摘要
目的:基于网络药理学初步预测加味升降散治疗免疫球蛋白(Ig)A肾病(IgAN)的活性成分、作用靶点及信号通路,通过分子对接及动物实验验证探讨其可能作用机制。方法:通过中药系统药理学数据库与分析平台(TCMSP),蛋白质数据库(UniProt Knowledge base)数据库,查阅文献及成分靶点预测数据库(SwissTargetPrediction)获取加味升降散药理作用的活性成分及相关靶点;通过基因数据库(GeneCards),人类孟德尔遗传病数据库(OMIM)获取IgAN相关靶点;使用Cytoscape 3.9.0软件数据库构建药物活性成分的相关靶点与疾病相关靶点的加味升降散-IgAN靶点调控网络,结合STRING数据库绘制蛋白相互作用网络;使用Metascape数据库对交集基因进行基因本体(GO)功能注释和京都基因与基因组百科全书(KEGG)通路富集分析。选取关键作用靶点与主要活性成分通过AutoDockTools 1.5.6软件进行分子对接验证;采用联合牛血清白蛋白(BSA)灌胃+尾静脉注射脂多糖(LPS)+皮下注射四氯化碳(CCl4)、蓖麻油的免疫复合的方法建立实验性IgA肾病大鼠模型,给予加味升降散和盐酸贝那普利干预4周。给药结束后处死大鼠,通过酶联免疫吸附测定法(ELISA)、免疫组织化学法、实时荧光定量聚合酶链式反应(Real-time PCR)、蛋白免疫印迹法(Western blot)检测大鼠血清及肾组织中转化生长因子-β(1 TGF-β1)、白细胞介素-6(IL-6)表达水平来进行动物实验验证。结果:经过口服生物利用度(OB)、类药性(DL)及查阅文献初步筛选获得加味升降散中活性成分105个;药物疾病交集基因124个,核心靶点59个,其中神经营养性酪氨酸激酶受体1型(NTRK1)、泛素化连接酶E3蛋白(CUL3)、肿瘤蛋白p53(TP53)、表皮生长因子受体(EGFR)、核输出蛋白1(XPO1)等靶点可能与IgAN密切相关,KEGG富集分析预测加味升降散治疗IgAN主要涉及磷脂酰肌醇3-激酶/蛋白激酶B(PI3K/Akt)信号通路、核转录因子-κB(NF-κB)信号通路、细胞因子-细胞因子受体相互作用(Cytokine-cytokine receptor interaction)等通路。分子对接分析,加味升降散中主要活性成分与核心作用靶点中的NTRK1、CUL3、TP53、EGFR、XPO1具有较为稳定的结合活性,其可能通过影响NTRK1、CUL3、TP53、EGFR、XPO1靶点蛋白进而调控炎症反应。动物模型验证结果:加味升降散能够显著降低IgAN大鼠血清及肾组织中细胞因子TGF-β1、IL-6表达水平,可能通过调控Cytokine-cytokine receptor interaction、PI3K/Akt、NF-κB等信号通路抑制过度纤维化、炎性反应和免疫应答。结论:加味升降散可能通过多靶点及多通路治疗IgAN,其发挥治疗作用的机制可能与影响NTRK1、CUL3、TP53、EGFR、XPO1等靶点蛋白的表达及调控Cytokine-cytokine receptor interaction、PI3K/Akt、NF-κB等信号通路进而抑制过度纤维化、炎症反应和免疫应答有关。
Objective:To preliminarily predict the active components,targets,and signaling pathways of modified Shengjiangsan in the treatment of immunoglobulin A nephropathy(IgAN)based on network pharmacology,and to explore its underlying mechanism through molecular docking and experimental verification on animals.Method:The active ingredients and related targets of modified Shengjiangsan were obtained from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),UniProt,SwissTargetPrediction,and literature review.IgAN-related targets were obtained from GeneCards and Online Mendelian Inheritance in Man(OMIM).Cytoscape 3.9.0 was used to construct the regulation network of the related targets of Shengjiangsan and IgAN,and the protein-protein interaction(PPI)network was plotted by STRING.The common genes were analyzed for gene ontology(GO)functional annotation and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment by Metascape.Key targets and main active ingredients were selected for molecular docking by AutoDockTools 1.5.6.The experimental model of IgAN was induced by bovine serum albumin(BSA,ig)combined with lipopolysaccharide(LPS,iv)and the complex of CCl4 and castor oil(sc)in rats.The model rats were treated with modified Shengjiangsan and benazepril hydrochloride for four weeks.The rats were sacrificed after drug administration.The levels of transforming growth factor-β1(TGF-β1)and interleukin-6(IL-6)in the serum and kidney tissues were detected by enzymelinked immunosorbent assay(ELISA),immunohistochemistry,Real-time quantitative polymerase chain reaction(Real-time PCR),and Western blot.Result:A total of 105 active ingredients were obtained according to oral bioavailability(OB),drug-likeness(DL),and literature screening.There were 124 common genes and 59 core targets.Neurotrophic tyrosine receptor kinase 1(NTRK1),cullin-3(CUL3),tumor protein 53(TP53),epidermal growth factor receptor(EGFR),exportin 1(XPO1),and other targets might be closely related to IgAN.As predicted by KEGG enrichment analysis,the treatment of IgAN with modified Shengjiangsan mainly involved the phosphoinositide 3-kinase(PI3K)/protein kinase B(Akt)signaling pathway,nuclear transcription factor-kappa B(NF-κB)signaling pathway,and cytokine-cytokine receptor interaction signaling pathway.As revealed by molecular docking,the main active ingredients in modified Shengjiangsan showed stable binding activities with NTRK1,CUL3,TP53,EGFR,and XPO1 in the core targets,indicating that it presumedly regulated inflammatory responses by affecting NTRK1,CUL3,TP53,EGFR,and XPO1 target proteins.The results of experimental verification on animals showed that the expression levels of cytokines TGF-β1 and IL-6 in the serum and kidney tissues of IgAN rats were significantly decreased by modified Shengjiangsan,suggesting that Shengjiangsan might inhibit excessive fibrosis,and inflammatory and immune responses by regulating signaling pathways such as cytokine-cytokine receptor interaction,PI3K/Akt,and NF-κB.Conclusion:Modified Shengjiangsan may treat IgAN through multiple targets and pathways.Its mechanism may be related to the inhibition of excessive fibrosis,and inflammatory and immune responses by affecting the expression of NTRK1,CUL3,TP53,EGFR,and XPO1 and the regulation of the cytokine-cytokine receptor interaction,PI3K/Akt,NF-κB,and other signaling pathways.
作者
张圆圆
靳培培
顾悦
郭登洲
ZHANG Yuan-yuan;JIN Pei-pei;GU Yue;GUO Deng-zhou(Graduate School,Hebei University of Chinese Medicine,Shijiazhuang 050091,China;The First Affiliated Hospital of Hebei University of Chinese Medicine,Hebei Provincial Hospital of Traditional Chinese Medicine,Shijiazhuang 050011,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第7期162-171,共10页
Chinese Journal of Experimental Traditional Medical Formulae
基金
河北省科技计划项目(16277765D)
河北中医学院科技能力提升项目(KTZ2019025)。
