基于体内外实验探讨蒙药七味清肝散对大鼠肝纤维化的作用及机制

Effect of Mongolian medicine Qiwei-Qinggan powder on liver fibrosis in rats and its mechanism

目的:探讨蒙药七味清肝散在大鼠肝纤维化(hepatic fibrosis,HF)模型及肝星状细胞(HSC-T6)中的作用及机制。方法:30只雄性Wistar大鼠随机分为对照组、模型组及低剂量(135 mg/kg)、中剂量(270 mg/kg)和高剂量(405 mg/kg)七味清肝散组,每组6只。模型组和各给药组大鼠灌胃50%四氯化碳(CCl4)溶液(2 mL/kg),同时各给药组每天予以相应剂量的七味清肝散灌胃8周。采用全自动生化分析仪检测肝血清生化指标[丙氨酸转氨酶(ALT),天冬氨酸转氨酶(AST)和碱性磷酸酶(ALP)]及羟脯氨酸(HYP)含量;HE和Masson染色观察肝组织损伤情况;Western blot检测肝组织中α-平滑肌肌动蛋白(α-SMA)、I型胶原蛋白(collagen type I,Col I)、磷酸酶与张力蛋白同源物(PTEN)、PI3K及Akt蛋白表达水平。将HSC-T6细胞分组并进行miR-21模拟剂(mimics)/抑制剂(inhibitor)转染,MTT法检测细胞活力;RT-qPCR检测HSC-T6细胞中miR-21的表达水平,及细胞转染后对α-SMA、Col I、PTEN、PI3K和Akt mRNA表达水平的影响;Western blot检测HSC-T6细胞中α-SMA、Col I、PTEN、PI3K和Akt的蛋白表达水平。结果:不同剂量的七味清肝散能改善肝功能,并减轻肝纤维化程度;与模型组相比,各剂量给药组的细胞A值显著降低(P<0.05,P<0.01)。七味清肝散显著下调HSC-T6细胞中miR-21表达(P<0.01);miR-21mimics显著升高α-SMA、Col I、PI3K和Akt的mRNA表达水平(P<0.01),下调PTEN的mRNA表达(P<0.05),而miR-21 inhibitor使上述指标mRNA表达水平显著逆转(P<0.05)。七味清肝散处理后大鼠肝组织和HSC-T6细胞中α-SMA、Col I、PI3K及Akt蛋白水平下调,PTEN蛋白水平上调(P<0.05)。结论:蒙药七味清肝散可能通过下调miR-21影响PTEN/PI3K/Akt信号通路来减轻大鼠的肝纤维化水平。

AIM:To study the effect and mechanism of Qiwei-Qinggan powder in liver fibrosis.METHODS:Thirty male Wistar rats were randomly divided into control group,model group,and low-dose(135 mg/kg),medium-dose(270 mg/kg),and high-dose(405 mg/kg)Qiwei-Qinggan powder groups,with 6 mice in each group.Rat liver fibrosis model was induced by 50%CCl4(2 mL/kg,gastric gavage twice a week).QiweiQinggan powder was administered by daily gastric gavage for 8 weeks.Alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP)and hydroxyproline(HYP)contents were detected by automatic biochemical analyzer.HE and Masson staining was used to observe liver tissue damage.Western blot was used to detect the protein level ofα-smooth muscle actin(α-SMA),collagen type I(Col I),phosphatase and tensin homolog deleted on chromosome(PTEN),PI3K and Akt.HSC-T6 cells were transfected with miR-21 mimics/inhibitors.MTT method was used to detect the viability.RT-qPCR was used to detect the expression level of miR-21 in HSC-T6 cells,and the effect of transfection onα-SMA,Col I,PTEN,PI3K and Akt mRNA expression.Western blot was used to detect protein levels ofα-SMA,Col I,PTEN,PI3K and Akt in HSCT6 cells.RESULTS:Qiwei-Qinggan powder improved liver function and reduced the degree of liver fibrosis.Compared with the control group,the viability of cells in each dose group was significantly decreased(P<0.05,P<0.01).Qiwei-Qinggan powder significantly down-regulated the expression of miR-21 in HSC-T6 cells(P<0.01).miR-21 mimics increased the mRNA expression levels ofα-SMA,Col I,PI3K and Akt(P<0.01),while down-regulated the mRNA expression of PTEN(P<0.05).The inhibitor of miR-21 significantly reversed the mRNA expression levels of the above indicators(P<0.05).The protein levels ofα-SMA,Col I,PI3K and Akt were all down-regulated,and the level of PTEN was up-regulated after treatment with QiweiQinggan powder(P<0.05).CONCLUSION:Qiwei-Qinggan powder alleviates liver fibrosis via miR-21/PTEN/PI3K/Akt signaling pathway in rats.