扶正化瘀复方抑制小鼠自噬的抗纤维化机制研究

摘要

目的 明确扶正化瘀复方抗肝纤维化的作用是否与其抑制自噬有关.方法 C57小鼠随机分为正常组(N组)和造模组,造模组采用四氯化碳腹腔注射诱导小鼠肝纤维化模型,正常组注射等剂量橄榄油,1周后将造模组随机分为模型(M)组、雷帕霉素(Rapa)组、雷帕霉素加氯喹(Rapa+CQ)组、雷帕霉素加丹参酚酸B(Rapa+Sal B)组、雷帕霉素加扶正化瘀方(Rapa+FZ)组,各药物组每日相应药物灌胃,N组、M组等剂量饮用水灌胃.5周后处死小鼠,HE和天狼猩红染色分别观察各组肝组织炎症和胶原沉积情况,碱水解法测定羟脯氨酸含量.蛋白质印迹法检测肝组织中自噬的表达变化和微管相关蛋白1轻链3Ⅱ/Ⅰ (LC3Ⅱ/Ⅰ)、p62、α-平滑肌肌动蛋白(α-SMA)和Ⅰ型胶原蛋白表达,冰冻切片免疫荧光染色观察各组肝组织α-SMA及LC3B免疫荧光定位.两独立样本间的比较用t检验,多个样本均数比较采用LSD检验或Dunnett T3检验. 结果 N组与M组小鼠体质量差异无统计学意义,各药物组小鼠体质量均明显下降(F=14.041,P＜0.001).M组和各药物组小鼠的肝质量/体质量比和脾质量/体质量比较N组均有明显增加(F值分别为26.992、6.589,P值均＜0.001).与M组比较,各用药组小鼠肝组织p62蛋白表达降低,其中Rapa组和Rapa+Sal B组差异有统计学意义(F=3.085,P值分别为0.039、0.003),Rapa+Sal B组则更低;与M组比较,Rapa组LC3BⅡ的表达显著升高(F=7.514,P=0.01).免疫荧光染色显示N组小鼠肝脏内未见LC3B与α-SMA共染细胞,M组小鼠肝内见到共染细胞,各药物组小鼠肝内共染细胞明显多于M组,其中Rapa+FZ组的共染细胞较少.与N组相比,M组和各药物组胶原显著增加,各药物组胶原沉积较M组减少,各药物组间差异无统计学意义.与N组(值为77.75±48.79)相比,M组小鼠的肝组织羟脯氨酸明显升高(值为293.48±84.43)(F=3.015,P=0.005),各药物组小鼠肝组织羟脯氨酸含量均较M组减少,但差异无统计学意义(F=0.750,P=0.573).与N组相比,M组α-SMA和Ⅰ型胶原蛋白的表达均明显增加(F值分别为27.718、18.893,P值均＜0.01).Rapa组和Rapa+Sal B组的α-SMA的表达与M组相似;而Rapa+CQ组和Rapa+FZ组较Rapa组和M组有显著降低(P值均＜0.01).Rapa+CQ组的Ⅰ型胶原蛋白表达明显高于Rapa组(P=0.017),而Rapa+FZ组的Ⅰ型胶原蛋白表达则明显低于M组(P=0.013).结论 四氯化碳诱导的肝纤维化模型中,观察到肝星状细胞的自噬;雷帕霉素能促进肝细胞和肝星状细胞的自噬;扶正化瘀方和丹参酚酸B均能对抗雷帕霉素促自噬的作用.%Objective To determine whether the ant-hepatic fibrosis effect of Fuzheng-Huayu formula is related to suppress autophagy in mice.Methods C57 mice were randomly divided into normal group (N group)and model group.The model group was induced by intraperitoneal injection of carbon tetrachloride to induce liver fibrosis in mice,and the normal group was injected with equal volume of olive oil.After 1 week,the model group was randomly divided into model (M) group,rapamycin (Rapa) group,rapamycin plus chloroquine (Rapa+CQ)group,rapamycin plus salvianolic acid B (Rapa+Sal B) group,rapamycin plus Fuzheng-Huayu fonnula (Rapa+FZ)group.Each drug group was administered corresponding drugs by gavage on a daily basis,and N group and M group were given the equal amount of drinking water by gavage.After 5 weeks,the mice were sacrificed,and HE and Sirius red staining were used to observe the inflammation and collagen deposition on liver tissue in each group.The hydroxyproline content was determined by alkaline hydrolysis method.Western blotting was used to detect changes in the expression of autophagy in liver tissue and microtubule-associated protein 1 light chain 3Ⅱ/Ⅰ (LC3Ⅱ/Ⅰ),p62,α-smooth muscle actin (α-SMA) and type Ⅰ collagen expression.Immunofluorescence staining was used to observe the immunofluorescence localization of o-SMA and LC3B in liver tissues of each group.).A t-test was used to compare the two independent samples.LSD or Dunnett's T3 test were used to compare the mean of multiple samples.Results There was no significant difference in N and M groups in terms of body weight.The body weight of the mice in each drug group decreased significantly (F=14.041,P ＜ 0.001).The liver/spleen /body weight ratios of each drug group and M group were significantly higher than the N group (F =26.992,6.589,P ＜ 0.001).The expression of p62 protein in the liver tissue of mice in each drug group was lower than M group,and the difference between Rapa group and Rapa+Sal B group (F =3.085,P =0.039,0.003) was statistically significant,while that of Rapa + Sal B group was lower.Compared with group M,the expression of LC3B Ⅱ in Rapa group was significantly higher (F =7.514,P =0.01).Imrnunofluorescence staining showed that LC3B and α-SMA CO-stained cells were absent in the liver of mice in N group,and co-stained cells were found in the liver of mice in M group.The co-stained cells in the liver of mice in each drug group were significantly higher than M group,and the co-stained cells in Rapa+FZ group were fewer.Compared with the N group,the collagen deposition of M group and each drug group was significantly increased;the collagen deposition of each drug group was lower than that of the M group.There was no statistically significant difference between each drug group.Compared with N group (77.75 + 48.79),hydroxyproline in liver tissue of mice in M group was significantly increased (293.48 + 84.43) (F =3.015,P =0.005),and the content of hydroxyproline in liver tissue of mice in each drug group was lower than M group,but the difference was not statistically significant (F =0.750,P =0.573).Compared with the N group,the expressions of α-SMA and type Ⅰ collagen in the M group were significantly increased (F =27.718,18.893,P ＜ 0.01).The expression of α-SMA in Rapa group and Rapa+Sal B group was similar to M group,while Rapa + CQ group and Rapa + FZ group were significantly lower than Rapa group and M group (P ＜ 0.01).The expression of type Ⅰ collagen in Rapa + CQ group was significantly higher than Rapa group (P =0.017),while the expression of type Ⅰ collagen in Rapa + FZ group was significantly lower than M group (P =0.013).Conclusion Autophagy of hepatic stellate cells was observed in carbon tetrachloride-induced liver fibrosis model.Rapamycin can promote autophagy in hepatocytes and hepatic stellate cells.Fuzheng-Huayu formula and Salvianolic Acid B might antagonize the effect of rapamycin on autophagy.