中药成分复方JY5通过抑制胆管反应抗胆汁性肝纤维化的研究

Study of the Chinese Herbal Compound JY5 Against Biliary Hepatic Fibrosis through Inhibition of Ductular Reaction

目的探讨源自扶正化瘀方的中药成分复方JY5(丹酚酸B、苦杏仁苷和五味子酯甲)对胆管结扎诱导的大鼠肝纤维化的作用及机制。方法将32只SD大鼠随机分为假手术组、模型组、JY5组和DAPT组,每组8只。采用胆管结扎术制备大鼠胆汁性肝纤维化模型。胆管结扎术后1周开始给药,JY5组给予JY5混悬液(丹酚酸B 16 mg·kg^(-1)+苦杏仁苷0.5 mg·kg^(-1)+五味子酯甲2 mg·kg^(-1))10 mL·kg^(-1)灌胃给药;DAPT组按照30 mg·kg^(-1)腹腔注射给药;假手术组、模型组灌胃给予0.3%CMC-Na;每日1次,连续给药3周。检测大鼠血清肝功能相关生化指标;采用苏木素-伊红(HE)及天狼星红(SR)染色法观察肝组织炎性损伤和胶原沉积情况;碱水解法测定肝组织羟脯氨酸(Hyp)含量;qPCR、Western Blot法检测肝组织CK19、CK7、EpCAM mRNA及蛋白表达;免疫组织化学法检测肝组织CK19、CK7、EpCAM、OV6蛋白表达情况;免疫荧光染色法观察肝组织中CK19、OV6共定位情况。结果与假手术组比较,模型组大鼠血清中的丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)、碱性磷酸酶(ALP)、γ-谷氨酰转肽酶(GGT)活性及总胆汁酸(TBA)、总胆红素(TBIL)、直接胆红素(DBIL)含量明显升高(P<0.01),白蛋白(ALB)含量明显降低(P<0.01);肝组织的炎性损伤和胶原沉积明显加重,肝脏SR染色阳性面积比和Hyp含量均明显升高(P<0.01);大鼠肝组织中CK19、CK7、EpCAM mRNA及蛋白表达量显著升高(P<0.01),OV6蛋白表达量显著升高(P<0.01);肝组织中OV6和CK19共表达的阳性细胞数显著增加。与模型组比较,JY5组大鼠血清的ALT、AST、ALP、GGT活性及TBA、TBIL、DBIL含量明显降低(P<0.01),ALB含量明显升高(P<0.01);肝组织的炎性损伤和胶原沉积明显减轻,肝脏SR染色阳性面积比和Hyp含量均明显降低(P<0.01);大鼠肝组织中CK19、CK7、EpCAM mRNA及蛋白表达量显著降低(P<0.01),OV6蛋白表达量显著降低(P<0.01);肝组织中OV6和CK19共表达的阳性细胞数明显减少。结论中药成分复方JY5可减轻胆管结扎诱导的胆汁性肝纤维化,其作用机制与抑制肝祖细胞来源的胆管反应有关。

Objective To investigate the effect and mechanism of the Chinese herbal compound JY5(salvianolic acid B,amygdalin and Schisantherin A)on bile duct ligation(BDL)-induced liver fibrosis in rats. Methods Thirty-two SD rats were randomly divided into sham-operation group,model group,JY5 group and DAPT group,with 8 rats in each group. BDL was used to prepare the rat model of biliary hepatic fibrosis. The JY5 group was administrated1 week after BDL with JY5 suspension(salvianolic acid B 16 mg·kg^(-1)+ amygdalin 0.5 mg·kg^(-1)+ Schisantherin A2 mg·kg^(-1))at 10 mL·kg^(-1)by gavage;DAPT group was administrated at 30 mg·kg^(-1)by intraperitoneal injection;the sham-operation group and model group were given 0.3% CMC-Na by gavage;the treatments in all the groups were administrated once per day for consecutive 3 weeks. The rats were tested for biochemical parameters related to serum liver function;the inflammatory damage and collagen deposition in liver tissue were observed by hematoxylin-eosin(HE) and Sirius red(SR) staining;the hydroxyproline(Hyp) content of liver tissue was detected by alkaline hydrolysis;the m RNA and protein expressions of CK19,CK7 and EpCAM in liver tissue were detected by qPCR and Western Blot;the protein expressions of CK19, CK7, EpCAM and OV6 in liver tissue were detected by immunohistochemistry;the co-localization of CK19 and OV6 in liver tissue was observed by immunofluorescence staining. Results Compared with the sham-operation group,the serum levels of ALT,AST,ALP,GGT and the levels of TBA, TBIL and DBIL were significantly increased(P<0.01) and the level of ALB was significantly decreased(P<0.01) in the model group;the inflammatory damage and collagen deposition in liver tissue were significantly increased, and the ratio of positive area of SR staining and Hyp content in liver were significantly increased(P<0.01);the mRNA and protein expressions of CK19, CK7, EpCAM in rat liver tissues were significantly increased(P<0.01)and OV6 protein expression was significantly increased(P<0.01);the number of positive cells co-expressed with OV6 and CK19 in liver tissues was significantly increased. Compared with the model group,activities of ALT,AST,ALP,GGT and contents of TBA,TBIL and DBIL in rat serum in the JY5 group were significantly decreased(P<0.01)and ALB contents were significantly increased(P<0.01);the inflammatory damage and collagen deposition in liver tissue were significantly reduced,and liver SR staining positive area ratio and Hyp content were significantly decreased(P<0.01). The mRNA and protein expressions of CK19, CK7,EpCAM in rat liver tissue was significantly decreased(P<0.01), and OV6 protein expression was significantly decreased(P<0.01);the number of positive cells co-expressed with OV6 and CK19 in liver tissue was significantly reduced. Conclusion The Chinese herbal compound JY5 exerts effect in alleviating BDL-induced liver fibrosis,and its mechanism of action is related to the inhibition of ductular reaction of hepatic progenitor cell(HPC)origin.