川芎嗪对肝星状细胞的增殖及凋亡的影响

Effects of tetramethylpyrazine on proliferation and apoptosis of hepatic stellate cells

目的:考察川芎嗪对肝星状细胞的细胞周期与凋亡的影响,并探讨潜在的分子机制。方法:以MTS试验分析川芎嗪对肝星状细胞增殖的影响;3H-TdR渗入法分析肝星状细胞的DNA合成速度;以LDH释放实验检测川芎嗪的毒性;以流式细胞术检测肝星状细胞周期;以Western blot检测肝星状细胞内Cyclin D1、p21、p27、p53蛋白的表达;以免疫荧光染色检测各时间点p53的表达;以JC-1试剂盒检测肝星状细胞线粒体膜电位;以Western blot检测川芎嗪作用后的肝星状细胞内Bcl-2、Bcl-x L、Bak、Bax、cleavedcaspase 8、cleaved-caspase 9、cleaved-caspase 3以及cleaved-PARP-1以及细胞质内Cyt c的表达。结果:MTS结果显示40μmol/L川芎嗪能显著抑制肝星状细胞活力;3H-TdR掺入法显示川芎嗪显著抑制肝星状细胞的DNA复制;LDH实验表明川芎嗪高达100μmol/L时可对肝星状细胞产生毒性;流式细胞术显示川芎嗪(10、20、40μmol/L)可诱导细胞周期停滞在G0/G1期;Western blot实验表明川芎嗪可抑制Cyclin D1而促进p21、p27和p53的蛋白表达;20μmol/L川芎嗪诱导p21与p27蛋白表达的作用可被p53抑制剂PFT削弱;JC-1线粒体膜电位检测结果显示川芎嗪能浓度(10~40μmol/L)依赖性地增强肝星状细胞绿色荧光强度;Western blot结果显示川芎嗪抑制Bcl-2和Bcl-x L表达而促进Bak、Bax、cleaved-caspase 8、cleaved-caspase 9、cleaved-caspase 3以及cleaved-PARP-1以及细胞质内Cyt c的蛋白表达。结论:川芎嗪抑制肝星状细胞增殖并促进其凋亡。

Objective： To investigate the effect of TMP on HSC proliferation and apoptosis. Methods： The effect of TMP on HSC proliferation was detected by MTS assay. TMP effect on DNA synthesis was detected by3 H-TdR incorporation. The cytotoxic effect of TMP was determined by LDH release assay. Flow cytometry was performed to detect cell cycle. Western blot was performed to detect protein levels of cyclin D1,p21,p27,and p53. Immunofluorescent staining with antibody against p53 was performed. A JC-1 kit was used to detect mitochondrial membrane potential. Western blot analyses were performed to detect Bcl-2,Bcl-x L,Bak,Bax,cleaved-caspase 8,cleaved-caspase 9,cleaved-caspase 3,cleaved-PARP-1,and Cyt c. Results： MTS assay showed that 40 μmol/L TMP obviously reduced cell viability.3 H-TdR incorporation showed that TMP inhibited DNA replication. LDH assay showed that 100 μmol/L TMP had cytotoxicity. Flow cytometry showed that TMP induced cell cycle arrest at G0/G1 checkpoint. Western blot assays showed that TMP inhibited cyclin D1 and induced p21,p27,and p53 expressions.Furthermore,the induction of TMP on p21 and p27 was blocked by p53 inhibitor PFT. Mitochondrial membrane potential detection by JC-1 kits showed that TMP（10 ～ 40 μmol/L） increased the green fluorescence intensity. Western blot assay showed that TMP inhibited Bcl-2 and Bcl-x L whereas induced Cyt c,Bak,Bax,cleaved-caspase 8,cleaved-caspase 9,cleaved-caspase 3,and cleaved-PARP-1. Conclusions： TMP inhibited proliferation and induced apoptosis of hepatic stellate cells.