基于网络药理学和实验验证探究藏药乌奴龙胆改善化学性肝损伤作用及机制

Exploring the effect and mechanism of Tibetan medicine Wunulongdan in improving chemical liver injury based on network pharmacology and experimental verification

关键词:  乌奴龙胆  网络药理学  保肝  化学性肝损伤  PI3K/AKT/NF-κB通路  凋亡

Key words:  Gentiana urnula H.Smith  Network pharmacology  Liver protection  Chemical liver injury  PI3K/AKT/NF-κB pathway  Apoptosis

摘要:

目的 网络药理学与实验验证结合探究藏药乌奴龙胆改善化学性肝损伤作用及机制。方法 文献检索结合Swiss ADME数据库平台筛选乌奴龙胆潜在活性成分，通过TargetPrediction和TCMSP数据库获得潜在活性成分的靶点，与从Genecards平台获取的肝损伤靶点取交集进行Veen分析，并建立“药物-成分-靶点”网络，获取其保肝活性成分及核心靶点。利用String平台与CytoScape软件构建核心靶点蛋白质互作(protein-protein interaction，PPI）网络，通过DAVID数据库进行GO及KEGG通路富集分析。将48只C57小鼠随机分为空白组、模型组、阳性药组、乌奴龙胆高、中、低剂量组，连续灌胃给药7d，腹腔注射0.5%CCl4橄榄油溶液建立小鼠急性肝损伤模型，观察肝脏表观并进行病理HE染色。取小鼠血清和肝组织检测肝功能指标ALT、AST、ALB、LDH，炎症指标IL-6、IL-1β、TNF-α及氧化应激指标MDA、SOD、GSH水平。Western blotting和RT-qPCR检测PI3K/AKT/NF-κB信号通路相关蛋白和基因表达，免疫组化检测NF-κB蛋白表达。建立CCl4诱导Hep-G2细胞损伤模型，筛选乌奴龙胆对细胞损伤保护活性部位。使用乙醇部位高、中、低剂量处理细胞24h后造模，CCK-8法检测细胞存活率，生化法检测ALT、AST、LDH水平。Western blotting和RT-qPCR检测PI3K/AKT/NF-κB信号通路相关蛋白和基因表达，流式细胞术检测细胞凋亡率。结果 从乌奴龙胆中筛选出29个成分和177个潜在保肝靶点，核心靶点包括AKT1、ALB、EGFR等，涉及PI3K/AKT/NF-κB等通路。动物实验显示，乌奴龙胆可改善CCl4诱导的小鼠肝组织广泛坏死和结构破坏，降低ALT、AST、LDH、TNF-α、IL-1β、IL-6、MDA水平，升高SOD、GSH，同时调控PI3K/AKT/NF-κB通路，抑制凋亡相关基因和蛋白的表达；细胞实验证实，乌奴龙胆乙醇部位可改善CCl4诱导的Hep-G2细胞损伤，降低ALT、AST、LDH水平并且调控PI3K/AKT/NF-κB信号通路，降低细胞凋亡率。结论 乌奴龙胆可以减轻氧化应激和炎症反应改善化学性肝损伤，其机制可能与调控PI3K/AKT/NF-κB通路抑制细胞凋亡有关。

Abstract:

OBJECTIVE Based on network pharmacology combined with experimental verification, this study explored the effect and mechanism of Tibetan medicine Gentiana urnula H.Smith in improving chemical-induced liver injury in mice. METHODS Based on literature retrieval and database integration with the Swiss ADME platform, potential active targets of Gentiana urnula H.Smith were screened. Targets of potential active components were obtained using the TargetPrediction and TCMSP databases, and Venn analysis was performed with liver injury targets obtained from the Genecards platform. A "drug-component-target" network was established to identify the main active components and core targets. The protein-protein interaction (PPI) network of core target proteins was constructed using the String platform and CytoScape software. GO and KEGG pathway enrichment analysis was performed through the DAVID database. Forty-eight C57 mice were randomly divided into a blank group, a model group, a positive control group, and high, medium, and low-dose Gentiana urnula H.Smith groups. Continuous gavage administration was performed for 7 days to establish a CCl4-induced acute liver injury model in mice, and the appearance and pathological HE staining of the liver were observed. Serum and liver tissues were collected to detect liver function indicators ALT, AST, ALB, LDH, inflammatory indicators IL-6, IL-1β, TNF-α, and oxidative stress indicators MDA, SOD, GSH levels. Western blotting and RT-qPCR were used to detect the expression of proteins and genes related to the PI3K/AKT/NF-κB signaling pathway, and immunohistochemistry was used to detect NF-κB protein expression. A CCl4-induced Hep-G2 cell injury model was established to screen the protective active fractions of Gentiana urnula H.Smith against cell injury. Cells were treated with high, medium, and low doses of ethanol fractions for 24 hours to establish the model, and cell survival rate was detected using the CCK-8 method. Biochemical methods were used to detect ALT, AST, and LDH levels. Western blotting and RT-qPCR were used to detect the expression of proteins and genes related to the PI3K/AKT/NF-κB signaling pathway, Flow cytometry was used to detect the apoptosis rate of cells. RESULTS From Gentiana urnula H.Smith, 29 components and 177 potential liver-protection targets were identified, with core targets including AKT1, ALB, EGFR, etc., involving pathways such as PI3K/AKT/NF-κB. Animal experiments showed that Gentiana urnula H.Smith can improve extensive necrosis and structural damage of liver tissue in mice induced by CCl4, reduce levels of ALT, AST, LDH, TNF-α, IL-1β, IL-6, SOD, and GSH,simultaneously regulating the PI3K/AKT/NF-κB pathway, it inhibits the expression of apoptosis-related genes and proteins. Cell experiments confirmed that the ethanol fraction of Gentiana rigescens can mitigate CCl4-induced Hep-G2 cell damage, reduce ALT, AST, and LDH levels, and modulate the PI3K/AKT/NF-κB signaling pathway, thereby decreasing the apoptosis rate.CONCLUSION Gentiana urnula H.Smith can alleviate oxidative stress and inflammatory responses, improving chemical liver injury in mice. Its mechanism may be related to its hepatoprotective effect through inhibiting apoptosis via the PI3K/AKT/NF-κB pathway, providing a reference for further research.