Bioinformatics-based Study On The Mechanism Of OA Methylation Differential Gene By Chemistry And Dampness Dispersal Formula

Objective: To screen the methylation differentially expressed genes(DEGs)associated with the treatment of osteoarthritis(OA)by Huayu Dispelling Dampness Formula using bioinformatics and data mining techniques,and to explore the potential mechanisms of action of the active ingredients,targets,signaling pathways and methylation differentially expressed genes of Huayu Dispelling Dampness Formula in order to provide a theoretical basis for clinical trials.Methods:(1)Obtaining the active ingredients and target genes of the formula for removing dampness by removing dampness from blood stasis The chemical components of the formula for removing dampness from blood stasis(Dan Shen 15 g,Angelica sinensis 10 g,Chuan Xiong 6g,Chuan Niu Kne 10 g,Atractylodes macrocephala 10 g,Radix Panax notoginseng 10 g,Chen Pi 10 g,Atractylodes macrocephala 10 g,Chen Pi 6g,)were searched through the TCMSP database using the standard Chinese or Latin names of the individual drugs in the formula.All the obtained chemical components were screened with the bioavailability(oral bioavailability,OB)≥ 30% and drug-likeness(DL)≥ 0.18 as the screening conditions to obtain the potentially active chemical components of the formula for removing dampness by resolving blood stasis.Based on the R language correlation package,Uniprot data were used to normalize the annotation of all drug target genes of Hua Yu Exorcising Dampness Formula soup.(2)OA-related gene chips were obtained using the keywords "OA" and "Normal" in the Gene Expression Omnibious(GEO)based search,and the study species was set as Homo sapiens" and "Expression profiling by array" as the study type,and the dataset was obtained by filtering.Similarly,a GEO search was conducted using the keywords "OA" and "Methylation",and the study type was changed to "Expression profiling by array ",and the OA methylation dataset was obtained by filtering.The dataset matrix files and platform annotation files were downloaded from GEO for differential analysis respectively.(3)Screening of OA differentially expressed genes and methylation differential genes In R language,R packages such as limma and impute were used to screen OA differential genes and OA methylation differential genes.Based on the obtained gene chips,OA differentially expressed genes were screened using corrected P< 0.05 and log FC absolute value > 1.5 as the screening conditions,where log FC > 1.5 was up-regulated differential genes(UPReg Genes)and log FC <-1.5 was down-regulated differential genes(Down Reg Genes);using corrected P(adjust P)< 0.05 and absolute value of t>2 were used to screen OA methylation differential genes,where t>2 was for Hyper Meth Genes and t<-2 was for Hypo Meth Genes.Based on the Venny online platform,the UPReg Genes and Down Reg Genes were concatenated,and the target proteins obtained from the chemistry and dampness elimination formula and the OA-related genes obtained from the Gene Cards database were intersected to obtain the target genes of the chemistry and dampness elimination formula for the treatment of OA.The UPReg Genes were intersected with Hypo Meth Genes,Down Reg Genes were intersected with Hyper Meth Genes,and then the two intersected genes were merged to obtain OA-specific methylation difference genes.Finally,the OA-specific differential methylation genes were obtained by intersecting the targets of the active ingredients of the formula for eliminating dampness with the OA-specific differential methylation genes,and the osteoarthritis differential methylation genes targeted by the formula for eliminating dampness.(4)GO and KEGG enrichment analysis of OA differential genes targeted by the formula for eliminating dampness Based on the David 6.8 database(https://david.ncifcrf.gov/)and setting P≤0.05 as the filtering condition,GO and KEGG enrichment analysis of OA differential genes targeted by the formula for eliminating dampness were performed,and the analysis results were imported into R and visualized in the form of bar graphs and visualized in the form of bubble plots.The pathways and biological processes involved in the OA differential genes of the Hua Yu Exorcism Formula and their effects on the targeted OA methylation differential genes were investigated.Results.(1)Eighty-nine active chemical components of the Hua Yu Exorcism Formula and their corresponding 236 target proteins were obtained based on the TCMSP database.(2)The GeneCards database was used to obtain 3262 genes associated with OA.Based on the GSE169077 microarray,the gene expression of synovial cells of 6 OA patients and 5 normal volunteers with bone joints were analysed and 624 differentially expressed genes of OA were obtained.(3)The 236 target proteins corresponding to the effective chemical components of the formula were intersected with the 3262 OA-related genes obtained from the Gene Cards database and the 624 differentially expressed genes obtained from GSE169077 to obtain 13 intersecting targets,i.e.the targets of the formula for targeting OA.In the bone synovial cells of six OA patients in the GSE169077 gene chip,the expression of 460 intersecting targets was up-regulated and 164 genes were down-regulated.(4)Thirteen intersecting genes targeting OA with the chemosynthesis and dispelling dampness formula were imported into String,and a PPI network was constructed with a minimum required interaction score of 0.9 as the screening condition.The analysis results using String database showed that the PPI network contained 13 nodes and 31 edges,and the average node degree value was 4.77.(5)In the R language,KEGG enrichment analysis was performed on 13 genes of OA intersection targets targeted by the Hua Yu Exorcism Formula,which included EGFR tyrosine-kinase inhibitor,Human cytomegalovirus infection The entries include EGFR tyrosine-kinase inhibitor,Human cytomegalovirus infection,Endocrine resistance,Viral protein interaction with cytokine and cytokine recepto,Calcium signaling pathway,interleukin-17(IL-17)and the development of OA are closely related.In the GO enrichment analysis,which contains entries for regulation of fibroblast proliferation,response to lipopolysaccharide,regulation of B cell apoptotic process,response to vitamin A,cytokine activity,prostaglandin receptor activity,Hsp70 protein binding,nuclear receptor activity,ligand-activated transcription factor activity,chemokine activity,and steroid hormone receptor activity.steroid hormone receptor activity,G protein-coupled receptor binding,etc.,in which the response to lipopolysaccharide,chemokine activity,and chemokine activity are listed.(6)Analysis of the Cytoscape,which has been closely linked to OA,includes entries on chemokine activity,fibroblast proliferation and other biological processes.(6)Analysis of the "herbal-component-target-pathway" network constructed by Cytoscape yielded five herbal components of the formula,namely Baicalin,Stigmasterol,Nobiletin,Quercetin,Salvia miltiorrhiza and Quercetin.Quercetin,and Tanshinone IIA(Tanshinone iia).(7)In the GEO database,3203 OA aberrantly methylated genes were obtained,including 1536 highly expressed methylated genes and 1667 hypomethylated genes.(8)The 164 differentially genes down-regulated by OA were intersected with 1536 highly expressed methylated genes to obtain 9 intersecting genes,460 differentially genes up-regulated by OA were intersected with 1667 low expressed methylated genes to obtain 38 intersecting genes,and the two intersecting genes were again taken together to obtain 47 OA differentially methylated genes,and finally,the chemosynthesis and dampness elimination formula was then targeted to 13 OA and 47 OA differentially methylated genes were intersected to obtain ERBB2 and MPO,two OA differentially methylated genes targeted by the formula for eliminating dampness.Conclusion.1.In the formula for removing dampness by removing blood stasis,five important effective herbal chemical components,namely baicalin,dousterol,chenoposide,quercetin and tanshinone IIA,can slow down the process of OA development through anti-inflammatory,anti-apoptotic,cartilage protection and delaying the degradation of cartilage extracellular matrix.2.This formula affects the progression of OA mainly through the calcium signalling pathway,IL-17 and other signalling pathways,as well as biological processes such as response to lipopolysaccharide,chemokine activity and fibroblast proliferation.3.The key methylation target genes of ERBB2 and MPO in the formula for eliminating dampness are involved in the inflammatory response to OA under the modifying effect of methylation.