Effect And Mechanism Of Sodium Butyrate Regulating Fibroblast Like Synovial Cells On Rheumatoid Arthritis

Objective:To investigate the characteristics of intestinal microbiota and the relationship between bacteria,lymphocyte subsets and cytokines in patients with rheumatoid arthritis(RA).To explore the impact of flora disorder on the occurrence and development of RA,and to find possible ways to improve the occurrence and development of RA.To further discover the effects of metabolite butyric acid of the screened different bacteria.The therapeutic effect of sodium butyrate on collagen induced arthritis(CIA)mice and the regulatory effect of sodium butyrate on the proliferation,apoptosis,migration and invasion of rheumatoid arthritis synovial fibroblasts(RA FLSs)were explored through experiments in vivo and in vitro,and the molecular mechanism of RA FLSs apoptosis induced by sodium butyrate was further explored.Methods:1.Fecal samples from 205 RA patients and 199 healthy controls(HCs)were collected for bacterial DNA extraction and 16S ribosomal RNA(r RNA)gene sequencing.The levels of T,B,CD4~+T,CD8~+T,NK cells,T helper cell 1(Th1),Th2,Th17 and regulatory T(Treg)cells in peripheral blood were detected by flow cytometry combined with standard absolute counting beads.The serum cytokines interleukin-2(IL-2),IL-4,IL-6,IL-10,IL-17,tumor necrosis factorα-α(TNF-α)and interferon-γ(INF-γ)level were detected by flow cytometry microsphere array(CBA).Bioinformatics analysis was used to explore theαandβdiversity of intestinal microbiota.Spearman rank correlation test was used to explore the relationship between intestinal flora,lymphocyte subsets and serum cytokines.2.Establishment of CIA model:DBA/1 mice were immunized with chicken type II collagen twice on day 0 and day 21,respectively.Mice were randomly divided into three groups:group A,control group,group B,CIA group and group C,which were treated with sodium butyrate.The severity of arthritis in mice was evaluated by clinical score and histopathological staining.On the 70th day,the end point of the experiment,serum cytokines such as IL-1-β,IL-2,IL-4,IL-6,IFN-γ,TNF-α,IL-17A and IL-10 were detected by flow cytometry bead array(CBA).The proportion of CD4~+T cell subsets such as Treg,Th1,Th2 and Th17 cells in spleen were measured by flow cytometry.3.Fresh synovial tissue from patients undergoing arthroscopic synovectomy or joint replacement were cultured by tissue block culture method,and cultured to 3-6 generations for follow-up experiments.CCK-8 experiment was used to detect the effect of sodium butyrate on the inhibition rate of RA FLSs.And the effect of sodium butyrate on RA FLSs apoptosis was detected by Annexin V-FITC PI method.Transwell plate was used to study the effect of sodium butyrate on the migration and invasion of RA FLSs.4.The experiment was carried out with RA FLSs cultured to 3-6 generations.The effects of sodium butyrate on the expression of AKT,mTOR,Bcl-2 and Bax m RNA in RA FLSs were analyzed by real-time fluorescence quantitative PCR.Western blot was used to analyze the effect of sodium butyrate on the expression of apoptosis related proteins Bcl-2and Bax in RA FLSs.Also,the effects of sodium butyrate on the expression and phosphorylation of AKT and mTOR were tested by western blot.And the effects of AKT inhibitor(MK-2206)on apoptosis related proteins were analyzed by western blot.Annexin V-FITC PI assay was used to detect the effect of MK-2206 on the apoptosis of RA FLSs.Results:1.The diversity and relative abundance of intestinal microbiota in RA patients were significantly lower than those of HCs.The abundance of Proteobacteria in patients with RA was more than that in HCs,while the abundance of Firmicutes was less than that in HCs(P<0.05).At the genus level,the relative abundance of dozens of bacteria such as Lachnospiracea＿incertae＿sedis,Prevotella and Clostridium＿Xl Va decreased in RA patients(P<0.05).Compared with the HCs,the relative abundance of Ruminococcus2 in RA patients increased and which was negatively correlated with the absolute count of Treg cells(P<0.05).And the relative abundance of Cloacibacillus in RA patients increased,which was positively correlated with the absolute count of Th17 cells(P<0.01).The relative abundance of Blautia and Clostridium＿XVIII decreased in RA patients.Blautia was negatively correlated with IL-6(P<0.05),and Clostridium＿XVIII was positively correlated with IL-10(P<0.05).Compared with the HCs,the bacteria producing short chain fatty acids in RA patients were significantly reduced,among which the bacteria related to acetic acid production were Akkermansia,Prevotella and Blautia.Among the bacteria related to propionic acid production,Dialist,Coprocccus and Roseburia decreased significantly.Among the bacteria related to butyric acid production,the bacteria significantly reduced include Coprococcus,Anaerostipes,Roseburia,Clostridium＿Xl Va and Clostridium＿XVIII.The results showed that the relative abundance of Blautia producing acetic acid was correlated with the absolute counts of B cells,CD4+T cells and Treg cells(P<0.05),and negatively correlated with IL-6(P<0.05).The relative abundance of Clostridium＿XVIII was positively correlated with IL-10(P<0.05).2.Sodium butyrate intervention can effectively reduce the joint inflammation of CIA mice and promote its remission.Compared with CIA group,the joint inflammation score and joint pathology score of NaB-CIA group decreased significantly(P<0.05 or P<0.01).CBA method was used to detect cytokines in peripheral blood of mice.The levels of IL-6,TNF-αand IFN-γin serum of CIA group were significantly increased compared with control group(P<0.05 or P<0.01).The expression levels of serum IL-4 and IL-10 in NaB-CIA group were significantly increased compared with control group and CIA group(P<0.05 or P<0.01).The expressions of Treg,Th1/2/17 and Tfh cells were detected by flow cytometry.Compared with control group,the expression of Treg cells in spleen of CIA group mice decreased significantly.However,the expression of Treg cells in spleen of NaB-CIA group was significantly increased compared with CIA group(P<0.05).3.The cultured cells were identified as RA FLSs by flow cytometry.The results of CCK-8 showed that sodium butyrate at the concentration of 16 mmol/L had an inhibitory effect on RA FLSs after 24 hours of intervention(P<0.05).Except for the concentration of0.25 mmol/L,all other concentrations(0.5,1,2,4,8,16 mmol/L)had an inhibitory effect on RA FLSs after 48 hours of intervention(P<0.01).Annexin V-FITC PI test showed that sodium butyrate(0.5 mmol/L and 16 mmol/L)could significantly induce RA FLSs apoptosis compared with the control group(P<0.05).Transwell migration experiment showed that compared with the control group,sodium butyrate group(0.5 mmol/L)could reduce the migration ability of RA FLSs in vitro(P<0.01).Transwell invasion experiment showed that compared with the control group,sodium butyrate group(0.5 mmol/L)could reduce the ability of RA FLSs to pass through matrix glue(P<0.01).4.Real-time fluorescence quantitative PCR showed that sodium butyrate could enhance the expression level of Bax m RNA in RA FLSs and reduce the expression of AKT,mTOR and Bcl-2 m RNA.There were significant differences between the above target genes and the blank control(P<0.05).Western blot analysis showed that compared with the control group,0.5mmol/L sodium butyrate intervention decreased the phosphorylation expression levels of AKT and mTOR proteins,and the difference was statistically significant(P<0.05).Sodium butyrate intervention in RA FLSs resulted in the decrease of Bcl-2 protein expression level,while the expression level of Bax was up-regulated,the difference was statistically significant(P<0.05),and the protein expression ratio of Bcl-2and Bax decreased significantly.AKT inhibitor MK-2206 enhanced the effect of sodium butyrate on RA FLSs.Compared with sodium butyrate group,MK-2206 inhibitor combined with sodium butyrate significantly decreased the expression level of Bc1-2protein,increased the expression level of Bax protein(P<0.05),and increased the apoptosis rate of RA FLSs(P<0.05).Conclusion:1.Richness and diversity of intestinal flora in RA patients were impaired,which might participate in the pathogenesis of RA by modulating the immune systems with lymphocyte subpopulations and cytokines.In addition,the bacteria producing short chain fatty acids in the intestinal flora of RA patients were significantly reduced,especially the bacteria producing butyric acid.Butyrate supplementation may provide a new idea for the treatment of RA.2.The intervention of sodium butyrate could promote the production of Treg cells and increase the level of serum anti-inflammatory cytokines,which was conducive to the alleviation of joint inflammation in CIA mice.3.Sodium butyrate effectively regulated the proliferation,apoptosis,migration and invasion of RA FLSs.Specifically,it was able to inhibit the proliferation,migration and invasion of RA FLSs,but promote RA FLSs apoptosis.4.Sodium butyrate induces apoptosis of RA FLSs through AKT/mTOR signaling pathway.