s2fieldsofstudy: Biology Medicine Chemistry

text:
Ginsenoside Rd Promotes Cardiac Repair After Myocardial Infarction by Modulating Monocytes/Macrophages Subsets Conversion

Abstract Purpose This study aimed to elucidate the potential molecular mechanisms by which GSRd improves cardiac inflammation and immune environment after MI. Materials and Methods The potential target genes of GSRd were predicted using the STITCH database. In vivo, MI mice models were established by left anterior descending ligation and were divided into the sham group, MI + Vehicle group, and MI + GSRd group. DMSO, DMSO, and GSRd 50 μL/day were intraperitoneally injected, respectively. After 28 days, echocardiography, Masson staining, immunofluorescence staining, flow cytometry, RT-PCR, and Western blot were performed. Mice peritoneal macrophages were extracted in vitro, and Western blot was performed after GSRd and/or Akt inhibitor MK2206 intervention. Results GSRd significantly improved mouse myocardial function, attenuated cardiac fibrosis, and inhibited inflammation and apoptosis in myocardial tissues after myocardial infarction. Meanwhile, GSRd increased non-classical Ly6Clow Mos/Mps while reduced of classical Ly6Chigh Mos/Mps at the same time in myocardial tissues. In addition, GSRd significantly reversed the activity of p-Akt and p-mTOR in the heart Mos/Mps after MI. In vitro studies showed that the activity of p-Akt and p-mTOR in peritoneal macrophages were significantly increased in a dose-dependent manner after GSRd treatment. Furthermore, the AKT inhibitor MK2206 was found to block the enhanced activity of p-Akt and p-mTOR induced by GSRd in peritoneal macrophages. Conclusion GSRd can enhance the transformation of Ly6Chigh Mos/Mps to Ly6Clow Mos/Mps in mice after MI by activating the Akt/mTOR signaling pathway, inhibiting cardiac dysfunction and promoting cardiac repair.

Introduction
Acute myocardial infarction (MI) is one of the major causes of death in coronary artery disease, and its incidence showed an upgraded trend in recent years. 1,2 In adult mammals, the limited regenerative capacity of the myocardium after myocardial infarction cannot compensate for a large number of necrotic cardiomyocytes while the lost cardiomyocytes are replaced by proliferating fibroblasts and secreting dysregulated extracellular matrix, leading to ventricular remodeling, arrhythmias, heart failure, and life-threatening events. 3 Therefore, how to inhibit ventricular remodeling after myocardial infarction, reduce the area of myocardial infarction and delay the occurrence of heart failure is an effective means to improve the clinical treatment effect and the living quality of patients.
After MI, monocytes/macrophages (Mos/Mps) are recruited into the heart to play key roles as inflammatory actors and central regulators of ventricular remodeling. 4,5 Two different Mos/Mps subpopulations, including Ly6C high and Ly6C low , were participated in ventricular remodeling after myocardial infarction at different periods. 6,7 During 1-3 days after myocardial infarction Early reactive Mos/Mps (Ly6C high ) exert phagocytic and proinflammatory effects by massively secreting interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), myeloperoxidase, and cathepsin in myocardial tissue. 8 And during 4-7 days after myocardial infarction, late responding Mos/Mps (Ly6C low ) plays an anti-inflammatory role and promotes cardiac repair by secreting repair cytokines including transforming growth factor-β1 (TGF-β1), interleukin-10 (IL-10), and vascular endothelial growth factor (VEGF). 7 Furthermore, Ly6C high Mos can be converted to Ly6C low Mos in circulation 9,10 and inflamed tissues including infarcted hearts. 8,11 Delayed conversions of Ly6C high to Ly6C low Mos/Mps may attenuate wound repair after myocardial infarction, as in animals with atherosclerosis, diabetes, or myocardial infarction. 7,12,13 Therefore, the enhanced transformation of Ly6C high Mos/Mps to Ly6C low Mos/Mps may help to slow down ventricular remodeling after MI and delay the development of heart failure.
Traditional Chinese Medicine (TCM) has a long history and remarkable efficacy in preventing and treating MI. The pathogenesis of MI is heart-Qi deficiency and failure to stimulate blood flow, then blood stasis, resulting in myocardial ischemic necrosis caused by transient or persistent occlusion of the distal coronary artery. Ginseng is one of the most valuable natural herbal medicines in China and worldwide and is a crucial herb for treating MI. 14 Ginsenosides have pharmacological and therapeutic effects on the immune, 15 cardiovascular, 16,17 endocrine, 18 and nervous systems. 19 Ginsenoside Rd (GSRd) is identified as one of the most important active ingredients in Ginseng and has anti-inflammatory effects on natural immune components. 20 GSRd has a wide range of applications in ischemic disease research. GSRd activated the Akt/GSK-3beta signaling pathway, increased the Bcl-2/Bax ratio, inhibited the mitochondria-dependent apoptotic pathway to attenuate myocardial ischemia-reperfusion injury in rats, and reduced infarct size. 21 GSRd enhances antioxidant enzyme activity, stabilizes mitochondrial membrane potential, increases intracellular ATP levels, and activates the PI3K/Akt signaling pathway to promote neuronal survival, and ameliorates ischemic brain injury. 22 GSRd protects against renal ischemia/reperfusion injury by inhibiting M1 macrophage polarization 23 and attenuates experimental autoimmune neuritis in mice by regulating monocyte subset conversion. 24 However, the role of GSRd in Mos/Mop phenotypic transformation after myocardial infarction and its molecular mechanism remain unclear.
In this study, we established the mouse model of myocardial infarction to evaluate the therapeutic effect of GSRd on fibrosis and cardiac function after acute myocardial infarction and explore its potential mechanism, providing a new choice and theoretical basis for the prevention and treatment of ventricular remodeling after myocardial infarction.

Materials and Methods Reagents
Ginsenoside Rd (Selleck, Houston, USA) standards (purity > 95%) were dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution for further investigation.

Animals
Eight-week-old male C57BL/6J mice (22-25 g) were purchased from SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and housed in an environment with controlled temperature (22°C ± 1°C) and relative humidity (50% ± 5%) on a 12-h:12-h light/dark cycle, with free access to sterile food and water. Mice were randomly divided into three groups: (a) sham group, (b) MI + Vehicle group, and (c) MI + GSRd group. Mice in the sham and MI groups were intraperitoneally injected with vehicle DMSO, 50 μL/day. Mice in the GSRd group received an intraperitoneal injection of GSRd, 50 μL/ day (GSRd dissolved in DMSO, 50 μg/kg/day). All procedures in this study were approved by the Experimental Animal Welfare and Ethics Committee of Shanghai University of Traditional Chinese Medicine (NO. PZSHUTCM210226013). All animal experiments were conducted following the animal experimental guidelines set by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

MI Mouse Model
Male mice aged 6-8 weeks underwent permanent ligation of the left anterior descending (LAD) branch coronary artery. 25 Briefly, Mice were anesthetized with 1-1.5% isoflurane gas and mechanically ventilated with rodent respirators. Left thoracotomy was performed to open the chest cavity and expose the heart so that the LAD could be seen and permanently ligated with 6-0 silk thread outside the left atrium. The presence of myocardial branches in the perfusion bed confirmed complete vascular occlusion. Mice that died within 24 hours of surgery were excluded from the experiment. The sham animals underwent the same procedure, but without coronary ligation.

Echocardiography
Transthoracic echocardiography was performed to assess mouse cardiac function after surgery using an echocardiograph (Vevo2100, Visual Sonics, USA) with M-mode analysis. All mice (8-10 weeks old) were anesthetized by isoflurane inhalation (1%-2%). As previously described, 26 the mitral valve lobe was visualized in a parasternal long-axis view to assess cardiac function.

Immunofluorescence
Cryosections of heart (7μm) were prepared and stained with CD68 monoclonal antibody (FA-11) (1:200, eBioscience, USA) overnight at 4 °C. Follow, the sections were incubated with Alexa Fluor 488-conjected secondary antibodies for 1 h in the dark. Nuclei were stained with DAPI (1:2000, Thermo Fisher, USA). All slides were mounted with ProLong TM Gold (Thermo Fisher, USA). Confocal microscope from Zeiss were used to take image. Image J software were applied to analysis the obtained images.

Quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR) Analysis
RNA was isolated from cells using TRIzol (Invitrogen, USA) and 1μg of RNA was used as a template for cDNA synthesis using reverse transcription kits (Takara, Dalian, China), then cDNA was subjected to real-time PCR analysis using TB green mixture (Takara, Dalian, China) on an ABI 7900 Q-PCR system from Life Technologies (Carlsbad, CA, USA) according to the manufacturer's protocol. All mRNA expression levels were normalized by comparing them to the housekeeping gene GAPDH. Sequences of primers for real-time PCR analysis of mice samples were verified against the National Center for Biotechnology Information database to confirm specificity and are listed in Table 1.

Heart Masson's Trichrome Staining
Heart samples were harvested and fixed in 10% phosphate-buffered formalin for 24 h, subsequently embedded in paraffin and cut into 5 μm slices. Histologic sections of tissues were stained with Masson's trichrome (Solaribio, Beijing, China). The mean cross-sectional area and fibrosis of the left ventricular apex up to the aortic valve leaflet attachment point were assessed by Image-Pro Plus software.

Cell Extract and Culture
Peritoneal macrophages were obtained from abdominal cavity and cultured in medium 1640 containing 10% fetal bovine serum. Single-cell suspensions of infarcted hearts were prepared. Briefly, the heart was dissected, minced with delicate scissors, and enzymatically digested with a digested with a collagenase mix (including 125 U/ml collagenase IX, 450 U/ ml collagenase I, 60 U/ml DNase I, and 60/U ml hyaluronidase I-S (Sigma -Aldrich, St Louis, MO, USA) for 1 h at 37°C with gentle agitation. After digestion, the tissues were triturated, passed through 70-mm cell strainers, washed, lysed red blood cells, and stained with antibodies for fluorescence activated cell sorting (FACS) analysis. For monocyte/macrophage sorting, cells were then stained with APC-Cy7-CD11b (

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FACS Aria flow cytometry system (BD Biosciences, San Jose, CA, USA) was used to perform flow cytometry, and FlowJo software (Tree Star, Ashland, OR, USA) were used to analysis the flow cytometry data.

Western Blot
Heart tissues and cells were extracted in lysis buffer containing protease inhibitors. Pierce BCA Protein Assay Kit (Pierce, Rockford, IL, USA) was used to assess protein concentration. Equal quantities of protein were denatured, dissolved in 10% SDS-PAGE gel, transferred to a nitrocellulose membrane, incubated with 5% skim milk for 1-1.5 h, and then incubated with primary antibody at 4° C overnight. Primary antibodies were diluted as follows: The membranes were then incubated at room temperature in a blocking buffer containing an HRP-labeled secondary antibody for 2 h at room temperature. The enhanced chemiluminescence reagent (Thermo Fisher Scientific, USA) were used to develop western blots.

Statistics Analysis
Data analysis were achieved by the GraphPad Prism version 5.0 (GraphPad Software, Inc.). Results are shown as mean ± standard error of the mean (SEM). Comparison between groups was performed using two-tailed Student's t-test and one-  or two-way analysis of variance with Bonferroni post-hoc analyses. p < 0.05 was considered statistically significant. According to the literature, 27 sufficient statistical sample size was designed in the experiment. Randomization and blinding strategies were used whenever possible.

GSRd Improves Mouse Cardiac Function After Myocardial Infarction
To explore the effect of GSRd ( Figure 1A) on cardiac contractile function in vivo, WT mice were injected with vehicle DMSO or GSRd (50 μg/kg body weight) and underwent sham or LAD artery ligation surgery for 4 weeks ( Figure 1B). The survival of mice was observed after 4 weeks ( Figure 1C). No mice in the sham group died, 55% of mice in the MI + vehicle group died, and 30% of MI mice treated with GSRd died. Survival was significantly decreased in the MI + vehicle group compared with the sham group; GSRd significantly improved survival compared with the MI + vehicle group. GSRd intervention significantly improved the survival of MI mice. Furthermore, a high-resolution echocardiography imaging system with M-model analysis revealed that ejection fraction and fractional shortening were significantly decreased in MI + vehicle group mice compared with the sham group, and GSRd treated mice exhibited a significant increase in ejection fraction and fractional shortening after MI  Figure 1D-F). These results indicated that the MI model mice modeling was successful, and GSRd could improve the cardiac function of mice after MI.

GSRd Attenuates Cardiac Fibrosis After Myocardial Infarction
To explore whether GSRd affects cardiac fibrosis following MI, Masson's trichrome staining (Figure 2A) was performed. As shown in Figure 2B and C, the infarct zone and border zone fibrosis area of MI + vehicle group mice increased significantly compared with the sham group. Compared with untreated MI mice, GSRd-treated MI mice significantly reduced the infarct zone and border zone fibrosis area. The mRNA levels of myocardial fibrosis markers (connective tissue growth factor (CTGF), collagen Iα1, collagen IIIα1, and fibronectin (Fn)) in the heart tissues with the infarct zone were measured in each group, as shown in Figure 2D. Compared with the sham group, the cardiac infarct zone mRNA levels of CTGF, collagen Iα1, collagen IIIα1, and Fn were significantly increased in MI + vehicle group mice. GSRdtreated MI mice significantly reduced cardiac infarct zone CTGF, collagen Iα1, collagen IIIα1, and Fn mRNA levels compared with untreated MI mice. These data suggest that GSRd therapy inhibits myocardial fibrosis after MI and alleviates cardiac remodeling.

GSRd Inhibits Inflammation and Apoptosis in Myocardial Tissues
As shown in Figure 3A and B, macrophages in the myocardial tissue were marked by CD68 immunohistochemical staining. Compared with the sham group, the cardiac macrophage infiltration in the MI + vehicle group was significantly increased. GSRd significantly reduced cardiac macrophage infiltration after MI compared with the MI + vehicle group.
Pro-inflammatory Ly6C high monocytes rely on Mcp-1/CCR2 to reach the inflammatory site of MI. As shown in Figure 3C and D, Western blot analysis was used to detect the effect of GSRd on Mcp-1/CCR2 protein expression in MI heart tissue. Compared with the sham group, Mcp-1/CCR2 protein expression was significantly up-regulated in the MI + vehicle group. Mcp-1/CCR2 protein expression was significantly down-regulated after GSRd treatment compared with the MI + vehicle group.
As shown in Figure 3E and F, Western blot analysis was used to detect the effect of GSRd on apoptosis-related protein Bcl-2/Bax of MI mice cardiomyocytes. Bcl-2 was an anti-apoptotic protein, and Bax was a pro-apoptotic protein.
Compared with the sham group, Bcl-2 protein expression was significantly down-regulated, and Bax protein expression was significantly up-regulated in the MI + vehicle group. Compared with the MI + vehicle group, Bcl-2 protein expression was significantly up-regulated, and Bax protein expression was significantly down-regulated after GSRd treatment.
As shown in Figure 3G, mRNA expression levels of pro-inflammatory mediators TNF-α, IL-1β, and IL-6 in MI heart tissue were detected by RT-PCR. Compared with the sham group, the mRNA expression levels of pro-inflammatory mediators TNF-α, IL-1β, and IL-6 in the MI + vehicle group were significantly increased. Compared with the MI + vehicle group, the mRNA expression levels of TNF-α, IL-1β and IL-6 decreased considerably after GSRd treatment.
These results indicate that GSRd inhibits inflammation and apoptosis in the myocardial tissues.

GSRd Modulating Mos/Mps Subsets Conversion Following MI
Circulating Mos/Mps have the important feature of being recruited to inflammatory sites, and subsets of different phenotypes show different functions. For instance, macrophages differentiated from monocytes can replenish the tissue macrophage pool and modulate the local inflammatory environment resulting in cardiac injury. In order to study the effect of GSRd on the in-situ transformation of Ly6C high -Ly6C low Mos/Mps, the mouse heart tissue was collected immediately for flow cytometry analysis and quantitative RT-PCR analysis after sacrifice. As shown in Figure 4A-D, the total Mos/Mps and Ly6C high Mos/Mps cardiac infiltration significantly increased in the MI + vehicle group compared with the sham group. In contrast, the Ly6C low Mos/Mps cardiac infiltration significantly decreased. Compared with the MI + vehicle group, GSRd significantly reduced the total Mos/Mps and Ly6C high Mos/ Mps cardiac infiltration after MI and significantly increased the cardiac infiltration of Ly6C low Mos/Mps. As shown in Figure 4E and F, the mRNA expressions of repairing and pro-angiogenic related cytokines (IL-13, TGFβ1, VEGF, and MMP9) and pro-inflammatory related cytokines (IL-1β, IL-12, and TNFα) in the infiltrating Mos/ Mps were detected by RT-PCR. Compared with the sham group, the mRNA expressions of repair and pro-angiogenic related cytokines (IL-13, TGFβ1, VEGF, and MMP9) were significantly down-regulated in the MI + vehicle group, and the mRNA expressions of pro-inflammatory related cytokines (IL-1β and TNFα) were significantly up-regulated. Compared with the MI + vehicle group, the mRNA expressions of repairable and pro-angiogenic related cytokines (IL-13, TGFβ1, VEGF, and MMP9) were significantly up-regulated after GSRd treatment, while the mRNA expressions of pro-inflammatory related cytokines were not significantly affected.

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Taken together, these results suggest that GSRd promotes the recruitment of Ly6C low Mos/Mps in the heart following MI.

GSRd Modulating Mos/Mps Subsets Conversion via Akt/mTOR Activity After MI
The potential target genes of GSRd were predicted using STITCH (http://stitch.embl.de/), 28 which identified AKT1, CYCS, and TRPM7 directly interacting with GSRd ( Figure 5A). It is noteworthy that mTOR, as a downstream molecule of PI3K/Akt activated by Toll-like receptors, has been previously reported to be critical in innate immune coordination of macrophages and monocytes. 29,30 As shown in Figure 5B and C, we examined the Akt/mTOR pathway activity in Mos/Mps in the heart of each group via western blot analysis. Compared with the sham group, the p-Akt, and p-mTOR activity were significantly decreased in the MI + vehicle group. Compared with the MI + vehicle group, p-Akt, and p-mTOR activity increased after GSRd treatment.
Gsrd regulates cardiac Mos/Mps subsets conversion after MI, and the possible mechanism is to activate Akt/mTOR pathway activity.

GSRd Regulates Akt/mTOR Activity of Peritoneal Macrophages
To further elucidate the effect of GSRd on the Akt/mTOR pathway activity in macrophages, mice peritoneal macrophages were extracted and treated with AKT activation inhibitor MK2206.
As shown in Figure 6A and B, compared with the blank control group, GSRd at 20μM and 50μM concentrations significantly increased the Akt/mTOR pathway activity in peritoneal macrophages, especially at 50μM concentrations. This dose-effect experiment suggested that the activity of the Akt/mTOR pathway in peritoneal macrophages depended on the concentration of GSRd.
As shown in Figure 6C and D, the Akt activation inhibitor MK2206 was used to further verify the Akt/mTOR pathway activity of GSRd peritoneal macrophages. Compared with the blank control group, GSRd(50μM) significantly increased the activity of the Akt/mTOR pathway, MK2206(5.5nM) significantly decreased the Akt/mTOR pathway activity, GSRd(50μM) combined with MK2206(5.5nM) significantly decreased the activity of Akt/mTOR pathway in peritoneal macrophages.
The Akt/mTOR pathway activity was increased in peritoneal macrophages dependent on GSRd concentration, while the Akt/mTOR pathway activity was inhibited by MK2206 (AKT inhibitor) treatment. Thus, the evidence supports the contention that GSRd converts the Mos/Mps subsets phenotype via Akt/mTOR activity following MI.

Discussion
This study found that GSRd treatment improved mouse myocardial function, attenuated cardiac fibrosis, and inhibited inflammation and apoptosis in myocardial tissues after MI. After the treatment of GSRd, increased non-classical Ly6C low Mos/Mps and reduced classical Ly6C high Mos/Mps in myocardial tissue after MI, and the phenotype of tissue Mos/Mps was transformed into a regression phase. Furthermore, in vitro studies confirmed that GSRd has a similar regulatory effect on monocyte/macrophage differentiation, which may be related to the AKT/mTOR pathway. As shown in Figure 7A, pathological manifestations of MI; As shown in Figure 7B, the pharmacological mechanism of GSRd action on macrophages is related to Akt/mTOR pathway.
Ginsenoside is the most bioactive pharmacological compound extracted from ginseng rhizomes and is used to treat several clinical diseases. 23 It is worth noting that the anti-oxidative stress, anti-apoptotic and anti-inflammatory effects of GSRd have attracted the attention of many researchers in recent years. 20,24,31 In particular, recent studies have shown that GSRd can mitigate experimental autoimmune nerve injury by regulating monocyte conversion. 24 GSRd has been widely used in the study of ischemic diseases and inflammatory diseases. MI is a typical ischemic disease and aseptic inflammatory disease. In this study, GSRd was selected as the research object to explore the possible mechanism of its treatment of MI.
Although the application of percutaneous coronary intervention has significantly reduced the mortality rate of AMI, 32  involved in ventricular remodeling, including cardiomyocyte hypertrophy, apoptosis, and myocardial fibrosis. 33 In particular, the myocardial fibrosis caused by excessive deposition of ECM protein in the myocardium distorts the myocardial structure and leads to the development of arrhythmia and cardiac dysfunction, thus affecting the clinical course and outcome of patients with heart failure. 34 Therefore, inhibition of myocardial fibrosis can be an effective means for the prevention and treatment of myocardial infarction. In this study, GSRd significantly inhibited MI myocardial fibrosis and its related markers (CTGF, Collagen I α1, Collagen III α1, Fn) mRNA expression. CTGF is one of the extracellular matrix proteins secreted by the CCN family, also known as CCN2. CTGF expression is elevated in human fibrotic diseases. 35 Treatment of MI mice with CTGF monoclonal antibody can improve survival rate, preserve left ventricular systolic function, reduce inflammation, inhibit myocardial fibrosis, and promote cardiac repair. 36 Collagen is the main component of the extracellular matrix, of which Collagen I and III account for 85% and 11% respectively. 37 Fn polymerization is a necessary condition for collagen matrix deposition. From 12 h to 14 days after MI, Fn was significantly increased in the infarct area and marginal area of MI patients. 38 Inhibition of Fn expression in ischemiareperfusion model mice significantly reduced cardiac injury fibroblast markers and neutrophil infiltration after 1 week. Cardiac dysfunction, pathological cardiac remodeling, and myocardial fibrosis were significantly improved after 4 weeks. 39

Figure 6
GSRd regulates Akt/mTOR activity of peritoneal macrophages. (A) total-Akt, p-Akt, total-mTOR and p-mTOR protein expression in peritoneal macrophage with GSRd treated detected by Western blot. (B) total-Akt, p-Akt, total-mTOR and p-mTOR protein expression in peritoneal macrophage relative protein expression to GAPDH. (C) total-Akt, p-Akt, total-mTOR and p-mTOR protein expression in peritoneal macrophage with MK2206 and/or GSRd treated detected by Western blot. Ginsenoside Rd; MI, myocardial infarction; MK2206, an AKT inhibitor. (D) total-Akt, p-Akt, total-mTOR and p-mTOR protein expression in peritoneal macrophage with MK2206 and/or GSRd relative protein expression to GAPDH. *P < 0.05 versus sham group; # P < 0.05 versus MI + vehicle group. MI is accompanied by excessive apoptosis of cardiomyocytes, which plays a role in regulating cell mass and structure in many tissues. 40 The Bcl-2 proto-oncogene family is essential for regulating apoptosis, and Bcl-2 exerts anti-apoptotic effects by binding to the Bax domain, which has pro-apoptotic effects and inhibiting Bax activation. 41 Clinical studies have shown overexpression of Bax in the infarcted area of MI patients and accelerated cardiomyocyte apoptosis; In the acute phase of MI, Bcl-2 is induced to express processes involved in anti-cardiomyocyte apoptosis. 42 The relative abundance of the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 determines the susceptibility of cells to death. 43 This study showed that GSRd ameliorated the excessive apoptosis of cardiomyocytes in MI by significantly upregulating anti-apoptotic Bcl-2 protein expression and downregulating pro-apoptotic Bax protein expression.
Mcp-1, also known as Chemokine ligand 2 (CCL2), regulates monocyte recruitment in inflammatory diseases. Inflammatory Ly6C high monocytes rely on Mcp-1/CCR2 to reach the inflammatory zone. 44 Mcp-1 or CCR2 gene deletion can reduce the inflammatory response in MI models. 44,45 Targeted silencing of CCR2 increased cardiac ejection fraction from 29% to 35% in mice 21 days after MI, reduced recruitment of Ly6C high monocytes to injured myocardium, alleviated inflammatory response after MI, and inhibited left ventricular remodeling. 46 Clinical studies have shown that intensive atorvastatin therapy down-regulates the expression of Mcp-1/CCR2 in patients with unstable angina pectoris and alleviates the inflammatory response of CD14 + monocytes recruitment. 47 In this study, GSRd significantly reduced cardiac Mcp-1/CCR2 protein expression and infiltration of cardio-inflammatory Ly6C high Mos/Mps in MI mice.
Monocyte and macrophage subsets are critical for cardiac fibrosis 48 and MI recovery. 49 Previous studies have shown that Mos/Mps could be divided into two subsets (pro-inflammatory Ly6C high and less inflammatory and patrolling Ly6C low ) according to Ly6C expression level in mice. 7 Meanwhile, CD14 + CD16 − Mos resembles mouse Ly6C high cells, while CD14 Dim Mos resembles mouse Ly6C low Mos in humans. 50 In myocardial infarction model mice, two types of monocytes/macrophages play an important role in cardiac healing. In the early stage (1-3 days after MI), Ly6C high (CD16-negative) pro-inflammatory monocytes lead to digestion of damaged tissue through phagocytosis and proteolysis, and in the late stage (3-5 days after MI), Ly6C low (CD16-positive) anti-inflammatory monocytes promote healing through myofibroblast aggregation, angiogenesis, and collagen deposition. 7 Consequently, regulating the monocyte /macrophage transformation from Ly6C high to Ly6C low to inhibit myocardial fibrosis is beneficial to the repair of the heart after myocardial infarction. In this study, after the treatment of GSRd, increased non-classical Ly6C low Mos/Mps and reduced classical Ly6C high Mos/Mps in myocardial tissue after MI, and the phenotype of tissue Mos/Mps was transformed into a regression phase.
Furthermore, in vivo and in vitro studies have confirmed that GSRd improves the cardiac immune environment after MI by enhancing cardiac Ly6C low Mos/Mps recruitment. The possible mechanism may be related to the activation of the Akt/mTOR pathway to regulate the transformation of macrophages from excessive autophagy M1 type to anti-inflammatory protective M2 type polarization, namely, Ly6C high to Ly6C low . The Akt/mTOR pathway is a major signal transduction pathway that regulates cell proliferation, survival, autophagy, and apoptosis. mTOR, a downstream molecule of Akt, is a crucial regulator of cellular protein synthesis, growth, expansion, autophagy, lysosomal function, and cellular metabolism. 51 Myocardial cell death after MI largely depends on inhibiting the Akt/mTOR signaling pathway. 52 Akt1 −/− macrophages reduce mTOR activity and induce M1-type polarization. 53 This study confirmed that GSRd regulates the Akt/mTOR pathway activity in peritoneal macrophages.

Conclusions
GSRd improves cardiac function, inhibits cardiac fibrosis and cardiomyocyte apoptosis in MI mice, suppresses cardiac inflammation by regulating the polarization of Mos/Mps subpopulations, enhances cardiac Ly6C low Mos/Mps recruitment to alleviate the immune environment in post-MI hearts, and promotes cardiac repair. The possible mechanism is related to activating the Akt/mTOR pathway to regulate the macrophage polarization from excessive autophagic M1 type to antiinflammatory protective M2 type, that is, the conversion of Ly6C high to Ly6C low .


s2fieldsofstudy: Biology Medicine

text:
Opioids alter paw placement during walking, confounding assessment of analgesic efficacy in a postsurgical pain model in mice

Abstract Introduction: Hind paw–directed assays are commonly used to study the analgesic effects of opioids in mice. However, opioid-induced hyperlocomotion can obscure results of such assays. Objectives: We aimed to overcome this potential confound by using gait analysis to observe hind paw usage during walking in mice. Methods: We measured changes in the paw print area after induction of postsurgical pain (using the paw incision model) and treatment with oxycodone. Results: Paw incision surgery reduced the paw print area of the injured hind paw as mice avoided placing the incised section of the paw on the floor. Surprisingly, oxycodone caused a tiptoe-like gait in mice, reducing the paw print area of both hind paws. Further investigation of this opioid-induced phenotype revealed that analgesic doses of oxycodone or morphine dose-dependently reduced the hind paw print area in uninjured mice. The gait changes were not dependent on opioid-induced increases in the locomotor activity; speed and paw print area had no correlation in opioid-treated mice, and other analgesic compounds that alter locomotor activity did not affect the paw print area. Conclusion: Unfortunately, the opioid-induced “tiptoe” gait phenotype prevented gait analysis from being a viable metric for demonstrating opioid analgesia in injured mice. However, this work reveals an important, previously uncharacterized effect of treatment with analgesic doses of opioids on paw placement. Our characterization of how opioids affect gait has important implications for the use of mice to study opioid pharmacology and suggests that scientists should use caution when using hind paw–directed nociceptive assays to test opioid analgesia in mice.

Introduction
Mice are widely used to study the behavioral effects of opioids, including analgesia, dependence, and respiratory depression. 8,13,29,32 Antinociceptive properties of mu-opioid agonists are often tested in mice using reflexive hot-plate or tail-flick assays. 13,32,36 To study opioid analgesia in tissue or nerve injury models in mice, injury-induced sensitization and its reversal with opioids are assessed by measuring sensitivity to stimuli directed to the affected body region, which is often the hind paw. 12 Most commonly used hind paw-directed assays entail the quantification of a withdrawal response to the application of noxious stimuli to the hind paw. 5,12 These assays reflect evoked responses to an exogenous stimulus and do not reflect naturalistic pain behaviors. 12,33 They also measure reflexive withdrawal responses, which may not accurately represent pain or the effects of analgesia. 15,19 In addition to having potentially flawed face validity, hind paw-directed evoked pain assays are particularly challenging to perform on opioid-treated mice because of increases in the locomotor activity induced by mu-opioid agonists. 10,35,36 Because of the shortcomings of evoked withdrawal tests, alternative approaches are needed to assess opioid analgesia in mice. We and others have used a variety of nonreflexive behavioral assays to assess pain sensitivity and analgesia in mice, such as voluntary wheel running, home cage lid hanging, and gait analysis. 22,31,33,38,42 The Noldus CatWalk XT gait analysis system was originally designed to quantify deficits in motor function seen in models of spinal cord injury. 16,17 More recently, others have used this system to demonstrate the effects of injury and analgesic treatment on gait in rats and mice. 1,22,31,38 Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
To achieve our goal of measuring behavior affected by postsurgical injury and reversed by opioids in mice, we used the CatWalk gait analysis system to observe hind paw usage during voluntary walking behavior. Gait analysis provides a means of assessing hind paw usage with a nonreflexive behavioral assay that would be technically feasible with a hyperactive mouse. Using the CatWalk system, we measured changes in the paw print area after induction of postsurgical pain with the paw incision injury model and treatment with oxycodone in mice. We hypothesized that paw incision injury would produce alterations in paw placement during walking that could be reversed with oxycodone. We found that paw incision injury reduces the print area of the injured paw. Surprisingly, however, analgesic doses of oxycodone induced a "tiptoe" phenotype that dose dependently reduced the print area of all paws. The reduction in the paw print area was independent of walking speed and was specific to opioids (oxycodone and morphine), as mice treated with the nonopioid analgesics fenobam and D 9 -tetrahydrocannibinol did not show altered paw print area. Overall, this work demonstrates the effect of opioid treatment on paw usage and has implications for the use of hind paw-directed sensory assays in opioid-treated mice.

Animals
All procedures used in this study were approved by the Institutional Animal Care and Use Committee at Washington University in St. Louis School of Medicine. C57BL/6 mice bred in-house were given food and water ad libitum in a facility with a 12-h light:dark cycle. Behavior was tested during the light phase at 8 to 12 weeks of age. Male and female mice were tested separately. Animals were randomly assigned to each condition. Different test groups (injury and drug treatment) had equal average weights. Cages had mixes of animals from different test groups, group-housed with 3 to 5 mice per cage. Experimenters were blinded to drug treatment or surgical condition until data were analyzed.

Paw incision surgery
Paw incision surgery was performed on the right hind paw (Fig.  1A), following previous published procedures. 11,41 While under isoflurane anesthesia, the surface of the right hind paw was sterilized and a 5-mm incision was made through the skin starting 2 mm from the heel using a type 11 scalpel blade (McKesson, Richmond, VA). The flexor digitorum brevis muscle was pulled up to isolate, and an approximately 3-mm vertical incision was made through the muscle. The skin was closed with 2 6-0 silk sutures (Ethicon, Guaynabo, Puerto Rico). Antibiotic ointment (McKesson) was applied. For the sham surgery, mice were anesthetized, the right hind paw was sterilized, and sutures were applied, but no incisions were made.

Electronic von Frey
Mechanical allodynia was measured using an electronic von Frey anesthesiometer (IITC Life Science, Woodland Hills, CA). 34 Mice were placed on an elevated stainless steel mesh platform and isolated in individual acrylic containers. Mice were acclimated for at least 1 day and were habituated for 1 hour before testing. Alternating hind paws, 5 measurements were taken per paw, with at least 1 minute between measurements for a single paw. On the day of testing postsurgery, approximately 15 minutes after acquiring baseline data, the animals were injected with the test compound and tested again for postdrug responses 30 minutes later. The blunt tip of the anesthesiometer was applied to the hind paw approximately 4 to 5 mm from the heel (corresponding to the space between the 2 sutures on the hind paw in sham and injured animals, Fig. 1A), and the amount of force applied before the animal withdrew their hind paw was recorded. Of the 5 measurements taken per paw, the highest and lowest measurements were excluded, and the remaining 3 measurements were averaged. The approximate size and location of the incision (magenta) and the 2 sutures that close the wound (black) are also shown, along with the area that the von Frey filament was placed during sensory testing (gray dashed circle). The area of the hind paw that makes contact with the ground is indicated (green). (B) A paw print image produced by the Noldus CatWalk software (white box surrounding the paw print added by the software) from a mouse walking without injury or drug treatment. Yellow outlines added to A and B indicate the outline of the paw print captured by the software and the corresponding hind paw anatomy that generated the print.

CatWalk gait analysis
The Noldus CatWalk XT gait analysis system (Noldus Information Technology, Leesburg, VA) was used to analyze the paw prints of mice. 1,22,31,33,38 In a dark room, mice walked across an elevated glass platform with a camera positioned underneath, facing the underside of the animal. Light illuminated the section of the paws that applied weight on the glass (Fig. 1A). Paw prints were imaged by the camera and measured using the Noldus CatWalk XT 10 software (Fig. 1B). The paw print area refers to the total area of the glass in contact with the paw, measured at the point in the step when the area of contact with the glass is maximal for that paw print. Analgesic studies in incision animals were performed in mice that had a relative paw print area in the ipsilateral paw that was no greater than 80% than of the contralateral paw; 10 of 46 mice were thus excluded based on this criterion.

Imaging mouse walking behavior
Mice walked across an elevated glass platform and were imaged with a Google Pixel 4 (Google, Mountain View, CA).

Statistics
Data were analyzed using GraphPad Prism 9.3.0 (GraphPad Software, San Diego, CA). Data were analyzed with one-way or two-way analysis of variance (ANOVA) tests, followed by Dunnett or Tukey multiple comparisons tests, respectively. When 2 groups were being compared, t tests were used (paired or unpaired, as indicated below). A simple linear regression was used to test the correlation between the paw print area and speed. Statistical values and tests used are described in the text below. Significant main effects are reported in the text, and significant post hoc test results are indicated in the figures.

Oxycodone reverses incision-induced mechanical allodynia but not reduced paw print area of the injured paw
Consistent with prior reports, we find that paw incision produces robust sensitization to mechanical stimuli in mice as assessed using von Frey 1 day after injury ( Fig. 2A). After treatment with oxycodone (10 mg/kg, s.c.) on day 1 postsurgery, the withdrawal thresholds were no longer significantly different from preinjury baseline values (F 6, 60 5 7.097, P , 0.0001, 2-way ANOVA interaction effect).
Paw incision alters paw placement by inducing paw guarding behavior in rats and mice. 4,41 Therefore, we hypothesized that the CatWalk gait analysis system, which evaluates paw placement during walking, would detect altered paw usage in injured mice. We performed paw incision surgery and analyzed the paw print area during walking using the CatWalk system 1 day postsurgery. Paw incision surgery reduced the paw print area of the injured (ipsilateral) hind paw, relative to the animal's contralateral paw (P , 0.0001, paired t test) or to sham animals' ipsilateral paws (P , 0.0001, unpaired t test; Fig. 2B). Viewing images of the paw prints shows that the reduction in the paw print area is due to an avoidance of placing weight on the proximal section of the paw, closer to the site of the incision (Fig. 2C).
Next, we assessed if oxycodone could reverse the incisioninduced reduction in the paw print area (Fig. 2D). As in Figure 2B, the paw print area was reduced in injured hind paws relative to contralateral paws 1 day postsurgery. We hypothesized that oxycodone would increase the contact area of the injured paws. Surprisingly, however, oxycodone (10 mg/kg) decreased the paw print area of both uninjured and injured hind paws (F 4, 31 5 10.64, P , 0.0001, 2-way ANOVA interaction effect). In other injury models, different doses of oxycodone were required to reverse sensitization phenotypes measured with different behavior tests. 26,40 Therefore, we sought to determine if a lower dose of oxycodone would reverse the impaired paw placement in the incised paws without affecting the contralateral paws (Fig. 2E, F). No dose of oxycodone significantly altered the paw print area of the injured paw relative to the vehicle (ie, 0 mg/kg of oxycodone) treatment (Fig. 2E); however, 3 and 10 mg/kg of oxycodone significantly decreased the area of the contralateral paw prints relative to the vehicle (F 4, 31 5 9.589, P , 0.0001, 1-way ANOVA; Fig. 2F).

Opioids dose-dependently decrease the paw print area during walking
Based on the finding of oxycodone's effect on the contralateral paw print area, we further investigated the effect of oxycodone on paw placement in uninjured mice. Using a range of analgesic doses of oxycodone, 2,14,26,30,39,40 we assessed paw placement during walking before and after oxycodone treatment, as in Figure 2D. We found that oxycodone caused dose-dependent reductions in the print area of the hind paws in both female (F 4, 45 5 12.48, P , 0.0001, 2-way ANOVA interaction effect; Fig. 3A) and male (F 4, 40 5 19.86, P , 0.0001, 2-way ANOVA interaction effect; Fig. 3B) mice, and the effect of oxycodone was greater than that of vehicle in females (F 4, 45 5 11.67, P , 0.0001, oneway ANOVA; Fig. 3C) and males (F 4, 40 5 26.65, P , 0.0001, one-way ANOVA; Fig. 3D), as well. In females, the hind paw print area was reduced after 3 or 10 mg/kg oxycodone treatment (Fig.  3A), and in males, the hind paw print area was reduced after 1, 3, or 10 mg/kg oxycodone treatment (Fig. 3B), relative to baseline print areas. The front paw print area was also significantly reduced by oxycodone in a dose-dependent fashion in both females (F 4, 45 5 11.27, P , 0.0001, 2-way ANOVA interaction effect; Fig. 3E) and males (F 4, 40 5 7.980, P , 0.0001, 2-way ANOVA interaction effect; Fig. 3F). Significant reductions relative to baseline were seen after 3 and 10 mg/kg oxycodone treatment in both female and male front paw print areas, and, as with the hind paws, the change in the paw print area induced by oxycodone was significantly greater than that induced by vehicle in females (F 4, 45 5 7.771, P , 0.0001, one-way ANOVA; Fig. 3G) and males (F 4, 40 5 7.539, P 5 0.0001, one-way ANOVA; Fig. 3H).
To assess how oxycodone alters paw placement to reduce the paw print area, we examined paw print images and images of animals walking after oxycodone treatment. Images of the paw prints when the maximum contact is made with the glass showed that mice favored the distal parts (toward the toes) of their feet after treatment with 10 mg/kg of oxycodone relative to their walking behavior at baseline (Fig. 4A). While observing mice at the moment when the hind paw first lands during a step, we saw that vehicle-treated mice (Fig. 4B) placed their full hind paw pad down on the glass and that 10 mg/kg oxycodone-treated mice (Fig. 4C) avoided placing the more proximal section of their paws (ie, their heels) down on the glass. Oxycodone-treated mice also showed rigid, vertically pointed tails and, by adopting a tiptoe-like posture while walking, were more elevated from the platform. The avoidance of heel placement seen after oxycodone treatment could thus produce a reduction in the paw print area.
To determine if the effect on paw placement found with oxycodone could be seen with another analgesic mu-opioid receptor agonist, we examined paw placement on the CatWalk before and after morphine treatment. Morphine also dosedependently decreased the paw print area. As with oxycodone, 10 mg/kg of morphine (s.c.) reduced the paw print area relative to baseline (F 4, 28 5 2.840, P 5 0.0428, 2-way ANOVA effect of drug; Fig. 5A, B).

Altered walking speed does not drive the reduced paw print area
The oxycodone doses used here significantly increase the distance covered in an open field, 10 and, thus, with time being a constant, average speed in an open field is increased. Therefore, we asked whether altered walking speed could cause the opioid-induced tiptoe like gait. We saw no correlation between the paw print area and the speed of the mice (Fig.  6A, B).
Nearly all vehicle-treated mice had slower average speeds on the CatWalk during their postdrug trials relative to their baseline trials in female mice (F 4, 45 5 9.736, P , 0.0001, 2-way ANOVA interaction effect; Fig. 6C), and both female and male mice (F 4, 40 5 6.671, P 5 0.0003, 2-way ANOVA interaction effect; Fig. 6D) were significantly slower during their post-10 mg/kg of oxycodone trials relative to baseline, as well. Notably, the reduction in speed during postdrug trials was significantly greater in the mice treated with 10 mg/kg of oxycodone relative to the change in speed in mice treated with the vehicle in both females (F 4, 45 5 12.88, P , 0.0001, one-way ANOVA; Fig. 6E) and males (F 4, 40 5 8.954, P , 0.0001, one-way ANOVA; Fig. 6F). Interestingly, in female mice, 3 mg/kg of oxycodone increased walking speed relative to baseline (Fig. 6E). The hind paw print area was decreased at both 3 and 10 mg/kg of oxycodone in females (Fig. 3A, C), but speed was increased at 3 mg/kg and decreased at 10 mg/kg of oxycodone. In addition, the effects of oxycodone on the hind paw area in males (Fig. 3B, D), particularly the equivalent effects of 3 and 10 mg/kg on paw placement, did not match the effects of oxycodone on walking speed (Fig. 6F), as only 10 mg/kg of oxycodone affected walking speed in male mice. Together, these data support the conclusion that the change in speed caused by oxycodone treatment did not drive the change in paw placement.
As an additional exploration into the effect of walking speed on paw placement, we tested the effects of 2 nonopioid analgesic drugs that differentially affect open field activity on the paw print area in female mice. Fenobam, an mGlu5 receptor negative allosteric modulator, reduces pain behaviors and increases open field activity. 27 D 9 -THC, a cannabinoid type 1 and type 2 receptor agonist, provides antinociception and decreases open field activity. 3,9,23 Neither fenobam (30 mg/kg, i.p.) nor D9-THC (3 mg/ kg, s.c.) affected the paw print area differently from their vehicletreated counterparts (Fig. 7A, B). Similarly to the oxycodone-treated mice, the effects on speed also did not match their respective effects on previously reported open field activity. Fenobam and vehicle treatment had equivalent effects on speed  (Fig. 7C), and mice treated with D 9 -THC did not have the same reductions in walking speed as the vehicle-treated mice (P 5 0.0192, unpaired t test; Fig. 7D).

Discussion
Overall, we found that paw incision surgery reduced contact of the incised section of the paw during walking on the CatWalk platform, resulting in a decrease in the paw print area of the injured hind paw. Treatment with analgesic doses of opioids induced a tiptoe like gait in injured and uninjured mice, causing reduced paw print area of both hind paws. Therefore, although the paw print area during walking may be used to as a phenotypic demonstration of postinjury sensitization, it is not one that can be reversed by opioids in mice, as opioid treatment presents a confounding phenotype.
Within the results showing that opioids induce tiptoe walking, notable differences were seen between some subgroups. At the same doses, morphine had smaller effects on the paw print area (Fig. 5A, B) than oxycodone did (Fig. 3A, C). Others have observed similar differences, as morphine has also been shown to be less analgesic in injured mice and has reduced antinociceptive efficacy in uninjured mice, relative to the same doses of oxycodone. 26,30 In addition, females and males responded differently to the same oxycodone doses. In males, 1 mg/kg of oxycodone reduced the hind paw print area relative to baseline (Fig. 3B), whereas females were only affected at a dose as low as 3 mg/kg of oxycodone (Fig. 3A). The sex differences in the effects of oxycodone on the paw print area echo the literature demonstrating the enhanced sensitivity of male mice to the effects of opioids relative to female mice, including opioidinduced antinociception, tolerance, and locomotor activity. 7,10,24 In addition to the effects of opioids on the paw print area, this work demonstrates an effect on walking speed that has not previously been quantified. Here, we show that male and female mice have a greater decrease in walking speed after treatment with 10 mg/kg of oxycodone than their vehicle-treated counterparts (Fig. 6C-F). This dose of oxycodone was previously reported to increase the total distance traveled in an open field in both sexes. 10 An increase in distance traveled is typically interpreted as an increase in average speed for the whole  Our results suggest that the 10 mg/kg oxycodoneinduced increase in distance covered may instead be driven by an increase in the percent of time spent mobile, with the walking speed (the average speed during mobile periods, not during the whole course of the test) actually being lower than that of vehicletreated mice. An increase in the percent of time spent walking would also lead to more distance covered, a result that is often interpreted as being due to faster walking. As researchers explore the side effects of opioids in mice, including locomotor sensitization, our results on the effects of oxycodone on speed may help people better evaluate their data. Similar differential effects were shown here for THC and fenobam; the average walking speed in CatWalk did not align with the average speed previously shown in an open field, as calculated using the total distance covered. CatWalk and open field experiments have different conditions (eg, floor material and lighting) that could contribute to different walking behaviors in each type of arena, but it would be interesting to parse out the effects on walking speed and time spent walking in an open field after treatment with these drugs as well.
One other mouse model that has shown tiptoe walking is the twy Yoshimura mouse, but the cause of tiptoe walking in this animal is a spinal injury arising from spinal ligament ossification and is unlikely to share a causal mechanism with acute opioidinduced tiptoe gait. 18 Others have anecdotally noted the presence of tiptoe like walking induced by buprenorphine, 20,21 but none have quantified this behavior, and the cause is unknown.
One known side effect of mu-opioid receptor agonist treatment that could cause altered paw placement is muscle rigidity. Rigid extension of the hind limbs resulting from subcutaneous morphine has been noted in mice 6 and was quantified in hind limb electromyographic activity studies using rats treated with subcutaneous alfentanil. 37 Muscle stiffness in the hind limbs could cause the back legs to be extended backward, preventing the heels of the hind paw from making contact with the walking surface, leading to a reduced hind paw print area. At higher oxycodone doses, this rigid extension may also occur in the fore limbs, leading to the reduced paw print area seen in the front paws. Similarly, muscle rigidity is seen in the tail (ie, Straub tail) in a dose-dependent fashion 6 that correlates well with the dose-dependent effect of tiptoe walking (Fig. 4C), supporting the potential conclusion that muscle rigidity drives this effect.
An interesting effect on speed is seen in females treated with oxycodone; compared with their baseline speed, postdrug walking speed dose dependently increases with oxycodone treatment up to 3 mg/kg but is significantly decreased with 10 mg/kg of oxycodone. If muscle rigidity begins between 3 and 10  mg/kg in females, perhaps the hyperlocomotion effect of oxycodone begins to be outweighed by the muscle rigidity effect at doses above 3 mg/kg. In other words, oxycodone may dosedependently increase the drive to be mobile (hence the increase in open field distance covered at 10 mg/kg 10 ), but by 10 mg/kg, the muscle rigidity effect is strong enough to slow down the walking speed so that the mice are not walking faster, they are simply walking for longer.
Ideally, there would be a dose of oxycodone that would provide analgesia in incised animals without inducing tiptoe walking. Indeed, the effective dose of oxycodone can vary for different phenotypes of a single injury model; for example, in a mouse model of burn pain, the oxycodone dose required to reverse injury-induced CatWalk phenotypes was higher than the dose required to reverse injury-induced von Frey effects. 40 However, in the paw incision mice tested here, the oxycodone doses that were low enough not to affect the paw print area in uninjured paws were not sufficient to reverse the paw print area phenotype in injured hind paws. The dose range at which analgesia is typically provided by oxycodone (1-20 mg/kg) 2,14,26,30,39,40 overlaps with the dose range for tiptoe walking shown here (1-10 mg/kg; Fig. 3). Therefore, although the paw print area is a robust metric for demonstrating gait alterations induced by paw incision, this metric cannot be used to evaluate oxycodone's ability to provide analgesia in this injury model.
Additional assessments are needed to confirm that the incision-induced paw print area reductions are indeed a painrelated phenotype. Opioid treatment was shown to reverse paw usage impairments (guarding behavior) caused by paw incision 41 and paw print area changes induced by other injury models 1,41 in rats. Furthermore, the paw print area changes induced by neuropathic injury were reversed by gabapentin in mice. 22 This suggests that altered paw placement is indeed a pain-related symptom that can be reversed by analgesics in rodents.
As a future direction, other metrics of paw usage could be analyzed to determine if they are altered by paw incision surgery and/or opioid treatment in mice. Whereas the CatWalk system measures 2-dimensional paw placement, the amount of weight placed on the paw can only be generally estimated based on the light intensity of the paw print. Oxycodone may not reverse the area of the injured paw that makes contact with the glass, but it may affect how much weight is being placed on that reduced foot print. Static or dynamic weight-bearing assays may be used as an alternative method to find a paw usage phenotype that is altered in an injured state and reversed by oxycodone. Having a better understanding of how paw usage varies with injury or oxycodone treatment, as well as how paw usage phenotypes manifest on test surfaces made of different materials, would also help determine if other paw-directed assays (eg, hot plate) are indeed appropriate to use in these contexts.
These results have interesting implications for how behavioral effects of opioids are studied in mice, including analgesia, muscle rigidity, and locomotion. Alternatives to hind paw-directed reflexive assays for testing opioid analgesia, particularly in a postsurgical pain model, are still needed, but analysis of paw placement during walking may be an interesting area to look for new approaches. Overall, this research will help refine approaches to evaluating opioids in preclinical research, particularly when applied to mouse pain models.

Disclosures
The authors have no conflict of interest to declare.


s2fieldsofstudy: Biology Chemistry Medicine

text:
The glucotoxicity protecting effect of honokiol in human hepatocytes via directly activating AMPK

Introduction Sustained hyperglycemia causes glucotoxicity, which has been regarded as a contributor to hepatocyte damage in type 2 diabetes (T2D) and its metabolic comorbidities. Honokiol is a natural biphenolic component derived from the dietary supplement Magnolia officinalis extract. This study aimed to investigate the effects of honokiol on glucose metabolism disorders and oxidative stress in hepatocytes and the underlying mechanisms. Methods HepG2 cells were treated with glucosamines (18 mM) to induce glucotoxicity as a diabetic complication model in vitro. Results and discussion Honokiol significantly increased glucose consumption, elevated 2-NBDG uptake, and promoted GLUT2 translocation to the plasma membrane in glucosamine-treated HepG2 cells, indicating that honokiol ameliorates glucose metabolism disorders. Furthermore, glucosamine-induced ROS accumulation and loss of mitochondrial membrane potential were markedly reduced by honokiol, suggesting that honokiol alleviated glucotoxicity-induced oxidative stress. These effects were largely abolished by compound C, an AMPK inhibitor, suggesting an AMPK activation-dependent manner of honokiol function in promoting glucose metabolism and mitigating oxidative stress. Molecular docking results revealed that honokiol could interact with the amino acid residues (His151, Arg152, Lys243, Arg70, Lys170, and His298) in the active site of AMPK. These findings provide new insights into the antidiabetic effect of honokiol, which may be a promising agent for the prevention and treatment of T2D and associated metabolic comorbidities.

Introduction: Sustained hyperglycemia causes glucotoxicity, which has been regarded as a contributor to hepatocyte damage in type diabetes (T D) and its metabolic comorbidities. Honokiol is a natural biphenolic component derived from the dietary supplement Magnolia o cinalis extract. This study aimed to investigate the e ects of honokiol on glucose metabolism disorders and oxidative stress in hepatocytes and the underlying mechanisms.
Methods: HepG cells were treated with glucosamines ( mM) to induce glucotoxicity as a diabetic complication model in vitro.

Results and discussion:
Honokiol significantly increased glucose consumption, elevated -NBDG uptake, and promoted GLUT translocation to the plasma membrane in glucosamine-treated HepG cells, indicating that honokiol ameliorates glucose metabolism disorders. Furthermore, glucosamine-induced ROS accumulation and loss of mitochondrial membrane potential were markedly reduced by honokiol, suggesting that honokiol alleviated glucotoxicity-induced oxidative stress. These e ects were largely abolished by compound C, an AMPK inhibitor, suggesting an AMPK activation-dependent manner of honokiol function in promoting glucose metabolism and mitigating oxidative stress. Molecular docking results revealed that honokiol could interact with the amino acid residues (His , Arg , Lys , Arg , Lys , and His ) in the active site of AMPK. These findings provide new insights into the antidiabetic e ect of honokiol, which may be a promising agent for the prevention and treatment of T D and associated metabolic comorbidities. KEYWORDS honokiol, glucotoxicity, glucose metabolism disorder, oxidative stress, type diabetes, AMPK

Introduction
The incidence and prevalence of type 2 diabetes (T2D) are on the rise worldwide and have reached alarming levels. It is estimated to increase from 536.6 million in 2021 to 783.2 million by 2045 (1). Microvascular (retinopathy, nephropathy, and neuropathy) and macrovascular (atherosclerosis) complications associated with diabetes are the major causes of disability and mortality among patients with T2D (2,3). Sustained hyperglycemia in T2D impairs insulin-stimulated glucose uptake and utilization by peripheral tissues, including the liver, which gives rise to insulin resistance (IR) (4,5). In the liver, IR impedes the normal glucose metabolism process, which elevates hepatic endogenous glucose production through increased gluconeogenesis and decreased glycogen synthesis (6). Accumulation of excess glucose in hepatocytes induces glucotoxicity (7). In a diabetic liver, glucotoxicity exacerbates oxidative stress, eliciting glucose metabolism disorders (8,9).
The nutrient-sensing hexosamine biosynthetic pathway (HBP), a glucose metabolic pathway branching off from main glycolysis, mediates glucotoxicity and is strongly associated with diabetic complications (10)(11)(12). As a metabolite of HBP, glucosamine increases HBP flux and promotes IR by inhibiting insulin-stimulated glycogen synthesis and glucose uptake in hepatocytes (13,14). It also impairs glucose transporter 2 (GLUT-2) translocation from an intracellular pool to the plasma membrane, reducing glucose uptake in hepatocytes (15). In addition, glucosamine-induced glucotoxicity impairs the mitochondrial function in hepatocytes, increases ROS production, and causes oxidative stress (16). Hence, it is of great significance to develop natural plant extracts that improve glucose metabolism disorders and alleviate oxidative stress in hepatocytes to prevent the occurrence and development of diabetes and its complications.
AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that regulates cellular energy homeostasis and plays an essential role in hepatic metabolism (17,18). Liver AMPK controls glucose homeostasis mainly by suppressing hepatic glucose production and decreasing the expression of genes involved in hepatic gluconeogenetic genes, which protects against hepatic IR (19,20). Under fasting conditions, AMPK activation enhances energy metabolism by upregulating mitochondrial function in the liver to respond to energy deficiency (21). Conversely, excess nutrients suppress AMPK phosphorylation in the liver and hepatocytes, which triggers increased HBP flux (22). In addition, activation of AMPK alleviates oxidative stress by reducing fatty acid synthesis, promoting NADPH synthesis, and limiting its consumption for maintaining cellular redox and metabolic homeostasis (23,24). Collectively, AMPK is regarded as a potential therapeutic target against hepatic glucose metabolism disorders and oxidative stress in T2D.
Natural products from plants are excellent sources of therapeutic agents (25). Magnolia officinalis, a Chinese medicinal herb, includes two major species, namely, M.  (27)(28)(29). It contains various nutrients and phytochemical components, such as lignans, phenylethanoid glycosides, phenolic glycosides, alkaloids, steroids, and essential oils, which enable it to display anti-oxidative, antiinflammatory, and anxiolytic effects (30). Phenylethanoid glycosides isolated from M. officinalis var. biloba fruits display free radical scavenging activities (28). Magnolol and its isomer honokiol, two lignan compounds, are major bioactive ingredients of M. officinalis (31). Magnolol exhibits multiple beneficial effects on T2D and its complications via improving glucose homeostasis, promoting lipid metabolism, and reducing oxidative stress and inflammation (32). Honokiol, an isolated dietary biphenolic natural product from the bark, leaves, and root of M. officinalis, demonstrated pleiotropic bioactivities such as anti-oxidation, anti-inflammation, hepatoprotection, anti-obesity, and antidiabetes (33-35). However, no previous studies have reported the glucotoxicity-protecting effect of honokiol in hepatocytes. Notably, some studies have reported that honokiol can activate AMPK phosphorylation, improving hepatocyte lipid metabolism. However, whether honokiol can directly bind to AMPK does not have computational molecular docking analyses. It is still unclear if honokiol ameliorates glucotoxicity-induced glucose metabolism disorders and oxidative stress is AMPK-dependent (36,37).
In this study, molecular docking was performed to investigate whether honokiol could directly bind to AMPK active sites. Glucosamine was used as an in vitro model of diabetic complications to assess the antidiabetic activity of honokiol. We examined changes in glucose consumption, 2-NBDG uptake, GLUT2 translocation, production of ROS, and mitochondrial membrane potential in HepG2 cells treated with glucosamine with or without honokiol. Application of compound C, a selective AMPK inhibitor, estimated whether these beneficial effects of honokiol were AMPK-dependent. This . /fnut. . study will provide new insights into the antidiabetic potential of honokiol.

HepG cell culture and viability assay
Human hepatic HepG2 cells were cultured in DMEM/high glucose supplement with 10% FBS, penicillin G (100 U/ml), and streptomycin (100 µg/ml) at 37 • C in a humidified atmosphere with 5% CO 2 . The CCK-8 assay was used to evaluate the viability of HepG2 cells according to the manufacturer's instructions. Briefly, HepG2 cells were seeded in 96-well plates. After overnight incubation, the cells were treated with honokiol (3.125-800 µM) for 18 h or 24 h. Then, 10 µl of CCK-8 was added to each well, and the cells were incubated under conditions of 37 • C and 5% CO 2 for 2 h. The absorbance was measured at 450 nm using the Tecan Spark multimode microplate reader (Tecan, Männedorf, Switzerland). The following formula was used to calculate the cell viability: cell viability (%) =

Diabetic complication model in vitro and drug treatment
The diabetic complication model was induced by glucosamine, as previously described (13). Briefly, HepG2 cells were cultured in high-glucose DMEM supplemented with 10% FBS. The medium was changed after reaching 75% confluence. HepG2 cells were treated with 18 mM glucosamine for 18 h in the serum-free DMEM medium with low glucose to induce glucotoxicity. Before drug treatment, the cells were starved for 4 h in a serum-free DMEM medium with low-glucose levels. HepG2 cells were then cultured with 18 mM glucosamine and co-treated with honokiol or rosiglitazone (positive drug control) for 18 h. The control group was administered the same amount of DPBS. To determine whether honokiol regulates phosphorylation of AMPK, HepG2 cells were pretreated with the AMPK inhibitor compound C (5 µM) for 30 min, followed by treatment with 18 mM glucosamine and co-treated with honokiol for 18 h.

Glucose consumption assay
HepG2 cells were treated with or without 18 mM glucosamine and co-treated with or without honokiol in 96-well plates for 18 h. The glucose content in the supernatant of each well was detected at 505 nm using a glucose assay kit (glucose oxidase method) according to the manufacturer's instructions. The glucose consumption in cell supernatant was calculated by subtracting the measured glucose concentration in the medium from the glucose concentration of the original DMEM media. Then, glucose consumption was normalized to the cell number using the CCK-8 assay in each well.

Glucose uptake assay
Glucose uptake was measured using the fluorescently labeled deoxyglucose analog, 2-NBDG, as described previously (38). Briefly, HepG2 cells were seeded into a 12-well plate and were maintained in a serum-free medium for 4 h. The test compound was added to each cell sample. Following treatments for 18 h, insulin (100 nM) was administered for 30 min. Next, HepG2 cells were treated with 200 µM 2-NBDG for 30 min incubation at 37 • C and then washed 3 times with PBS. 2-NBDG-treated .
HepG2 cells were transferred into a black 96-well plate. The fluorescence was measured using the Tecan Spark multimode microplate reader (Tecan, Männedorf, Switzerland), set at an excitation wavelength of 488 nm and an emission wavelength of 542 nm. For 2-NBDG visualization analyses, HepG2 cells were cultured in a chamber slide. The cell nuclei were stained with Hoechst 33342 staining solution for live cells for 10 min. The image was taken using a fluorescent microscope (Leica DM4000, Germany). The mean fluorescence intensity was quantified using the StrataQuest software module (TissueGnostic, Vienna, Austria). The fluorescence intensity was normalized to the cell number with Hoechst 33342. The 2-NBDG-uptake of cells was expressed as a percentage of control cells.

Reactive oxygen species (ROS) assays
Intracellular ROS levels were generally measured using a ROS assay kit following the manufacturer's instructions. HepG2 cells were seeded in a 12-well plate and were serumstarved for 4 h. After appropriate treatment, HepG2 cells were washed 3 times with PBS and incubated with 10 µM DCFH-DA for 30 min at 37 • C. DCFH-DA can freely diffuse through the cell membrane and be hydrolyzed into DCFH by ester enzymes after entering cells. Intracellular ROS can convert DCFH to fluorescent DCF. The detection of DCF fluorescence represents the intracellular level of active oxygen. HepG2 cells were washed with PBS twice (pH 7.4), and the cell nuclei were stained with Hoechst 33342 staining solution for live cells for 10 min. DCF fluorescence images were acquired using a fluorescence microscope (Leica DM 4000, Germany). ROS levels were quantified using the StrataQuest software module (TissueGnostic, Vienna, Austria). The ROS levels in cells are expressed as a percentage of control cells.

Mitochondrial membrane potential measurement
The mitochondrial membrane potential of HepG2 cells was determined with a Mitochondrial Membrane Potential Assay Kit with JC-1. HepG2 cells were cultured with 18 mM glucosamine and co-treated with honokiol for 18 h. HepG2 cells were incubated with JC-1 at 37 • C for 30 min in the dark and then washed two times with PBS. The image was taken using a fluorescent microscope (Leica DM4000, Germany). The JC-1 red/green fluorescence intensity ratio of each sample was calculated.

Immunofluorescence assays
Membrane translocation of GLUT2 from intracellular vesicles to the plasma membrane in HepG2 cells was observed by immunofluorescence staining. Briefly, HepG2 cells were seeded on sterile glass coverslips and were gently washed 3 times with PBS. Cells were fixed in freshly prepared 4% paraformaldehyde at room temperature for 25 min. Coverslip was rinsed with PBS 3 times for 3 min each. For 30 min, 10% goat serum was used to block at room temperature, and then the goat serum was discarded. Anti-GLUT2 antibody was added, and the cells were incubated at 37 • C for 1 h protected from light. After washing, cells were incubated with the Alexa Fluor 488-conjugated Donkey anti-Goat IgG (H+L) at 37 • C for 1 h protected from light. The coverslips were mounted on microscope slides and sealed with an antifade solution, and nuclei were stained with DAPI. Images were captured using a fluorescence microscope (Leica DM 4000, Germany).

Western blot analysis
The treated HepG2 cells were washed two times with PBS (pH 7.4) and lysed in RIPA lysis buffer to detect total AMPK and phosphorylated AMPK. The membrane proteins were extracted using a Membrane and Cytosol Protein Extraction Kit, following the manufacturer's instructions. Protein concentration was measured with the BCA Kit. A 5 × SDS loading buffer was added. Protein samples were separated by SDS-PAGE and transferred to a PVDF membrane. Then the membranes were incubated with specific primary antibodies, followed by incubation with the appropriate secondary horseradish peroxidase-conjugated antibodies. Immunoblots were captured using the iBright 1500 imaging system (Thermo Fisher). The levels of protein were normalized to GAPDH expression. Quantification of immunoblotting band intensity was performed using Image J.

Molecular docking
Molecular docking was performed using Discovery Studio 2021 (Dassault Systèmes BIOVIA, San Diego, CA), following the standard procedures (39). The co-crystal structure of AMPK with an AMPK agonist was obtained from the RSCB Protein Data Bank (PDB ID: 4ZHX). 4ZHX is a structure of AMPK complexed with its agonist (Pubchem ID: 49870838), which facilitates the precise location of AMPK protein activity sites. Honokiol was docked into the active sites on the docking pocket, which was created according to the position of the agonist of AMPK. The x, y, and z coordinates of the active pocket box center of AMPK were set to 77.525835, 13.612198, and 33.299745, respectively. The radius of the active pocket was set to 17 Å. The docking study employed CDOCKER, an effective method for docking, to conduct a semiflexible docking algorithm based on molecular dynamics simulations. CDOCKER is an accurate docking technology in Discovery  compared with the control cells; *p < . , **p < . , *** p < . compared with the glucosamine-induced cells, ns indicates no significant di erence compared with the glucosamine-induced cells.
Studio. The binding modes were analyzed by Discovery Studio 2021.

Statistical analysis
All of the experiments were performed using at least three independent experiments per sample, and the data were presented as the means ± SEM. Differences among multiple groups were analyzed using one-way ANOVA, and two groups were compared by a two-tailed Student t-test by GraphPad Prism 8. The p-values of less than 0.001 were considered highly statistically significant, while p-values of less than 0.01 or 0.05 were considered statistically significant.
by honokiol at concentrations of 6.25 µM and 12.5 µM ( Figure 1A). The diabetic complication model in vitro was induced by high concentrations of glucosamine in HepG2 cells and confirmed by glucose consumption and the 2-NBDG uptake assay. HepG2 cells were treated with 18 mM glucosamine for 18 h, reducing the viability to 85.08% compared with the control group. However, the combined treatment of honokiol and .

FIGURE (Continued)
mM glucosamine + . µM honokiol + µM compound C. The nuclei were stained with Hoechst . Scale bar, µm. (B) Relative mean fluorescence intensities of ROS were quantified using StrataQuest software. Data are presented as the means ± SEM (n = ). ### p < . compared with the control cells; *p < . , **p < . , ***p < . compared with the glucosamine-induced cells. Abbreviation: CON, control; GLU, glucosamine; GLU+HON, glucosamine + honokiol; GLU+HON+C, glucosamine + honokiol+ compound C. glucosamine did not affect the cell viability compared with glucosamine-treated cells ( Figure 1B). Glucose consumption was significantly reduced after glucosamine treatment in HepG2 cells. Honokiol and rosiglitazone (positive drug control) markedly increased glucose consumption in glucosamineinduced HepG2 cells ( Figure 1C). In addition, the effect of honokiol on insulin-stimulated glucose uptake was determined by 2-NBDG. The results demonstrated that honokiol remarkably increased the glucose uptake in the glucosamine-induced HepG2 cells ( Figure 1D). These results suggested that honokiol promoted glucose metabolism to alleviate glucosamine-induced glucotoxicity in HepG2 cells.
Honokiol was predicted to bind to AMPK Docking analysis revealed that honokiol could bind to the same active site of AMPK agonist (40). The estimated binding free energy is −14.2637 kcal/mol. Furthermore, honokiol formed interactions with residuals of His151, Arg152, and Lys243, which was in accordance with a reported AMPK agonist (Pubchem ID: 49870938). Honokiol also formed other interactions, including hydrogen bonds with Arg70 and Pialkyl interaction with Lys170 and His298 ( Figure 2). These data suggested that honokiol is a potent activator of AMPK.

Honokiol ameliorates glucose metabolism disorder by directly activating AMPK in glucosamine-induced HepG cells
It was known that liver AMPK activation improved hepatic IR in hepatocytes (41,42). Therefore, to further explore whether honokiol improved glucosamine-induced IR by activating AMPK, the HepG2 cells were pretreated with or without compound C (5 µM), a specific AMPK inhibitor, for 30 min. Then, HepG2 cells were treated with 18 mM glucosamine to induce IR and co-treated with 6.25 µM honokiol for 18 h. After treatment with compound C and glucosamine alone, the phosphorylated AMPK levels in HepG2 cells were significantly reduced. However, treatment with honokiol upregulated phosphorylation of AMPK, and compound C significantly decreased honokiol-induced AMPK activation in glucosamine-induced HepG2 cells (Figures 3A,B). Accordingly, compound C essentially abolished the beneficial effects of honokiol on glucose consumption and uptake in glucosamineinduced HepG2 cells, indicating that honokiol ameliorates glucotoxicity-induced IR by activating AMPK (Figures 3C-F).

Honokiol promoted GLUT translocation from cytoplasm to the plasma membrane in an AMPK-dependent manner in glucosamine-induced HepG cells
To further investigate whether honokiol elevated glucose uptake by promoting the translocation of GLUT2 and whether the beneficial effect was related to AMPK activation, the translocation of GLUT2 from the cytoplasm to the membrane was examined by immunofluorescence assays. As shown in Figure 4, glucosamine treatment caused an apparent blockage of GLUT2 translocation in HepG2 cells compared with the control group, and honokiol significantly promoted the glucosamine-induced GLUT2 translocation, thus restoring glucose uptake in glucosamine-induced HepG2 cells. Furthermore, the honokiol-mediated GLUT2 translocation effect was largely blocked by compound C in glucosamineinduced HepG2 cells, indicating that honokiol enhanced glucosamine-induced GLUT2 translocation from the cytoplasm to the membrane by activating AMPK.

Honokiol inhibited ROS overproduction via directly activating AMPK in glucosamine-induced HepG cells
Oxidative stress was strongly associated with hepatic IR. The increased level of ROS, which led to oxidative stress, was one of the primary causes of IR (43,44). To determine whether honokiol could eliminate excessive ROS induced by glucosamine in HepG2 cells, ROS production was determined using DCF-DA labeling, and the mean DCF fluorescence intensity was determined by image analysis. Glucosamine treatment led to increased production of ROS compared with the control group, and honokiol markedly reduced the glucosamine-induced intracellular ROS level. Treatment with compound C largely eliminated the decrease in honokiol-mediated ROS production ( Figure 5).
Honokiol prevented glucosamine-induced loss of mitochondrial membrane potential in HepG cells via directly activating AMPK Cellular ROS levels are closely related to mitochondrial function. Overproduction of ROS leads to the loss of mitochondrial membrane potential (45, 46). To further investigate whether honokiol could protect mitochondrial function under glucosamine-induced stress, the mitochondrial membrane potential was determined by JC-1 staining. Glucosamine alone treatment resulted in the loss of mitochondrial membrane potential compared with the control group. Honokiol co-treatment significantly reversed the loss of glucosamine-elicited mitochondrial membrane potential, indicating that honokiol had a protective effect on the disruption of mitochondrial function caused by glucosamine. The beneficial effect of honokiol on mitochondrial function was largely abrogated by inhibiting AMPK activity with AMPK inhibitor compound C, indicating that honokiol inhibited glucosamine-induced loss of mitochondrial membrane potential by activating AMPK in HepG2 cells ( Figure 6). Taken together, honokiol had a positive effect on reducing oxidative stress by directly activating AMPK in glucosamine-induced HepG2 cells.

Discussion
Fasting hyperglycemia is attributed to the increase in hepatic endogenous glucose production and gluconeogenesis caused by hepatic IR at an early stage in the natural history of T2D (47). During diabetes progression, sustained hyperglycemia induces glucotoxicity, which leads to diabetic complications (48). A limitation of diabetic complication studies is the absence of suitable in vitro models for rapidly screening potential antidiabetic agents. Glucosamine, an endogenous metabolite of HBP, is thought to be a promising inducer for in vitro models of diabetic complications studies. Persistent fasting and postprandial hyperglycemia in diabetic complications increase flux through the HBP and associated protein O-GlcNAcylation, which results in increased endogenous glucosamine that impairs insulin action and triggers oxidative stress (11,49,50). Exogenous glucosamine is transported into hepatocytes by GLUT-2 and further metabolized to N-acetylglucosamine (GlcNAc), the end product of HBP, which is used as a donor for protein O-GlcNAcylation (51,52). Using in vitro models of glucosamine-induced hepatic IR and oxidative stress can effectively characterize in vivo metabolic burdens in patients with diabetic complications, which would provide some insights into the role of antidiabetic agents. In our study, exposure of HepG2 cells to a high concentration of glucosamine alone (18 mM) inhibited glucose uptake and translocation of GLUT2 from the cytoplasm to the cell membrane, which agreed with the previous study (15,53). Meanwhile, glucosamine induces the accumulation of ROS and loss of mitochondrial membrane potential. These findings mimicked glucotoxicity-induced hepatic IR and oxidative stress in diabetic complications. Based on this model, we explored the antidiabetic effect and the mechanisms of honokiol, an active component of the dietary supplement M. officinalis extract.
officinalis extract has been used as a new food ingredient and is classified as "generally regarded as safe" (GRAS) by the United States Food and Drug Administration (FDA) following independent recommendation (55,56). For example, dietary supplements with M. officinalis extract possessed mild anxiolytic and anti-stress effects by reducing cortisol exposure and perceived daily stress in moderately stressed subjects (27, 57). In fact, the addition of 4% M. officinalis extract to chewing gum inhibited plaque formation (58).
Honokiol, as one of the primary active ingredients in M. officinalis, has been shown to exert antidiabetic abilities in multiple animal diabetic models. Hyperglycemia-induced oxidative stress contributes significantly to the development of diabetes and diabetic complications (15). Oxidative stress impairs mitochondrial function and is strongly associated with increased ROS and loss of mitochondrial membrane potential (59-61). There is considerable evidence supporting the antioxidant functionality of honokiol in various diseases, such as cancer, cardiovascular diseases, and neurodegenerative diseases, which has been attributed to the presence of phenolic functions in its structure (62-64). Honokiol prevented the development of diabetes and its multiple complications. As a ROS scavenger, it improved pancreatic β-cell function . /fnut. . by attenuating oxidative stress through Nrf2/ARE pathway in diabetic rats (37) and ameliorated diabetic myocardial ischemia/reperfusion (MI/R) injury by reducing oxidative stress by activating the SIRT1-Nrf2 signaling pathway (65). Furthermore, it rescued myocardial energetic dysfunction in diabetes by activating SIRT3-mediated deacetylation of mitochondrial proteins (66) and alleviated lipid-induced hepatic IR by activating SIRT3-mediated lipophagy (37,67). Collectedly, the antidiabetic effects of honokiol may be attributed to its anti-oxidative property and activation of the deacetylase . /fnut. .

SIRT3.
In this study, we showed that honokiol ameliorated glucosamine-induced glucose metabolism disorder by reversing the reduction of glucose consumption, glucose uptake, and translocation of GLUT2 in human hepatocytes in vitro.
A high concentration of glucosamine triggers glucotoxicity, leading to oxidative stress. Honokiol mitigated oxidative stress by reducing ROS accumulation and mitochondrial membrane potential loss in glucosamine-induced HepG2 cells.
Overall, these results confirmed the protective effects of honokiol against glucotoxicity-induced glucose metabolism disorder and oxidative stress using an in vitro diabetic complications model. AMPK is a vital nutrient-sensing enzyme that mediates cellular energy homeostasis, insulin sensitivity, and oxidative metabolism (68, 69). Dysregulation of AMPK activity leads to impaired glucose uptake and mitochondrial metabolism in studies of animals and humans with T2D (70,71). High glucose inhibited AMPK phosphorylation in the liver and HepG2 hepatocytes (72, 73). Notably, an increase in protein O-GlcNAcylation is considered glucotoxicity and diabetic complications because GlcNAc is produced by HBP, a nutrient-sensing pathway (74). Increased HBP flux reduces AMPK activity, and the inhibition of O-GlcNAcylation has an antidiabetic effect through AMPK activation (75). As expected, our results in this study indicate that glucosamine treatment significantly downregulated AMPK phosphorylation in HepG2 cells.
Therapeutic interventions targeting AMPK represent one of the most effective strategies for treating T2D (41, 76). AMPK activation can improve insulin sensitivity and metabolic health (68). Metformin, a prescribed insulin-sensitizing clinic drug for T2D treatment, improves hepatic glucose metabolism for alleviating IR by activating AMPK (77,78). However, metformin is often accompanied by side effects such as diarrhea, abdominal or stomach discomfort, and congenital disabilities in offspring (79,80). M. officinalis, a Chinese medicinal herb, is widely used to treat gastrointestinal disorders, including diarrhea. As a digest-beneficial dietary supplement, its extract, combined with metformin, might represent a new and promising treatment regimen for T2D. Here, we sought to investigate the role of AMPK in the effects of honokiol on glucose metabolism disorders and oxidative stress in glucosamine-induced HepG2 cells. In our study, honokiol effectively activated AMPK in glucosamine-induced HepG2 cells. The molecular docking result showed that honokiol binds to the same active site of AMPK agonist (Pubchem ID: 49870938), suggesting that honokiol, as a potential AMPK agonist, can directly bind to AMPK. Therefore, we postulated that activation of AMPK might be involved in the protective effects of honokiol on hepatic IR and oxidative stress. To test this possibility, HepG2 cells were pretreated with an AMPK inhibitor, compound C. As expected, compound C significantly inhibited the effects of honokiol on glucose consumption, GLUT2 translocation, ROS accumulation, and damage of mitochondrial membrane potential in glucosamine-induced HepG2 cells. Our results suggest that AMPK is required for honokiol to ameliorate glucosamine-induced glucose metabolism disorders and oxidative stress in HepG2 cells.

Conclusion
This study provides the first experimental evidence for the glucotoxicity-protecting effect of honokiol in glucosamine-induced hepatocytes, an in vitro diabetic complication model. Honokiol ameliorated glucose metabolism disorder by increasing glucose consumption, insulin-stimulated 2-NBDG uptake, and translocation of GLUT2 and alleviated oxidative stress by reducing ROS accumulation and loss of mitochondrial membrane potential in glucosamine-induced HepG2 cells. Molecular docking analysis suggested that honokiol could bind to AMPK directly. Honokiol activated AMPK activity in glucosamine-induced HepG2 cells. These effects of honokiol on promoting glucose metabolism and alleviating oxidative stress were largely eliminated by AMPK inhibitor compound C (Figure 7). These findings provide new insights into the antidiabetic effect of honokiol, which may be a promising agent for preventing and treating T2D and its complications.

Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

Author contributions
DL: conceptualization, funding acquisition, project administration, resources, and writing-review and editing. HL and WL: data curation, investigation, and visualization. WL and XK: formal analysis. HL, WL, and JL: methodology. WL and JL: software. XK and LY: supervision. JY, TZ, and LS: validation. WL, HL, and XK: writing-original draft. All authors have read and agreed to the published version of the manuscript.

Funding
This study was supported by the National Natural Science Foundation of China (81773850) and the Science and Technology Major Project of Hunan Province (2017SK1020).