膈下逐瘀汤治疗酒精性肝病的作用机制探讨

Mechanism of Gexiazhuyu decoction in treatment of alcoholic liver disease

目的基于网络药理学、分子对接的方法分析膈下逐瘀汤治疗酒精性肝病的作用机制,并对其进行动物模型实验验证。方法基于TCMSP数据库筛选膈下逐瘀汤潜在活性化合物和作用靶点,并通过GeneCards、OMIM、PharmGkb、TTD和DrugBank数据库检索酒精性肝病疾病相关靶点,二者取交集,获得膈下逐瘀汤治疗酒精性肝病的关键靶点。对关键靶点进行功能富集分析和分子对接。通过STRING平台和Cytoscape软件构建膈下逐瘀汤治疗酒精性肝病的潜在活性化合物—靶点调控网络和蛋白互作网络,并对网络进行拓扑分析,得到最终核心网络和靶点。取SPF级雄性SD大鼠60只,随机取12只作为正常对照组,其余48只以乙醇灌胃的方法制备酒精性肝病模型,将其分为模型对照组、水飞蓟素组、膈下逐瘀汤低剂量和高剂量组各12只。膈下逐瘀汤低剂量组和高剂量组分别给予6.2、12.4 g/kg膈下逐瘀汤水煎液灌胃,水飞蓟素组予以水飞蓟素0.2g/kg灌胃,模型对照组和正常对照组予以同等体积蒸馏水灌胃,连续灌胃6周。取大鼠腹主动脉血检测血清丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、甘油三酯(TG)、γ谷氨酰转肽酶(GGT);取肝脏组织,HE染色法观察肝脏组织病理形态,Western blotting法检测关键通路相关蛋白。结果共获得膈下逐瘀汤治疗酒精性肝病253个关键靶点,关键靶点主要富集于PI3K/AKT等信号通路。经拓扑分析后,获得核心靶点19个。分子对接显示,各核心靶点蛋白均可与膈下逐瘀汤中的对应的活性化合物结合,且结合自由能低于-0.5 kcal/mol。动物实验显示,膈下逐瘀汤高低剂量组大鼠肝脏组织病理形态较模型组明显改善,血清ALT、AST、TG、GGT水平下降,PI3K/AKT信号通路相关蛋白p-PI3K、p-AKT表达升高(P均<0.01)。结论膈下逐瘀汤对酒精性肝损伤具有良好的保护作用,其主要机制可能与调节PI3K/AKT信号通路中p-PI3K和p-AKT等关键蛋白的表达有关。

Objective To investigate the mechanism of action of Gexiazhuyu decoction in the treatment of alcoholic liver disease based on network pharmacology and molecular docking technology,and to validate it through the animal ex⁃periments.Methods Based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)database,we screened the potential active compounds and targets of Gexiazhuyu decoction,and searched the GeneCards,Online Mendelian Inheritance in Man(OMIM),PharmGkb,Therapeutic Target Database(TTD)and Drug⁃Bank databases for the targets related to alcoholic liver disease,and obtained the key targets of Gexiazhuyu decoction for al⁃coholic liver disease after the intersection of the two.Functional enrichment analysis and molecular docking were per⁃formed on key targets.The STRING platform and Cytoscape software were used to construct the potentially active com⁃pound-target regulatory network and protein interactions network of Gexiazhuyu decoction in the treatment of alcoholic liver disease,and topological analysis of the network was performed to obtain the core network and targets.Sixty specific patho⁃gen free(SPF)-grade male Sprague-Dawley(SD)rats were taken,12 were randomly selected as the normal control group,and the remaining 48 rats were prepared as alcoholic liver disease models by ethanol gavage,and then were divided into the model control group,silymarin group,low-dose and high-dose Gexiazhuyu decoction groups,with 12 in each.The rats in the low-dose and high-dose Gexiazhuyu decoction groups were treated with 6.2 and 12.4 g/kg of Gexiazhuyu decoction,respectively;the rats in the silymarin group were gavaged with 0.2 g/kg of silymarin;the rats in the model control group and normal control group were gavaged with the same volume of distilled water for 6 weeks.Serum alanine aminotransfer⁃ase(ALT),aspartate aminotransferase(AST),triglyceride(TG)and gamma-glutamyl transpeptidase(GGT)were mea⁃sured in the abdominal aorta blood of rats;liver tissues were stained with HE to observe the histopathological morphology,and Western blotting was used to detect key pathway-related proteins.Results A total of 253 key targets were obtained for the treatment of alcoholic liver disease by Gexiazhuyu decoction,which were mainly enriched in PI3K/AKT and other signaling pathways.After topological analysis,19 core targets were obtained.The molecular docking showed that all the core target proteins could bind to the corresponding active compounds in Gexiazhuyu decoction,and the binding free ener⁃gy was below-0.5 kcal/mol.Animal experiments showed that the pathological morphology of liver tissues in rats of the low-dose and high-dose Gexiashuyu decoction groups was significantly improved in comparison with that of the model group.Serum levels of ALT,AST,TG,and GGT decreased,and the expression of PI3K/AKT signaling pathway-related proteins p-PI3K and p-AKT increased(all P<0.01).Conclusion Gexiazhuyu decoction has protective effect on rats with alcoholic liver injury,and its mechanism of action may be related to the regulation of the expression of key proteins such as p-PI3K and p-AKT in the PI3K/AKT signaling pathway.