Astragaloside IV Research Articles

Astragaloside IV protects against ischemia/reperfusion (I/R)-induced kidney injury based on the Keap1-Nrf2/ARE signaling pathway.

BackgroundThis study sought to investigate the protective effects of Astragaloside IV (AS-IV) on ischemia-reperfusion (I/R) renal injury based on the keap1-Nrf2/ARE signaling pathway.Methods A total of 36 male Sprague-Dawley rats (aged: 8 weeks; weighing: 200–220 g) were randomly divided into the following 6 groups (n=6 per group): (I) the control group; (II) the sham operation group; (III) the kidney I/R injury group (the I/R group); (IV) the kidney I/R injury group treated with 2 mg/kg of AS-IV (the low-dose group); (V) the kidney I/R injury group treated with 5 mg/kg of AS-IV (the mid-dose group); and (VI) the kidney I/R injury group treated with 10 mg/kg of AS-IV (the high-dose group). Serum creatinine (CRE), serum urea, malondialdehyde (MDA), and superoxide dismutase (SOD) were examined using enzyme-linked immunoassay kits. Cell apoptosis and the pathological damage to the renal tissue were determined by terminal deoxynucleotidyl transferase dUTP nick end labeling, hematoxylin and eosin, and periodic acid-Schiff (PAS) staining. The immunohistochemistry, reverse transcription-polymerase chain reaction (RT-PCR), and western blot methods were used to determine the expression of Nrf2, HO-1, Bcl-2 and Bax in the kidney tissue.ResultsIn the I/R rat model, the serum CRE level was increased. In the acute kidney injury (AKI) and chronic kidney disease (CKD) models, AS-IV treatment significantly decreased serum CRE levels in a dose-dependent manner. AS-IV treatment also reduced the injury of renal tubular epithelial cells, increased the expression levels of Nrf2 and HO-1, decreased the rate of apoptosis, downregulated the level of MDA, and elevated the activity of SOD. In the CKD rat model, the AS-IV treatment groups had reduced renal tubular epithelial cell injury, increased expression levels of Nrf2 and HO-1, decreased MDA levels, and increased SOD activity compared to the I/R group.ConclusionsAS-IV induced the expression of Nrf2, enhanced the activity of antioxidant enzymes, reduced apoptosis, protected against renal I/R injury, and prevented AKI from transforming into CKD. These findings suggest that AS-IV is a promising drug for treating kidney injury.

Analysis of astragaloside IV metabolism to cycloastragenol in human gut microorganism, bifidobacteria, and lactic acid bacteria.

This study investigated different gut bacteria in an anaerobic environment to identify specific candidates that could transform astragaloside IV (AIV) to cycloastragenol (CA). Two representative gut microbes, lactic acid bacteria (LAB) and bifidobacteria, could metabolize AIV to CA. Multiple screenings showed two metabolic pathways to metabolize AIV in two groups of bacteria. LAB metabolized AIV initiated by removing the C-6 glucose, whereas bifidobacteria indicated the initial removal of C-3 xylose. The final products differed between the two groups as bifidobacteria showed the production of CA, whereas LAB demonstrated preferential production of 20R, 24S-epoxy-6α, -16β, -25-trihydroxy-9, -19-cycloartan-3-one (CA-2H).

Astragalus polysaccharides and astragaloside IV alleviate inflammation in bovine mammary epithelial cells by regulating Wnt/β-catenin signaling pathway.

The Wnt/β-catenin signaling regulates cell renewal and repair and is closely associated with inflammation. Astragalus polysaccharides (APS) and astragaloside IV (AS-IV), which are the main active substances extracted from Radix Astragali, protect cells by regulating Wnt signaling in cells, exerting antiinflammatory, antioxidant, and antistress effects. However, the mechanisms by which APS and AS-IV interact with Wnt signaling to achieve their therapeutic effects in bovine mammary epithelial cells (BMECs) are not understood. In this study, we used lipopolysaccharide (LPS)-stimulated BMECs as an in vitro model of inflammation to investigate the effects of APS and AS-IV on Wnt signaling in inflamed BMECs. Drug concentrations were screened using the CCK-8 method, the effect on protein expression was analyzed using immunoblotting, the effect on inflammatory factors using enzyme-linked immunosorbent assay, and the effect on oxidative factors using enzyme labeling and flow cytometry. LPS activated the expression of inflammatory and oxidative factors in cells and inhibited Wnt/β-catenin signaling. APS and AS-IV antagonized the inhibitory effect of LPS, protecting BMECs. They inhibited the expression of the IL-6, IL-8, and TNF-α inflammatory factors, and that of the MDA oxidative factor, and activated Wnt signaling in LPS-stimulated BMECs. Silencing of β-catenin abolished the protective effect of APS and AS-IV against LPS-stimulated BMECs. Thus, APS and AS-IV mediate protective effects in inflammatory BMECs model through activation of the Wnt signaling pathway. Wnt signaling pathway is one of the targets of the inhibitory effects of APS and AS-IV on inflammation.

Protective effect of Astragalus membranaceus and Astragaloside IV in sepsis-induced acute kidney injury.

Background: Acute kidney injury (AKI) is the most common target organ damage in sepsis. Sepsis-associated AKI (SA-AKI) may be characterized by damage to the renal tubular epithelium. In this study, the pharmacological mechanisms of Astragalus membranaceus and its active monomer Astragaloside IV (AS-IV) were predicted based on a network pharmacology approach and validated both in vitro and in vivo using the SA-AKI model.Method: We constructed an in vivo sepsis model using a mouse cecum ligation puncture (CLP) and HK-2 cells were treated with lipopolysaccharide (LPS) to mimic Gram (–) induced sepsis to assess the renal-protective efficacy of Astragalus membranaceus and AS-IV.Results: The findings demonstrated that Astragalus membranaceus and AS-IV attenuate renal tubular injury in mice with polymicrobial sepsis, including vacuolization, loss of brush border, mitochondrial ultrastructural changes, and increased staining of kidney injury molecule-1 (KIM-1). AS-IV protected human proximal tubular epithelial (HK-2) cells against LPS induced cell viability loss. Both Astragalus membranaceus and AS-IV activated the PI3K/AKT pathway both in vitro and in vivo, as shown by Western blot and immunohistochemistry analysis.Conclusion: The findings demonstrate that Astragalus membranaceus and AS-IV protect against sepsis-induced kidney tubular injury by activating the PI3K/AKT pathway.

Astragaloside IV ameliorates metabolic disorder in db/db obese mice as a PPARγ antagonist

Metabolic disorder is highly related to obesity, insulin resistance, hypertension, and hyperlipidemia. The present study found that astragaloside IV (ASI) attenuated metabolic disorder related symptoms and modulated hepatic lipid metabolism associated gene mRNA expression in db/db mice. ASI inhibited rosiglitazone-induced adipocyte differentiation of 3T3-L1 cells, and lipid accumulation in palmitic acid (PA)-induced HepG2 cells with down-regulated mRNA expression of lipogenesis-related genes. In addition, it was predicted to bind to the ligand binding domain (LBD) of PPARγ and inhibit its transactivity. Collectively, our study suggested that ASI improves lipid metabolism in obese mice probably through suppressing PPARγ activity.

Astragaloside IV Protects Detrusor from Partial Bladder Outlet Obstruction-Induced Oxidative Stress by Activating Mitophagy through AMPK-ULK1 Pathway.

Aims Bladder outlet obstruction (BOO) and the consequent low contractility of detrusor are the leading causes of voiding dysfunction. In this study, we aimed to evaluate the pharmacological activity of astragaloside IV (AS-IV), an antioxidant biomolecule that possess beneficial effect in many organs, on detrusor contractility and bladder wall remodeling process. Methods Partial BOO (pBOO) was created by urethral occlusion in female rats, followed by oral gavage of different dose of AS-IV or vehicle. Cystometric evaluation and contractility test were performed. Bladder wall sections were used in morphology staining, and bladder tissue lysate was used for ELISA assay. Primary smooth muscle cells (SMCs) derived from detrusor were used for mechanism studies. Results Seven weeks after pBOO, the bladder compensatory enlarged, and the contractility in response to electrical or chemical stimuli was reduced, while AS-IV treatment reversed this effect dose-dependently. AS-IV also showed beneficial effect on reversing the bladder wall remodeling process, as well as reducing ROS level. In mechanism study, AS-IV activated mitophagy and alleviated oxidative stress via an AMPK-dependent pathway. Conclusion Out data suggested that AS-IV enhanced the contractility of detrusor and protected the bladder from obstruction induced damage, via enhancing the mitophagy and restoring mitochondria function trough an AMPK-dependent way.

Astragaloside IV attenuate MI-induced myocardial fibrosis and cardiac remodeling by inhibiting ROS/caspase-1/GSDMD signaling pathway

ABSTRACT Astragalus membranaceus is a traditional Chinese medicine and has been widely used in treating cardiovascular diseases (CVDs), such as asthma, edema, and chest tightness. Astragaloside IV (AS-IV), one of the major active components extracted from Astragalus membranaceus, has a series of pharmacological effects, including inhibiting inflammation, regulating energy metabolism, reducing oxidative stress and apoptosis. However, the effect of AS-IV on myocardial infarction (MI) and the underlying molecular mechanism remains unclear. The purpose of our study is to investigate the effects of AS-IV on MI-induced myocardial fibrosis and cardiac remodeling and to elucidate its underlying mechanisms. MI was induced by ligation of the left anterior descending (LAD) coronary artery. Echocardiography was used to evaluate cardiac function in mice. Pathological changes in cardiac tissues were analyzed with hematoxylin and eosin (H&E) staining, Masson staining, and wheat germ agglutinin (WGA) staining. Immunohistochemistry was used to detect the expression of fibrosis and inflammation-related proteins. Immunofluorescence and flow cytometry were used to detect ROS level. The expressions of α-SMA, Collagen I, NLRP3, cleaved cas-1, cleaved IL-18, cleaved IL-β, GSDMD-N, and cleaved caspase-1 were examined using western blot. The results of cardiac ultrasound showed that AS-IV could improve poor ventricular remodeling, myocardial pathological staining showed that AS-IV could significantly reduce the myocardial fibrosis and myocardial hypertrophy, ROS levels were also significantly reduced, and the protein expression of NLRP3/Caspase-1/GSDMD signaling pathway was remarkably decreased in the AS-IV group. Furthermore, immunohistochemical staining results showed that the expression of myocardial macrophages and neutrophils in AS-IV group decreased significantly, to further investigate whether the reduction of myocardial pyroptosis by AS-IV is related to the regulation of macrophages, in vitro, AS-IV was selected to stimulate bone marrow-derived macrophages (BMDMs). Our findings indicated that AS-IV protective effect of the heart might be related to the reduction of macrophage pyroptosis. These results demonstrate that AS-IV alleviated MI-induced myocardial fibrosis and cardiac remodeling by suppressing ROS/Caspase-1/GSDMD signaling pathway, AS-IV should be further studied in the future.

Astragaloside IV regulates autophagy-mediated proliferation and apoptosis in a rat model of PCOS by activating the PPARγ pathway.

Objective(s):Astragaloside IV (AS-IV) is a bioactive saponin with a wide range of pharmacological effects. This study was aimed at investigating its potential effect on polycystic ovary syndrome (PCOS). Materials and Methods:Female Sprague-Dawley rats were randomly divided into five groups (control, PCOS, PCOS+AS-IV 20 mg/kg, PCOS+AS-IV 40 mg/kg, and PCOS+AS-IV 80 mg/kg). The pathological injury level of rat ovary was observed with hematoxylin-eosin (H&E) staining; enzyme-linked immunosorbent assay (ELISA) kit was utilized to measure the levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone in rat serum; western blot detected autophagy-associated or peroxisome proliferator-activated receptor γ (PPARγ) pathway-related protein expression; immunofluorescence was performed to observe LC3 level in rat ovarian tissue. After co-treatment with AS-IV and PPARγ inhibitor, the proliferation in ovarian granulosa cell line KGN was examined employing cell counting kit-8 (CCK-8), EdU staining, and colony formation; cell apoptosis was observed with TdT-mediated dUTP nick-end labeling (TUNEL); apoptosis-related protein expression was assayed by western blot. Results:Treatment with AS-IV inhibited the ovarian pathological damage in PCOS rats. It also promoted the level of autophagy and activated PPARγ signaling in the rat PCOS model. In KGN cells, the level of autophagy and expression of PPARγ-related proteins were also elevated by AS-IV treatment. Furthermore, AS-IV facilitated autophagy, thus inhibiting KGN cell proliferation and promoting its apoptosis, through activating the PPARγ signaling pathway. Conclusion:AS-IV-activated PPARγ inhibits proliferation and promotes the apoptosis of ovarian granulosa cells, enhancing ovarian function in rats with PCOS.

Astragaloside Ⅳ alleviates ulcerative colitis by regulating the balance of Th17/Treg cells

BackgroundRestoring immune homeostasis by targeting the Th17/Treg response is a potentially valuable therapeutic strategy for ulcerative colitis (UC). Astragaloside IV (AS-Ⅳ) is a phytochemical naturally occurring in Astragalus membranaceus that has good anti-inflammatory, anti-oxidant and anti-stress properties. However, the effects of AS-IV on the homeostasis of Th17/Treg cells in colitis mice remains unknown. PurposeTo investigate the protective effects and potential immunomodulatory mechanisms of AS-IV on UC. MethodsThis study was constructed for DSS-induced acute colitis and recurrent colitis, with AS-IV administered prophylactically and therapeutically, respectively. The balance of Th17/Treg cells was analyzed by flow cytometry, their specific nuclear transcription factors were detected by RT-PCR as well as their secreted inflammatory cytokines were detected by ELISA and RT-PCR. Notch signaling-related proteins were detected by RT-PCR and Western blotting. Oxidative stress indicators were measured by biochemical technology. ResultsIn this study, AS-IV treatment not only effectively prevented and alleviated the clinical symptoms of DSS-induced colitis mice, including weight loss, DAI soaring, colon length shortening and colon weight gain, but also significantly improved ulcer formation, inflammatory cell infiltration and index, and regulated the expression of inflammatory cytokines in colon tissues. Importantly, the efficacy of high-dose AS-IV (100 mg/kg/day) in mice with recurrent colitis in this study was comparable to that of 5-ASA. AS-IV early administration was able to reshape the homeostasis of Th17/Treg cells in mice with acute colitis; meanwhile, AS-IV inhibited Th17 cell responses and promoted Treg cell responses in mice with recurrent colitis. Moreover, AS-IV not only inhibited the activation of Notch signaling pathway in colitis mice, but also prevented and ameliorated DSS-induced oxidative stress injury. ConclusionIn conclusion, AS-IV effectively prevented and alleviated UC by reshaping Th17/Treg cell homeostasis and anti-oxidative stress.

Investigation on the potential targets of Astragaloside IV against intracerebral hemorrhage based on network pharmacology and experimental validation

Intracerebral hemorrhage (ICH) is a life-threatening type of stroke that affects millions of individuals worldwide. Astragaloside IV (AS-IV), the main bioactive ingredient in Radix Astragali, has been linked to a variety of pharmacologic actions, including stroke. However, the effects and potential mechanisms of AS-IV on hematoma absorption after ICH are still unknown. The study aims to identify potential targets and regulation mechanisms of AS-IV on hematoma absorption after ICH. Network pharmacology, molecular docking, pharmacodynamic study, and western blot were used in this study to explore the potential mechanisms. The results showed that AS-IV could improve the hematoma absorption and neurological outcomes in collagenase VII induced rat ICH models. Molecular docking results had shown that PI3K and AKT were the potential targets of AS-IV against ICH. The experimental validation showed that AS-IV could reduce phosphorylation expression of PI3K and AKT, thereby inhibiting the NF-κB and increasing CD36 expression. This study demonstrated that AS-IV could play a critical role on hematoma absorption after ICH by regulating the PI3K/AKT signaling pathway and promoting CD36 phagocytosis, which provided a new thought for the drug development of ICH.

Astragaloside IV improved antioxidative stress capacity and related gene expression of the Keap1-Nrf2 pathway in grass carp (Ctenopharyngodon idella) hepatocytes under heat stress.

This study aims to investigate the protective effects of astragaloside IV (AS-IV) on the hepatocytes of grass carp (Ctenopharyngodon idella) on heat stress. Cultured cells were treated with AS-IV (0, 50, 100 and 200 μg/ml) at 28°C for 24 h and then exposed to heat stress by increasing the culturing temperature (32 ± 0.5°C) for 6h. The increased temperatures significantly reduced cell viability and superoxide dismutase (SOD) activity, and increased malondialdehyde (MDA) levels in the 0μg/ml AS-IV treatment group at 32°C, but the grass carp hepatocytes treated with 100 and 200 μg/ml AS-IV had significantly increased cell viability and SOD activity and decreased MDA levels. The mRNA levels of keap1a, keap1b, nrf2, gsh-px, cat, cu-zn sod, mgst1 and il-6 were significantly lower in the 0μg/ml AS-IV treatment group at 32°C, while those of keap1a, nrf2, gsh-px, cat, cu-zn sod, gstp1, ho-1 and il-6 were significantly higher in cells treated with 100 or 200 μg/ml AS-IV. Our findings indicate that AS-IV could enhance the antioxidative stress capacity of grass carp hepatocytes under heat stress, and its mechanism may be associated with the activation of the Keap1-Nrf2 pathway. Thus, these results provide new insights into how to alleviate heat stress in grass carp.

Preparation of astragaloside IV (AS-IV) nanoparticles via SAS process for anticancer efficacy: Optimization based on Box-Behnken Design

Despite significant advances in drug formulation, the clinical application of most active pharmaceutical ingredients from traditional Chinese herbs remained a significant challenge due to their poor solubility. To address this crucial issue, astragaloside-IV (AS-IV) was selected as a model drug to prepare nanoparticles with a large specific surface area via a constant, steady, and well-organized supercritical antisolvent (SAS) method. Initially, the optimal range of nanoparticle nucleation was determined by a single-factor investigation under altered critical conditions. Under the optimized conditions obtained from the Box-Behnken Design model analysis (AS-IV solution flow rate (F): 0.85 mL/min, Temperature (T): 42 ℃, Pressure (P): 120 bar), the SAS process resulted in nanospheres with smooth surface and narrow particle size distribution. After the SAS process treatment, AS-IV with altered physical states presented improved solubility and enhanced in vitro antiproliferative effects compared to untreated AS-IV, illustrating the potential application of the SAS method in promoting the bioavailability of low water-soluble drugs.

Astragaloside IV ameliorates pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension by restraining the T follicular helper cell response and expanding T follicular regulatory cell response

BackgroundPulmonary hypertension (PH) is a progressive disorder lacking a validated and effective therapy which characterized by elevated pulmonary arterial pressure, vascular remodeling and eventual death. FDA approved sildenafil is being used as a first-line drug for PH, however, neither survival rates nor quality of life have been improved because of side effects and patient noncompliance. Thus, the exploration of novel therapeutic drugs is urgently needed. Astragaloside IV (ASIV) exhibits a protective effect on HPH, but its mechanisms of action is unclear. HypothesisCD4+T cell subsets, Tfh and Tfr cells, may contribute to the development of chronic hypoxia-induced PH (HPH). We hypothesized that ASIV could effectively ameliorates pulmonary vascular remodeling of HPH by restraining the Tfh cell response and expanding Tfr cell response. Methods and resultsHPH mice model was established by exposure to chronic hypoxia for 21 days. Mice were randomly assigned to six groups: NaCl group, model group, SN group (100 mg/kg of sildenafil), low-dose group (20 mg/kg of ASIV), medium-dose group (40 mg/kg of ASIV) and high-dose group (80 mg/kg of ASIV). Primary culture and identification of distal pulmonary artery smooth muscle cells (PASMCs) in mice were established. Here, we demonstrated that ASIV treatment could significantly ameliorate the increase of mean PAP, RV/ (LV+S) ratio and PAMT in HPH mice. ASIV inhibited Tfh cell differentiation and IL-21 production, but promoted Tfr cell differentiation and TGF-β, IL-10 production. Chronic hypoxia promoted germinal center B cell responses, which inhibited by ASIV. ASIV regulated Tfh and Tfr cell differentiation by inhibiting the phosphorylation of mTOR signaling pathway, and the effect of ASIV-H was better than that observed in the SN group. ASIV inhibited the proliferation, migration and adhesion of PASMCs in vitro. Moreover, ASIV significantly downregulated the protein level of RhoA and upregulated the protein level of p27 in PASMCs under hypoxic condition. ConclusionCollectively, ASIV may regulate Tfh and Tfr cell responses to subsequently repress pulmonary vascular remodeling and hypoxic pulmonary hypertension.

Astragaloside IV attenuated TGF-β1- induced epithelial-mesenchymal transition of renal tubular epithelial cells via connexin 43 and Akt/mTOR signaling pathway

IntroductionThe objective of the study was to observe whether connexin 43 (Cx43) could regulate epithelial mesenchymal transformation (EMT) of renal tubular epithelial cells (RTECs) by influencing Akt/mTOR signaling pathway, and whether ASV could inhibit the development of renal interstitial fibrosis by regulating Cx43. MethodsLentivirus infection was transfected into RTECs with the final concentration of 50 ×PFU/ cell to regulate the expression of Cx43. And RTECs were intervened by different doses of Astragaloside IV (ASV). After synchronous culture of RTECs in each group，the expression levels of EMT-related indicators and Cx43 were detected by fluorescence microscope and Western-Blotting (WB), even the protein expressions and phosphorylation levels of AKT and mTOR in different groups were detected by WB. ResultsWhen the expression of Cx43 in RTECs was regulated by lentivirus infection, the degree of EMT induced by TGF‑β1 and the phosphorylation level of Akt and mTOR were changed accordingly, indicating that Akt/mTOR pathway might be a downstream molecular mechanism by which Cx43 could regulate EMT. After intervention with different doses of ASV, the expression level of Cx43 increased with obvious concentration dependence, and the expression levels of p-Akt and p- mTOR were significantly altered, suggesting that ASV could effectively increase the protein expressions of TGF‑β1-induced Cx43 in RTECs and inhibit the phosphorylation levels of Akt and mTOR. ConclusionCx43 were the main material basis of RTECs’ injury, and ASV could inhibit TGF-β1- induced RTECs’ transdifferentiation. In-depth study of the mechanism might provide a broad application prospect for the treatment of renal interstitial fibrosis.

A comprehensive review of cycloastragenol: Biological activity, mechanism of action and structural modifications

Cycloastragenol (CAG) is a sapogenin of Astragaloside IV (AG-IV), isolated from the dried roots of legumes Astragalus mongolica or Astragalus membranaceus. Cycloastragenol has a steroidal skeleton of tetracyclic triterpene and possess diverse pharmacological activities such as anti-aging, anti-inflammatory, anti-fibrosis, pro-wound healing, liver protection and endothelial protection. In addition, cycloastragenol is the only telomerase activator reported in natural products, which is closely related to MAPK and PI3K/Akt signaling pathways. This review provides a theoretical basis for the development of cycloastragenol into the candidate for the treatment of multi-factorial diseases in clinic, focusing on the extensively biological activities as well as the mechanism of action and structural modification and aiming to attract researchers to conduct in-depth studies on cycloastragenol. Meanwhile, the pharmacokinetics and toxicology studies of CAG are also described for further druggability exploration to provides valuable reference.

Astragaloside IV Ameliorates Isoprenaline-Induced Cardiac Fibrosis in Mice via Modulating Gut Microbiota and Fecal Metabolites.

AimGut microbiota is of crucial importance to cardiac health. Astragaloside IV (AS-IV) is a main active ingredient of Huangqi, a traditional edible and medicinal herb that has been shown to have beneficial effects on cardiac fibrosis (CF). However, it is still uncertain whether the consumption of AS-IV alleviates cardiac fibrosis through the gut microbiota and its metabolites. Therefore, we assessed whether the anti-fibrosis effect of AS-IV is associated with changes in intestinal microbiota and fecal metabolites and if so, whether some specific gut microbes are conducive to the benefits of AS-IV.MethodsMale C57BL-6J mice were subcutaneously injected with isoprenaline (ISO) to induce cardiac fibrosis. AS-IV was administered to mice by gavage for 14 days. The effects of AS-IV on cardiac function, myocardial enzyme, cardiac weight index (CWI), and histopathology of ISO-induced CF mice were investigated. Moreover, 16S rRNA sequencing was used to establish gut-microbiota profiles. Fecal-metabolites profiles were established using the liquid chromatograph-mass spectrometry (LC-MS).ResultsAS-IV treatment prevented cardiac dysfunction, ameliorated myocardial damage, histopathological changes, and cardiac fibrosis induced by ISO. AS-IV consumption increased the richness of Akkermansia, Defluviitaleaceae_UCG-011, and Rikenella. AS-IV also modulated gut metabolites in their feces. Among 141 altered gut metabolites, amino acid production was sharply changed. Furthermore, noticeable correlations were found between several specific gut microbes and altered fecal metabolites.ConclusionsAn increase of Akkermansia, Defluviitaleaceae_UCG-011, and Rikenella abundance, and modulation of amino acid metabolism, may contribute to the anti-fibrosis and cardiac protective effects of Astragaloside IV.

Mitogen-activating protein kinase kinase kinase kinase-3, inhibited by Astragaloside IV through H3 lysine 4 monomethylation, promotes the progression of diabetic nephropathy by inducing apoptosis

ABSTRACT Astragaloside IV (AS-IV) is a bioactive saponin extracted from the Astragalus root and has been reported to exert a protective effect on diabetic nephropathy (DN). However, the underlying mechanism remains unclear. Herein, we found that AS-IV treatment alleviated DN symptoms in DN mice accompanied by reduced metabolic parameters (body weight, urine microalbumin and creatinine, creatinine clearance, and serum urea nitrogen and creatinine), pathological changes, and apoptosis. Epigenetic histone modifications are closely related to diabetes and its complications, including H3 lysine 4 monomethylation (H3K4me1, a promoter of gene transcription). A ChIP-seq assay was conducted to identify the genes regulated by H3K4me1 in DN mice after AS-IV treatment and followed by a Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis. The results showed that there were 16 common genes targeted by H3K4me1 in normal and AS-IV-treated DN mice, 1148 genes were targeted by H3K4me1 only in DN mice. From the 1148 genes, we screened mitogen-activating protein kinase kinase kinase kinase-3 (MAP4K3) for the verification of gene expression and functional study. The results showed that MAP4K3 was significantly increased in DN mice and high glucose (HG)-treated NRK-52E cells, which was reversed by AS-IV. MAP4K3 silencing reduced the apoptosis of NRK-52E cells under HG condition, as evidenced by decreased cleaved caspase 3 and Bax (pro-apoptotic factors), and increased Bcl-2 and Bcl-xl (anti-apoptotic factors). Collectively, AS-IV may downregulate MAP4K3 expression by regulating H3K4me1 binding and further reducing apoptosis, which may be one of the potential mechanisms that AS-IV plays a protective effect on DN.

Hepatoprotective Effects of Astragaloside IV and Formononetin Against Oxidative Injury

The mitochondrial SIRT3 is a major deacetylase protein that regulates important mitochondrial biology, including metabolic pathways, reactive oxygen species detoxification, mitochondrial dynamics, and the mitochondrial unfolded protein response. We found that astragalus aqueous extract increased the activity of SIRT3 in vitro. We further explore the potential antioxidative effects of Astragaloside IV (AST‐IV) and Formononetin (FMR) on induced oxidative stressed AML12 (alpha mouse liver 12) cells. The AML12 cell line was established from healthy hepatocytes from a mouse (CD1 Strain, Line MT42) transgenic for human TFG alpha.We first tested whether AST‐IV or formononetin increases SIRT3 expression in cell model. We cultured AML12 and treated with various concentrations of AST‐IV or formononetin in parallel with untreated control, negative control, and positive control. We found that AST‐IV and formononetin induced SIRT3 expression through Western Blotting (WB) analysis.In addition, we assessed rescue effects of AST‐IV and formononetin on t‐BHP‐induced oxidative injury in AML12 hepatocytes under three separate conditions: pre‐treatment with AST‐IV or formononetin, post‐treatment with AST‐IV or formononetin, and both pre‐treatment and post‐treatment with AST‐IV and formononetin. Through CCK‐8 analysis, we identified that both pre‐treatment and post‐treatment significantly increased cell proliferation on induced‐oxidative stress AML12 cells compared to no treatment and control. Pre‐treatment of AML12 cells before induction of oxidative stress with AST‐IV and formononetin also showed cell proliferation but was less significant compared to AML12 cells that received both pre‐treatment and post‐treatment with AST‐IV or formononetin. Post‐treatment AST‐IV or formononetin after induction of oxidative stress on AML12 cells did not significantly show any difference in cell proliferation compared to non‐treatment and control. Significantly AST‐IV and formononetin showed induction of the SIRT3 protein on tert‐butyl hydroperoxide (t‐BHP) induced oxidative stressed AML12 hepatocytes by WB.Taken together, the increase in cell proliferation is indicative of the antioxidative effects of AST‐IV and formononetin through induction of SIRT3 protein. Further investigation is currently conducted to provide evidence of AST‐IV and formononetin's mechanistic effect on inducing the expression of the SIRT3 protein‐linked downstream pathways for their antioxidative benefit to hepatocytes. The ultimate goal is to utilize these SIRT3 modulators as pharmacologic agents in treating oxidative injury hepatocytes.

Astragaloside IV Alleviates Renal Tubular Epithelial-Mesenchymal Transition via CX3CL1-RAF/MEK/ERK Signaling Pathway in Diabetic Kidney Disease.

BackgroundEpithelial–mesenchymal transition (EMT) plays an important role in interstitial matrix deposition and renal fibrosis in diabetic kidney disease (DKD). It has been verified that Astragaloside IV (AS-IV) is beneficial for ameliorating DKD. However, the underlying mechanisms of AS-IV on regulating EMT in DKD are yet to be established. Accumulated evidence has suggested that C-X3-C motif ligand 1 (CX3CL1) plays a significant role in the progression of EMT.PurposeWe aimed to investigate whether AS-IV could alleviate EMT by regulating CX3CL1 in DKD and reveal its underlying mechanisms.MethodsFor the in vivo study, mice were divided into the following five groups (n=10): db/m+vehicle, db/db+vehicle, db/db+AS-IV-L (10mg/kg/d), db/db+AS-IV-M (20mg/kg/d), db/db+AS-IV-H (40mg/kg/d). After 12 weeks of treatment, the renal injuries were assessed based on the related parameters of urine, blood and histopathological examination. Immunohistochemistry and Western blotting were used to detect relative proteins levels. Then in HK-2 cells, the molecular mechanism of AS-IV attenuating the EMT in mice with DKD through the CX3CL1-RAF/MEK/ERK pathway was studied.ResultsIn the present study, we found that AS-IV reduced urinary protein levels and improved renal pathological damage in DKD mice. Moreover, AS-IV ameliorated the renal tubular EMT induced by hyperglycemia or high glucose (HG), and decreased the expression of CX3CL1 and inhibited the activation of the RAF/MEK/ERK pathway in vivo and in vitro. In HK-2 cells, downregulation of CX3CL1 suppressed the stimulation of the RAF/MEK/ERK pathway and EMT induced by HG. However, CX3CL1 overexpression eliminated the benefits of AS-IV on the RAF/MEK/ERK pathway and EMT.ConclusionIn summary, we indicated that AS-IV alleviates renal tubular EMT through the CX3CL1-RAF/MEK/ERK signaling pathway, indicating that CX3CL1 could be a potential therapeutic target of AS-IV in DKD.

Astragaloside IV drug-loaded exosomes (AS-IV EXOs) derived from endothelial progenitor cells improve the viability and tube formation in high-glucose impaired human endothelial cells by promoting miR-214 expression.

The high glucose changes caused by diabetes mellitus (DM) can damage the vascular system. Astragaloside IV (AS-IV) can improve diabetes and promote angiogenesis. Exosomes (EXOs) help to carry specific drugs into cells efficiently. However, whether AS-IV loaded EXOs (AS-IV EXOs) can improve damaged endothelial cells through miR-214 remains to be determined. We prepared and identified AS-IV EXOs derived from endothelial progenitor cells (EPCs) and high glucose stimulated endothelial cell models to investigate whether AS-IV EXOs can improve damaged endothelial cells through miR-214. We used a transmission electron microscope (TEM) and DAPI staining to identify the morphology and characteristic expression of EPCs and EXOs, and then prepared AS-IV EXOs. Cell function tests were performed to detect the cloning, proliferation, and migration capabilities of cells. Western blot (WB) and real-time quantitative polymerase chain reaction (qRT-PCR) were used to assess the expression level of Tie-2, Ang-1, and PI3K/Akt-related protein. The DAPI staining results showed that inducing human umbilical vein endothelial cells (HUVECs) could effectively absorb AS-IV EXOs. The results of plate clone formation assay, CCK-8, cell adhesion, and transwell assay of HUVECs stimulated by high glucose showed that AS-IV EXOs had a damage relief effect. By the detection of WB and qRT-PCR, it was found that AS-IV EXOs promoted the expression of miR-214 and proteins related to blood vessel growth. After transfection of miR-214 to pre-treat HUVECs under high glucose stimulation, AS-IV EXOs promoted the tube formation of HUVECs by regulating the level of miR-214. By promoting the expression of miR-214, AS-IV EXOs significantly improved the activity and tubularization of HUVECs under high glucose stimulation.