柴胡-黄芩药对的不同分离部位对H_2O_2刺激肝星状细胞增殖的影响

The Effect of Different Fractions from Bupleurium-Scutellaria on the Proliferation of Hepatic Stellate Cells Stimulated by H_2O_2

目的:研究柴胡-黄芩药对的不同分离部位对过氧化氢(H_2O_2)刺激的肝星状细胞增殖的影响,探讨柴胡-黄芩药对抗肝纤维化主要药效物质基础。方法:柴胡-黄芩水提液经D101型大孔吸附树脂吸附,依次用水、30%、60%和90%的乙醇梯度洗脱,分别得到水洗脱部位(A)、30%乙醇洗脱部位(B)、60%乙醇洗脱部位(C)及90%乙醇洗脱部位(D)。体外培养肝星状细胞,采用不同浓度的H_2O_2刺激肝星状细胞增殖,筛选最佳造模浓度,然后用柴胡-黄芩药对水提液及A、B、C、D的高、中、低剂量组进行干预24 h。采用MTT法测定各组肝星状细胞的抑制情况;应用ELISA法检测细胞上清液中透明质酸酶(hy-aluronidase,HA)、层黏连蛋白(laminin,LN)、Ⅲ型前胶原(procollagenⅢ,PCⅢ)、Ⅳ型胶原(collagen typeⅣ,Ⅳ-C)的浓度。结果:结果显示,5μmol/L的H_2O_2能显著刺激肝星状细胞增殖,柴胡-黄芩药对及其各分离部位对H_2O_2刺激的肝星状细胞增殖均具有一定的抑制作用,而且能够将低HA、LN、PCⅢ、Ⅳ-C的细胞释放水平,具有剂量相关性,其中B、C高剂量组对其抑制作用最为明显,有统计学意义(P<0.01)。结论:柴胡-黄芩药对及其30%、60%乙醇洗脱部位对H_2O_2刺激的肝星状细胞增殖均具有一定的抑制作用,30%、60%乙醇洗脱部位可能是柴胡-黄芩药对抗肝纤维化主要有效部位。

Objective To study the effect of different fractions of Bupleurium-Scutellaria on the proliferation of hepatic stellate cells stimulated by hydrogen peroxide(H2O2).In order to seek for the basis of the main pharmacodynamic substances of Bupleurium-Scutellaria in anti-hepatic fibrosis.Methods Bupleurium-Scutellaria water extraction liquid was adsorbed by D101 macroporous resin,after being eluted by water,30%,60%and 90%ethanol,respectively get water-eluted fraction(A),30%ethanol-eluted fraction(B),60%ethanol-eluted fraction(C)and 90%ethanol-eluted fraction(D).The HSC of rat was cultured in vitro,with different concentrations of H2O2 to stimulate the proliferation of hepatic stellate cell,screening the optimum concentration,using the high,medium and low dose group of Bupleuri-Scutellariae water extraction liquid and A,B,C,D to intervene for 24 h.To measure the inhibition of hepatic stellate cells by MTT assay.The HA,LN,PCⅢ,Ⅳ-C concentration in the cell supernatant was detected by ELISA.Results The experimental results show that the tendency towards 5μmol/L H2O2 could significantly stimulate the proliferation of hepatic stellate cell,Bupleurium-Scutellaria and its different fractions have certain inhibition to the proliferation of hepatic stellate cell by H2O2 stimulated,and it can reduce the release level of HA,LN,PCⅢ,Ⅳ-C in cell.There was a certain dose correlation.among them,B and C high dose group of the inhibitory effect is most obvious,There were a certain statistically significant(P<0.01).Conclusion Bupleuri-Scutellariae and its 30%,60%ethanol-eluted fractions have certain inhibitory effect on the proliferation of hepatic stellate cells stimulated by H2O2.30%ethanol-eluted fraction and 60%ethanol-eluted fraction may be the main effective part for Bupleuri-Scutellariae in anti-hepatic fibrosis.