Fan Er-Yan, He Song-Qi, Wen Bin, Sun Hai-Tao, Jia Wen-Yan, Chen Guan-Xin
Zhongguo Zhong Xi Yi Jie He Za Zhi. 2016 Aug;36(8):960-966.
Objective To observe the possible mechanism of Biejiajian Pill (BP) in fighting a- gainst hepatic fibrosis of hepatic stellate cell T6 ( HSC-T6) by studying effect of BP containing serum on inhibiting proliferation and inducing apoptosis of HSC-T6. Methods Forty Wistar rats were randomly di- vided into the negative control group (NC) , the positive drug control group (P) , high, middle, and low dose groups (H, M, L) , 8 in each group. BP suspension was administered by gastrogavage to rats in Group H, M, L at 21. 87, 43. 75, and 87. 50 mg/mL, respectively. Rats in Group NC were administered with equal volume of normal saline. Rats in Group P were administered with 0. 01 mg/mL colchicine solu- tion by gastrogavage. Each rat received 2 mL corresponding solution, twice per day, with an interval of 12 h gastrogavage, a total of 7 successive times to prepare drug containing serum. HSC-T6 cells were then randomly divided into drug containing serum groups (group H/M/L/NC) , colchicine positive control group (group P) , and the blank control group (BC). Cells in Group H/M/L/NC/P were fed with correspond- ing drug containing serums, while those in-Group BC were cultured with free drug serum. The proliferation inhibition rate of HSC-T6 was detected using CCK8 method at 24, 48, and 72 h, respectively. The apop- totic rate and cell cycle were detected using flow cytometry. Protein expressions of B cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax) were detected using Western blot. Results Compared with Group NC, 24-h proliferation inhibition rate of HSC-T6 was obviously elevated in Group M, H, P (P < 0. 05). Compared with Group NC, 48- and 72-h proliferation inhibition rate of HSC-T6 was obviously ele- vated in Group L, M, H, P (P <0. 05). But there was no statistical difference in 24-, 48-, and 72-h prolif- eration inhibition rate of HSC-T6 among Group L, M, H, P (P >0. 05). Compared with Group NC and BC, early-and late-stage apoptosis rates of HSC-T6 obviously increased in Group M, H, P (P<0. 05) ; G,/G1 phase cell number obviously increased in Group M, H, P (P <0. 05) ; S phase and G2/M phase cell num- bers obviously decreased in Group L, M, H, P (P <0. 05). There was no statistical difference in Bcl-2 protein expression among each group (P>0. 05). Compared with Group NC, Bax protein expression ob- viously increased Group L, M, H, P (P <0. 01). Conclusion The mechanism of BP for fighting against hepatic fibrosis might be associated with inhibiting proliferation of HSC-T6 and inducing apoptosis.
目的 通过研究鳖甲煎丸含药血清对肝星状细胞T6（HSC-T6）增殖的抑制作用及诱导凋亡的影响，探讨鳖甲煎丸抗肝纤维化的可能机制。方法 将40只Wistar大鼠随机分为阴性对照组（NC）、阳性药物对照组（P）、高、中、低剂量组（H、M、L），每组8只。分别按21.87、43.75、87.50mg/mL的剂量给H、M、L组大鼠灌胃鳖甲煎丸混悬液，NC组大鼠灌胃等体积生理盐水，P组大鼠灌胃0.01mg/mL秋水仙碱溶液。每只大鼠每次灌胃2mL相应溶液，每天2次，间隔12h，连续灌胃7次制备含药血清。将HSC-T6细胞随机分为含药血清组（H/M/L/NC组）、秋水仙碱阳性对照组（P组）和空白对照组（BC）。H/M/L/NC/P组细胞用相应含药血清培养，BC组细胞用不含药物的血清培养。分别于24、48、72h用CCK8法检测HSC-T6的增殖抑制率，用流式细胞术检测细胞凋亡率及细胞周期，用Western blot法检测B细胞淋巴瘤-2（Bcl-2）和Bcl-2相关X蛋白（Bax）的蛋白表达。结果 与NC组比较，M、H、P组HSC-T6细胞24h增殖抑制率明显升高（P<0.05）；与NC组比较，L、M、H、P组HSC-T6细胞48、72h增殖抑制率明显升高（P<0.05），但L、M、H、P组之间HSC-T6细胞24、48、72h增殖抑制率差异无统计学意义（P>0.05）。与NC组和BC组比较，M、H、P组HSC-T6细胞早期和晚期凋亡率明显升高（P<0.05）；M、H、P组G0/G1期细胞数明显增加（P<0.05）；L、M、H、P组S期和G2/M期细胞数明显减少（P<0.05）。各组Bcl-2蛋白表达差异无统计学意义（P>0.05）；与NC组比较，L、M、H、P组Bax蛋白表达明显增加（P<0.01）。结论 鳖甲煎丸抗肝纤维化的机制可能与抑制HSC-T6细胞增殖及诱导凋亡有关。
