仙鹤草不同提取部位抗肝纤维化作用的研究

Study on the different extraction fractions of Agrimonia pilosa against hepatic fibrosis

目的研究仙鹤草不同提取部位的抗肝纤维化作用。方法以大鼠肝星状细胞HSC-T6为研究对象,检测不同质量浓度(0.5、5、50、500、5 000μg/mL,均以生药量计)的仙鹤草不同提取部位(总提取物、乙酸乙酯部位、石油醚部位和正丁醇部位)对该细胞增殖的影响(作用24、48、72 h后),并计算半数抑制浓度(IC50)。采用血小板衍生生长因子(PDGF-BB)刺激HSC-T6细胞以复制肝纤维化细胞模型,采用流式细胞仪检测仙鹤草不同提取部位对HSC-T6细胞凋亡的影响,采用酶联免疫吸附试验检测细胞培养上清液中Ⅰ型胶原蛋白(Col-Ⅰ)表达水平,采用Western blot法检测细胞中α-平滑肌肌动蛋白(α-SMA)、Col-Ⅰ、B细胞淋巴瘤2(Bcl-2)、Bcl-2相关X蛋白(Bax)、胱天蛋白酶3(caspase-3)的表达水平。结果仙鹤草总提取物、乙酸乙酯部位、石油醚部位和正丁醇部位均可显著升高HSC-T6细胞凋亡率(P<0.01);作用24 h时,上述各部位的IC50分别为50.17、20.75、5.82、4.09μg/mL。经PDGF-BB干预后,HSC-T6细胞培养上清液中Col-Ⅰ和细胞中Col-Ⅰ、α-SMA、Bcl-2、Bax、caspase-3蛋白表达水平均显著升高(P<0.01);经仙鹤草不同提取部位干预后,HSC-T6细胞培养上清液或细胞中上述蛋白表达水平大部分均较PDGF-BB组显著逆转(P<0.05或P<0.01),其中仙鹤草正丁醇部位干预作用最为显著。结论仙鹤草不同提取部位均可抑制HSC-T6细胞增殖,并诱导其凋亡;正丁醇部位可能是仙鹤草发挥抗肝纤维化作用的有效部位。

OBJECTIVE To study different extraction fractions of Agrimonia pilosa against hepatic fibrosis. METHODS Using hepatic stellate cells HSC-T6 of rats as objects,the effects of different extraction fractions(total extract,ethyl acetate fraction,petroleum ether fraction and n-butanol fraction)with different concentrations(0.5,5,50,500,5 000 μg/mL,calculated by raw drug) of A. pilosa on the proliferation of HSC-T6 cells were detected(after treated for 24,48,72 h);median inhibition concentration(IC50)was also caculated. Platelet-derived growth factor(PDGF-BB)was used to induce the activation of HSC-T6 cells to establish hepatic fibrosis cell model. Flow cytometry was used to detect the effects of different extraction fractions of A.pilosa on apoptosis of HSC-T6 cells. The expression of collagen Ⅰ (Col-Ⅰ) in the supernatant was detected by enzyme linked immunosorbent assay. The expressions of α-smooth muscle actin(α-SMA),Col-Ⅰ,B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax) and caspase-3 were detected by Western blot assay. RESULTS Total extract,ethyl acetate fraction,petroleum ether fraction and n-butanol fraction of A. pilosa could significantly increase the apoptotic rate of HSC-T6 cells(P<0.01). After treated for 24 h,IC50 of above fractions were 50.17,20.75,5.82,4.09 μg/mL,respectively. After intervened with PDGF-BB,the expression of Col-Ⅰ in supernatant of HSC-T6 cells as well as protein expression of Col-Ⅰ,α-SMA,Bcl-2,Bax and caspase-3 in HSC-T6 cells were increased significantly(P<0.01). After intervened with different extraction fractions of A. pilosa,most of the expressions of above proteins in HSC-T6 cell culture supernatant or cells were significantly reversed compared with PDGF-BB group(P<0.05 or P<0.01), and the intervention effect of n-butanol fraction of A. pilosa was the most significant.CONCLUSIONS Different extraction fractions of A. pilosa can inhibite the proliferation of HSC-T6 cells and induce their apoptosis;n-butanol fraction from A. pilosa may be an effective fraction to exert the effect of anti-hepatic fibrosis.