Review of the pharmacological effects of astragaloside IV and its autophagic mechanism in association with inflammation

Astragalus membranaceus Bunge, known as Huangqi, has been used to treat various diseases for a long time. Astragaloside IV (AS-IV) is one of the primary active ingredients of the aqueous Huangqi extract. Many experimental models have shown that AS-IV exerts broad beneficial effects on cardiovascular disease, nervous system diseases, lung disease, diabetes, organ injury, kidney disease, and gynaecological diseases. This review demonstrates and summarizes the structure, solubility, pharmacokinetics, toxicity, pharmacological effects, and autophagic mechanism of AS-IV. The autophagic effects are associated with multiple signalling pathways in experimental models, including the PI3KI/Akt/mTOR, PI3K III/Beclin-1/Bcl-2, PI3K/Akt, AMPK/mTOR, PI3K/Akt/mTOR, SIRT1–NF-κB, PI3K/AKT/AS160, and TGF-β/Smad signalling pathways. Based on this evidence, AS-IV could be used as a replacement therapy for treating the multiple diseases referenced above.


INTRODUCTION
Traditional Chinese medicine (TCM) has been used to treat various diseases with great effects for a long time [1]. Radix Astragali (Huangqi) is a prevalent TCM. Compared to western medicine, TCM has significantly fewer side effects [2]. In addition to polysaccharides, flavones and amino acids, the primary components are astragalosides [3]. Astragaloside IV (AS-IV) is the primary active astragaloside, whose pharmacological effects have been reported in various diseases in vitro and in vivo. A search strategy was performed of published studies from 2001 to present using keyword searches "(astragaloside IV) and (inflamm)" in PubMed. Then we included the studies related to autophagy and excluded the studies not related to autophagy. In this review, we summarize the protective effects and autophagic mechanism of AS-IV.

PHARMACOKINETICS AND TOXICITY OF AS-IV
The Chinese Pharmacopoeia lists AS-IV as a quality test for Astragalus membranaceus [4,5]. Figure 1 shows the structural formula of AS-IV. As a cycloartane triterpene saponin (CAS number 84687-43-4), the molecular formula and molecular weight of AS-IV are C 14 H 68 O 14 ( Figure 1) and 784.97, respectively [4,6]. AS-IV is easily soluble in ethanol, methanol or acetone [7]. The methods of reflux, ultrafiltration, highspeed centrifugation, water extraction, ultrasonic extraction, and alcohol precipitation are combined to extract and isolate AS-IV [8].
Tissues, including kidney, lung, spleen, liver, skin, adipose tissue, heart, muscle, duodenum, brain, stomach and ovary, in rats after intravenous AS-IV, can detect AS-IV [9]. The highest concentration of AS-IV is in the liver and kidney, followed by the lung, heart and spleen [10], with limited distribution in the brain. Plasma proteins combined with AS-IV, and in urine and faeces, 50% AS-IV was recovered [9]. The linear pharmacokinetic features of AS-IV have been characterized in rats [9]. AS-IV lacks first-pass elimination and is little metabolised in the liver. The absolute bioavailability of oral administration AS-IV is 3.66% in rats and only 7.4% in beagle dogs [11], which restricts its oral administration. AS-IV causes no obvious adverse reactions or preclinical toxicity. Liver and renal function were not affected after oral administration [3,12]. In rats, no maternal toxicity appeared at doses from 0.25 to 1.0 mg/kg, but caution should be taken when using AS-IV to treat cardiovascular disease (CVD) in pregnant women at the dose of 1.0 mg/kg/d [13,14]. AS-IV exhibits maternal toxicity (intravenously guttae 1.0 mg/kg) and foetal toxicity (≥ 0.5 mg/kg) in rats, but there are no teratogenic effects in rats or rabbits [13]. The reproductive toxicity of AS-IV includes a delay in fur development, the cliff parry reflex of pups, and eye-opening under 1.0 mg/kg AS-IV for 4 wk [14]. Hence, AS-IV should be used with caution in pregnant women.  [20]. Decreased inflammation was confirmed by decreased expression of NLR family, pyrin domain containing 3 (NLRP3) and interleukin (IL)-1β [20]. Activated autophagy is demonstrated by increased microtubule-associated protein light chain 3 (LC3II) expression and decreased p62 levels. AS-IV alleviated myocardial ischaemia-reperfusion (I/R) injury by attenuating I/R-induced autophagosome accumulation, which is regulated by reactive oxygen species (ROS). AS-IV upregulated SOD 2 and downregulated O 2 in myocardial I/R injury in vivo and H9C2 (a subclone of the original clonal cell line which exhibits many of the properties of skeletal muscle) cardiomyocyte injury in vitro [21]. AS-IV ameliorated autophagosome numbers in H9C2 cells caused by I/R injury and decreased autophagy-related genes (Beclin-1 and LC3 II) [22]. AS-IV improves cardiomyocyte injury by inhibiting high glucose-induced oxidative stress and autophagy via the miR-34a/Bcl2/(LC3II/LC3I) and pAKT/Bcl2/(LC3II/LC3I) pathways [23]. AS-IV protected against heart damage in rats induced by doxorubicin through autophagy regulation and PI3K/Akt pathway activation [24].

Protective effect of AS-IV on the brain and nervous system
Neurological disorders include stroke, dementia, cerebral ischaemia, epilepsy, Alzheimer's disease and Parkinson's disease [25]. Among them, stroke is the leading cause of death worldwide [26]. Ischaemic stroke causes I/R, leading to brain injury, in which autophagy plays an important role. Huangqi has been used to treat stroke, especially in China, where it has been used for thousands of years [22]. AS-IV can cross the blood-brain barrier [26] and alleviate brain injury due to I/R through autophagy, which has been reported in several studies. AS-IV decreased cerebral I/R injury in middle cerebral artery occlusion rats. AS-IV increased autophagy in HT22 cells in vitro after oxygen and glucose deprivation/reoxygenation (OGD/R). Expression levels of the autophagy marker p62 were reduced, and the ratio of LC3II/LC3I was augmented [27]. However, Xu et al [28] found that AS-IV decreased the increased autophagy markers LC3 II, Beclin-1, and autophagy-related gene ATG12. Moreover, AS-IV (intraperitoneal injection 10 mg/kg/d) may promote functional recovery after spinal cord injury and stimulate autophagy in neuronal cells in vitro [29]. AS-IV reduced autophagic injury in PC12 cells, demonstrating that cell survival was increased and lactate dehydrogenase leakage was decreased by OGD/R. The protective mechanism is related to decreased autophagy, indicating reduced autophagosomes, reduced LC3 II/LC3 I ratio, and p62 protein upregulation. The autophagy-related mechanisms may be associated with PI3K III/Beclin-1/Bcl-2 and the PI3KI/Akt/mTOR signalling pathways [30]. AS-IV suppressed astrocyte senescence of LPS/MPP + induced premature senescence in vitro and loss of dopamine neurons in a murine model of Parkinson's disease. The mechanism included promotion of mitophagy, which reduced generation of mitochondrial ROS and accumulation of damaged mitochondria [31]. Table 2 lists the protective effect of AS-IV on the brain and nervous system.

Protective effect of AS-IV on lung disease
As-IV has been reported to exert protective effects in various diseases, including non-small cell lung cancer (NSCLC), lung toxicity caused by fine particulate matter (PM2.5, < 2.5 μm), and acute respiratory distress syndrome (ARDS), which are related to autophagic mechanisms (Table 3). Lung cancer is one of the most serious diseases worldwide [32]. AS-IV not only suppresses lung cancer in vivo by enhancing immune responses [33], but also increases the chemosensitivity of NSCLC [34]. For NSCLC cells treated with cisplatin, AS-IV inhibited the increased autophagy of proteins Beclin1 and LC3 I/II [35]. Serious lung toxicity is caused by prolonged exposure to PM2.5, and no effective prevention or treatment measures currently exist [36,37]. PM2.5-induced lung toxicity in a rat model in vivo was established using PM2.5 dust suspension through intratracheal instillation. AS-IV increased autophagic flux and inflammation by activating the AMPK/mTOR pathway in rat models [36].

Protective effect of AS-IV on diabetes
As a metabolic disorder, diabetes is marked by high levels of glucose, which can damage the kidneys, heart, eyes, gastric mucosa, and even cause coma and death [11]. AS-IV can lower blood glucose levels and can alleviate diabetic complications in diabetic mice [11]. One of the chronic diabetes complications is diabetic peripheral neuropathy (DPN), whose prevention and treatment have become a hot research topic [38]. Several studies have shown that AS-IV protects against DPN through an autophagic mechanism (Table 4) . AS-IV also exerted neuroprotection by alleviating peripheral nerve myelin sheath injury by inhibiting autophagy and upregulating PI3K/Akt/mTOR signalling pathways [41]. A diabetic liver injury model in type 2 diabetes mellitus (T2DM) rats was induced by highfat diets/streptozotocin. ASIV treatment improved liver injury and suppressed liver autophagy in T2DM rats, which was correlated with upregulation of AMPK/mTORmediated autophagy [42] (Table 4).

Protective effect of AS-IV on organ injury
Many vital roles of the liver, such as drug metabolism and detoxification, cause drug-induced liver Xu et al [28] Ischemic stroke OGD/R in PC12 cells (-) Excessive autophagy and damage in PC12 cells The PI3K I/Akt/mTOR and PI3K III/Becline-1/Bcl-2 signalling pathways Huang et al [30] Spinal cord injury  Lung injury PM2.5-induced lung toxicity in rats (-) PM2.5-induced lung toxicity; (+) autophagic flux (+) AMPK/mTOR pathway Wang et al [36] Lung injury PM2.5 in rats and rat alveolar macrophages  (Table 5). LO 2 cells were treated with an overdose of iron dextran, which was selected as a liver injury in the cell model. Excessive autophagy of hepatocytes was induced in this model, primarily causing hepatocyte damage. AS-IV reduced the growing number of autophagosomes, which was induced by iron dextran. LC3II/I was also significantly downregulated, and p62 was increased in the cell model. Hence, AS-IV reversed this damage by inhibiting excess autophagy [43]. Liver and kidney injuries were induced by cisplatin in rats. AS-IV plays a protective role by inducing autophagy and limiting the expression of proinflammatory mediators [44]. We found that AS-IV alleviated liver injury in acute liver failure induced by Dgalactosamine/lipopolysaccharide by reducing the autophagy levels of monocytes/macrophages (data not shown).

Protective effect of AS-IV on kidney disease
Kidney diseases for which AS-IV has been tested include chronic glomerulonephritis (CGN), and glomerular diseases, which are associated with autophagy treated by AS-IV (Table 6). As an immunemediated disease, CGN is the most common glomerular disease [45]. AS-IV protects against CGN through autophagy activation via the PI3K/AKT/AS160 pathway, which is demonstrated by improved kidney function and ameliorated kidney lesions [46]. AS-IV not only alleviated fibrosis but also improved renal function and morphology in diabetic KK-Ay mice [47] (Table 6). Many glomerular diseases related to renal fibrosis are associated with mesangial cell activation. AS-IV inhibited mesangial cell activation and enhanced autophagy via the SIRT1/NF-κB p65 pathway, and these effects were eliminated by an autophagy inhibitor [47]. October 6, 2022 Volume 10 Issue 28

Protective effect of AS-IV on gynaecological diseases
The gynaecological diseases treated by AS-IV through an autophagic mechanism include triple-negative breast cancer, cervical cancer, and vulvar squamous cell carcinoma (VSCC) ( Table 7). Triple-negative breast cancer seriously threatens women's health worldwide. AS-IV is one of the four active ingredients of SANT (a novel Chinese herbal monomer combination) treatment. The efficacy and safety of SANT as an antitumor agent were evaluated in mouse models. SANT administration exerted significant antitumor efficacy, which enhanced autophagic flux and increased gene expression levels of ATG16L1, ATG9B and ATG4D [48]. The survival rate of advanced cervical cancer remains low. AS-IV decreased the tumour growth curves and suppressed cell invasion through autophagy induction. The autophagy regulatory proteins mRNA-decapping enzyme 1A (DCP1A) and thymosin beta-4 (TMSB4X) were increased in cervical cancer cells in response to AS-IV. Hence, by inducing autophagy, AS-IV inhibits cervical cancer invasion [49]. AS-IV also inhibited the proliferation of VSCC (SW962 cells) by increasing autophagic activity, as evidenced by increased Beclin-1 and LC3 II and decreased p62. AS-IV decreased apoptosis after autophagy inhibition by 3-methyladenine [50].

Other diseases
AS-IV also has beneficial effects on gastric mucosa, flap survival, Graves' orbitopathy (GO), and so on. AS-IV protected the gastric mucosa in a rat model and decreased expression levels of the autophagic proteins Beclin1, p62, ATG5 and ATG12 [51]. AS-IV may have beneficial functions for flap survival. AS-IV increased the flap survival area and reduced tissue oedema. AS-IV promoted survival of skin flaps decreased tissue oedema by activating autophagy in a rat model [52]. AS-IV significantly decreased IL-1β secretion in influenza A (H1N1) infection by activating autophagy [53]. AS-IV decreased inflammatory responses and reduced excessive autophagy induced by heat in vitro and in vivo [54]. AS-IV or rapamycin played an antiapoptotic role by increasing autophagic activity in IL-1β-treated chondrocytes [55]. GO is a disease affecting the cornea of the eye. AS-IV protects against GO orbital inflammation by suppressing autophagy induced by IL-1β[56].

THE AUTOPHAGY PROMOTING OR INHIBITORY EFFECTS OF AS-IV
AS-IV promotes or inhibits autophagy to exert beneficial effects on various diseases. The related signalling pathways of the autophagic mechanism are shown in Table 8. AS-IV promotes or inhibits autophagy to alleviate inflammation and disease severity (Figure 2). AS-IV also promotes or inhibits autophagy to alleviate disease severity not associated with inflammation ( Figure 2).

The autophagy-promoting effects of AS-IV are associated with inflammation
AS-IV has beneficial effects on various diseases by promoting autophagy to alleviate inflammation. These diseases include lung injury induced by PM2.5, liver injury in type 2 diabetic rats, acute spinal cord injury, myocardial hypertrophy, H1N1 infection, and flap survival. AS-IV protected against PM2.5-induced lung toxicity in rats and in vitro through increasing autophagic flux and inhibiting severe inflammation [36,37]. The inflammatory mediators granulocytemacrophage colony-stimulating factor, intercellular adhesion molecule 1, tumour necrosis factor (TNF) α, IL 6, IL-18 and C-reactive protein were inhibited by AS-IV [36,37]. The associated mechanism includes the AMPK/mTOR or PI3K/Akt/mTOR pathway [36,37]. AS-IV induced autophagic flux depending on AMPK activation and inhibiting phosphorylation of mTOR [36]. AS-IV primarily restored autophagic flux through autophagosome degradation and increased autophagosome-lysosome fusion [37]. AS-IV significantly inhibited the protein expression of phosphorylated phosphatidylinositol 3-kinase (p-PI3K), p-Akt and phosphorylated mechanistic target of rapamycin (p-mTOR) [37].
ASIV promoted autophagy through the AMPK/mTOR signalling pathway to further inhibit expression of TNFα and IL6 and protect against liver injury in T2DM rats [42]. ASIV reversed the suppression of the AMPK/mTOR pathway, elevated the pAMPK/AMPK ratio and reduced the pmTOR/mTOR ratio [42]. AS-IV induced autophagy to protect against liver and kidney injury in rats induced by cisplatin, inhibiting the expression of inflammatory mediators, such as the NLRP3 inflammasome [44]. AS-IV may promote functional recovery in vivo after spinal cord injury through a mechanism related to autophagy promotion and inflammatory inhibition, such as the downregulated expression of inducible NO synthase, cyclo-oxygenase-2 and TNF-α. AS-IV may promote autophagy in neuronal cells to inhibit apoptosis and polarize towards an M2 phenotype in microglial cells to attenuate neuroinflammation [29]. AS-IV activated autophagy and reduced the inflammatory mediators NLRP3 and IL-1β in cardiomyocytes, preventing hypertrophy and improving cardiac function induced by mechanical stress [20]. AS-IV activated autophagy triggered by H1N1 infection to reduce secretion of the inflammatory mediator IL-1β [53]. AS-IV promoted skin flap survival via autophagic activation in the rat McFarlane flap model [52]. The related mechanism also involved inflammatory inhibition, which was evidenced by decreased expression levels of TNF-α, IL-1β and IL6 and inhibited leukocyte infiltration October 6, 2022 Volume 10 Issue 28 Table 8 The autophagy promotion or inhibition effects of astragaloside IV

The autophagy-promoting effects of AS-IV are not associated with inflammation
Diseases that benefit from the autophagic mechanism of AS-IV are not related to inflammation and include cancer, diabetes, nervous system diseases, and CVDs. AS-IV exerted anticancer effects on lung cancer, lung adenocarcinoma cells, VSCC, and the gastric mucosa. AS-IV promoted autophagy mediated by the p53/AMPK/mTOR signalling pathway and was favourable in lung cancer [32]. AS-IV enhanced autophagy levels associated with the AKT and mTOR pathways, which was inhibited by bevacizumab in lung adenocarcinoma cells [57]. AS-IV induced autophagy and inhibited cell proliferation in VSCC, which was associated with the TGF-β/Smad pathway [50]. AS-IV promoted the expression level of the autophagy proteins Beclin1, p62, ATG5, and ATG12 to have a beneficial effect on gastric mucosa in vivo [51].
AS-IV exerted beneficial effects on diabetic complications, such as DPN and liver injury. AS-IV activated autophagy to improve renal fibrosis and function in diabetic KK-Ay mice, while an autophagy inhibitor abrogated the effect of AS-IV[40,47]. AS-IV induced autophagy promoted by AMPKα to inhibit DPN progression [39]. AS-IV enhanced autophagy to alleviate myelin sheath injury in DPN induced by Schwann cell apoptosis. Enhanced autophagy was associated with inhibition of the PI3K/Akt/mTOR signalling pathway [41]. ASIV promoted autophagy mediated by the AMPK/mTOR pathway to alleviate liver injury and insulin resistance in diabetic rats [42] .
AS-IV exerts a protective role against senescent astrocytes, ischaemic stroke, CVD and arthritis. AS-IV induced mitophagy to reduce astrocyte senescence, which is involved in PD [31]. I/R caused by ischaemic stroke leads to brain injury, and autophagy reduction plays a role in its pathology, which can be reversed by AS-IV [27]. AS-IV-induced mitochondrial autophagy has favourable effects on mitochondrial dysfunction in rat vascular smooth muscle cells induced by Ang II [58]. Autophagy was promoted by AS-IV or rapamycin to reduce chondrocyte apoptosis caused by IL-1β[55] .

The autophagy inhibition effects of AS-IV are associated with inflammation
AS-IV has beneficial effects on various diseases through autophagy inhibition to alleviate inflammation. These diseases include acute respiratory distress syndrome, kidney disease, GO, and thermal injury. For the pulmonary endothelial ARDS cell model stimulated by LPS, AS-IV inhibited inflammation and apoptosis to limit autophagy initiation, which was evidenced by the downregulated expression of autophagy proteins [34]. AS-IV-activated autophagy is associated with inhibition of the PI3K/ AKT/AS160 pathway to reduce kidney injury and inhibit inflammation in CGN rats [45]. AS-IV suppresses autophagy to reduce the expression of inflammatory mediators such as IL-6, IL-8, TNF-α, and monocyte chemoattractant protein-1, thus protecting against GO[56]. AS-IV alleviated excessive autophagy and inflammatory responses induced by heat injury in vitro and in vivo. Autophagy induced by heat stress can activate and cross talk with the PERK/eIF2α pathway [54].

The autophagy inhibition effects of AS-IV are not associated with inflammation
AS-IV exerts beneficial effects on various diseases through autophagy inhibition, which is not associated with inflammation. These diseases include heart diseases, CVD, nerve injury, liver injury, lung injury, and cancer.
AS-IV can reduce ROS-mediated autophagosome accumulation and myocardial injury caused by I/R [21]. AS-IV inhibits the cardiotoxicity of rats via autophagy inhibition and PI3K/Akt pathway activation [21]. AS-IV decreased apoptosis and autophagosome number, which were increased in hypoxia/ reoxygenation-injured H9C2 cells [21]. AS-IV inhibited autophagy and improved heart dysfunction induced by LPS [19]. AS-IV inhibited HG-induced autophagy and improved cardiac dysfunction[23]. AS-IV inhibited autophagy and decreased mineralization in in vitro and in vivo models of atherosclerosis [59]. AS-IV reversed the upregulation of autophagic proteins, such as Beclin-1, LC3II and ATG12, caused by the intestinal microbiota [28]. AS-IV blocked excessive autophagy injury in PC12 cells in response to OGD/R [30]. AS-IV inhibited mitophagy and excessive autophagy in Schwann cells induced by high glucose [38]. AS-IV reduced the number of autophagosomes and excessive autophagy, alleviating the damage to L02 hepatocytes induced by iron overload [43]. AS-IV degraded autophagosomes to alleviate lung injury caused by PM2.5 in vivo and in vitro [37]. AS-IV inhibited autophagy to counteract chemoresistance in NSCLC cells treated with cisplatin [35]. AS-IV induces autophagy to inhibit the invasion of cervical cancer[49].

CONCLUSION
AS-IV, the primary active astragaloside from Radix Astragali, has been reported to have pharmacological effects on various diseases. The pharmacokinetics characterization revealed that AS-IV was detected in 12 tissues, including the liver and kidney. AS-IV not only promotes but also inhibits autophagic activity through a variety of signalling pathways to improve various diseases. These pathways include the PI3K I/Akt/mTOR, PI3K III/Beclin-1/Bcl-2, PI3K/Akt, AMPK/mTOR, PI3K/Akt/mTOR, SIRT1-NF-κB, PI3K/AKT/AS160, and TGF-β/Smad signalling pathways. The suggested pathway in this literature review is that the autophagic proteins Atg7/Atg12 are mediated by DCP1A and TMSB4X. When the level of Atg7/Atg12 is reduced, the level of DCP1A and TMSB4X is also decreased. The autophagy-related proteins include Beclin-1, LC3II, p62, ATG16L1, ATG9B and ATG4D. AS-IV is distributed widely in various tissues and the autophagic mechanism of AS-IV is a basic biological mechanism. Hence, AS-IV is an effective therapeutic drug for various diseases. According to existing research, AS-IV possesses the potential for broad application in many diseases, and the autophagy mechanism deserves further investigation.