基于网络毒理学的鬼画符致大鼠肝脏损伤作用机制研究

Mechanism of Liver Injury in Rats Induced by Breynia fruticosa Based on Network Toxicology

本研究拟探讨鬼画符(Breynia fruticosa)致大鼠肝毒性损伤的潜在作用机制,以高、低剂量鬼画符提取物(5 g/kg、2.5 g/kg)连续3个月和6个月对大鼠灌胃给药,检测血清谷草转氨酶(AST)和谷丙转氨酶(ALT)水平,以明确鬼画符的毒性靶器官。通过网络毒理学方法,构建鬼画符化学成分库和肝毒性靶标库,利用蛋白互作和网络分析手段筛选鬼画符化学成分致肝毒性的关键靶标,并对关键靶标进行生物功能富集及通路分析,采用免疫组化技术验证鬼画符对大鼠肝脏诱导型一氧化氮合酶(iNOS)、缺氧诱导因子-1α(HIF-1α)、白介素-1β(IL-1β)和白介素-6(IL-6)蛋白表达的影响。结果显示,与对照组相比,鬼画符给药6个月可诱导大鼠肝细胞变性和纤维组织增生的病理变化,ALT、AST水平出现一定程度偏离,但不具有统计学意义。网络毒理学研究初步揭示鬼画符中有17种化合物可能是诱导肝毒性发生的潜在成分,可能通过IL-6、肿瘤蛋白p53(TP53)、丝裂原活化蛋白激酶1(MAPK1)、血管内皮生长因子A(VEGFA)、肿瘤坏死因子(TNF)等17个关键靶点诱导肝损伤。富集结果显示,鬼画符致肝毒性的关键靶标主要富集于糖尿病并发症相关的AGE-RAGE信号通路、IL-17信号通路、TNF信号通路、Toll样受体信号通路、HIF-1α信号通路等,这些关键蛋白以及通路与细胞内氧化应激、炎症反应密切相关。免疫组化分析结果也验证了鬼画符对大鼠给药6个月可明显升高肝脏中IL-1β、iNOS、IL-6、HIF-1α表达水平,表明鬼画符在一定剂量范围内具有肝毒性,可诱导肝细胞变性,其机制可能与促进肝脏氧化应激损伤和炎症反应,升高氧化应激和炎性相关蛋白的表达有关。

This study intends to explore the potential mechanism of hepatotoxic damage induced by Breynia fruticosa in rats.Rats were intragastrically administered with high and low doses of Breynia fruticosa extract(5 g/kg,2.5 g/kg)for 3 and 6 months,and the serum levels of aspartate aminotransferase(AST)and alanine aminotransferase(ALT)were detected to identify the toxic target organs of B.fruticosa.The chemical composition library and hepatotoxicity target library of B.fruticosa were constructed by network toxicology method.The key targets of hepatotoxicity induced by chemical composition of B.fruticosa were screened by protein interaction and network analysis.The biological function enrichment and KEGG pathway analysis of the key targets were carried out.The effects of B.fruticosa on the protein expression of iNOS,HIF-1α,IL-1βand IL-6 in rat liver were verified by immunohistochemistry.The results showed that compared with the control group,the pathological changes of liver cell degeneration and fibrous tissue hyperplasia in rats were induced after 6 months of administration,and the levels of ALT and AST were deviated to some extent,but not statistically significant.The network toxicological study preliminarily revealed that 17 compounds in B.fruticosa might be potential components in inducing hepatotoxicity,which may induce liver injury through 17 key targets such as IL-6,tumor protein p53(TP53),mitogen-activated protein kinase 1(MAPK1),vascular endothelial growth factor A(VEGFA)and tumor necrosis factor(TNF).These key targets might have potential enrichment in AGE-RAGE signaling,HIF-1αsignaling,TNF signaling and Toll-like receptor signaling,etc.The enrichment results showed that the key targets of liver toxicity induced by B.fruticosa were mainly enriched in AGE-RAGE signaling pathway,IL-17 signaling pathway,TNF signaling pathway,Toll-like receptor signaling pathway and HIF-1αsignaling pathway related to diabetic complications.These key proteins and pathways were closely related to intracellular oxidative stress and inflammatory response.The results of immunohistochemical analysis also verified that the expression levels of IL-1β,iNOS,IL-6 and HIF-1αin the liver of rats were significantly increased after 6 months of administration of B.fruticosa,indicating that B.fruticosa had hepatotoxicity in a certain dose range and could induce hepatocyte degeneration.The mechanism might be related to promoting oxidative stress injury and inflammatory response in the liver,and increasing the expression of oxidative stress and inflammatory related proteins.