截断逆挽方对慢加急性肝衰竭大鼠cyclin D-E2F1信号通路及转录因子活化的机制研究

Study on the mechanism of Jieduan Niwan formula on cyclin D-E2F1 signaling pathway and transcription factor activation of rats with acute-on-chronic liver failure

目的探寻"截断逆挽方"对cyclin D-E2F1信号通路上相关转录因子的作用及该通路对慢加急性肝衰竭(acute-on-chronic liver failure,ACLF)模型大鼠的作用机制。方法将105只Wistar大鼠随机分为正常组5只(生理盐水注射)、造模组100只予人血清白蛋白诱导肝纤维化联合D-氨基半乳糖(D-galactosamine,D-GalN)/脂多糖(lipopolysaccharide,LPS)急性攻击,建立ACLF成模大鼠30只。将成模大鼠随机分为模型5、10、15天组(24小时后予生理盐水连续灌胃)和截断逆挽方5、10、15天组(24小时后予截断逆挽方连续灌胃),每组5只。检测各组大鼠血浆凝血酶原活动度(prothrombinase activity,PTA)、谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、总胆红素(total bilirubin,TBIL)水平,用HE染色观察肝脏病理学变化,用蛋白免疫印迹法检测转录因子DP1蛋白表达,用Real-time PCR检测cyclin D1、cyclin D2、cyclin D3、细胞周期蛋白依赖性激酶4(cyclin-dependent kinase4,CDK4)及细胞周期蛋白依赖性激酶6(cyclin-dependent kinase6,CDK6) mRNA的表达。结果与正常组相较,模型组大鼠血浆PTA水平显著降低,ALT、AST、TBIL水平明显升高;与同一时间模型组相较截断逆挽方10天、15天组血浆PTA明显升高,ALT、AST有所降低,cyclin D1、cyclin D2、cyclin D3、CDK4、CDK6 mRNA、DP1蛋白表达均有不同程度的上调(P<0.05)。结论截断逆挽方减轻ACLF模型大鼠肝损伤,恢复肝脏功能的作用机制可能与其上调了cyclin D-E2F1信号通路上肝细胞再生相关转录因子介导的肝细胞增殖有关。

Objective To investigate the mechanism of Jieduan Niwan formula treating model rats with acute-on-chronic liver failure(ACLF),by regulating related transcription factors in the cyclin D-E2F1 signaling pathway.Methods 105 Wistar rats were randomly divided into the normal group and the modeling group.The normal group contains 5 rats,which were injected with normal saline.While the modeling group contains 100 rats,given with human serum albumin to induce liver cirrhosis and accompanied by D-GalN and lipopolysaccharide acute attack,to establish 30 model rats successfully.Then rats successfully modeled were randomly divided into model groups for 5,10 and 15 days(continuous gavaged of normal saline after 24 hours)and Jieduan Niwan formula groups for 5,10 and 15 days(continuous gavaged of Jieduan Niwan formula after 24 hours),5 rats in each group.The levels of prothrombinase activity(PTA),alanine aminotransferase(ALT),aspartate aminotransferase(AST)and total bilirubin(TBIL)were detected.The pathological changes of liver were observed by HE staining.The expression level of transcription factor DP1 protein was detected by western blot.The mRNA expression levels of cyclin D1,cyclin D2,cyclin D3 and cyclin-dependent kinase 4(CKD4)and cyclin-dependent kinase 6(CKD6)were detected by real-time PCR.Results Compared with the normal group,the PTA level in the model group is decreased significantly,while the levels of ALT,AST and TBIL are increased significantly.When compared with the model group at the same time,the plasma PTA levels in Jieduan Niwan formula groups for 10 and 15 days decreased significantly,while the levels of ALT and AST decreased.The mRNA levels of cyclin D1,cyclin D2,cyclin D3 CDK4 and CDK6 and the protein level of DP1 were up-regulated at different degrees(P<0.05).Conclusion Jieduan Niwan formula can relieve liver injury and restore liver function in ACLF model rats,its mechanism may be related to the promotion of various transcription factors in cyclin D-E2F1 signaling pathway which induces liver cell reproduction.