基于网络药理学的益气解毒复方对实验性自身免疫性重症肌无力大鼠的免疫治疗效果及机制

Effect and mechanism of Yiqi Jiedu decoction based on network pharmacology on the immunotherapy of experimental autoimmune rats with myasthenia gravis

目的基于网络药理学探讨益气解毒复方对实验性自身免疫性重症肌无力(MG)大鼠免疫治疗的效果及机制。方法运用中医药分子机理生物信息学分析软件(BATMAN-TCM)筛选益气解毒复方相关靶点信息,利用在线人类孟德尔遗传(OMIM)数据库以及Genecards数据库查询MG疾病有关靶点的信息;通过注释、可视化和综合发现数据库(DAVID)、Metascape数据库对益气解毒复方治疗MG的潜在靶点进行蛋白-蛋白互作网络(PPI)分析、基因本体(GO)功能富集及京都基因组百科全书(KEGG)相关信号通路分析;制备鼠源性乙酰胆碱受体(AChR)Rα97-116肽段抗原乳剂并皮下注射至30只无特定病原体(SPF)级雌性Lewis大鼠的尾基部、后肢垫部及背部(第1次免疫注射),分别于第30天和第45天时进行加强免疫(第2、3次免疫注射),第3次免疫注射结束后1周判断实验性自身免疫性MG(EAMG)大鼠模型是否成功,造模成功后EAMG大鼠随机均分为模型组、0.945 g/(kg·d)益气解毒复方(低剂量)组、1.890 g/(kg·d)益气解毒复方(中剂量)组、3.780 g/(kg·d)益气解毒复方(高剂量)组及5.400 mg/(kg·d)醋酸泼尼松片剂组,另取6只SPF级雌性Lewis大鼠作为正常组,各药物组大鼠分别予对应药物、正常组和模型组大鼠分别予等体积纯净水,连续灌胃干预30 d,采用电子天平和Lennon评分检测各组大鼠灌胃干预前和干预第30天时的体质量和肌力,同时采用酶联免疫吸附试剂盒检测各组大鼠血清AChR抗体(AChR-Ab)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)及肿瘤坏死因子-α(TNF-α)的水平,采用流式细胞术方法检测各组大鼠灌胃干预第30天时外周血中白细胞分化抗原4(CD4^(+))T细胞、白细胞分化抗原8(CD8^(+))T细胞的百分比及T淋巴细胞亚群CD4^(+)/CD8^(+)比率;灌胃干预30 d后处死各组大鼠,取骨骼肌组织,采用苏木精-伊红染色(HE)评估骨骼肌组织的形态学特征。结果筛选到益气解毒复方治疗疾病相关基因168个,PPI分析预测IL-6、TNF、丝氨酸/苏氨酸蛋白激酶1(AKT1)、肿瘤蛋白(TP53)等可能是其治疗MG的重要靶点;GO功能富集结果表明,细胞组成(CC)、生物过程(BP)及分子功能(MF)涉及354个、4365个及61个条目,KEGG富集分析搜集到有关作用通路257条;灌胃干预第30天,与模型组相比,低、中、高益气解毒复方剂量组及醋酸泼尼松组大鼠体质量增加(P<0.01),Lennon评分降低(P<0.01),血清AChR-Ab、IL-1β、IL-6及TNF-α水平降低(P<0.05);EAMG大鼠血清中CD4^(+)T细胞分布及CD4^(+)/CD8^(+)比值减少(P<0.01),CD8^(+)T细胞分布增高(P<0.01),骨骼肌组织改善,肌纤维恢复,细胞核排列更规则,炎性细胞减少,其中益气解毒复方高剂量组效果较为明显。结论益气解毒复方可减轻EAMG大鼠炎症反应、减轻骨骼肌组织的损伤及促进免疫系统恢复,其机制与血清AChR-Ab水平降低及平衡CD4^(+)、CD8^(+)T细胞两者的比例有关。

Objective To explore the effect and mechanism of Yiqi Jiedu decoction on the immunotherapy of experimental autoimmune rats with myasthenia gravis(MG)based on network pharmacology.Methods Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM)was used to screen the target information related to Yiqi Jiedu decoction,and the Online Mendelian Inheritance in Man(OMIM)database and Genecards database was utilized to search for target information related to MG.The protein-protein interaction(PPI)network analysis,gene ontology(GO)functional enrichment,and Kyoto encyclopedia of genes and genomes(KEGG)pathway analysis were performed through annotation,visualization,and comprehensive discovery in the Database for Annotation,Visualization and Integrated Discovery(DAVID)and Metascape database for potential targets of Yiqi Jiedu decoction in the treatment of MG.The rat acetylcholine receptor(AChR)Rα97-116 peptide antigen emulsion was prepared and subcutaneously injected into the tail base,hind paw,and back of 30 specific pathogen-free(SPF)female Lewis rats(as the first immunization);and booster immunity was carried out on the 30 th day and 45 th day respectively(as the second and third immunizations).Whether the rat model of experimental autoimmune MG(EAMG)was successfully established was assessed one week after the third immunization.After successful modelling,EAMG rats were divided into the model group,Yiqi Jiedu decoction groups with doses of 0.945 g/(kg·d)as the low dose group,1.890 g/(kg·d)as the medium dose group and 3.780 g/(kg·d)as the high dose group,and the prednisone acetate tablet group with a dose of 5.400 mg/(kg·d),while other 6 SPF female Lewis rats were collected as the normal group.The drug groups were treated with corresponding drugs,and the normal and model groups were just given pure water,all intervened by gavage for 30 days.The body weight and muscle strength of each group were measured by the electronic balance and Lennon score,and tail vein blood was extracted from each group before intervention and on Day 30 of intervention,serum levels of acetylcholine receptor antibody(AChR-Ab),interleukin-1β(IL-1β),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay.The flow cytometry was used to detect the percentage of cluster of differentiation 4(CD4^(+))T cells,cluster of differentiation 8(CD8^(+))T cells,and the CD4^(+)/CD8^(+)ratio of T lymphocyte subgroups in peripheral blood of all the rats on Day 30 of gastric perfusion intervention.Thirty days of gavage intervention later all the rats were euthanized,and their skeletal muscle tissues were taken to evaluate the morphological characteristics by Hematoxylin-Eosin(HE)staining.Results Identified 168 disease-related genes for the Yiqi Jiedu decoction were screened out with PPI network analysis predicting IL-6,TNF,AKT1,TP53,and other potential key targets for the treatment of MG.GO functional enrichment results indicated involvement of 354 cellular component(CC),4,365 biological process(BP),and 61 molecular function(MF)entries.KEGG enrichment analysis collected 257 relevant pathways.Compared with the model group,rats in the low,medium,and high dose groups as well as the prednisone acetate group showed that 30 days after oral intervention,their body weight increased(P<0.01),Lennon scores decreased(P<0.01),serum levels of AChR-Ab,IL-1β,IL-6,and TNF-αwere reduced(P<0.05),distribution of CD4^(+)T cells and CD4^(+)/CD8^(+)ratio in EAMG rat serum decreased(P<0.01),distribution of CD8^(+)T cells increased(P<0.01),skeletal muscle tissues were improved,muscular fiber was recovered,nuclear arrangement became more regular,and inflammatory cells increased.During the treatment Yiqi Jiedu decoction with the high dose showed more significant effects.Conclusion Yiqi Jiedu decoction can reduce the inflammatory response of rats with EAMG,promote the recovery of the immune system,increase the weight of rats,effectively improve clinical symptoms,lower serum AChR-Ab levels,balance the ratio of CD4^(+)and CD8^(+)T cells,and reduce damage to skeletal muscle tissues,thereby playing a certain therapeutic role in EAMG.