基于WGCNA和动物实验探究骨坏死康复丸治疗激素性股骨头坏死的作用机制

Exploring the Mechanism of Action of Guhuaisi Kangfu Pill for Treating Steroid-induced Osteonecrosis of the Femoral Head Based on WGCNA and Animal Experiment Validation

目的探讨骨坏死康复丸治疗激素性股骨头坏死(Steroid-induced osteonecrosis of the femoral head,SONFH)的主要药理学基础和作用机制。方法通过中药系统药理学数据库与分析平台(Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform,TCMSP)等数据库对骨坏死康复丸的有效成分和靶点进行筛选。基于人类在线孟德尔遗传数据库(Online Mendelian Inheritance in Man,OMIM)和GeneCards数据库筛选SONFH的靶点。采用加权基因共表达网络分析(Weighted gene co-expression network analysis,WGCNA)对SONFH基因模块和枢纽基因进行鉴定。取三者的交集,获得骨坏死康复丸治疗SONFH的潜在靶点,并利用Cytoscape软件构建的“活性成分-靶点”网络筛选关键活性成分;然后,利用STRING数据库构建蛋白相互作用网络,筛选关键靶点。进行基因组百科全书(Kyoto Encyclopedia of Genes and Genomes,KEGG)信号通路富集分析,探索“关键活性成分-关键靶点-关键信号通路”之间的关系。最后,进行了关键活性成分与关键靶点的分子对接,并使用SONFH大鼠模型进行实验验证。结果得到骨坏死康复丸的146个化合物和相应的346个靶点,获得了4187个SONFH靶点。此外,基于WGCNA筛选出SONFH的12个基因模块和2556个枢纽基因,得到槲皮素、木犀草素和山柰酚是骨坏死康复丸治疗SONFH的关键活性成分,涉及PI3K/AKT等多条信号通路。分子对接显示关键活性成分与关键靶点之间存在较好的结合活性。动物实验结果证明骨坏死康复丸可通过上调AKT1、PI3K、RUNX2,下调Caspase-3和IL-6的表达改善骨生物学改变(P<0.01),验证了网络药理学部分预测结果。结论通过网络药理学和动物实验分析了骨坏死康复丸治疗SONFH的潜在作用机制,可为进一步研究其药理基础和靶点提供参考。

Objective To investigate the main pharmacological basis and mechanism of action of Guhuaisi Kangfu Pill(GHSKF)in the treatment of steroid-induced osteonecrosis of the femoral head(SONFH).Methods The active constituents and targets of GHSKF were screened by using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP)and other databases.The speculative targets of SONFH were screened out based on GeneCards and Online Mendelian Inheritance in Man(OMIM)databases.The gene modules and hub genes of SONFH were identified using a weighted gene co-expression network analysis(WGCNA).The intersection of the two targets and the result of WGCNA was taken to obtain the potential targets of GHSKF for the treatment of SONFH.The key active constituents were screened with the“active constituent-target”network,which was constructed by the Cytoscape software.Then,the STRING database was used to construct the protein interaction network to screen the key targets.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of key targets was performed,and the relationship between key active constituent,key targets and key signaling pathways was explored.Finally,the molecular docking between key active constituents and key targets was verified.In addition,the SONFH rat model was used for experimental verification.Results A total of 146 compounds and the corresponding 346 targets were identified based on the TCMSP database.A total of 4187 targets of SONFH were obtained based on GeneCards and OMIM databases.In addition,twelve gene modules and 2556 hub genes of SONFH were screened out based on WGCNA.Quercetin,luteolin and kaempferol were key active ingredients for the treatment of SONFH.Various signaling pathways such as PI3K/AKT were involved.Molecular docking showed the key active ingredients had good binding activity with the key targets.The results of animal experiments demonstrated that GHSKF could improve bone biological alterations by up-regulating AKT1,PI3K,RUNX2,and downregulating the expression of Caspase-3 and IL-6(P<0.01),which verified some results of the network pharmacology prediction.Conclusion We analyzed the potential mechanism of action of GHSKF for the treatment of SONFH using network pharmacology and animal experiments,which may provide a reference for further research on its pharmacological basis and targets.