四逆散含药血清诱导大鼠肝脏干细胞分化及其调控机制

Effect of drug-containing serum of Si-Ni-San on inducing differentiation of rat's hepatic stem cells and its regulatory mechanism

目的:研究四逆散含药血清对大鼠肝脏干细胞分化的影响并探讨其治疗肝病的作用机制。方法:采用HPLC-MS-MS法,梯度洗脱,测定血清中四逆散的有效成分;以四逆散含药血清处理WB-F344细胞后,采用CCK-8法观察细胞增殖情况,血糖仪检测细胞培养液葡萄糖含量,乳酸测定试剂盒检测细胞中乳酸含量,荧光素-荧光素酶法检测ATP含量,蛋白质印迹法观察表皮生长因子受体(epidermal growth factor receptor,EGFR)、纤维母细胞生长因子受体(fibroblast growth factor receptor,FGFR)、转化生长因子受体(transforming growth factor beta receptor typeⅠ,TGF-βRⅠ)、肝细胞生长因子受体(hepatocyte growth factor receptor,HGFR)蛋白表达情况。结果:在四逆散血清中,检测到以原型入血芍药苷、橙皮苷等成分,并检测到橙皮素葡萄糖醛酸等部分代谢物。各浓度含药血清作用WB-F344细胞48h后,对细胞均无毒性作用,10%含药血清显示出较好的促进细胞增殖的效果。随着药物血清作用时间的延长,WBF344细胞葡萄糖消耗量、乳酸生成量降低,ATP生成增加,EGFR、FGFR、TGF-βRⅠ、HGFR蛋白表达水平上升。结论:四逆散含药血清能够促进肝脏干细胞WB-F344的增殖并诱导其分化,分化机制可能与影响数种细胞因子受体的表达有关。

Objective:To study the effect of drug-containing serum of Si-Ni-San on differentiation of rat' s hepatic stem cells and its mechanism,and explore the scientific basis for treatment of liver disease.Methods:HPLC-MS-MS method was used to detect drug-containing serum of Si-Ni-San; WB-F344 cells were treated with drug-containing serum,the activity of cells was detected by CCK-8 assay,the glucose consumption was assessed by glucometer,generation of lactic acid was determined by LA kit,ATP content was measured by luciferin luciferase method,and the expressions of epidermal growth factor receptor(EGFR),fibroblast growth factor receptor(FGFR),transforming growth factor beta receptor type Ⅰ (TGF-βR Ⅰ),hepatocyte growth factor receptor(HGFR) were observed by western blotting.Results:In drug-containing serum of Si-Ni-San,some ingredients in prototype like paeoniflorin and aurantiamarin,others in metabolite type such as naringenin-glucuronic acid and glycyrrhetinic acid.Different concentrations of drugcontaining serum showed no toxic effects on WB-F344 cells after been incubated with drug-containing serum,10％ concentration of drug-containing serum could significantly promote proliferation of WB-F344 cells.The levels of glucose consumption and lactic acid decreased with the time of differentiation,but ATP took on a up-regulating trend.the expressions of EGFR,FGFR,TGF-βRI and HGFR also increased as drug-containing serum incubation.Conclusion:The Si-Ni-San containing serum could promote hepatic stem cells' proliferation and induce their differentiation,and maybe the regulatory mechanism is related to cytokine receptors,such as EGFR,HGFR.