益肾健脾化瘀汤对糖尿病大鼠肝脏氧化应激、凋亡和纤维化的影响及机制OA

Objective:To investigate the effect of Yishen Jianpi Huayu(益肾健脾化瘀)Decoction on liver injury in diabetic rats and reveal the underlying mechanism.Methods:The rat model of diabetes was established by a high-fat diet combined with a single intraperitoneal injection of streptozotocin(STZ,35 mg/kg).The successfully modeled dia-betic rats were randomly assigned into model control,Yishen Jianpi Huayu Decoction(16.8 g/kg and 33.6 g/kg),and positive control(canagliflozin,9 mg/kg)groups and received drug intervention for 8 weeks.The rats being fed a stand-ard maintenance diet were included as the normal control group.Biochemical methods were used to determine the levels of blood glucose,blood lipids,liver function indicators,oxidative stress indicators,inflammatory factors.The chemilumi-nescent assay was employed to measure the levels of four liver fibrosis indicators.The liver index was calculated.Hema-toxylin-eosin staining,periodic acid-Schiff staining,Masson staining,and transmission electron microscopy were employed for pathological analysis of the liver tissue.TdT-mediated dUTP nick-end labeling(TUNEL)was employed to detect the apoptosis of rat hepatocytes.Collagen deposition in hepatocytes was detected by immunohistochemical staining.The ex-pression levels of proteins in the Kelch-like ECH-associated protein 1/nuclear factor erythroid 2-related factor 2/heme oxygenase-1(Keap1/Nrf2/HO-1)signaling pathway,apoptosis-associated proteins,and proteins in the transforming growth factor-β1(TGF-β1)/Smad signaling pathway in the liver were analyzed by Western blotting.Results:Compared with the normal control group,the model control group showed decreased body weight,increased blood glucose level,blood lipid levels,and liver index(P<0.05 or P<0.01),elevated serum levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)(P<0.05 or P<0.01),steatosis and fibrosis in the liver tissue,lowered serum levels of superoxide dismutase(SOD),glutathione peroxidase(GSH-PX),and catalase(CAT)(P<0.05 or P<0.01),raised se-rum level of malondialdehyde(MDA)(P<0.01),up-regulated protein level of Keap1(P<0.05),and down-regulated protein levels of Nrf2 and the downstream HO-1(P<0.05 or P<0.01).In addition,the model control group showed ele-vated serum levels of tumor necrosis factor(TNF)-α and interleukin(IL)-1β(P<0.01),raised serum levels of hyalu-ronidase(HA),laminin(LN),collagen type IV(CIV),and procollagen type III(PCIII)(P<0.05 or P<0.01),in-creased TUNEL-stained positive area in the liver(P<0.01),down-regulated protein levels of cysteinyl aspartate-specific proteinase 3(caspase3)and B-cell lymphoma-2(Bcl2)(P<0.05),and up-regulated protein level of Bcl2-associated X protein(BAX)(P<0.05).In addition,the liver tissue in the model control group presented elevated levels of hepatic fibrosis-related factors such as α-smooth muscle actin(α-SMA),TGF-β1,fibronectin,and collagen type I(Collagen-1)(P<0.01)and significantly up-regulated expression of proteins in the TGF-β1/Smad signaling pathway.Compared with the model control group,Yishen Jianpi Huayu Decoction increased the body weight of rats,decreased the blood glucose level,blood lipid levels,and liver index(P<0.05 or P<0.01),lowered the serum levels of ALT and AST(P<0.05 or P<0.01),alleviated the pathological damage in the liver tissue,raised the serum levels of SOD,GSH-PX,and CAT(P<0.01),inhibited the production of MDA(P<0.05 or P<0.01),Keap1 protein expression was inhibited(P<0.05),up-regulated the protein levels of Nrf2 and downstream HO-1(P<0.01).In addition,the decoction reduced the serum lev-els of TNF-α and IL-1β(P<0.05 or P<0.01),significantly decreased the serum levels of HA,LN,CIV,and PCIII and the TUNEL-stained positive area(P<0.05 or P<0.01),up-regulated the protein levels of caspase3 and Bcl2(P<0.05),down-regulated the protein level of BAX(P<0.05).Furthermore,the decoction down-regulated the expression levels of α-SMA,TGF-β1,fibronectin,and collagen-1(P<0.05 or P<0.01)and the proteins in the TGF-β1/Smad sig-naling pathway to inhibit liver fibrosis(P<0.05 or P<0.01).Conclusion:Yishen Jianpi Huayu Decoction can alleviate glucose and lipid metabolism disorders and liver injury in diabetic rats by inhibiting oxidative stress,apoptosis,and fibro-sis.