Analysis Of Differential MRNA Expression In Peripheral Blood Of Rheumatoid Arthritis With Blood Stasis Syndrome And Study On The Therapeutic Mechanism Of Rabdosia Rbescens To Rheumatoid Arthritis

ObjectiveRheumatoid Arthritis(RA)is an autoimmune disease with chronic joint inflammation.Blood stasis is a major pathogenic factor in the pathogenesis of RA in TCM,and "blood stasis syndrome" is one of the common syndromes of RA.Biotechnology has become a new research tool for TCM syndromes.Currently,there are few reports on the genetic level of RA blood stasis syndromes,so it is clinically important to investigate the mRNA expression of RA blood stasis syndromes at the transcriptional level and to explore new traditional Chinese medicines for the treatment of RA blood stasis syndromes.In this study,we screened the differential mRNA expression profiles of RA blood stasis syndrome by clinical high-throughput transcriptome sequencing and analyzed the biological information to investigate the intrinsic connection between RA blood stasis syndrome and mRNA expression profiles;we established an inflammation model of RA blood stasis syndrome in mice,and investigated the effects of the traditional Chinese medicine Rabdosia rubescens(RR)and its main active ingredient Oridonin(ORI)on the treatment of RA blood stasis syndrome.The effects of the Chinese medicine RR and its main active ingredient ORI on the inflammation model of mice with RA blood stasis syndrome were investigated,and the effects and molecular mechanisms of ORI on RA fibroblast-like synoviocytes(RA-FLSs)were further studied,which provided the theoretical basis for the treatment of RA blood stasis syndrome by RR.We also investigated the role of Vascular Endothelial Growth Factor A(VEGFA)in RA blood stasis through clinical and basic experimental studies.Methods1.Peripheral blood was collected from RA blood stasis syndrome,RA non-blood stasis syndrome group,normal human blood stasis syndrome and normal human non-blood stasis syndrome group,and peripheral blood mononuclear cells were isolated and total RNA was extracted for transcriptome sequencing,and mRNA expression of different genes in RA blood stasis syndrome and RA non-blood stasis syndrome and the enrichment pathway of the two different genes was analyzed by using Bioinformatics software,and then the genes that were differentially expressed were verified by RT-PCR.2.Thirty six C57BL/6 female mice were randomly divided into control group,model group.low concentration RR group(L-RR group),high concentration RR group(H-RR group),low concentration ORI group(L-ORI group),and high concentration ORI group(H-ORI group),with 6 mice in each group.In addition to the control group,the remaining five groups of mice were injected with 10 μL of fully emulsified complete Freund’s adjuvant into the foot pads of the hind limbs bilaterally,and then subcutaneously injected with 0.1 mg/kg of epinephrine hydrochloride injection on the back for 2 h.The mice were also placed in 4℃ cold water and swam in the cold water for 5 min(once per day)for 7 consecutive days,to establish an RA blood stasis syndrome mouse inflammation model.From the 7th day of modeling,the L-RR and H-RR groups were gavaged with 240 mg/Kg/d and 480 mg/Kg/d of RR solution(two per day),and the L-ORI and H-ORI groups were intraperitoneally injected with 20 mg/Kg/d and 40 mg/Kg/d of ORI solution(once per day)for 14 days.The thickness of the hindfoot paw was measured every 3 days,and the mice were weighed after 14 days of treatment to assess the joint index scores,ELISA method was used to detect the concentrations of vascular endothelial growth factor A(VEGFA),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and interleukin-6(IL-6)in the toes,and HE staining and fanconi solid green method were used to detect the synovial proliferation,inflammation,and cartilage destruction of the ankle joints.infiltration and articular cartilage destruction.3.We Cultured and identification of primary RA-FLSs,different concentrations of ORI intervened RA-FLSs,CCK8 assay detected the survival of RA-FLSs,LDH assay detected the release of lactate dehydrogenase from RA-FLSs,Hochest/PI assay detected the death of RA-FLSs,Endoplasmic reticulum probe detected the structural alteration of endoplasmic reticulum in RA-FLSs,high-throughput transcriptome sequencing and Bioinformatics software to analyze differential mRNA expression,enrichment and prediction of activated signaling pathways in RA-FLSs,RT-PCR,immunoblotting to verify the expression of genes and proteins related to the endoplasmic reticulum stress pathway of apoptosis in RA-FLSs.Results1.In this study,we screened 5 cases each of RA blood stasis syndrome,RA non-blood stasis syndrome,normal human blood stasis syndrome and normal human non-blood stasis syndrome samples that met the criteria for sequencing,and the transcriptome sequencing and biosignature analysis showed that there were 31 differential mRNA expression profiles in the RA blood stasis syndrome compared with that in the RA non-blood stasis syndrome group(13 up-regulated and 19 down-regulated).Compared with the normal human non-blood stasis syndrome,there was 1 differential gene mRNA expression profile(1 upregulation)in the normal human blood stasis syndrome group.The GO enrichment analysis of differential mRNAs in the RA blood stasis syndrome group and non-blood stasis syndrome group showed that 16 biological processes,10 molecular functions and 7 cellular components were mainly enriched,and the KEGG enrichment analysis of differential mRNAs in the RA blood stasis syndrome group and non-blood stasis syndrome group showed a significant difference in the expression of natural killer cell-mediated cytotoxicity,cytotoxicity.and cellular components.The KEGG enrichment of mRNAs of different genes in the RA blood stasis syndrome group and non-blood stasis syndrome group was analyzed in the signaling pathways of natural killer cell-mediated cytotoxicity,antigen presentation,rheumatoid arthritis,and type I diabetes and other signal pathways.The mRNAs of vascular endothelial growth factor A(VEGFA)and the immune-related gene,HLA-DQAI,were verified to be up-regulated in the RA blood stasis syndrome group by RT-PCR(P<0.05).2.A mouse model of RA blood stasis inflammation was successfully established,and after 14 days of RR and ORI treatment,compared with the model group,joint swelling symptoms were reduced in the low and high concentration RR and ORI groups,and the thickness of the hindfoot paw was reduced in the H-RR,L-ORI,and H-ORI groups(P<0.05),and The joint index scores of the H-RR and H-ORI groups decreased(P<0.05);the H-RR,L-ORI H-RR,L-ORI,and H-ORI groups reduced the concentration of inflammatory factors TNF-α,IL-6,and IL-1β(P<0.O5);H-RR,L-ORI,and H-ORI groups reduced the concentration of VEGFA(P<0.05);and RR and ORI reduced the infiltration of inflammatory cells in the ankle joints,synovial proliferation,and inhibited cartilage destruction.3.Human RA-FLSs were identified and cultured,and ORI at 15,20.25,and 30 μM inhibited the cellular activity of RA-FLSs(P<0.05),with a half inhibitory concentration(IC50)of 20.21)μM.20,25,and 30 μM of ORI promoted the release of LDH(P<0.05).15,20.and 25 μM of ORI induced the death of RA-FLSs(P<0.05).After ORI intervention in endoplasmic reticulum,the endoplasmic reticulum structure was significantly altered from long spindle shape to round shape condensed and distributed around the nucleus.1451 genes were upregulated and 1042 genes were downregulated in RA-FLSs after 20 μM ORI intervention in RA-FLSs.The upregulation of 1451 genes was significantly enriched in the 20 pathways of the GO bioprocesses,of which.endoplasmic reticulum Folding protein response was highly enriched.GSEA analysis revealed high expression of genes for endoplasmic reticulum-to-cytoplasmic transport.KEGG software analysis revealed up-regulation of endoplasmic reticulum stress-related genes ATF4,eIF2α,and CHOP expression.RT-PCR quantification revealed up-regulation of genes related to the endoplasmic reticulum signaling pathway.including PERK,eIF2α,ATF4,and CHOP(P<0.05).Immunoblotting demonstrated that endoplasmic reticulum stress-related genes were up-regulated(P<0.05).blotting demonstrated that the expression levels of endoplasmic reticulum stress pathway p-PERK,p-eIF2α,ATF4,CHOP and apoptotic Bax protein were upregulated(P<0.05).Conclusion1.The RA blood stasis syndrome group was genetically differentiated from the RA non-blood stasis syndrome group,with differential gene mRNAs mainly enriched in antigen presentation,rheumatoid arthritis,and natural killer cell-mediated cytotoxicity.RA can affect the expression of blood stasis syndrome,which was associated with the up-regulation of VEGFA and HLA-DQA1 genes.2.RR and ORI improve joint swelling,posterior foot thickness,joint index score,and the concentration of cytokine TNF-α,IL-6,IL-1β and VEGFA in RA mouse models with blood stasis syndrome.RR and its main active ingredient ORI can reduce inflammation cells,synovial hyperplasia,and cartilage damage in the ankle joint,indicating that RR and its main active ingredient ORI have a therapeutic effect on the inflammation model of RA with blood stasis syndrome.VEGFA can be used as a therapeutic evaluation indicator for RA blood stasis syndrome.3.ORI may activate endoplasmic reticulum stress PERK/eIF2 α/The CHOP signaling pathway induces apoptosis of RA-FLSs,and Rabdosia rubescens may treat RA with blood stasis syndrome through its main active ingredient,oridonin.