Effect Of Zhuang Medicine Stereo Comprehensive Therapy On The Expression Of CircRNA、IL-1、IL-6 In Peripheral Blood Of Patients With Rheumatoid Arthritis

Objective:The purpose of this study is explore the effect of Zhuang medicine Stereo Comprehensive therapy on the expression levels of circRNA,IL-1 and IL-6 in peripheral blood of patients with rheumatoid arthritis through high-throughput sequencing technology and bioinformatics methods,screen out differentially expressed circRNA and predict the biological processes and signal pathways involved in the genes derived from differentially expressed circRNA,in-depth understanding of the mechanism of differentially expressed circRNA in the treatment of rheumatoid arthritis in Zhuang medicine Stereo Comprehensive therapy,at the same time,find out the effects of Zhuang medicine Stereo Comprehensive therapy on the inflammatory factors IL-1 and IL-6 expression and regulation levels,further explore the effect of Zhuang medicine Stereo Comprehensive therapy on rheumatoid arthritis inflammation control and provide objective scientific basis for the clinical promotion and application of Zhuang medicine Stereo Comprehensive therapy.Methods:According to the diagnostic criteria of Western medicine for rheumatoid arthritis and the diagnostic criteria of Zhuang medicine,10patients with rheumatoid arthritis who meet the criteria of this study were selected,and SPSS25.0 software was used for complete random grouping,they were divided into control group and observation group with 5 cases each.At the same time,5 healthy normal persons were included as the normal group.Among them,the control group was treated with Western Medicine（（1）methotrexate 10mg/QW;（2）Leflunomide tablets（10mg/QD）plus oral administration of Zhuang medicine longzuantongbi granules（15g/bag,twice a day）were used in the treatment group.The observation group was also treated with western medicine basic treatment（（1）methotrexate 10mg/QW;（2）Leflunomide tablets 10mg/QD）plus Zhuang medicine Stereo comprehensive therapy（oral Zhuang medicine longzuantongbi granules,external Zhuang medicine thermosensitive point probing acupuncture therapy combined with Zhuang medicine Fawang food therapy prescription）was given to the normal group,and no treatment was given to the normal group.The control group and the observation group were treated for 2 courses（8weeks）.During the treatment,the peripheral venous blood of patients with rheumatoid arthritis before and after treatment was collected for testing.In the normal group,blood was drawn together for testing in the control group and observation group after the treatment.Among them,circRNA uses high-throughput sequencing technology for detection.Sequencing data results using python 2.7,R 3.5.0,Fast QC 0.11.7,String Tie 1.3.3,STAR-2.5.2b and other software for data sorting and differential expression circRNA screening,using DAVID database to screen the differentially expressed circRNA the source genes are analyzed for GO enrichment and pathway enrichment analysis.IL-1 and IL-6 were detected by double-antibody sandwich enzyme-linked immunosorbent assay（ELISA）,and the detection was carried out in strict accordance with the kit instructions.Results:1.Circ RNA test results:（1）Comparison between groups:（1）Compared between the control group and the observation group and the normal group,1803 circRNAs were differentially expressed,286 circRNAs were significantly differentially expressed,including 156 circRNAs up-regulated The expression of 130 circRNAs was down-regulated,the most significantlyup-regulatedcircRNAwas chr9:116155726-116155914:-（log₂FC=2.27）,and the most significant down-regulated circRNA was chr2:98165881-98166077:-（log₂FC=-3.09）.The GO enrichment analysis of genes derived from these significantly differentially expressed circRNAs yielded 123 entries,which mainly included the positive regulation of RNA polymerase II promoter transcription,the negative regulation of polymerase II promoter transcription,viral processes,cell proliferation,and DNA as a template for the positive regulation of transcription,etc.;KEGG enrichment analysis yielded 15 entries,mainly including protein processing in the endoplasmic reticulum,endocytosis and protein export signaling pathways.（2）Compared with the control group after treatment,there were 1949 differentially expressed circRNAs and 427significantly differentially expressed circRNAs,including 219 circRNAs were up-regulated and 208 circRNAs were down-regulated.The most significant up-regulated circRNA was chr9:116155726-116155914:-（log₂FC=2.99）,the mostsignificantlydown-regulatedcircRNAis chr2:98165881-98166077:-（log₂FC=-5.27）.The GO enrichment analysis of genes derived from these significantly differentially expressed circRNA yielded 220 entries,mainly including ER-related misfolded protein catabolism,protein autophosphorylation and protein sulfonylation,etc.;KEGG enrichment analysis yielded 14 This item mainly includes TGF-beta signaling pathway,Fox O signaling pathway,MAPK signaling pathway and Wnt signaling pathway.（3）Compared with the normal group after treatment,the observation group had 1915 differentially expressed circRNAs and 186significantly differentially expressed circRNAs,including 94 circRNAs were up-regulated and 92 circRNAs were down-regulated.Among them,the most significantlyup-regulatedcircRNAwaschr8:86253827-86254037:-（log₂FC=2.43）,the most significantly down-regulated circRNA is chr2:98165881-98166077:-（log₂FC=-3.20）.The GO enrichment analysis of these significantly differentially expressed genes derived from circRNA yielded 81 entries,mainly including bone marrow dendritic cell differentiation,intracellular protein transport and cell adhesion,etc;KEGG enrichment analysis yielded 4 entries,mainly Including m RNA monitoring pathways,protein export pathways,and protein processing pathways in the endoplasmic reticulum.（4）Compared between the control group and the observation group after treatment,there were 1930 differentially expressed circRNAs and 140 significantly differentially expressed circRNAs,including74 circRNA expression up-regulation and 66 circRNA expression down-regulation.Among them,the most significantly up-regulated circRNA was hsa＿circ＿0000233（log₂FC=2.38）,the most significantly down-regulated circRNA is hsa＿circ＿0000713（log₂FC=-2.04）.The GO enrichment analysis of these significantly differentially expressed genes derived from circRNA yielded 66 entries,which mainly included cell division,peptidylserine phosphorylation and cell response to nerve growth factor stimulation;KEGG enrichment analysis yielded 9 entries It mainly includes PI3K-Akt signaling pathway,Th17 cell differentiation,insulin resistance and MAPK signaling pathway.（2）Intra-group comparison:（1）Compared before and after treatment in the control group,there are 1907 differentially expressed circRNAs and 45significantly differentially expressed circRNAs,including 23 circRNA expression up-regulation and 22 circRNA expression down-regulation,of which the most significant up-regulation The circRNA is chr9:128103455-128104578:+（log₂FC=1.94）,and the most significantly down-regulated circRNA is chr5:162898190-162898469:+（log₂FC=-2.16）.The GO enrichment analysis of genes derived from these significantly differentially expressed circRNAs yielded 32 entries,mainly including the positive regulation of proteasome ubiquitin-dependent protein catabolism,Ras protein signal transduction,and nucleotide excision repair during DNA cleavage;KEGG enrichment analysis yielded 3 entries,mainly including HIF signaling pathway,TNF signaling pathway and extracellular matrix（ECM）-receptor interaction pathway.（2）Compared before and after treatment in the observation group,there were 1930 differentially expressed circRNA and 114 significantly differentially expressed circRNA,including 69up-regulated circRNA expression and 45 down-regulated circRNA expression.Among them,the most significantly up-regulated circRNA was hsa＿circ＿0017817（log₂FC=2.38）.）,the most significantly down-regulated circRNA is hsa＿circ＿0002279（log₂FC=-2.20）.The GO enrichment analysis of genes derived from these significantly differentially expressed circRNAs yielded 64 entries,including mi RNA gene silencing,mi RNA-mediated translation repression,and chromatin covalent modification.The KEGG enrichment analysis yielded 3 entries.It mainly includes transcription disorders in cancer,protein processing in the plasma reticulum,and glycerophospholipid metabolism pathways.2.IL-1/IL-6 test results:Compared with the normal group,the levels of IL-1 and IL-6 in the serum before treatment in the observation group and the control group are higher than those in the normal group（P<0.05）,the difference is statistical learn meaning.Compared with before treatment,the serum levels of IL-1 and IL-6 in the observation group and the control group decreased significantly after treatment（P<0.05）,and the difference was statistically significant.Comparing the control group with the observation group after treatment,the serum levels of IL-1 and IL-6 in the observation group were significantly lower than those in the control group（P<0.05）,and the difference was statistically significant.Conclusion:1.The Stereo comprehensive therapy of Zhuang medicine can up regulate the expressed level of hsa＿circ＿0037353、hsa＿circ＿0083861、hsa＿circ＿0039161 ect and down regulate the expressed level of hsa＿circ＿0000603、hsa＿circ＿0002601、hsa＿circ＿0000396 ect.What is more,these regulations are maybe related to many biological processes such as DNAtemplated positive transcription regulation of cell proliferation,protein auto-phosphorylation and protein sulfonylation,and many signal pathways such as TNF signaling pathway,extracellular matrix（ECM）-receptor interaction pathway and PI3K-Akt signaling pathway to exert therapeutic effects.2.According to this study,we found that there are a large number of differentially expressed circrna in the peripheral blood of patients with rheumatoid arthritis,and these differentially expressed circrna may have a close relationship with the pathogenesis of rheumatoid arthritis,which is a new idea and direction to further explore the mechanism of three-dimensional comprehensive therapy of Zhuang medicine in the treatment of rheumatoid arthritis in the future.3.The Stereo comprehensive therapy of Zhuang medicine has a positive regulatory effect on inflammatory factors IL-1 and IL-6 in peripheral blood of patients with rheumatoid arthritis,which can effectively reduce the level of inflammatory factors in peripheral blood of patients with rheumatoid arthritis.It shows that the Stereo comprehensive therapy of Zhuang medicine maybe has a good anti-inflammatory effect on rheumatoid arthritis,which is worthy of clinical application.