黄芪总黄酮对肝硬化大鼠肝组织转化生长因子βⅠ型膜受体表达的影响

The effect of total flavonoids of Astragalus on expression of TGFβR1 in hepatic cirrhotic rat liver tissue

目的:研究黄芪总黄酮对肝硬化大鼠肝组织转化生长因子βⅠ型膜受体(TGFβR1)表达的影响。方法:雄性SD大鼠随机分为正常组、模型组、高剂量组、低剂量组等4组。除正常组外其余3组大鼠均给予二甲基亚硝胺(DMN)腹腔注射造肝硬化模型,共4周。两治疗组从第3周起各予高、低剂量的黄芪总黄酮灌胃,共4周。实验结束后处死大鼠采集标本,肝组织经甲醛固定、石蜡包埋后4m切片,经脱蜡、洗涤、微波抗原修复、5%BSA封闭后,与TGFβR1抗体37℃孵育,以PBS代替一抗做阴性对照,使用二步法免疫组化检测试剂盒,采用SABC法进行检测。肝组织总蛋白使用RIPA裂解液进行抽提,10%SDS聚丙烯酰胺凝胶电泳分离蛋白,采用Western blot检测目标蛋白表达水平,采用GAPDH进行校正。结果:免疫组化染色结果显示,与模型组相比,两黄芪总黄酮治疗组TGFβR1染色显著减少,尤以高剂量组为明显。Western blot检测结果显示,与模型组相比,两治疗组大鼠TGF R1蛋白表达均降低(P<0.05,P<0.01),高剂量组表达水平显著低于低剂量组存(P<0.05)。结论:黄芪总黄酮能够抑制肝硬化大鼠肝组织TGFβR1表达。

Objective： To study the effect of total flavonoids of Astragalus on TGFβR1 expression in hepatic cirrhotic rat model.Methods： Fifty-three male SD rats were allocated randomly into control group （10 rats） and model group （43 rats）, rats in model group were given DMN by intraperitoneal injection for 4 weeks.At the 3rd experimental week the model rats were randomly divided into model group （14 rats） and treatment groups （14 rats in low-dose group, 15 rats in high-dose group）, rats in treatment groups were given total flavonoids of Astragalus at different dosage correspondingly for 4 weeks.At the end of ex-periment rats were sacrificed, liver tissue were collected, fixed in formalin, paraffin embedded, 4 m slices prepared, dewaxed, washed, microwave antigen retrieval and 5%BSA blocked.Then slices were incubated with TGFβR1 antibody at 37＆#176;C, using PBS instead of primary antibody as negative control.SABC method was adopted by using two-step immunohistochemical detec-tion kit.Total the proteins were isolated with RIPA reagents and separated by 10% sodium dodecyl sulfate -polyacrylamide gelelectrophoresis;Western blot was adopted to determine protein levels.GAPDH was used as internal control.Results： Com-pared with model group, immunohistochemical staining results showed that TGFβR1 staining in treatment groups reduced marked-ly, especially in high dosage group.Western blot detection showed that TGF R1 protein in treatment groups reduced significantly compared with that in model group （P〈0.05, P〈0.01）, and there was significant difference between the high and low dos-age groups （P〈0.05）.Conclusion： Total flavonoids of Astragalus can inhibit the expression of TGFβR1 in rat hepatic cirrho-sis model.