白花丹对大鼠肝星状细胞增殖、凋亡及细胞周期的影响

Effects of plumbago zeylanica on the proliferation, apoptosis and cell cycle of HSC-T6

目的:观察白花丹含药血清对大鼠肝星状细胞(HSC-T6)增殖、凋亡细胞周期的影响,探讨白花丹抗肝纤维化的作用及其机制.方法:SD大鼠用白花丹水煎液灌胃(大、中、小剂量),取得含药血清.含药血清按不同浓度(50、100、200mL/L)分组与大鼠HSC-T6孵育.设立空白对照组,并以秋水仙碱和复方鳖甲软肝片含药血清为阳性对照组.孵育后各组采用MTT比色法检测各组HSC-T6的增殖;流式细胞仪检测细胞凋亡及各细胞周期DNA含量的百分比.结果:HSC-T6白花丹含药血清各组与空白组比较,增殖抑制率和细胞凋亡率明显增高,有显著性差异(P<0.01);并且含药血清浓度增高时,HSC-T6的抑制率、凋亡率也逐渐增强;血清高浓度组的增殖抑制率和细胞凋亡率明显高于秋水仙碱组(P<0.01),与复方鳖甲软肝片组相当;各组的G2/M期细胞百分比无明显变化.HSC-T6白花丹含药血清各组与空白组比较,G0/G1期的细胞百分比明显增高(55.23%±2.83%,52.60%±2.26%,48.75%±1.37%vs44.08%±1.41%,均P<0.01)、S期细胞百分比明显降低(31.47%±1.26%,34.14%±1.17%,40.28%±1.62%vs47.36%±1.35%,均P<0.01).并且含药血清浓度下降时,G0/G1期的细胞百分比逐渐下降,S期细胞百分比逐渐增高.同一血清浓度下,与秋水仙碱组比较,白花丹组的G0/G1期细胞百分比明显较高(P<0.01),S期细胞百分比明显较低;而与复方鳖甲软肝片组无显著性差异.结论:白花丹含药血清可以抑制HSC-T6增殖,诱导其凋亡,且作用具有剂量依赖性.白花丹含药血清抑制HSC-T6增殖的机制可能是使HSC-T6细胞周期停滞于G0/G1期,阻止其通过G1/S关卡.

AIM： To observe the effects ot me plumbago -containing serum on proliferation, apoptosis and cell cycle of the rat hepatic stellate cells （HSC-T6） so as to explore the plumbago＇s antihepatic fibrosis effects and its mechanism.METHODS： Plumbago-containing serum was obtained by intragastric administration to the SD rats with plumbago decoction. HSC-T6 was incubated at different concentrations of the plumbago -containing serum. The colchicine and Fufang compound biejia containing serum were used as positive control group. MTT were applied to measure the HSC-T6 proliferation and flow cytometry to measure the cell apoptosis, and the percentage of DNA content in different groups.RESULTS： Compared with blank group, all the plumbago-containing serum groups had a significantly higher proliferation inhibition rate and apoptosis rate （P 〈 0.01）. When the plumbago- containing serum concentration was increased, proliferation inhibition rate and apoptosis rate to HSC-T6 reached the highest. Its action intensity was the same as compound biejia but stronger than that of Colchicine＇s. Cell percentage in G2/M phase had no significant change in each group. Compared with blank control group, the cell percentage in G0/G1 phase was significantly increased and significantly decreased in S phase in all plumbago -containing serum groups,（55.23% ± 2.83%, 52.60% ± 2.26%, 48.75% ± 1.37% vs 44.08% ± 1.41%, all P 〈 0.01; 31.47% ±1.26%, 34.14% ± 1.17%, 40.28% ± 1.62% vs 47.36% ±1.35%, all P 〈 0.01）. When the plumbago-containing serum concentration was decreased, the cell percentage was significantly decreased in G0/G1 phase and significantly decreased in S phase. Compared with colchicine group at the same serum concentration, the cell percentage was significantly increased in G0/G1 phase and significantly decreased in S phase in all plumbago-containing serum groups, compared with compound biejia group, but there was no significant difference. CONCLUSION： The plumbago-containing serum can inhibit the proliferation and induce apoptosis to HSC-T6, in a dose-dependent manner. Its action intensity is the same as compound biejia but stronger than that of Colchicine＇s. The possible mechanism to inhibit the proliferation of HSC-T6 may be related with HSC-T6＇s stagnation in G0/G1 phase due to plumbago＇s action to prevent HSC-T6 to pass G1/S checkpoint.