| BackgroundRheumatoid arthritis(RA)is a chronic,systemic autoimmune disease characterized by synovitis and destruction of articular bone and cartilage which bone destruction usually begins in the early stage.50%of patients with rheumatoid arthritis develop bone destruction within 2 years,and once bone destruction occurs,it progresses irreversibly.In traditional Chinese medicine RA belongs to "Wangbi".Dampness-heat and blood stasis is not only the core pathogenesis of active RA,but also the pathological key of bone destruction of RA,so its treatment should be based on clearing heat and promoting dampness,promoting blood circulation and dredging collaterals.Qingre Huoxue decotion is a commonly used prescription as an intervention for RA in our department.Previous research results show that Qingre Huoxue decotion affects the formation and differentiation of osteoclasts and osteoblasts via regulating Th/RANKL pathway and DKK/Wnt pathway,which reduce the activity of RA disease,delay bone destruction of RA,and play the role of bone protection.The balance of the osteoblasts and osteoclasts is the key to maintain normal bone mass.For the two opposite processes of bone formation and bone resorption,Qingre Huoxue decotion regulates the balance of bone formation and bone resorption by regulating which factors of the body.In recent years,the in-depth study of exosomes has made it possible to deal with the problem.Exosomes are small vesicles which are fused by intracellular bodies and cell membranes and released into extracellular vesicles,which contain biological information such as miRNA,which can transport them to target cells and regulate signal transduction pathways and gene expression MiRNA is a class of highly conserved non-coding RNA molecules that regulate multiple biological pathways by inhibiting the expression of genes encoded by post-transcriptional proteins.It has been found that exosome miRNA plays an important role in the occurrence and development of RA.It not only participates in the inflammation-related process of RA,but also affects bone metabolism by affecting osteoblasts,osteoclasts and chondrocytes.Therefore,the scientific hypothesis of this study is put forward:Qingre Huoxue decotion may delay bone destruction and play a role in bone protection by regulating exosome miRNA pathway in the treatment of RA,.What is the specific way of regulation and control?Qingre Huoxue decotion,as a compound prescription of traditional Chinese medicine,has multi-channel and multi-target function.In order to further study the targeting effect of the drug,the compound medicine was optimized according to the principle of "assistant and guide ".We hypothesized that simplified compound prescription which is extracted from the core drug composition of the compound have the same curative effect as the Qingre Huoxue decotion.This study explored through the following three studies:through high-throughput sequencing analysis of serum exosome in patients with RA,the differential expression of miRNA involved in bone destruction of RA was screened,and adopted Qingre Huoxue decotion to interfere with CIA rat model.Combined with the results of study 1,to explore the intervention effect of Qingre Huoxue decotion on exosome miRNA,and investigated its possible regulatory network;Compare the curative effect difference and related mechanism between Qingre Huoxue decotion and simplified compound prescription,explore the further optimization of prescription and medicine,and lay the foundation for follow-up research.Part 1:Study on miRNA expression profile of peripheral serum exosome bodies in patients with rheumatoid arthritisObjective:To compare the differential expression of miRNA in peripheral serum exosome bodies between RA patients with dampness-heat stasis syndrome and healthy volunteers to explore the possible pathway and mechanism of miRNA involved bone destruction of RAMethods:8 RA patients with dampness-heat stasis syndrome and 8 healthy volunteers were selected.8 ml peripheral blood was extracted from the subjects on an empty stomach.The exosome was extracted by kit method after the serum was separated.The exosome was identified using particle size determination,transmission electron microscope photography,Western Blot and other methods.We used high-throughput sequencing technique to screen the miRNA which differentially expressed exosome.The target genes of differential miRNA were predicted by RNAhybrid,miRanda,TargetScan database,and the intersection predicted by three databases was selected as candidate target genes.Through GO enrichment and KEGG Pathway enrichment analysis,to explore its participation in biological processes and signal pathways.Results:The general characteristics of the two groups of subjects were comparable.The particle size,morphological determination and surface specific protein of the exosome were consistent with those reported in the literature.The number of known miRNA in the normal group was 474 and the number of miRNA in the RA group was 370.A total of 150 differentially expressed miRNA,in the two groups were up-regulated and down-regulated compared with the normal group.Compared with the normal group,33 of the differentially expressed miRNA,were up-regulated and 117 were down-regulated in the two groups,of which 5 were significantly differentially expressed miRNA,hsa-miR-214-3p up-regulated,hsa-miR-193b-3p,hsa-miR-376a-3p,hsa-miR-30a-5p and hsa-miR-140-3p down-regulated.In general,there were 4 differentially expressed miRNA,while hsa-miR-3074-5p,hsa-miR-27a-5p,hsa-miR-496 and hsa-miR-429 were down-regulated.A total of 2338 target genes were screened by target gene prediction analysis of 9 miRNA with significant differential expression.There are 425 GO functions,including 116cellular components and 102molecular functions,including protein binding and transferase activity,and 207 biological processes,including protein phosphorylation,autophagy,intracellular signal transduction and so on.There were three pathways with significant difference,that is,the target genes of differentially expressed miRNA were most significantly enriched in Autophagy,Mitophagy and MAPK signal pathwayConclusion:We validated hsa-miR-214-3p,hsa-miR-376a-3p,hsa-miR-30a-5p,hsa-miR-140-3p involved in the pathogenesis of RA bone destruction of damp-heat stasis syndrome,and have become biomarkers of potential application value of RA bone destruction.MAPK signal pathway plays a pivotal role in the process of RA bone destruction.Abnormal functions such as protein phosphorylation,autophagy,intracellular signal transduction and mitochondria may be the mechanism of RA bone destruction.Part 2:Study on the intervention effect of Qingre Huoxue recipe on exosome miRNA of Rheumatoid ArthritisObjective:To explore the intervention effect of Qingre Huoxue decotion on rat serum exosome miRNA and to find out its possible exosome miRNA regulatory network.Methods:12 female SD rats were randomly divided into normal group,model group and Qingre Huoxue decotion group with 4 rats in each group.Drug intervention began 10 days after the first immunization,and the treatment period was 4 weeks.The rat serum exosome was extracted and the differential miRNA expression was calculated by DEGseq software based on MA-plot.The differential expression was considered to be differential expression when the absolute value of Fold Change>1 and Q value<0.01.The exosome was extracted from rat serum.The RNAhybrid,miRanda,TargetScan database was used to predict the target gene,and the intersection predicted by the three databases was selected as the candidate target gene.The main biological functions of the target genes corresponding to differentially expressed miRNA can be determined by GO functional significance enrichment analysis.Pathway with Qvalue<0.05 is defined as Pathway significantly enriched in differentially expressed genes.The most important biochemical metabolic pathway and signal transduction pathway involved in differentially expressed miRNA target genes can be determined by significant enrichment of Pathway.Results:Compared with Normal group,there were 355 miRNA differentially expressed in serum exosomes of rats in Model group,of which 145 were up-regulated miRNA and 210 were down-regulated miRNA.Compared with Model group,there were 486 miRNA differentially expressed in serum exosomes in QRHX group,of which 358 were up-regulated miRNA and 128 were down-regulated miRNA.Model/Normal and QRHX/Model have reverse effect.There are 49 miRNA in the database,37 miRNA down-regulated miRNA in Model group compared with Normal group under the intervention of QRHX group,and 12 up-regulated miRNA in Model group compared with Normal group under the intervention of QRHX group.Combined with the results of the study,the target genes of the screened miR-214-3p and miR-30a-5p were predicted.The target genes of miR-214-3p are ATF4,RUNX3,STAT6,CADM1,and so on.The target genes of ADAM 19,FOXD1,BCL-2,SEPT7,and so on.Target genes mainly play a role in biological processes such as protein modification,ion binding,cell differentiation,activation of nucleic acid binding transcription factors,cytoskeleton dependent intracellular transport and so on.The target genes are significantly enriched in MAPK signaling,Endocytosis,Ras signal pathway,Rapl signal pathway,Tight junction,Calcium signal pathway,Oxytocin signal pathway and other signal pathways.Conclusion:The abnormal expression of exosome miRNA is involved in the pathological process of bone destruction in CIA rat model.MiR-214-3p and miR-30a-5p may be the initial part or central node in the pathogenesis of RA.Qingre Huoxue decotion may interfere with the pathological process of bone destruction of RA through multi-pathway and multi-target regulation axes such as miR-214-3p-ATF4 and miR-30a-5p-ADAM19Part 3:Optimization of Qingre Huoxue decotion in the treatment of rheumatoid arthritisObjective:To explore the difference and related mechanism between Qingre Huoxue decotion and simplified compound prescription in the treatment of bone destruction in RA,and to explore the further optimization of compound medicineMethods:60 SD female rats were randomly divided into normal group,model group and Qingre Huoxue decotion group,Qingre Huoxue simplified compound group and Etanercept group with 10 rats in each group.The drug intervention began 10 days after the first immunization,and the treatment period was 4 weeks.Four rats were selected for serum exosome sequencing analysis,combined with the results of the second study,the exosome miRNA,of simplified compound intervention on RA was screened and compared with the miRNA regulated by Qingre Huoxue decotion.The pathological section of the joint,the swelling volume and swelling score of both hindlimbs,the change of body weight,the number of osteoclasts in joint tissue and the bone mineral density of hindlimb were observed.The serum levels ofTNF-?,IL-1?,IL-17,MMP-9,TRAP,ALP and OC were detected by ELISA methodResults:The swelling of both hind limbs of CIA rats in each group began to appear on the 10th day after the first immunization,and obvious swelling of both forelimbs was also seen in the severe ones gradually.It reached the peak on the 21st day,decreased on the 24th day,increased again on the 28th day,and then decreased gradually.Compared with the model group,the simplif-ied compound of clearing away heat and activating blood circulation upregulated 9 miRNA,and downregulated 25 miRNA.There were 22 miRNA with the same regulatory effect of Qingre Huoxue decotion and simplified compound prescription,of which 4 were up-regulated and 18 were down-regulated.Pathological sections in the model group showed that the joint space of rats narrowed obviously,a large number of inflammatory cells infiltrated,synovium tissue proliferated obviously,and articular cartilage and articular surface were destroyed in varying degrees.The proliferation of synovial tissue,infiltration of inflammatory cells and destruction of articular cartilage in Qingre Huoxue decotion group were less than those in model group,and the joint structure was relatively complete.In the simplified compound group,the synovial tissue proliferated,part of it fused with articular cartilage,bone erosion appeared and the joint space narrowed.Compared with the model group,there was significant difference in the number of osteoclasts between the Qingre Huoxue decotion group and Etanercept group(P<0.001),but there was no significant difference between the simplified compound group and the Qingre Huoxue decotion group(P<0.05).The difference in the simplified compound group was statistically significant(P<0.05).The bone mineral density of CIA rats in each model group was significantly lower than that in the normal group,and compared with the model group,the bone mineral density of rats in Qingre Huoxue decotion group,simplified compound prescription group and Etanercept group increased significantly(P<0.05),but there was no significant difference among the three groups(P>0.05).Qingre Huoxue decotion decreased the levels of TRAP,MMP-9,IL-1?,IL-17 and TNF-? better than those in the simplified compound group,but there was no significant difference in increasing the levels of OC and ALP between the Qingre Huoxue decotion group and the simplified compound group.Conclusion:Both Qingre Huoxue decotion and simplified compound prescription can interfere with the pathological process of RA bone destruction by affecting exosome miRNA.Both Qingre Huoxue decotion and simplified compound prescription can reduce joint swelling,inhibit joint inflammatory cell infiltration and synovial hyperplasia,inhibit the expression of pro-inflammatory cytokines such as TNF-?,IL-17,IL-1?,reduce the degree of inflammation,inhibit the production of osteoclasts,delay bone compensatory overgrowth in bone destruction and joint destruction,and play the role of "bone protection".The overall curative effect of Qingre Huoxue decotion is better than that of simplified compound prescription. |
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