紫草素通过调控ERK1/2-NF-κB通路改善2型糖尿病小鼠肝损伤

Shikonin ameliorate the hepatic injury of type 2 diabetes mellitus mice by regulating ERK1/2-NF-κB signaling pathway

目的本研究利用网络药理学及分子对接技术,探究紫草素改善2型糖尿病(T2DM)小鼠肝损伤靶点及机制。方法利用靶点数据库及疾病数据库挖掘紫草素药物靶点及T2DM肝损伤疾病靶点,韦恩交集法收集紫草素-T2DM肝损伤的共有靶点,String数据库构建蛋白互作(PPI)网络,Glue-GO及Metascape数据库分析Biological Process及KEGG。AutoDock进行分子对接。ELISA检测血清丙二醛(MDA)、天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)含量,HE染色观察肝脏形态,Q-PCR检测炎症因子及抗氧化酶基因表达,Western blot检测细胞外调节激酶1/2(ERK1/2)、p-ERK1/2、磷酸化核转录因子-κB(p-NF-κB)、NF-κB等蛋白表达。结果紫草素与T2DM肝损伤的共有靶点105个,36%共有靶点富集在“regulation of inflammatory response”生物学过程,KEGG信号通路主要涉及“AGE-RAGE signaling pathway in diabetic complications”等炎症通路。紫草素与ERK1/2、c-Jun氨基末端激酶(JNK)、蛋白激酶B(AKT)有较好的对接活性。体内动物实验结果显示,与模型组相比,紫草素显著减少肝脏组织炎性颗粒细胞浸润,抑制脂质空泡形成,减少血清MDA、AST、ALT含量(P<0.05),上调SOD1、SOD2、CAT抗氧化酶基因表达(P<0.05),下调p-ERK1/2/ERK1/2、p-NF-κB/NF-κB及IL-1β等炎症因子表达(P<0.01)。结论紫草素能通过增加抗氧化酶基因表达及抑制ERK1/2-NF-κB信号通路改善T2DM肝损伤。

Objective The study used the network pharmacology and molecular docking to explore the potential targets and the possible mechanisms of Shikonin for improving the type 2 diabetes mellitus(T2DM)with liver injury.Method The targets of Shikonin and the disease targets of hepatic injury of T2DM were collected by different databases,and the Wenn was used to collect the overlap common targets of Shikonin-hepatic injury of T2DM,and String database was used to build the protein and protein interaction(PPI)network,and Clue-GO and Metascape database were used to analysis the Biological Process and KEGG pathways,and AutoDock was used for molecular docking.ELISA was used to detect the level of malondialdehyde(MDA),aminotransferase(AST)and aminotransferase(ALT)in serum,and HE staining was used to observe liver histomorphology,and Q-PCR was used to detect the mRNA expression of inflammation factors and anti-oxidant enzymes,and Western blot was used to detect the protein expression of extracellular signal regula-ted kinase1/2(ERK1/2),p-ERK1/2,phosphorylated nuclear factor-κB(p-NF-κB),NF-κB and so on.Result The common tar-gets of Shikonin and T2DM with hepatic injury was 105,and 36%of common targets was enriched at the Biological Process of inflam-matory response regulation,and the common targets mainly enriched at the related inflammation KEGG pathway,including“AGE-RAGE signaling pathway in diabetic complications”and so on.There was good docking binding activity between Shikonin and c-Jun N-terminal kinase(JNK),protein kinase B(AKT),ERK1/2.In the model of T2DM mice,compared with the model group,Shikonin significantly decreased the level of MDA,AST and ALT in serum(P<0.05),and inhibited the inflammatory particle cell infiltration and reduced lipid vacuolation in liver tissue,and upregulated the mRNA expression of anti-oxidant enzymes SOD1,SOD2 and CAT(P<0.05),and downregulated the protein expression of p-ERK1/2/ERK1/2,p-NF-κB/NF-κB,IL-1βand so on(P<0.05).Conclusion Shikonin can significantly improve the hepatic injury in type 2 diabetic mice by inhibiting ERK1/2-NF-κB signaling path-way and upregulating the gene expression of anti-oxidant enzymes.Shikonin might be a potential drug for protecting liver.