROCK, BCR::ABL-mediated aberrant signaling was disrupted and inhibited cell growth of BCR::ABL+ cell lines, even those bearing the T315I mutation that confers resistance to all available TKIs, except ponatinib [8]. Nevertheless, no ROCK inhibitors have entered clinical trials so far.
LIMK1 and LIMK2 are two isoforms of serine/threonine kinases and their activation is caused by phosphorylation at threonine 508 by ROCK [9,10]. The downstream targets of LIMK1 and LIMK2 are members of factors of depolymerisation of the actin-(ADF) family called cofilin [11]. In its inactivated state, cofilin is phosphorylated at serine 3 by LIMK1 or LIMK2, and dephosphorylation caused by inhibiting LIMK1/2 leads to the activation of cofilin, and consequently to actin reorganization and mitochondrial apoptosis in cancer cells [12,13]. The precise mechanisms that links LIMK inhibition to apoptosis are not yet fully elucidated. The inhibition of LIMK leads indirectly to mitochondrial apoptosis, as activated LIMK inactivates cofilin by phosphorylation. As a result of the inactivation of LIMK, cofilin is activated by dephosphorylation, which causes mitochondrial translocation of cofilin, leading to mitochondrial apoptosis characterized by cytochrome c release and subsequent activation of caspase-3 [13,14]. For pediatric t(8;21) RUNX1-RUNX1T1 AML, it was shown that overexpression of the Ras homolog family member (RHOB) correlated with resistance to chemotherapy and relapse [15]. RHOB re-organizes the actin cytoskeleton by activating the ROCK-LIMK-cofilin pathway, which increases blast adhesion with stress fiber formation. This leads to reduced mitochondrial apoptotic cell death after exposure to chemotherapeutic agents. Furthermore, LIMK inhibition leads to centrosome fragmentation caused by the disruption of proper kinetochore-microtubule interactions at the onset of anaphase [16]. This leads to impaired Aurora-A kinase activation, resulting in inhibition of cell growth and apoptosis in leukemic cells.
ROCK phosphorylation is also triggered by FLT3-ITD mutations in AML. Based on this rationale, we recently reported preclinical activity of a highly specific small molecule LIMK1 and LIMK2 inhibitor (LIMKi) CEL_Amide in FLT3-ITD+ cell lines and in a xenograft mouse model [17]. LIMKi had antiproliferative effects in FLT3-ITD+ cells and decreased constitutive phosphorylation of cofilin. LIMKi yielded synergy with different FLT3-ITD+ inhibitors in vitro and in vivo. Notably, exposure to CEL-Amide had no significant toxicity for CD34+ cells obtained from healthy donors, which is encouraging for future potential of clinical testing [17].
Here, we show preclinical activity of LIMKi CEL_Amide in different BCR::ABL+ models: CEL_Amide had antiproliferative effects in BCR::ABL+ cells and in vivo activity in a CDKN2A ko /BCR::ABL1+ mouse model that abrogated the inactivating phosphorylation of cofilin. Furthermore, CEL_Amide synergized with commercially available TKIs and backbone chemotherapy, delineating a novel treatment strategy for BCR::ABL+ ALL.

Compounds
CEL_Amide (632.1 g·mol −1 as sodium salt) was donated from Cellipse. It was resuspended in water as a 5 mM stock solution and stored at −20 • C. Imatinib was diluted in DMSO as a 6 mM stock solution. Dasatinib and nilotinib were diluted in DMSO as a 50 mM stock solution. Vincristin was resuspended in DMSO as a 100 mM stock solution. Dexamethasone and ponatinib were resuspended in DMSO as a 150 mM stock solution. The drugs were obtained from Clinisciences (Nanterre, France), except ponatinib, which was obtained from Euromedex (Souffelweyersheim, France); all were stored at −20 • C.

Cell Lines and Primary Patient Cells
BV-173 and TOM-1 were purchased from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Braunschweig, Germany). Cell lines were grown in RPMI 1640 (Life Technologies, Courtaboeuf, France), supplemented with 20% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 IU/mL penicillin, and 100 µg/mL streptomycin (Dominique Dutscher, Brumath, France), at 37 • C with 5% CO 2 . Patients and healthy donors provided informed consent prior to bone marrow aspiration or blood sample collection, following the Declaration of Helsinki. MNC were obtained after gradient centrifugation with a separation medium for lymphocytes (Eurobio, Courtaboeuf, France).

Quantitative-Real Time Polymerase Chain Reaction (RT-qPCR)
Extraction of total RNA was performed with Maxwell ® RSC simplyRNA kit (Promega, Charbonnières Les Bains, France). Then, RNA was titrated with the NanoDrop 2000 c UV-Vis spectrophotometer (Thermo Fisher Scientific, Courtaboeuf, France) and frozen at −80 • C. Complementary DNA (cDNA) was synthesized from 500 ng RNA with a reverse transcriptase PrimeScriptTM kit (TaKaRa, Ozyme, Saint-Cyr-l'École, France). RT-qPCR was performed for LIMK1 and LIMK2 in 10 µL from one-tenth of the cDNA volume (10 ng RNA), using a thermocycler StepOnePlusTM (Thermo Fisher Scientific, Courtaboeuf, France) with SYBR ® Green reagent (Thermo Fisher Scientific, Courtaboeuf, France) in standard mode (1 cycle of 30 s at 95 • C, followed by 40 cycles of 5 s at 95 • C, then 30 s at 60 • C), with a supplementary melting curve step for SYBR ® Green assays. Primers (Eurogentec, Angers, France) are listed in Supplementary Table S1. GAPDH served as a control gene for normalization of mRNA levels.

MTS Assay, Apoptosis Assessment, and Cell Cycle Analysis
For the MTS assay, cells were incubated in 96-well plates at 20,000 cells per well, and treated with a broad range of CEL_Amide concentrations (78 nM-10 µM) for 72 h. An amount of 20 µL per well of MTS solution (Promega, Charbonnières Les Bains, France) were placed into each well and incubated in the dark at 37 • C for 4 h. Absorbance was read at 490 nm using GloMax Explorer (Promega, Charbonnières Les Bains, France). Three independent experiments were run for each cell line, and untreated cells were used as negative controls. The half maximal inhibitory concentration (IC50) values were calculated with Prism ® v6 software (GraphPad Software LLC, La Jolla, CA, USA).
For apoptosis analysis, 1 × 10 6 cell lines were resuspended in 1 mL culture medium and treated with a range of CEL_Amide concentrations 250-1500 nM or with CEL_Amide 1500 nM, nilotinib 25 nM, or ponatinib 1 nM for 72 h. Cells were stained with 1 µg/mL PI and Annexin-V-FITC (Becton Dickinson, Le Pont de Claix, France), according to the manufacturer's instructions for 15 min at room temperature. Apoptotic cells were defined as Annexin V+ with or without PI uptake.
Cell cycle distribution and apoptosis were determined by cytofluorometric analysis using a CytoFLEX flow cytometer (Beckman Coulter, Villepinte, France) and analyzed with the FlowJo flow cytometry software (FlowJo LLC, Ashland, OR, USA).

Drug-Dose-Response Experiments and Combenefit Analysis
CEL_Amide was combined with different agents, including the TKIs imatinib, dasatinib, nilotinib, ponatinib, and vincristin or dexamethasone. Drugs were added simultaneously to cultured cell lines and relative cell numbers were measured at 72 h with MTS assays. IC50 values were computed with Prism ® v6 software (GraphPad Software LLC, La Jolla, CA, USA). The dose-response matrix was calculated with the Combenefit Software v.2.021 (Cancer Research UK Cambridge Institute, Cambridge, UK) by using the Loewe independence model [18].

Mouse Model
This study was carried out in accordance with the EC Directive 86/609/EEC for animal experiments and was approved by the Committee for Experimental Animal Studies of the University of Paris Institute Board Ethics. Animals were housed and bred at our animal facility (Institut de Recherche Saint-Louis, Saint Louis Hospital, Paris, France).
On day 15, nilotinib or ponatinib treatment was stopped. On day 25, CEL_Amide treatment was stopped. On days 8, 15, 22, and 29, a blood test was performed to measure the GFP percentage by flow cytometry analysis using a CytoFLEX flow cytometer (Beckman Coulter, Villepinte, France) and analyzed with the FlowJo flow cytometry software v.10 (FlowJo LLC, Ashland, OR, USA).

Biological Effects of LIMKi in BCR::ABL+ Cell Lines
As BCR::ABL+ activates the ROCK-LIMK axis, we sought to investigate the sensibility of BCR::ABL+ B ALL to LIMK inhibition [8]. Thus, we determined effects of CEL_Amide in the BCR::ABL+ B-ALL cell lines TOM-1 and BV-173. Cell viability after CEL_Amide exposure to increasing doses was measured with MTS assay in the cell lines. Significant inhibition of cell growth was found in TOM-1 (580 nM) and BV-173 (1090 nM) ( Figure 1A). We also found dose-dependent apoptosis induction upon exposure to increasing doses of CEL_Amide (250-1500 nM) as detected by phosphatidylserine exposure and PI uptake up to 45% (TOM-1) and 50% (BV-173) at 48 h ( Figure 1B). Furthermore, CEL_Amide caused cell cycle arrest in the G1/S transition ( Figure 1C).
As BCR::ABL+ activates the ROCK-LIMK axis, we sought to investigate the sensibility of BCR::ABL+ B ALL to LIMK inhibition [8]. Thus, we determined effects of CEL_Amide in the BCR::ABL+ B-ALL cell lines TOM-1 and BV-173. Cell viability after CEL_Amide exposure to increasing doses was measured with MTS assay in the cell lines. Significant inhibition of cell growth was found in TOM-1 (580 nM) and BV-173 (1090 nM) ( Figure 1A). We also found dose-dependent apoptosis induction upon exposure to increasing doses of CEL_Amide (250-1500 nM) as detected by phosphatidylserine exposure and PI uptake up to 45% (TOM-1) and 50% (BV-173) at 48 h ( Figure 1B). Furthermore, CEL_Amide caused cell cycle arrest in the G1/S transition ( Figure 1C).

LIMK1/2 Are Expressed in BCR::ABL+ Cell Lines and CEL_Amide Inhibits Constitutive Phosphorylation of Cofilin
Furthermore, we measured mRNA expression of the two isoforms LIMK1 and LIMK2 in BV-173 and TOM-1 cell lines. LIMK1 and LIMK2 mRNA were expressed at the same level in the 2 BCR::ABL+ cell lines (Figure 2A). We also found LIMK1 and LIMK2 protein expression in BV-173 and TOM-1 cell lines ( Figure 2B). Upon treatment with CEL_Amide, we observed a dose-dependent decrease in LIMK1 protein expression in both tested BCR::ABL+ cell lines. As inhibition of LIMK1/2 results in activating dephosphorylation of cofilin, we determined total and phosphorylated protein levels of cofilin ( Figure 2B). As expected, we observed a significant decrease in the phosphorylated form of cofilin in BCR::ABL+ cell lines, whereas total protein of cofilin was not modified. We also compared CD34+ enriched cells from three healthy donors with bone marrow blasts derived from four BCR::ABL+ patients and found protein expression of LIMK1 and phosphorylated cofilin in BCR::ABL+ blast cells, indicating constitutively increased cofilin inactivating activity of LIMK1 in patient-derived cells ( Figure 2C). Patient characteristics are summarized in Supplementary Table S2.
Furthermore, we measured mRNA expression of the two isoforms LIMK1 and LIMK2 in BV-173 and TOM-1 cell lines. LIMK1 and LIMK2 mRNA were expressed at the same level in the 2 BCR::ABL+ cell lines (Figure 2A). We also found LIMK1 and LIMK2 protein expression in BV-173 and TOM-1 cell lines ( Figure 2B). Upon treatment with CEL_Amide, we observed a dose-dependent decrease in LIMK1 protein expression in both tested BCR::ABL+ cell lines. As inhibition of LIMK1/2 results in activating dephosphorylation of cofilin, we determined total and phosphorylated protein levels of cofilin ( Figure 2B). As expected, we observed a significant decrease in the phosphorylated form of cofilin in BCR::ABL+ cell lines, whereas total protein of cofilin was not modified. We also compared CD34+ enriched cells from three healthy donors with bone marrow blasts derived from four BCR::ABL+ patients and found protein expression of LIMK1 and phosphorylated cofilin in BCR::ABL+ blast cells, indicating constitutively increased cofilin inactivating activity of LIMK1 in patient-derived cells ( Figure 2C). Patient characteristics are summarized in Supplementary Table S2. 3.3. BCR::ABL-Targeting TKIs Synergize with CEL_Amide for Treatment of BCR::ABL+ ALL Cell Lines As BCR::ABL-targeting TKIs, used with chemotherapy and corticosteroids, are the standard of care of BCR::ABL+ B-ALL, we were interested to test combinations of CEL_Amide with commercially available TKIs, including imatinib, dasatinib, nilotinib, and ponatinib, in BV-173 and TOM-1 cell lines. First, we tested the impact of nilotinib and ponatinib on protein expression of LIMK1 and phospho-cofilin and both TKIs had no effect on pro-tein levels, indicating an absence of off-target effects in combination experiments with CEL_Amide (Supplementary Figure S1). We found significant synergies of all TKIs tested in combination with CEL_Amide for both BCR::ABL+ cell lines ( Figure 3A). Furthermore, we tested indicated fixed doses for nilotinib and ponatinib and CEL_Amide and we observed significant increases in apoptosis for combinations in BV-173 cells from 40-50% up to 60-70% with CEL_Amide and nilotinib, and up to 80-90% for CEL_Amide with ponatinib ( Figure 3B). Combination treatment of CEL_Amide with nilotinib or ponatinib enhanced cell cycle arrest in the G1/S transition in BV-173 and TOM-1 cell lines (Supplementary Figure S2A,B). Interestingly, we also found significant synergies for combination treatments of BV-173 and TOM-1 cell lines by CEL_Amide with either vincristin or dexamethasone (Supplementary Figure S3).

BCR::ABL-Targeting TKIs Synergize with CEL_Amide for Treatment of BCR::ABL+ ALL Cell Lines
As BCR::ABL-targeting TKIs, used with chemotherapy and corticosteroids, are the standard of care of BCR::ABL+ B-ALL, we were interested to test combinations of CEL_Amide with commercially available TKIs, including imatinib, dasatinib, nilotinib, and ponatinib, in BV-173 and TOM-1 cell lines. First, we tested the impact of nilotinib and ponatinib on protein expression of LIMK1 and phospho-cofilin and both TKIs had no effect on protein levels, indicating an absence of off-target effects in combination experiments with CEL_Amide (Supplementary Figure S1). We found significant synergies of all TKIs tested in combination with CEL_Amide for both BCR::ABL+ cell lines ( Figure 3A). Furthermore, we tested indicated fixed doses for nilotinib and ponatinib and CEL_Amide and we observed significant increases in apoptosis for combinations in BV-173 cells from 40-50% up to 60-70% with CEL_Amide and nilotinib, and up to 80-90% for CEL_Amide with ponatinib ( Figure 3B). Combination treatment of CEL_Amide with nilotinib or ponatinib enhanced cell cycle arrest in the G1/S transition in BV-173 and TOM-1 cell lines (Supplementary Figure S2A,B). Interestingly, we also found significant synergies for combination treatments of BV-173 and TOM-1 cell lines by CEL_Amide with either vincristin or dexamethasone (Supplementary Figure S3).

Impact of Combined LIMKi and TKI Treatment on Survival in a BCR::ABL+ Mouse Model
To explore efficacy of the combination of CEL_Amide and BCR::ABL-targeting TKIs in vivo, we next sought to explore the therapeutic potential in a BCR::ABL+ mouse model. C57Bl/6 mice were transplanted with GFP+ CDKN2A ko /BCR::ABL1+ B-ALL cells. Mice were treated with CEL_Amide alone, nilotinib, ponatinib, or a combination of LIMKi and TKI, compared with vehicle-treated mice ( Figure 4A). Untreated mice died from this aggressive leukemia within 11-12 days ( Figure 4B,C). CEL_Amide monotherapy had no impact on survival, whereas nilotinib and ponatinib monotherapy significantly extended mouse survival to 17 and 28 days, respectively (p = 0.0003 and p = 0.0005). Interestingly, combination therapy of CEL_Amide + nilotinib extended survival to 33 days (p = 0.0003) and CEL_Amide + ponatinib to 50 days (p = 0.0005). Leukemic GFP+ cells were scored once weekly in peripheral blood by flow cytometry and mice were considered leukemic if >1% GFP+ cells were detected in peripheral blood. CEL_Amide combined with nilotinib or ponatinib significantly delayed detection of GFP+ BCR::ABL+ leukemic cells in peripheral blood (Supplementary Figure S4A,B).

Impact of Combined LIMKi and TKI Treatment on Survival in a BCR::ABL+ Mouse Model
To explore efficacy of the combination of CEL_Amide and BCR::ABL-targeting TKIs in vivo, we next sought to explore the therapeutic potential in a BCR::ABL+ mouse model. C57Bl/6 mice were transplanted with GFP+ CDKN2A ko /BCR::ABL1+ B-ALL cells. Mice were treated with CEL_Amide alone, nilotinib, ponatinib, or a combination of LIMKi and TKI, compared with vehicle-treated mice ( Figure 4A). Untreated mice died from this aggressive leukemia within 11-12 days (Figure 4 B,C). CEL_Amide monotherapy had no impact on survival, whereas nilotinib and ponatinib monotherapy significantly extended mouse survival to 17 and 28 days, respectively (p = 0.0003 and p = 0.0005). Interestingly, combination therapy of CEL_Amide + nilotinib extended survival to 33 days (p = 0.0003) and CEL_Amide + ponatinib to 50 days (p = 0.0005). Leukemic GFP+ cells were scored once weekly in peripheral blood by flow cytometry and mice were considered leukemic if >1% GFP+ cells were detected in peripheral blood. CEL_Amide combined with nilotinib or ponatinib significantly delayed detection of GFP+ BCR::ABL+ leukemic cells in peripheral blood (Supplementary Figure S4A,B).

Discussion
In our study, we report the anti-leukemic effects of CEL_Amide, a novel highly specific small molecule inhibitor of LIMK1/2, in BCR::ABL+ ALL cells and in a BCR::ABL+

Discussion
In our study, we report the anti-leukemic effects of CEL_Amide, a novel highly specific small molecule inhibitor of LIMK1/2, in BCR::ABL+ ALL cells and in a BCR::ABL+ mouse model, which, to our knowledge, is the first report of preclinical activity of a LIMKi in BCR::ABL+ B-ALL. Interestingly, CEL_Amide significantly synergized with commercially available TKIs in vitro and in vivo. The outcomes of BCR::ABL+ B-ALL have dramatically improved in recent years due to the introduction of BCR::ABL-targeting TKIs and antibodies, including the bispecific monoclonal antibody blinatumomab and the conjugated antibody inotuzumab ozogamicin, either frontline, in combination with classical chemotherapy, or in relapsed and refractory disease [1]. Nevertheless, 30-40% of patients are relapsing or are non-responders, especially elderly patients who are ineligible for allogenic hematopoietic stem cell transplantation [1,19,20]. Thus, the combination of LIMKi and TKI warrants further investigation, as this treatment strategy is synergistic in vitro and in vivo preclinical models where LIMKi act as a sensitizer to apoptosis. This strategy may potentially overcome resistance to BCR::ABL-targeting TKIs, which can emerge after point mutations of the breakpoint cluster region-ABL due to prolonged exposure to those drugs [6,7,21]. This hypothesis is supported by the observation that inhibition of ROCK or LIMK hyper-stabilizes mitotic spindles and impairs Aurora kinase A activation, suggesting the implication of the ROCK-LIMK-cofilin axis in Aurora kinase A activation via actin dynamics [16]. It was recently shown that Aurora kinase A is constitutively activated in BCR::ABL+ cells [22]. Furthermore, knockdown of Aurora kinase A enhanced activity of BCR::ABL-targeting TKIs, including leukemic cells harboring BCR::ABL mutations that conferred resistance to TKIs. Thus, the synergy of LIMKi and BCR::ABL+-targeting TKIs may be due to indirect Aurora kinase A inhibition, and constitutes a suitable target to overcome TKI resistance.
In accordance with data obtained for ROCK inhibitors, which is a kinase that activates LIMK1 and 2 by phosphorylation, we could also show that LIMK1/2 inhibition had antiproliferative effects in BCR::ABL+ cells [8]. Thus, targeting the ROCK-LIMK-cofilin axis constitutes a promising target in BCR::ABL+ ALL.
It was shown that LIMK inhibition could sensitize cells to cell death induction, and here, we showed that CEL_Amide was synergistic in combination with BCR::ABL+targeting TKIs [17]. It is possible that LIMKi by CEL_Amide monotherapy was not sufficient, as it was shown to only have modest activity in FLT3-ITD+ AML [17]. Nevertheless, CEL_Amide monotherapy had significant biological effects, including strong apoptosis induction at higher doses in BCR::ABL+ cells, but it is not known what plasma levels for CEL_Amide may be achievable in vivo. Thus, combination strategies may be the future direction for developing LIMKi, such as CEL_Amide and others, in cancer treatment [10].
We found that the two isoforms, LIMK1 and 2, were expressed in BCR::ABL+ ALL cell lines and primary patient cells, whereas no significant expression of LIMK isoforms could be detected in CD34+ cells from healthy donors, underpinning their potential role in leukemic transformation. Upon treatment of BV-173 and TOM-1 cell lines with CEL_Amide, we observed degradation of LIMK1, whereas the LIMK2 protein was left unaffected, suggesting a possible role of LIMK1 in leukemic maintenance in BCR::ABL+ ALL. Nevertheless, the precise mechanisms of LIMK-mediated leukemogenesis are not clearly elucidated and it was recently reported that upregulation of either LIMK1 or LIMK2 was observed to be dependent on the cellular system for AML cells [23]. This suggests that loss of one family member can be partially compensated for by the other, as a potential mechanism of resistance, encouraging the development of LIMK1/2 inhibitors to ensure inhibition of downstream signaling [23].
LIMK1 and LIMK2 phosphorylate cofilin, which leads to its inactivation and subsequent modulation and reorganization of the cytoskeleton, leading to the abrogation of mitochondrial apoptosis [13,24]. Here, we demonstrated that LIMK inhibition by CEL_Amide led to dephosphorylation of cofilin in BCR::ABL+ ALL cell lines and patient cells. In turn, no significant amount of phospho-cofilin could be detected in cells derived from healthy donors. These findings underline the on-target effects of LIMKi. In future clinical trials, monitoring of phospho-cofilin in patient-derived cells could serve as a potential biomarker for efficient LIMK inhibition in vivo.

Conclusions
In conclusion, we report for the first time that the small molecule LIMK1/2 inhibitor CEL_Amide has anti leukemic activity in BCR::ABL+ cells, leading to activation of cofilin and subsequent apoptosis and cell cycle arrest. Furthermore, combinations of CEL_Amide with BCR::ABL-targeting TKIs yield synergy in vitro and in vivo, encouraging further preclinical and clinical testing of LIMK1/2 inhibitors in BCR::ABL+ B-ALL.

Supplementary Materials:
The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/jcm11226761/s1, Table S1: PCR primers used in this study; Table S2: Characteristics of patients; Figure S1: LIMK1, LIMK2, cofilin, and phospho-cofilin protein expression in BCR::ABL+ B-ALL cell lines after TKI exposure; Figure S2: Effect of drug combinations with TKI on cell cycle in BCR::ABL+ ALL cell lines; Figure S3: Drug combinations of LIMKi with vincristin and dexamethasone in BCR::ABL ALL cell lines; Figure S4: Detection of GFP positive BCR::ABL+ blast cells in C57BL/6J mice.  Institutional Review Board Statement: Patients and healthy donors provided informed consent prior to bone marrow aspiration or blood sample collection, following the Declaration of Helsinki. This study has been carried out in accordance with the EC Directive 86/609/EEC for animal experiments and was approved by the Committee for Experimental Animal Studies of the University of Paris Institute Board Ethics.


Mechanism of protective role of miR-874-3p in intervertebral disc degeneration

Background The intervertebral disc can increase the amplitude of spinal motion, withstand pressure, buffer vibration, and protect the brain and spinal cord. It is also the main reason why height changes, but the regulatory mechanism is still unclear, and this study mainly explored the role of miR-874-3p in intervertebral disc degeneration (IDD). Methods The mechanism and perform correlation analysis of miR-874-3p and the pathological degree and prognosis of patients with IDD. miR-874-3p is involved in the progression of several diseases, such as cell differentiation, proliferation, apoptosis and extracellular matrix degradation, overexpressing cell line GV369-miR-874-3p-NP was obtained by infected nucleus pulposus (NP) cells, and the empty vector GV369-NP group was set with the blank group. The expression of the tag protein (green fluorescent protein, GFP) was visualized by fluorescence microscopy, followed by real-time polymerase chain reaction (PCR) method to detect miR-874-3p expression, apoptosis by flow cytometry, luciferase reporter analysis for verifying the targeting relationship between miR-874-3p, caspase-3, B-cell lymphoma-2 (Bcl-2) and Bax in cells, and examined the changes in cellular mitochondrial membrane potential using kits. Results The expression of miR-874-3p was significantly reduced in IDD patients, and was negatively correlated with matrix metallopeptidase 2 (MMP2) and downregulated matrix metallopeptidase 3 (MMP3) in the NP cells. In addition, western blot revealed that overexpression of miR-874-3p increased the aggregation protein level in the NP cells. Conclusions miR-874-3p can inhibit the cell death of IDD, and not only participate in caspase-3 and Fas-associating protein with a novel death domain (FADD)-mediated apoptosis through targeted regulation of exogenous MMP2/MMP3 pathway, but also play a role in cell apoptosis through mitochondrial pathway.

Introduction
Intervertebral disc degeneration (IDD) affects the fibrocartilage disc (i.e., the intervertebral disc), causing instability (1). IDD is decreased musculoskeletal function, but its pathogenesis is not fully defined (2). Therefore, the intervertebral disc (IVD) is the fibrocartilage tissue connecting the two adjacent vertebrae. The stability provided by the IVD is important for the whole spine, and the central part of the IVD is the nucleus pulposus (NP), forming a hydrogel-like nucleus in the IVD (3). NP is mainly composed of proteoglycans and collagen fibers, and its elastic function can be distributed in all directions (4). Mymyeloid cells have two cell types, chordate and mature myeloid cells (5). The NP is surrounded by a fiber ring (A nnulus Fibrosus, AF). Excessive destruction of the outer disc extracellular matrix (ECM), plays a crucial role in the development of IDD and the major catabolic enzymes mediating this destruction are the matrix metalloproteinases (MMPs). In the process of disc degeneration, the disc

Original Article
Mechanism of protective role of miR-874-3p in intervertebral disc degeneration has complex biochemistry and molecular level changes, including the reduction of proteoglycan content, type collagen to collagen I transition and NP cell density (6). These changes directly lead to the reduction of IVD machinery and eventually lead to structural damage, such as fiber ring rupture, NP cell protrusion, etc. In sharp contrast to the factors causing the extracellular matrix breakdown are the synthetic factors that stimulate the extracellular matrix, and they play a role in regeneration (7)(8)(9). However, the regulators of matrix metallopeptidase 2 (MMP2) and matrix metallopeptidase 3 (MMP3) and their underlying mechanisms are still unknown (10). miRNA was formally recognized as a short-segment noncoding RNA in 2001 as one of the classical gene regulators in eukaryotic cells (11). miRNA is an endogenous small RNA that plays an important role in cell proliferation, development, and metabolism by acting on other genes (12). miRNA, as a single-stranded RNA in a noncoding region composed of 18 to 22 nucleotides, is transcribed by miRNA (13). It is generally believed that pri-miRNA has two sources: (I) genes encoded by special miRNA are transcribed by type RNA polymerase, and then these pri-miRNA pass more in the nucleus Proteins cleave together, which contain an anchor protein DGCR8 that contributes to pre-miRNA composed of about 70 nucleotides; (II) transcribed from intrinsic mRNA fragments, and their maturation process does not require Drosha/DGCR8 from lariat debranching enzyme (Ldbr) and co-transcribed with host proteincoding genes to form hairpin pre-miRNA, these intrinsic miRNA will often appear in the same biological pathway as the host protein-encoded genes (14)(15)(16), and a very recent study found that miR-874-3p targets and inhibits MMP2 in non-small cell lung and gastric cancer . The role of tumor suppression.
With the deepening of IDD development mechanism research in recent years, it is found that degeneration-related genes could activated the development of IDD, but their interaction and their abnormal expression mechanism remain unclear. Small RNA (microRNA), as one of the important regulatory molecules of gene expression, has been shown to play a key role in multiple disease initiation and progression stages, so it is believed that it may also play an important role in disc degeneration. Current studies of miRNA affecting disc degeneration have found that many kinds of miRNA are mainly available Participate in influencing the development of IDD through its action on apoptosis, inflammatory signaling response, and ECM components.
Significant abnormalities in the expression of many miRNA in the nuclear medullary tissue of the degenerative disc, suggesting that miRNA may be involved in the pathophysiology of IDD (17). Although increasingly it is being reported that abnormal miRNA leads to nuclear myeloid cell decline in IDD patients, and if miR-874-3p expression levels change in IDD (18). Whether there is a MMP2 alveolar pathway that also functions in the nuclear myeloid cells of IDD, and whether there are other relevant target genes are unknown (19,20). In this study, the correlation of disease severity in patients with IDD expressing miR-874-3p, and a possible protective role of miR-874-3p may play through inhibition of MMP2 and MMP3 expression were investigated. I present the following article in accordance with the MDAR reporting checklist (available at https://atm. amegroups.com/article/view/10.21037/atm-22-91/rc).

Sample collection
Fifty-four clinical NP tissue samples were collected from either the Department of Pathology or the operating room with the informed consent of all subjects. Of the 54 patients, 28 had significant disc degeneration and underwent laminectomy fusion. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Orthopaedic Clinical Research Ethics Committee of the Yingkou Central Hospital (2021-146) and informed consent was taken from all the patients.

Real-time quantitative and qualitative polymerase chain reaction (qRT-PCR) analysis
For mRNA detection, cDNA was obtained using a RT kit (Promega) with oligomeric-dt primers (21). The qRT-PCR analysis was performed using SYBRGreen PCRMas systems (Applied Biosystems) using standard protocols and GAPDH served as control group. miR-874-3p is significantly upregulated in cervical disc degeneration, and miR-874-3p may serve as a new therapeutic target in cervical disc degeneration (22).

Isolation and culture of NP cells
Cells were cultured in medium containing fetal bovine serum (23). NP cells from a patient with immunofluorescence assay characteristics for subsequent experiments. Human IDD NP cells from log growth period were spread on 48-well cell culture plates. After overnight culture, appropriate Dulbecco's modification of Eagle's medium containing 10% fatal bovine serum containing different gradient concentrations of puromycin was added to each well, and fluid was changed every 48 h for 1 week to observe cell growth, and the concentration of all death was the minimum screening concentration.

Lentiviral infection
m i R -8 7 4 -3 p i n h i b i t o r y l e n t i v i r a l v e c t o r ( LV ) contained the anti-miR-874-3p target sequences (TCGGTCCGGGGCAG) (24). Human IDD NP cells prepared and stored in this laboratory were stored in 6-well plates for cell growth at 37 ℃ and 5% CO 2 overnight. When cells grew to about 75% the next day, GV369-miRNA-326 lentiviral medium and human IDD NP cells for 8 h, changed to culture for 2 d and observed green fluorescent label expression under a fluorescence microscope.

Carrier construct and double-luciferase assay
The pmiR-report vector construct that amplified the MMP3 gene and cloned the downstream luciferase gene. We again performed the reporter gene activity measurements using the dual-luciferase assay system (Promega). The target fragments were amplified by polymerase chain reaction (PCR), digested and purified using a gel recycling kit. Connecting the linearized lentiviral expression vector GV369, transformed into E. coli competent cell DH5, coated with LB plates with puromycin resistance, colonies were picked for identification, then positive clones were expanded in culture after plasmid extraction.

Apoptosis detection and cell counting kit-8 (CCK-8) detection
CCK-8 is a rapid and highly sensitive Kit based on WST-8 that is widely used in Cell activity and cytotoxicity detection. We also examined apoptosis using flow cytometry. Cell proliferation or cell viability (CCK-8 assay) (Beyotime, China) was analyzed.

Cellular immunoassay analysis
Human IDD NP cells infected with lentivirus were collected, and total protein was extracted from the cells by using the SD-001/SD-002 Animal Cell/Tissue Total Protein Extraction Kit, and were subjected to SDS-PAGE gel electrophoresis, transferred to a polyvinylidene difluoride membrane and blocked with a blocking solution containing 5% bovine serum albumin for 2 h at room temperature. Membranes were washed three times in Phosphate Buffer Saline Tween (PBST) for 10 min each, supplemented with HRP-labeled sheep anti-rabbit IgG secondary antibody, incubated at room temperature for 2 h, counterstained with PBST, washed with ECL, and photographed with dark chamber exposure.

Statistical analysis
Our study analyzed relevant experimental data by SPSS software (23.0), measurement data were expressed as mean ± standard deviation (x±s), one-way ANOVA, LSD-t test for pairwise comparisons, P<0.05 was statistically significant.

Expression of miR-874-3p and MMP2 in IDD
The level of miR-874-3p differed from those in normal subjects, the qRT-PCR results suggested that in IDD patients compared with the controls, the expression level of miR-874-3p were obviously decreased. The MMP2 expression levels in the IDD and control patients were also evaluated and the results showed they were elevated in the IDD patients as compared with the control group. There was also a significant increase in MMP3 as another important enzyme for ECM degradation in the IVD, lower expression levels of MMP2 and MMP3 ( Figure 1).

Downregulation of MMP2 in IDD
The experimental results showed that the cells had the markers. Lentivirus expressing miR-874-3p, lentivirus with miR-874-3p inhibition and negative control (NC) lentivirus were generated and activated by the infected NP cells' response, ensuring that infection efficiency was sufficiently high. By qRT-PCR analysis, lentiviral infection with miR-874-3p led to significantly downregulated level of expression of MMP2 ( Figure 2).

Downregulation of MMP3 in IDD
miR-874-3p is an extremely important regulator of gene expression regulation, which is involved in various aspects of cell function such as development, aging and disease. miRNAs work by regulating mRNA expression levels. Abnormalities of immune system are closely related to miRNA, which can lead to the occurrence and development of a variety of diseases. Two homologous miRNAs, MMP3 and miR-874-3p, were found to play an important role in maintaining joint homeostasis and inhibiting the pathogenesis of osteoarthritis. At present, it is quite clear that miRNA will intervene in the occurrence, development and maintenance of diseases in a variety of ways, which is of great help for us to master and treat diseases. It is known from the latest research progress that the regulatory network of MMP3 is extremely complex and penetrates into all aspects of the body response. It is extremely difficult to fully identify target-related regulatory genes in IDD (Figure 3).

Role of abnormal miR-874-3p expression in IDD pathology
Decomposition of the polymerization glue and collagen, which mainly correlates. After finding that miR-874-3p can regulate cell death and that there is decreased miR-874-3p in IDD patients, whether miR-874-3p affects the development of IDD was further explored using western blot, which found that overexpression of miR-874-3p increases aggregated protein levels in NP cells. On the other hand, the inhibition of miR-874-3p reduced the expression of Aggr in NP tissue. More importantly, no significant degradation of collagen was found. It is precisely because the reporter gene test results are affected by a variety of factors, such as carrier state, cell state, transfection quantity, transfection efficiency, lysis efficiency, sample addition accuracy, and detection process, that improper processing of a certain detail may lead to experimental failure or inaccurate results. In this study, the results of the dual-luciferase reporter gene test to detect the effect of miR-874-3p on MMP2 were not ideal, so the results were not further analyzed in the results section. The changes were consistent and suggested that regulation of miR-874-3p expression may play a role in developmental disorders such as IDD. Therefore, apoptosis of NP cells expressing miR-874-3p or with miR-874-3p infection was analyzed by flow cytometry. These experimental data suggested that  (Figure 4).

Discussion
IDD disease has become a common and high-incidence of chronic disease, suffering from both middle-aged and elderly people and young people (25)(26)(27). IDD can cause a variety of symptoms, such as waist and leg pain, spinal stenosis, protruding disc and compression of nerve roots leading to pain in other parts. Currently, age, drinking, overwork and so on are considered to be extrinsic factors contributing to disc degenerative diseases (28). There are also numerous views on the molecular mechanisms and transduction pathways of its pathogenesis, and inflammatory cytokines, genetic genes, oxidative stress, Nitric oxide induction, and miRNA play important roles in IDD (29). miR-874-3p plays an important role in cell and mitochondrial energy metabolism. With the deepening of research, more and more studies have found that miR- 874-3p not only participates in the control of energy metabolism, but also is closely related to the abnormal electronic respiratory chain in gene transcription, protein synthesis, mitochondrial oxidative stress, and the activity of intracellular phosphorylation of miR-874-3p is strongly correlated with the aging state of chondrocytes. Although miR-874-3p has been previously stated to be involved in cell energy generation, some studies have shown that miR-874-3p is involved in inflammatory response mediated by NF-kB signaling pathway. There was no significant difference between miR-874-3p gene level and miR-874-3p regulatory protein level in early cartilage stage, but the phosphorylation level of miR-874-3p gradually decreased with aging. Gene energy metabolism is related to oxidative stress in mitochondria (30). We speculated that miR-874-3p signaling pathway not only interferes with energy metabolism, but also mediates the corresponding oxidative system and antioxidant system in mitochondria. Abnormal miR-874-3p is closely related to cellular stress. Moreover, studies have shown that there is a strong correlation between the phosphorylation activation of miR-874-3p and the formation or degradation of cartilage. Studies on osteoarthritis have found that the activation of miR-874-3p is significantly reduced, and corresponding inhibitors or gene knockout can effectively alleviate the progression and inflammatory expression of osteoarthritis. miR-874-3p regulates the interaction of not only mTOR pathway but also HIF and other signaling pathways, which is closely related to the differentiation and maturation of cartilage endplate. Therefore, miR-874-3p is an important regulatory pathway in the formation, maintenance and aging of joint and cartilage endplates (31). The results also suggest biomarkers for the diagnosis of IDD, because miR-874-3p is significantly reduced in IDD, and also point to future exploration of restoring miR-874-3p expression in the NP cells of patients with IDD. The function of miR-874-3p is important for IDDrelated phenotypes, and warrants further exploration. A limiting factor in this study was the relatively small number of samples from patients (32). miR-874-3p mediates cervical disc degeneration via MMP2/MMP3, thereby affecting protein synthesis and function. Studies have shown that the MMP2/MMP3, mammalian target of rapamycin and protein kinase B (Akt) activators proteins in hypothalamus of rainbow trout were found to have corresponding changes in rainbow trout fed with different nutrient levels of diets. It was found that the p-MMP2/MMP3 protein level of rainbow fish fed with high fat diet for 3 hours was significantly reduced. However, p-MTOR protein content increased significantly, reflecting the negative regulation of MMP2/MMP3 and mTOR. Relevant studies have shown that miR-217 regulates the proliferation and differentiation of A549 and H1299 cells through the MMP2/MMP3/ mTOR pathway (33). Cervical and lumbar intervertebral disc degeneration is the most common degenerative degeneration of spine, and is also one of the important factors causing neck, low back pain. The endplate cartilage is a thin layer of hyaline cartilage located at both ends of the vertebral body anatomically. We have already explained that the endplate cartilage not only plays an important role in the nutritional supply of the intervertebral disc, but also plays an important role in the mechanical transmission between the vertebral body and the intervertebral disc.
The intervertebral disc has been identified as the largest unvascular occluded structure in the body. As the embryo develops mature, the blood vessels in the intervertebral disc degenerate rapidly, and the circulation of the nucleus pulposus in the intervertebral disc is isolated from the attachment and becomes covert antigen. Endplate cartilage degeneration is the early morphological change of intervertebral disc degeneration (34). MMP2/MMP3 has been identified as an extremely important protein kinase in eukaryotes. It is expressed in multiple tissues closely related to metabolism and has tissue specificity. Different tissues have different subtypes. Studies have shown that MMP2/MMP3 is not only involved in the occurrence and development of tumors, but also closely related to the occurrence and development of diseases, such as the related pathways of protein synthesis and mitochondrial energy metabolism mentioned above (35,36). All of the above miRNA molecules are involved in the regulation of the apoptotic pathway in myeloid cells, but none of the apoptosis-related pathways studied above were significantly disease-specific. Whether the above miRNA molecules are representative of IDD and whether they can respond to specific changes of IDD disease is still worth further research and analysis, and strive to find IDD specific changes of molecules and clear mechanism to provide important theoretical basis for future application research (37). MMP-mediated degradation of ECM promotes the metastasis of tumor cells from their primary site (38). Cazzanelli et al. (39) found that the content of chondroitin sulfate (CS) decreased significantly, while the five chondrothiol sulfate glycosyltransferases in the degenerative nuclear myeloid tissue was somewhat different (40). After comprehensive database analysis, it was believed that miR-29b, miR-194-515, miR-2355 may be able to act on chondroitinase sulfate and only miR-194 and miR-515 Significant increase, significant decrease in chondroitinase sulfate protein levels after miR-194 and miR-515 overexpression, no mRNA levels, miR-194 and miR-515 inhibitors without mRNA levels. Ethical Statement: The author is accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All procedures performed in this study involving human participants were in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Orthopaedic Clinical Research Ethics Committee of the Yingkou Central Hospital (2021-146) and informed consent was taken from all the patients.
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