黄芪注射液对肝纤维化抑制作用的实验研究

目的探讨黄芪注射液对大鼠肝星状细胞(HSC)和肝纤维化的作用。方法体外细胞实验: 用不同浓度黄芪注射液(0、25、50、100、200、400 mg/ml)作用HSC不同时间(24、48、72h)后,采用四甲基偶氮唑盐法检测其活化增殖;流式细胞术检测HSC增殖周期;溴乙锭/吖啶橙荧光染色和流式细胞术检测HSC凋亡。动物实验:用40%四氯化碳和5%乙醇制备大鼠肝纤维化动物模型,实验分为正常对照组、模型组和黄芪注射液组。黄芪注射液组和模型组在模型制备的同时分别给予黄芪注射液(800 mg·kg-1·d-1)和等渗盐水腹腔注射,第8周时测定血清透明质酸(HA),层黏连蛋白(LN)水平及肝组织中超氧化物歧化酶(SOD)活性,丙二醛(MDA)含量,免疫组织化学方法观察肝组织LN的表达,苏木素-伊红、苦味酸-酸性品红染色观察肝组织病理改变。结果在体外细胞实验中,与0 mg/ml组比较,黄芪注射液其它浓度组明显抑制了HSC增殖,并呈剂量和时间依赖性;HSC增殖周期被抑制在G2-M期;黄芪注射液各浓度组荧光染色法和流式细胞术均未检测到HSC凋亡。在体内实验中,血清HA、LN含量:模型组分别为(114.3±25.6)μg/L和(78.8±11.7)μg/L,黄芪注射液组分别为(85.6±37.3)μg/L和(66.8±17.6)μg/L,P <0.05;肝组织SOD活性黄芪注射液组为(75.9±5.9)NU/mg,模型组为(49.6±5.7)NU/mg,P< 0.01;而MDA含量黄芪注射液组为(2.4±0.2)μmol/g,模型组为(3.7±0.4)μmol/g,P<0.01。显微镜下黄芪注射液组肝纤维化程度明显轻于模型组,免疫组织化学结果黄芪注射液组肝组织LN表达明显减少。结论黄芪注射液可延缓肝纤维化的发生,其机制除可直接抑制HSC增殖外,还有抗氧化、抗脂质过氧化、减少LN产生,防止肝窦毛细血管化等作用。

Study on the inhibitive effect of Astragalus Injection solution on hepatic fibrosis in rats

OBJECTIVE: To investigate the effect of Astragalus Injection solution on rat hepatic stellate cells (HSC) and hepatic fibrosis. METHODS: HSCs of rats were incubated with various concentrations of Astragalus Injection solution (0 mg/ml, 25 mg/ml, 50 mg/ml, 100 mg/ml, 200 mg/ml, 400 mg/ml) for 24, 48 and 72 hours. Cell proliferation was detected with 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphennyltetrazolium bromide (MTT) colorimetric assay. Cell cycle was detected with flow cytometry. Cell apoptosis was detected with acridine orange/ethidium bromide (AO/EB) fluorescent staining and flow cytometry. In vivo, rats were randomly allocated into a normal control group, a model control group and an Astragalus Injection group. Astragalus Injection (800 mg.kg-1.d-1) was administered to rats of the Astragalus Injection group. Rats of the model control group received saline. Serum concentrations of hyaluronic acid (HA) and laminin (LN), hepatic tissue activity of superoxide dismutase (SOD), and hepatic tissue contents of malondialdehyde (MDA) were measured in these groups at 8 weeks. Hepatic tissue expression of LN was assessed by using immunohistochemistry. The pathological changes of hepatic tissues were examined by hematoxylin-eosin (HE) and van Gieson (VG) staining of their histological slides. RESULTS: In vitro, compared with the 0 mg/ml group, the proliferation of HSCs in other concentration groups was significantly inhibited by Astragalus Injection solution in a dose and time dependent manner, the cell proliferation cycle of HSCs was blocked in the G2-M phase, there was no apoptosis of HSCs in AO/EB fluorescent staining and flow cytometry. In vivo, compared with rats of the model control group, the rats of the Astragalus Injection solution treated group had remarkably decreased serum HA and LN levels (114.3+/-25.6) microg/L vs (85.6+/-37.3) microg/L and (78.8+/-11.7) microg/L vs (66.8+/-17.6) microg/L, P < 0.05, and liver MDA level (3.7+/-0.4) micromol/g protein vs (2.4+/-0.2) micromol/g protein, P < 0.01, but had increased activity of liver SOD (49.6+/-5.7) NU/mg protein vs (75.9+/-5.9) NU/mg protein, P < 0.01. Microscopic studies revealed that the livers of rats receiving Astragalus Injection solution showed decreases in fibrosis and in expression of LN. CONCLUSIONS: Astragalus Injection solution has an inhibitive effect on experimental hepatic fibrogenesis. The mechanisms of its effects might possibly be associated with its antioxidant activity, expression of decreasing LN and its inhibition of HSCs proliferation.