盐酸川芎嗪影响HSC-T6凋亡的作用机制研究

The Effect of Ligustrazine Hydrochloride on Hepatic Stellate Cell Apoptosis and Its Possible Mechanism

目的研究盐酸川芎嗪对大鼠肝星状细胞系T6(HSC-T6)凋亡的影响及其作用机制。方法采用细胞乳酸脱氢酶(LDH)试剂盒观察药物对乳酸脱氢酶活性的影响;~3H-TdR掺入实验检测药物对细胞增殖的影响;流式细胞仪Annexin-V/PI双染方法检测药物对细胞凋亡率影响;应用Western blotting和Real-time PCR方法观察药物对凋亡相关细胞因子Bcl-2、Bax和caspase-3蛋白及mRNA表达的影响。结果细胞毒性实验发现当盐酸川芎嗪作用终浓度低于80μmol·L^(-1)时,不具有明显细胞毒作用;~3H-TdR掺入实验研究发现,药物浓度为50和70μmol·L^(-1)具有抑制细胞增殖的作用;流式细胞仪检测药物对细胞凋亡率的影响,发现盐酸川芎嗪具有促进HSC凋亡的作用,且呈量效关系;Western blotting结果显示,盐酸川芎嗪通过抑制Bcl-2,促进Bax和caspase-3蛋白表达促进细胞凋亡;进一步采用Real-time PCR研究发现,盐酸川芎嗪能够从mRNA水平抑制Bcl-2、促进Bax和caspase-3表达。结论盐酸川芎嗪具有促进HSC-T6凋亡的作用,这种作用的发挥主要是通过抑制Bcl-2、促进Bax和caspase-3蛋白和mRNA的表达实现的。

OBJECTIVE To observe the effect of ligustrazine hydrochloride on HSC-T6 apoptosis and to explore the possible mechanism. METHODS LDH kit was used to detect the effect of ligustrazine hydrochloride on LDH activity. 3 H-TdR incorporation assay was used to detect the effect of drug on HSC-T6 proliferation. Apoptosis was measured by using flow cytometry （FCM） analysis with Annexin-V/PI dual staining for apoptotic cell ratio. Western blotting and Real-time PCR were used to detect the protein and mR- NA expression of Bcl-2, Bax and caspase-3. RESULTS No obvious cytotoxic effect was found when the final concentration of ligus- trazine hydrochloride was less than 80 p^mol ~ L-I. 3 H-TdR incorporation assay showed that ligustrazine hydrochloride inhibited HSC-T6 proliferation at the concentrations of 50 and 70 p,mol ~ L-l. FCM detection showed that ligustrazine hydrochloride dose-dependently stimulated apoptosis of I-ISC-T6. Western blotting results showed that ligustrazine hydrochloride promoted HSC-T6 apoptosis by inhibi- ting Bcl-2, and increasing Bax and caspase-3 protein expression. Further use of real-time PCR showed that ligustrazine hydrochloride inhibited Bcl-2 mRNA expression, and promoted the mRNA expression of Bax and caspase-3. CONCLUSION Ligustrazine hydro- chloride has the function of promoting the apoptosis of HSC-T6 by inhibiting Bcl-2, and promoting Bax and caspase-3 at both protein and mRNA levels.