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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 study the effect of ligustrazine hydrochloride on the apoptosis of rat hepatic stellate cell line T6 (HSC-T6) and its mechanism. Methods Lactate dehydrogenase (LDH) kit was used to observe the effects of drugs on lactate dehydrogenase activity; ~ 3H-TdR incorporation assay was used to detect the effect of drugs on cell proliferation; flow cytometry Annexin-V / PI double staining The effects of drugs on the apoptosis rate were observed. The effects of drugs on the expression of Bcl-2, Bax and caspase-3 proteins and mRNA were observed by Western blotting and Real-time PCR. Results Cytotoxicity experiments showed that when the final concentration of ligustrazine hydrochloride was less than 80 μmol·L -1, no obvious cytotoxicity was observed. The experimental results of 3H-TdR incorporation showed that the drug concentration was 50 and 70 μmol·L -1, (-1) could inhibit cell proliferation. Flow cytometry was used to detect the effect of drugs on the apoptosis rate. It was found that tetramethylpyrazine hydrochloride could promote the apoptosis of HSC in a dose-response manner. Western blotting showed that ligustrazine hydrochloride Triazine enhanced apoptosis by inhibiting Bcl-2 and Bax and caspase-3 protein expression. Real-time PCR further showed that tetramethylpyrazine hydrochloride could inhibit Bcl-2 and Bax and caspase-3 expression at mRNA level. Conclusion Tetramethylpyrazine hydrochloride can promote the apoptosis of HSC-T6. This effect is mainly through inhibiting Bcl-2, promoting the expression of Bax and caspase-3 protein and mRNA.