| BackgroundRheumatoid arthritis(RA)is an autoimmune disease.C y tokines,including IL-17 A and IL-17 F which produced b y T helper cell 17(Th17)are involved in its pathogenesis.Th17 contributes to bone and cartilage destruction in RA patients b y initiating joint inflammation [1].Both Th17 cells and Treg cells have spec ific functional genes,which are derived from the same naive CD4+T cells but developed in different cytokine environments[2].Typical pro-inflammatory Th17 cells lead to autoimmune tissue inflammation and joint damage through induction of pro-inflammatory cytokines.In addition,Treg cells inhibited the activit y of Th17 cells and other effector T cells.Treg cells have an immunosuppressive effect b y producing anti-inflammatory cytokines,which can maintain the bod y's self-tolerance and suppress autoimmunity [3].The association between Th17/Treg cell imbalance and the production of pro-inflammatory or anti-inflammatory cytokines is related to the progression of RA disease,which in turn closely related to autoimmunit y,chronic inflammation and joint destructi on [4-5].TAZ(transcriptional coactivator with PDZ-binding motif)is a downstream effector of Hippo signaling pathway [6-7].TAZ has been shown to play a vital role in cell migration,invasion and epithelial-mesench ymal transition(EMT)in some human cance rs and immune s ystem diseases[8-9].TAZ is involved in autophagy of RA FLs and the differentiation of Th17/Treg [10-11].Previous studies of our group confirmed that Leonurine can regulate the activation,migration and invasion of fibroblast s ynovial in rh eumatoid arthritis,and reduce arthritis s ymptoms in CIA mice [12].However,the effect of leonurine on TAZ in RA disease development has not been reported.Therefore,we made the h ypothesis that leonurine regulates the secretion of cytokines,activation,m igration and invasion of FLS in the inflammatory response through TAZ.Therefore,this stud y intends to clarify the effect of leonurine on the expression of TAZ,the regulation of inflammatory cytokines,and the inhibitory effect on the migration and invas ion of activated FLS.Objective(1)To analyze the differential gene expression in peripheral blood lymphocytes of RA patients downloaded from NCBI Gene Expression Omnibus.(2)Purification and identification of CD4+ T cells.To study the regulatory effects of TAZ on CD4+ T cells differentiation and production of inflammation-related cytokines.(3)To investigated the effect of leonurine on Th17/Treg balance.To investigated the effect of leonurine on the production of inflammation-related cytokines.To investigate d the effect of leonurine on the expression of TAZ.(4)To study the molecular mechanism of leonurine regulating the inflammatory migration and invasion of fibroblast-like s ynovial induced b y interleukin-6.Methods(1)Raw data on gene expression(CEL files)were downloaded from the NCBI Gene Expression Omnibus(http://www.ncbi.nlm.nih.gov/geo).Affymetrix Human Genome U133 Plus 2.0 Array(GP L570)was anal yzed using the Affymetrix Transcriptome Anal ysis Console.Differentiall y expressed m RNAs were identified consi dering p-value<0.001 and |fold change(FC)|>1.5.Heatmap and volcano plot were drawn for the differentiall y expressed m RNAs.GO and KEGG pathway enrichment anal ysis of differentiall y expressed m RNAs was performed using R cluster Profiler(version 3.10.1)fr om Aipufu.(2)Peripheral blood of normal SD rats was collected,PBMC was separated b y gradient centrifugation method,and cells were purified b y magnetic bead sorting method.The obtained cells were anal yzed and identified b y flow cyt ometry.The target gene w as transfected with lentivirus.The expression of target gene was detected b y real-time quantitative PCR,and the expression of target protein was detected b y Western blot.The effect of target gene on CD4+ T cell polarization was detected b y flow cytometr y.The effect of target gene on the production of cytokines was detected b y enz yme-linked immunosorbent assay.(3)MTT assay were used to evaluate cell proliferation.Flow cytometry was used to detect the effect of leonurine on CD4+ T cell polarization in the group with high expression of TAZ.Enzyme-linked immunosorbent assay was used to detect the effect of leonurine on cytoki ne levels in the group with over expression of TAZ.Real-time quantitative PCR and Western blot were used to detect the effects of le onurine on TAZ m RNA and protein expression.(4)Primary FLS were obtained from synovial of SD rats by collagenase digestion.IL-6 was used to induce FLS inflammation.The in vitro migration and invasion assay was used to detect the abilit y of cell migration and invasion,and the effect of leonurine on the migration and invasion of inflammatory FLS.Real-time quantitative PCR and Western blot were used to detect the molecular mechanism of the effect of leonurine on FLS function.Results(1)3425 m RNAs were found to be differentially expressed between RA patients and health y individuals.The expression of TAZ was upregulated in CD4+ T cells of RA patients.GO pathway enrichment anal ysis showed that m RNA was enriched in biological process,l ike negative regulation of cytokine production,neutrophil activation involved in immune response and mucosal immune response.KEGG pathway enrichment anal ysis showed that m RNA was enriched in various disease pathways.(2)The purity of Lymphocytes extracted from rat peripheral blood was above 90%.The q RT-PCR results showed that the m RNA expression level of TAZ in the experimental group was significantl y increased after virus transfection(P<0.05),and there was no significant difference in the m RNA expression level of TAZ in the negative control group and the normal group(P>0.05).WB results showed that the level of TAZ protein in the experimental group was increased(P<0.05).The m RNA expression level of TAZ in negative control group was not significantl y d ifferent from that in normal group(P>0.05).The results of flow cytometry on the transfected cells phenot ype showed that the proportion of Th17 cells in the experimental group was higher than that in the normal group(P<0.05),while the proportion of Treg cells was lower than that in the normal group(P<0.05).There was no significant difference in the ratio of Th17 cells to Treg cells between the negative control group and the normal group(P>0.05).The expression of IL-1?,IL-17 and TNF-? were increased(P<0.05),and the expression of IL-10 was decreased(P<0.05)using ELISA to anal yze supernatant.There was no significant difference between the negative control group and the normal group(P>0.05).(3)The results of cell proliferation test showed that the proliferation of CD4+ T cells was not significantly affected by 10?M and 20?M Leonurine(P>0.05).Flow cytometry results showed that the differentiation of Th17 cells in the experimental grou p was decreased(P<0.05)and differentiation of Treg cells was increased(P<0.05)after the intervention of Leonurine.In addition,the proportion of Treg cells increased and the proportion of Th17 cells decreased in the normal group treated with Herb Leon urine compared with the normal group without Leonurine(P<0.05).The results were dose-dependent.ELISA results showed that compared with the untreated group,the expression of IL-1?,IL-17 and TNF? decreased(P<0.05),and the expression of IL-10increased(P<0.05)in the treated group.The results of real-time quantitative PCR and western blot showed that the m RNA and protein expression of TAZ were decreased in the intervention group with and without the addition of Leonurine(P<0.05).In addition,the m RNA and protein expression of TAZ in normal group were reduced b y Leonurine(P<0.05).(4)The results of invasion and migration of FLS in vitro showed that under the stimulation of IL-6,the number of migration and invasion of FLS was increased compared with the normal group(P<0.05).The number of migration and invasion of FLS in the groups treat ed with 10?M and 20?M leonurine was decreased compared with that in the 0?M leonurine group(P<0.05).The results showed that the m RNA and protein expressions of TAZ,RANK L and RANK in FLS were increased after IL-6 stimulation(P<0.05).The m RNA and protei n expressions of TAZ,RANK L and RANK in FLS were decreased in 10 and 20 ?M leonurine groups compared with 0?M leonurine group(P<0.05).Moreover,the results were dose-dependent.Conclusion(1)TAZ is highly expressed in CD4+ T cells of RA patients.(2)The aimed gene was successfully transfected.TAZ expression can polarize CD4+ T cells,promote Th17 differentiation,and inhibit Treg differentiation.The expression of TAZ can promote the production of pro-inflammatory cyt okines and inhibit the expression of anti-inflammatory cytokines in the inflammatory response.(3)Leonurine promote the differentiation of CD4+ T cells into Treg cells;inhibit the differentiation of Th17 cells.Leonurine inhibit the production of pro-inflammatory cytokines and stimulate the product ion of anti-inflammatory cytokines.The effect of leonurine on CD4+ T cells differentiation is through inhibiting the expression of TAZ m RNA and protein.(4)Leonurine can reverse the IL-6-induced FLS inflammation,and regulate the migration and invasion of F LS in vitro b y inhibiting the TAZ/RANKL/RANK signaling pathway... |
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