摘要
目的基于网络药理学和分子对接技术预测川楝子肝毒性的物质基础及毒性机制,并结合体内、外实验验证炮制减毒作用。方法利用中药系统药理学数据库与分析平台(TCMSP)与小分子药物预测作用靶点的在线平台(Swiss Target Prediction)分别收集川楝子的毒性成分和作用靶点。检索GeneCards和OMIM数据库获取肝损伤疾病靶点,绘制Venny图获取川楝子毒性成分和肝相关疾病的共同靶标后,利用STRING数据库进行蛋白-蛋白相互作用(PPI)网络分析,并对GO功能和KEGG通路进行富集分析,预测其可能的毒性机制。使用AutoDockTools和PyMOL软件对川楝子肝毒性的关键成分与关键蛋白结合位点及结合作用进行分子对接验证及可视化。体内实验通过检测小鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)的活力,苏木精-伊红染色法(HE)观察肝组织病理形态,比较川楝子不同炮制品致小鼠急性肝毒性的大小;测定小鼠肝脏中超氧化物歧物酶(SOD)、丙二醛(MDA)、还原型谷胱甘肽(GSH)、谷胱甘肽过氧化物酶(GSH-P_X)的水平,从氧化损伤的角度分析川楝子不同炮制品致小鼠肝毒性大小差异的原因。结果网络药理学表明川楝子肝毒性成分为苦楝子酮、苦楝素、印楝波灵A等,其肝损伤核心靶点为肿瘤蛋白p53(TP53)、白细胞介素-6(IL-6)、半胱天冬蛋白酶-3(CASP3)等;GO富集分析到转录和基因表达的正调控、细胞凋亡过程的负调控、炎症反应等生物过程,胞外区、线粒体外膜、线粒体等细胞组分,蛋白质结合、蛋白质同源二聚活性、酶结合等分子功能;KEGG通路分析筛选到的肿瘤坏死因子(TNF)、磷脂酰肌醇3激酶(PI3K-Akt)、白细胞介素-17(IL-17)、缺氧诱导因子-1(HIF-1)、细胞凋亡等是川楝子引起肝毒性的关键信号通路。分子对接结果表明,川楝子主要毒性成分与核心靶点能较好结合。体内实验结果显示,与川楝子生品相比,醋炙品、炒焦品、盐炙品均能显著降低小鼠血清中ALT和AST的活性(P<0.05),酒炙品的ALT、AST均显著升高;HE染色结果显示,醋炙品、炒焦品、盐炙品可减少小鼠肝组织炎症细胞浸润及肝细胞的凋亡,提示川楝子炮制后具有减轻肝损伤的作用,但酒炙品的肝毒性要大于生品。此外,醋炙品、炒焦品、盐炙品可以降低生品造成的脂质过氧化,从而达到炮制减毒的效果,酒炙品则相反。结论醋炙、炒焦、盐炙炮制方法可能通过改变川楝子对TP53、IL-6和CASP3等肝毒性靶点的影响,减轻炎症反应,抑制细胞膜脂质过氧化,从而降低肝毒性,但酒炙方法毒性增强,在临床上应该避免。
Objective Based on network pharmacology and molecular docking techniques to predict the material basis and toxicity mechanism of Chuanlianzi(Toosendan Fructus)hepatotoxicity and to validate the reducing effects after processing in combination with in vivo and in vitro experiments.Methods The toxic components and targets of Chuanlianzi(Toosendan Fructus)were collected using the systematic pharmacology analysis platform of traditional Chinese medicine(TCMSP)and the Swiss Target Prediction database,respectively.Liver injury related targets were obtained through GeneCards and OMIM databases.After the common targets were screened by Venny diagram,PPI analysis was carried out by STRING database,and enrichment analysis on GO function and KEGG pathway were conducted to predict their possible toxicity mechanisms.Molecular docking validation and visualization of key components of Chuanlianzi(Toosendan Fructus)hepatotoxicity with key protein binding sites and binding effects were performed using AutoDockTools and PyMOL software.In vivo experiments were conducted to compare the magnitude of acute hepatotoxicity of different processed products of Chuanlianzi(Toosendan Fructus)in mice by detecting the viability of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum and observing the pathologic morphology of liver tissues with HE staining method and to determine the levels of superoxide dismutase(SOD),malondialdehyde(MDA),glutathione(GSH),and glutathione peroxidase(GSH-P_X)and analyzed the reasons for the differences in the hepatotoxicity of different processed products of Chuanlianzi(Toosendan Fructus)in mice from the perspective of oxidative damage.Results Network pharmacology showed that Chuanlianzi(Toosendan Fructus)hepatotoxicity components were melianone,Toosendanin and Nimbolin A,and its core targets for liver injury were TP53,IL-6 and CASP3.GO enrichment was analyzed to the biological processes,such as positive regulation of transcription and gene expression,negative regulation of the apoptotic process,and inflammatory response,cellular components,such as extracellular region,outer mitochondrial membrane,mitochondria,protein binding,protein homodimerization activity,enzyme binding and other molecular functions.KEGG pathway analysis screened TNF,PI3K-Akt,IL-17,HIF-1,apoptosis and other key signaling pathways of Chuanlianzi(Toosendan Fructus)-induced hepatotoxicity.The molecular docking results showed that the main toxic components of Chuanlianzi(Toosendan Fructus)could bind well to the core targets.In vivo experimental results showed that compared with the raw Chuanlianzi(Toosendan Fructus),the vinegar-processing,stir-frying to brown and salt-processing products all significantly reduced the activities of ALT and AST in the serum of mice(P<0.05),and the wine-processing product showed a significant increase in both ALT and AST.HE staining showed that the vinegar-processing product,stir-frying to brown and salt-processing products reduced the infiltration of inflammatory cells in the liver tissues and apoptosis of hepatocytes of mice,which suggested that Chuanlianzi(Toosendan Fructus)had the effect of alleviating liver injury after processing.This suggested that Chuanlianzi(Toosendan Fructus)had the effect of reducing liver injury,but the hepatotoxicity of the wine-processing product was greater than that of the raw product.This result suggested that the mechanism of liver toxicity induced by raw Chuanlianzi(Toosendan Fructus)in mice was related to the disruption of their antioxidant defense system,reduced ability to scavenge free radicals and subsequent excessive accumulation of free radicals in the body,which induced cell membrane lipid peroxidation.Conclusion The vinegar-processing,stir-frying to brown and salt-processing products may reduce hepatotoxicity by altering the effects of Chuanlianzi(Toosendan Fructus)on hepatotoxic targets such as TP53,IL-6 and CASP3,reducing inflammatory responses and inhibition of cell membrane lipid peroxidation,but the wine-processing product is more toxic and should be avoided in clinical practice.
作者
曾春晖
邢丽蓉
陈海鹏
林群峰
章昱
陈泓
杨柯
ZENG Chunhui;XING Lirong;CHEN Haipeng;LIN Qunfeng;ZHANG Yu;CHEN Hong;YANG Ke(College of Pharmacy,Guangxi University of Chinese Medicine,Nanning 530200,Guangxi,China)
出处
《中华中医药学刊》
北大核心
2025年第4期1-6,I0001-I0003,共9页
Chinese Archives of Traditional Chinese Medicine
基金
国家中医药管理局“中药炮制传承技术传承基地”项目(桂中医药发[2022]9号)
广西中医药大学桂派杏林拔尖人才资助项目(桂中医大学[2022]23号)
广西中医药大学桂派中医药传承创新团队项目(2022B005)
广西中医药大学硕士研究生科研创新重点项目(YCSZ2018015)。
关键词
川楝子
生物信息学
肝毒性
炮制减毒
实验验证
Chuanlianzi(Toosendan Fructus)
bioinformatics
hepatotoxicity
reducing toxin after processing
experiment validation
