Notoginsenoside R1 targets PPAR-γ to inhibit hepatic stellate cell activation and ameliorates liver fibrosis.

BACKGROUND: Hepatic fibrosis, a common pathological process that occurs in end-stage liver diseases, is a serious public health problem and lacks effective therapy. Notoginsenoside R1 (NR1) is a small molecule derived from the traditional Chinese medicine Sanqi, exhibiting great potential in treating diverse metabolie disorders. Here we aimed to enquired the role of NR1 in liver fibrosis and its underlying mechanism in hepatoprotective effects. METHODS: We investigated the anti-fibrosis effect of NR1 using CCl4-induced mouse mode of liver fibrosis as well as TGF-ß1-activated JS-1, LX-2 cells and primary hepatic stellate cell. Cell samples treated by NR1 were collected for transcriptomic profiling analysis. PPAR-γ mediated TGF-ß1/Smads signaling was examined using PPAR-γ selective inhibitors and agonists intervention, immunofluorescence staining and western blot analysis. Additionally, we designed and studied the binding of NR1 to PPAR-γ using molecular docking. RESULTS: NR1 obviously attenuated liver histological damage, reduced serum ALT, AST levels, and decreased liver fibrogenesis markers in mouse mode. Mechanistically, NR1 elevated PPAR-γ and decreased TGF-ß1, p-Smad2/3 expression. The TGF-ß1/Smads signaling pathway and fibrotic phenotype were altered in JS-1 cells after using PPAR-γ selective inhibitors and agonists respectively, confirming PPAR-γ played a pivotal protection role inNR1 treating liver fibrosis. Further molecular docking indicated NR1 had a strong binding tendency to PPAR-γ with minimum free energy. CONCLUSIONS: NR1 attenuates hepatic stellate cell activation and hepatic fibrosis by elevating PPAR-γ to inhibit TGF-ß1/Smads signalling. NR1 may be a potential candidate compound for reliving liver fibrosis.

Gentianella acuta-derived Gen-miR-1 suppresses myocardial fibrosis by targeting HAX1/HMG20A/Smads axis to attenuate inflammation in cardiac fibroblasts.

BACKGROUND: Continuous activation and inflammation of cardiac fibroblasts (CFs) are essential for myocardial fibrosis. Gentianella acuta (Michx.) Hiitonen (G. acuta), that contains xanthones with cardioprotective properties, a typical healthful herb extensively used to treat cardiovascular diseases in Inner Mongolia region of China. However, it remains unknown whether or not G. acuta-derived miRNAs can shield CFs from activation by inflammatory stimulation. Therefore, we tend to investigated the role and core mechanism of G. acuta-derived Gen-miR-1 in regulating fibrosis and inflammation induced by TGF-ß1. METHODS: An animal model for myocardial infarction was built by subcutaneous injections of ISO and treated with Gen-miR-1 using intragastric administration. The protective effect of Gen-miR-1 on the heart was assessed by pathomorphological analysis of myocardial fibrosis. Using loss- and gain-of-function approaches, Gen-miR-1 regulation of HAX1/HMG20A/Smads axis was investigated by utilizing luciferase assay, Western blot, co-immunoprecipitation, etc. RESULTS: Screened and identified Gen-miR-1 from G. acuta. Gen-miR-1 can enter the mouse body, and markedly inhibit myocardial infarction induced by ISO in mice, as well as suppresses fibrosis in CFs and attenuates the inflammatory response elicited by TGF-ß1 in vitro. Gen-miR-1 downregulates HCLS1-related Protein X-1 (HAX1) expression through direct binding to the 3' UTR of HAX1, which in turn relieves HAX1 from promoting the expression of high-mobility group protein 20A (HMG20A), whereas HMG20A downregulation restrains the activation of TGF-ß1/Smads signaling pathways, subsequently resulting in a decrease of fibrosis and in facilitating CFs anti-inflammatory effects induced by Gen-miR-1 in the context of CFs activation induced by TGF-ß1. CONCLUSIONS: Our results first uncovered unique bioactive components in G. acuta and elucidated the molecular mechanism by which G. acuta-derived Gen-miR-1 suppress inflammation and myocardial fibrosis. These findings expand our understanding of G. acuta's therapeutic properties and bioactive constituents. Gen-miR-1-regulated HAX1/HMG20A/Smads axis will be one potential therapeutic target for cardiac remodeling.

Minnelide combined with Angptl3 knockout completely protects mice with adriamycin nephropathy via suppression of TGF-ß1-Smad2 and p53 pathways.

Minimal change disease (MCD) is the common type of nephrotic syndrome in children. There is an urgent need to explore new treatment methods as current treatments have many drawbacks and cause significant side effects. Our group found that Angiopoietin-like protein 3 (Angptl3) is closely related to renal disease and Angptl3 knockout significantly alleviated proteinuria in mice with adriamycin nephropathy (AN), however, some proteinuria was still present. Minnelide is a water-soluble prodrug of triptolide which has been used for the treatment of glomerular diseases. Therefore, this study aimed to investigate whether minnelide, combined with Angptl3 knockout, could completely protect mice with AN and its mechanism. AN was induced in B6;129S5 female mice by tail vein injection of 25 mg/kg of Adriamycin (ADR), and treatment with 200 ug/kg/d of minnelide. The results showed that minnelide combined with Angptl3 knockout completely reduced proteinuria and restored the foot processes in mice with AN. Moreover, in Angptl3 knockout mice with AN, minnelide restored the distribution of nephrin, podocin and cd2ap and reduced inflammatory factors (Tumor necrosis factor alpha (TNF-α), Interleukin-6 (IL-6) and Interleukin-1ß (IL-1ß)). Through RNA sequencing and related experiments, we found minnelide could ameliorate fibrosis and apoptosis by inhibiting TGF-ß1-Smad2 and p53 pathways in Angptl3 knockout mice with AN, respectively. In Angptl3 knockout primary podocytes, triptolide alleviates ADR-induced decreases in nephrin, podocin and cd2ap, upregulation of Bax and downregulation of Bcl-2. Overall, our study shows that minnelide combined with Angptl3 knockout completely protects mice with AN by inhibiting the TGF-ß1-smad2 and p53 pathways.

Aluminum exposure induces nephrotoxicity via fibrosis and apoptosis through the TGF-ß1/Smads pathway in vivo and in vitro.

Aluminum (Al), the most common element in nature, can enter the body through various routes. Unfortunately, excessive accumulation of Al in the body can cause chronic toxicity. In this study, rats were randomly allocated to 4 groups and intraperitoneally injected with AlCl3 solution at 0, 5, 10, and 20 mg/(kg·d), respectively, for 4 weeks. The kidney function of rats and Al contents in the kidney were measured, and the pathological structural changes and apoptosis of the kidney were observed. Meanwhile, the expression of fibrosis- and apoptosis-related proteins was detected with western blot. For the in vitro assay, HK-2 cells were used to construct a model to evaluate the effects of Al exposure on cell viability, cell apoptosis, and the expression of fibrosis- and apoptosis-related proteins. Additionally, the TGF-ß1/Smads pathway was also altered in HK-2 cells, followed by the measurement of changes in apoptosis and fibrosis-related proteins. The results revealed that Al could accumulate in kidney tissues, then leading to histopathological changes and kidney function impairment, promoting renal tubular cell apoptosis and renal collagen fiber deposition, and also elevating the expression of TGF-ß1/Smads pathway-related proteins. In vitro experiments also exhibited that Al exposure increased apoptosis and the expression of fibrosis-related factors in HK-2 cells, accompanied by activation of the TGF-ß1/Smads pathway. Further modulation of the TGF-ß1/Smads pathway manifested that activation of the TGF-ß1/Smads pathway facilitated Al-induced apoptosis and fibrosis-related factor expression, while inhibition of the pathway negated this effect of Al. In conclusion, the findings of the present study illustrate that Al exposure damages kidney function and facilitate apoptosis and kidney fibrosis, which may be achieved through the activation of the TGF-ß1/Smads pathway. This study provides a new theoretical basis for the study of nephrotoxicity induced by excessive Al exposure.

Triptolide Improves Renal Injury in Diabetic Nephropathy Rats through TGF-ß1/Smads Signal Pathway.

OBJECTIVE: To investigate the therapeutic effect and mechanism of Triptolide on renal injury in diabetic nephropathy rats. METHODS: A total of 15 male SD rats aged 8 weeks were randomly divided into five groups (3 rats in each group): control group, model group, Triptolide low-dose (Triptolide-L) group, Triptolide medium- dose (Triptolide-M) group, Triptolide high-dose (Triptolide-H) group. The rat models of diabetic nephropathy (DN) were established by a single intraperitoneal injection of STZ after being fed with high-fat and high-sugar diet for 4 weeks, and the fasting blood glucose (FBG) concentration of rats was detected. After 4 weeks, HE-staining was used to evaluate the renal pathological damage in rats; biochemical analysis was used to determine the blood urea nitrogen (BUN), serum creatinine (SCr), total cholesterol (TC), triglyceride (TG); ELISA was used to measure the serum inflammatory factor levels; Western blot (WB) was used to detect the expression of TGF-ß1/Smads pathway proteins. RESULTS: In the four FBG tests (once a week), the FBG concentration in the model group was significantly higher than that in the control group, while Triptolide-treated rats were significantly lower than that in the model group. Rats in Model group showed obvious renal injury, and Triptolide significantly improved the renal injury in DN rats. Compared with the control group, the expression of BUN, SCr, TC, TG, inflammatory factors TNF-α, IL-6 and IL-1ß in the model group increased significantly. WB results showed that the expressions of TGF-ß1, Smad3, α -SMA and vimentin in the kidney significantly increased, while the Smad7 expression significantly decreased. Triptolide significantly reduced the levels of BUN, SCr, TC, TG and TNF-α, IL-6, IL-1ß in diabetic rats, decreased the expression of TGF-ß1, Smad3, α-SMA, vimentin, and increased the Smad7 expression. In different doses of Triptolide treatment group, its effect showed a significant concentration dependence. CONCLUSION: Triptolide alleviates renal injury in diabetic rats by inhibiting the TGF-ß1/Smads signaling pathway.

Therapeutic Targeting of Nrf2 Signaling by Maggot Extracts Ameliorates Inflammation-Associated Intestinal Fibrosis in Chronic DSS-Induced Colitis.

Intestinal fibrosis is induced by excessive myofibroblast proliferation and collagen deposition, which has been regarded as a general pathological feature in inflammatory bowel disease (IBD). Therefore, identifying clinical markers and targets to treat and prevent intestinal fibrosis is urgently needed. The traditional Chinese medicine maggot, commonly known as "wu gu chong", has been shown to reduce oxidative stress and alleviate inflammation in chronic colitis. This study investigated the mechanisms underlying the effects of maggot extract (ME) on inflammation-associated intestinal fibrosis in TGF-ß1-stimulated human intestinal fibroblasts (CCD-18Co cells) and dextran sodium sulphate (DSS)-induced chronic colitis murine model. To assess the severity of inflammation and fibrosis, histological and macroscopic evaluation were carried out. The results showed that ME was a significant inhibitor of body weight loss and colon length shortening in mice with chronic colitis. In addition, ME suppressed the intestinal fibrosis by downregulating TGF-ß1/SMADs pathway via upregulation of Nrf2 expression at both protein and mRNA levels. ME markedly increased the expression of Nrf2, thus resulting in a higher level of HO-1. After treatment with Nrf2 inhibitor (ML385) or siRNA-Nrf2 for deactivating Nrf2 pathway, the protective effects of ME were abolished both in vitro and in vivo. Moreover, the histopathological results for the major organs of DSS mice treated with ME showed no signs of clinically important abnormalities. Treatment with ME had no effect on the viability of CCD-18Co cells, suggesting its low in vitro cytotoxicity. Furthermore, ME could mediate intestine health by keeping the balance of the gut microbes through the enhancement of beneficial microbes and suppression of pathogenic microbes. In conclusion, this is the first ever report demonstrating that ME ameliorates inflammation-associated intestinal fibrosis by suppressing TGF-ß1/SMAD pathway via upregulation of Nrf2 expression. Our findings highlight the potential of Nrf2 as an effective therapeutic target for alleviating intestinal fibrosis.

Forsythiaside B inhibits myocardial fibrosis via down regulating TGF-ß1/Smad signaling pathway.

Forsythiaside B is the major ingredient of Callicarpa kwangtungensis Chun, and has been proven to protect myocardium from ischemia-reperfusion injury to achieve myocardial protection. However, the effect of forsythiaside B on adverse myocardial fibrosis remains unclear. In the present study, the myocardial fibrosis animal models were established induced by isoproterenol (ISO) to investigate whether forsythiaside B exhibited antifibrotic actions. Forsythiaside B was found to significantly improve the cardiac ejection fraction and fractional shortening rate of myocardial fibrosis mice compared with the normal saline group. In addition, forsythiaside B could lower the level of TGF-ß1, the expression of α-SMA and collagen III. Forsythiaside B down-regulated the expression of Smad4 and the phosphorylation level of Smad3, which indicates that forsythiaside B could suppress myocardial fibrosis by inhibiting the TGF-ß1/Smad signaling pathway. These results demonstrated that forsythiaside B could prevent myocardial fibrosis in ISO-induced mice, and may be a potentially rational therapeutic approach for the treatment of myocardial fibrosis.

Bellidifolin Ameliorates Isoprenaline-Induced Myocardial Fibrosis by Regulating TGF-ß1/Smads and p38 Signaling and Preventing NR4A1 Cytoplasmic Localization.

Myocardial fibrosis is closely related to high morbidity and mortality. In Inner Mongolia, Gentianella amarella subsp. acuta (Michx.) J.M.Gillett (G. acuta) is a kind of tea used to prevent cardiovascular diseases. Bellidifolin (BEL) is an active xanthone molecule from G. acuta that protects against myocardial damage. However, the effects and mechanisms of BEL on myocardial fibrosis have not been reported. In vivo, BEL dampened isoprenaline (ISO)-induced cardiac structure disturbance and collagen deposition. In vitro, BEL inhibited transforming growth factor (TGF)-ß1-induced cardiac fibroblast (CF) proliferation. In vivo and in vitro, BEL decreased the expression of α-smooth muscle actin (α-SMA), collagen Ⅰ and Ⅲ, and inhibited TGF-ß1/Smads signaling. Additionally, BEL impeded p38 activation and NR4A1 (an endogenous inhibitor for pro-fibrogenic activities of TGF-ß1) phosphorylation and inactivation in vitro. In CFs, inhibition of p38 by SB203580 inhibited the phosphorylation of NR4A1 and did not limit Smad3 phosphorylation, and blocking TGF-ß signaling by LY2157299 and SB203580 could decrease the expression of α-SMA, collagen I and III. Overall, both cell and animal studies provide a potential role for BEL against myocardial fibrosis by inhibiting the proliferation and phenotypic transformation of CFs. These inhibitory effects might be related to regulating TGF-ß1/Smads pathway and p38 signaling and preventing NR4A1 cytoplasmic localization.

Discovery of 9O-Substituted Palmatine Derivatives as a New Class of anti-COL1A1 Agents via Repressing TGF-ß1/Smads and JAK1/STAT3 Pathways.

Twenty 9O-substituted palmatine derivatives were prepared and tested for their biological effect against collagen α1 (I) (COL1A1) promotor in human hepatic stellate LX-2 cells. The structure-activity relationship (SAR) indicated that the introduction of a benzyl motif on the 9O atom was favorable for activity. Among them, compound 6c provided the highest inhibitory effect against COL1A1 with an IC50 value of 3.98 µM, and it also dose-dependently inhibited the expression of fibrogenic COL1A1, α-soomth muscle actin (α-SMA), matrix metalloprotein 2 (MMP2) in both mRNA and protein levels, indicating extensive inhibitory activity against fibrogenesis. A further primary mechanism study indicated that it might repress the hepatic fibrogenesis via inhibiting both canonical transforming growth factor-beta 1 (TGF-ß1)/Smads and non-canonical janus-activated kinase 1 (JAK1)/singal transducer and activator of transcription 3 (STAT3) signaling pathways. Additionally, 6c owned a high safety profile with the LD50 value of over 1000 mg·kg-1 in mice. These results identified palmatine derivatives as a novel class of anti-fibrogenic agents, and provided powerful information for further structure optimization.

Huoxin pill attenuates myocardial infarction-induced apoptosis and fibrosis via suppression of p53 and TGF-ß1/Smad2/3 pathways.

Huoxin Pill (HXP), a Traditional Chinese Medicine, is used widely to treat patients with coronary heart disease and angina pectoris in China. However, the underlying protective mechanism of HXP on cardiac apoptosis and fibrosis has never been evaluated. Therefore, the aim of this study was to investigate the role of HXP in a myocardial infarction (MI) mouse model. The mice were randomly divided into 3 groups and subjected to surgical ligation of the left anterior descending (LAD) coronary artery or sham surgery (n = 6 for each group) and treated with HXP (50 mg/kg/day) or saline by gavage for 2 weeks. At 2 weeks post MI, we found that HXP significantly enhanced myocardial function and attenuated the increase of heart weight index (HWI) and pathological changes in MI mice. RNA-sequencing and KEGG pathway analyses identified 660 differentially expressed genes and multiple enriched signaling pathways including p53 and TGF-ß. In support of these findings, HXP attenuated cardiac apoptosis and decreased p53 and Bax protein expression, while increasing Bcl-2 protein expression in cardiac tissues of MI mice. Furthermore, HXP treatment inhibited cardiac fibrosis and significantly down-regulated TGF-ß1 protein expression and Smad2/3 phosphorylation in cardiac tissues. In summary, HXP can improve cardiac function in mice after MI by attenuating cardiac apoptosis and fibrosis partly via supression of the p53/Bax/Bcl-2 and TGF-ß1/Smad2/3 pathways.

Methyl helicterilate ameliorates alcohol-induced hepatic fibrosis by modulating TGF-ß1/Smads pathway and mitochondria-dependent pathway.

This study aimed to investigate the effect and underlying mechanism of Methyl helicterilate from Helicteres angustifolia (MHHA) on alcohol-induced hepatic fibrosis. The results showed that MHHA treatment markedly alleviated alcohol-induced liver injury and notably reduced collagen deposition in liver tissue. It significantly enhanced the activity of alcohol dehydrogenase and aldehyde dehydrogenase. Moreover, MHHA treatment markedly decreased the content of inflammatory cytokines, alleviated collagen accumulation, and inhibited the expression of TGF-ß1 and Smad2/3 in liver tissue. The experiments in cells showed that MHHA significantly inhibited HSC activation by blocking TGF-ß1/Smads signaling pathway. Additionally, it notably induced HSC apoptosis by modulating the mitochondria-dependent pathway. The present study demonstrates that MHHA treatment significantly ameliorates alcoholic hepatic fibrosis and the underlying mechanism may be ascribed to the inhibition of the TGF-ß1/Smads pathway and regulation of the mitochondria-mediated apoptotic pathway.

HBOA ameliorates CCl4-incuded liver fibrosis through inhibiting TGF-ß1/Smads, NF-κB and ERK signaling pathways.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-ß1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl4-induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-ß1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-ß1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-ß1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis.

Methyl helicterte ameliorates liver fibrosis by regulating miR-21-mediated ERK and TGF-ß1/Smads pathways.

Methyl helicterate (MH) has been reported to have protective effects against CCl4-induced hepatic injury and fibrosis in rats, but its protective mechanism, especially on hepatic stallete cells (HSCs), remains unclear. Recently, our pilot experiment showed that MH could inhibit miR-21 expression in HSC-T6 cells, suggesting that miR-21 may be one of the targets of MH to intervene liver fibrosis. To verify the hypothesis, the present study would focus on the regulatory effect of MH on the miR-21-mediated ERK and TGF-ß1/Smads pathways. Briefly, rats were intraperitoneally injected with 0.5 ml porcine serum (PS) twice a week for 24 weeks to induce liver fibrosis, and meanwhile, the rats were treated with MH from weeks 16 to 24. In vitro experiment, miR-21 expression in HSC-T6 cells was up- or down-regulated using lentiviral transfection assay. Collagen accumulation, inflammatory cytokines, cell apoptosis, miR-21 expression, and activation of the ERK and TGF-ß1/smad2/3 pathways were then assessed. The results showed that MH treatment markedly alleviated PS-induced liver injury, as evidenced by the attenuation of histopathological changes and the decrease in serum alanine and aspartate aminotransferases activity. MH significantly decreased the content of inflammatory cytokines and recruited the anti-oxidative defense system. Moreover, MH treatment significantly decreased miR-21 expression and inhibited the activation of the ERK and TGF-ß1/smad2/3 pathways in liver tissues. In vitro experiments showed that MH strongly inhibited HSC-T6 cell activation and reduced collagen accumulation. Interestingly, miR-21 overexpression significantly promoted HSC-T6 cell proliferation, reduced HSC apoptosis, and increased collagenation, while these abnormal changes induced by miR-21overexpression were significantly reversed by MH treatment. Furthermore, miR-21 overexpression notably activated the ERK and TGF-ß1/Smads pathways via repressing SPRY2 and Smad7 expression respectively, however, these effects were largely abolished by MH treatment. In conclusion, our study demonstrates that MH significantly alleviates PS-induced liver injury and fibrosis by inhibiting miR-21-mediated ERK and TGF-ß1/Smads pathways.

Alleviation of hepatic fibrosis and autophagy via inhibition of transforming growth factor-ß1/Smads pathway through shikonin.

BACKGROUND AND AIM: Liver fibrosis is a worldwide clinical challenge during the progression of chronic liver disease to liver cirrhosis. Shikonin is extracted from the root of Lithospermum erythrorhizon with antioxidant, anti-inflammatory, anticancer, and wound-healing properties. The study aims to investigate the protective effect of shikonin on liver fibrosis and its underlying mechanism. METHODS: Two liver fibrosis models were established in male C57 mice by intraperitoneal injection of CCl4 or bile duct ligation. Shikonin was administered orally three times weekly at a dose of 2.5 or 5 mg/kg. Protein and mRNA expressions were assayed by quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemical staining. RESULTS: Shikonin significantly inhibited activation of hepatic stellate cells and extracellular matrix formation by downregulating the transforming growth factor-ß1 expression and maintaining the normal balance between metalloproteinase-2 and tissue inhibitor of metalloproteinase-1. Shikonin also decreased hepatic stellate cell energy production by inhibiting autophagy. CONCLUSIONS: The results confirmed that shikonin attenuated liver fibrosis by downregulating the transforming growth factor-ß1/Smads pathway and inhibiting autophagy.

Astragaloside IV improves renal function and fibrosis via inhibition of miR-21-induced podocyte dedifferentiation and mesangial cell activation in diabetic mice.

BACKGROUND: Podocyte dedifferentiation and mesangial cell (MC) activation play an important role in many glomerular diseases associated with fibrosis. MicroRNA-21 (miR-21) is closely linked to renal fibrosis, but it is unknown whether and how miR-21 promotes podocyte dedifferentiation and MC activation and whether astragaloside IV (AS-IV) improves renal function and fibrosis through the regulation of miR-21. MATERIALS AND METHODS: Cultured MCs, primary mouse podocytes, and diabetic KK-Ay mice were treated with AS-IV. Cell transfection, Western blot, real-time PCR, immunofluorescence assay, immunohistochemical assay, and electronic microscopy were used to detect the markers of podocyte dedifferentiation and MC activation and to observe the renal morphology. RESULTS: Our data showed that miR-21 expression was increased and that AS-IV decreased miR-21 levels in cells, serum, and kidney. Overexpressed miR-21 promoted podocyte dedifferentiation and MC activation, and treatment with AS-IV reversed this effect. Furthermore, the overexpression of miR-21 activated the ß-catenin pathway and the transforming growth factor (TGF)-ß1/Smads pathway in the process of podocyte dedifferentiation and MC activation, which was abolished by AS-IV treatment. In addition, both the Wnt/ß-catenin pathway inhibitor XAV-939 and the TGF-ß1/Smads pathway inhibitor SB431542 reversed the effect of AS-IV. Furthermore, AS-IV improved renal function and fibrosis in diabetic KK-Ay mice. CONCLUSION: Our results indicated that AS-IV ameliorates renal function and renal fibrosis by inhibiting miR-21 overexpression-induced podocyte dedifferentiation and MC activation in diabetic kidney disease. These findings pave way for future studies investigating AS-IV as a potential therapeutic agent in the management of glomerular diseases.