五灵胶囊工艺优化前后对四氯化碳诱导的肝纤维化大鼠保护作用的比较

Protective effects of Wuling capsule on CCl4-induced hepatic fibrosis in rats before and after process optimization

目的比较五灵胶囊工艺优化前后对四氯化碳(CCl_(4))诱导的肝纤维化大鼠的保护作用。方法将SD大鼠随机分为9组:对照组,模型组,水飞蓟素阳性药组,五灵胶囊新工艺低(0.25 g/kg)、中(0.5 g/kg)、高(1.0g/kg)剂量组,五灵胶囊原工艺低(0.25 g/kg)、中(0.5 g/kg)、高(1.0 g/kg)剂量组,每组10只。除对照组外,其余各组大鼠腹腔注射40%CCl_(4)(橄榄油配制,剂量为2 mL/kg),每周2次,连续8周,构建肝纤维化模型。给药组于第2周开始灌胃给药,持续6周。实验结束后称取大鼠肝脏、脾脏湿重并计算脏器指数;检测血清生化指标丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)、总胆汁酸(TBA)、白蛋白(ALB)含量和超氧化物歧化酶(SOD)活性;采用H-E染色和Masson染色进行肝组织病理结构和纤维化程度观察;应用免疫组织化学染色检测肝组织中α-平滑肌肌动蛋白(α-SMA)水平;采用ELISA测定血清中炎症因子IL-6、IL-1β和TNF-α含量;采用qPCR法测定肝脏中胶原蛋白(COL)Ⅰ、COLⅢ、COLⅣ和NF-κB mRNA的相对表达量。结果五灵胶囊优化工艺前后均改善了模型大鼠的肝脏病理结构、降低了肝纤维化程度。与模型组相比,五灵胶囊新工艺和原工艺组大鼠血清中ALT、AST、ALP和TBA含量均降低(P<0.05);血清中ALB含量和SOD活性均升高(P<0.05);肝脏组织中α-SMA阳性表达面积均减少(P<0.05);血清中IL-6、IL-1β和TNF-α含量均降低(P<0.05);肝脏组织中COL I、COLⅢ、COLⅣ和NF-κB的mRNA表达水平均下调(P<0.05),其中五灵胶囊新工艺组TBA、α-SMA和COLⅢ优于原工艺组。结论五灵胶囊新工艺和原工艺对CCl_(4)诱导的大鼠肝纤维化均具有保护作用,但新工艺的部分指标优于原工艺。

Objective To compare the protective effects of Wuling capsule on CCl_(4)-induced hepatic fibrosis rats before and after process optimization.Methods SD rats were randomly divided into 9 groups:control group,model group,positive control group(silymarin)and low(0.25 g/kg)-,medium(0.5 g/kg)-and high(1.0 g/kg)-dose of Wuling capsule new process and original process groups,with 10 rats in each group.The rat model with liver brosis was established by intraperitoneal injection of CCl_(4)solution(dissolved in olive oil,at a dosage of 2 mL/kg),twice a week for 8 weeks except control group.The rats in the treatment groups were administered by gavage at the second week for 6 consecutive weeks.At the end of the experiment,the wet weights of the rat liver and spleen were examined and the viscera indexes were calculated.Serum biochemical indexes(alanine aminotransferase[ALT],aspartate aminotransferase[AST],alkaline phosphatase[ALP],total bile acid[TBA],and albumin[ALB])and superoxide dismutase(SOD)activity were detected.Hematolyxin-eosin staining and Masson staining were used to observe the pathological structure and brosis degree of liver tissues.The level ofα-smooth muscle actin(α-SMA)in rat liver tissues was detected by immunohistochemistry.The levels of interleukin(IL)-6,IL-1βand tumor necrosis factorα(TNF-α)in serum were determined by enzyme-linked immunosorbent assay.The mRNA relative expression levels of collagen(COL)Ⅰ,COLⅢ,COLⅣand nuclear factorκB(NF-κB)in the liver were determined by quantitative polymerase chain reaction(qPCR).Results Both the new and original process groups of Wuling capsule improved the liver pathological structure of CCL4-induced hepatic brosis rats and reduced the degree of hepatic brosis.Compared with the model group,the contents of ALT,AST,ALP and TBA in serum were signi cantly decreased(P<0.05);serum ALB content and SOD activity were signi cantly increased(P<0.05);theα-SMA positive area ratio in liver tissues decreased(P<0.05);the contents of serum IL-6,IL-1βand TNF-αwere signi cantly decreased(P<0.05);and the mRNA expression levels of COLⅠ,COLⅢ,COLⅣand NF-κB in liver tissues were signi cantly down-regulated(P<0.05).The experimental results of TBA,α-SMA and COLⅢin the new process group were signi cantly better than those in the original process group.Conclusion Both the new technology and original technology of Wuling capsule have protective effects on CCl_(4)-induced hepatic brosis in rats,but some indexes of the new technology are better than those of the original technology group.