摘要
目的通过研究鳖甲煎丸药物血清对大鼠肝星状细胞(HSC-T6)的增殖抑制作用及诱导其凋亡的影响,探讨鳖甲煎丸抗肝纤维化的可能作用机制。方法 Wistar大鼠40只,系统随机抽样(抓阄法)分为阴性对照组(NC组)、阳性药对照组(P组)、鳖甲煎丸高(H)、中(M)、低(L)剂量组,每组8只。L、M、H剂量组分别按21.87、43.75、87.50mg/mL灌服鳖甲煎丸混悬液。P组灌胃0.01mg/mL秋水仙碱溶液,NC组灌予等量的生理盐水。每只大鼠每次灌胃2mL,每日2次。每次灌胃间隔12h,连续灌胃7次,制备药物血清。将HSC-T6分为药物血清组(H/M/L/NC组)、秋水仙碱对照组(P组)及空白对照组(BC组),H、M、L、NC及P组给予对应药物血清进行培养,BC组为不含药物血清的培养基。采用CCK8法检测HSC-T6细胞24、48、72h时增殖抑制率,流式细胞术检测细胞凋亡率及细胞周期,Western blot法检测凋亡蛋白B淋巴细胞瘤-2(B-cell lymphoma-2,Bcl-2)、Bcl-2相关X蛋白(Bax)表达水平。结果与NC组比较,M、H、P组24h细胞增殖抑制率明显增高(P<0.05);48、72h时与NC组比较,L、M、H、P组细胞增殖抑制率明显增高(P<0.05)。但24、48、72h各时间点之间L、M、H、P组细胞增殖抑制率差异无统计学意义(P>0.05)。与NC组、BC组比较,M、H、P组的HSC-T6早期凋亡与晚期凋亡率明显升高(P<0.05),M、H、P组G0/G1期细胞数明显增多(P<0.05),L、M、H、P组S期与G2/M期细胞数明显减少(P<0.05)。各组间Bcl-2蛋白表达比较,差异无统计学意义(P>0.05)。与NC组比较,P、H、M、L组的Bax蛋白表达明显升高(P<0.01)。结论鳖甲煎丸抗肝纤维化的作用机制可能与其抑制肝星状细胞增殖并诱导其凋亡有关。
Objective To observe the possible mechanism of Biejiajian Pill （BP） in fighting against 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 divided 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 solution 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 corresponding 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 apoptotic 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 elevated in Group L, M, H, P （P 〈0.05）. But there was no statistical difference in 24-, 48-, and 72-h proliferation 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）; G0/G1 phase cell number obviously increased in Group M, H, P （P 〈0.05） ; S phase and G2/M phase cell numbers 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 obviously 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.
出处
《中国中西医结合杂志》
CAS
CSCD
北大核心
2016年第8期960-966,共7页
Chinese Journal of Integrated Traditional and Western Medicine
基金
国家自然科学基金资助项目(No.81373807)
广东省中医药局资助课题(No.2013265)
