Effect And Mechanism Of Regulatory B Cells Induced By Luteolin On Rheumatoid Arthritis

ObjectiveRheumatoid arthritis is an immune disease caused by abnormal immune system attacking the body,which not only damages joints,but also causes many complications and high disability rate.At present,the progress of arthritis is mainly delayed by reaching the standard treatment in clinic.Regulatory B cells,which have negative immunomodulatory effects in potential therapeutic targets of rheumatoid arthritis,have been concerned by scholars.Studies have found that the number and function of regulatory B cells in patients with rheumatoid arthritis are impaired.In the animal experiment of rheumatoid arthritis induced by periodontitis in mice,we found that down-regulated regulatory B cells may be an important factor in the enhancement of rheumatoid arthritis by Porphyromonas gingivalis-mediated periodontitis,and immunotherapy by injecting regulatory B cells and inducing regulatory B cells with specific drugs may be effective in treating rheumatoid arthritis.Luteolin(LUT),as a flavonoid,is widely found in people’s daily diet and has anti-inflammatory properties.The purpose of this study is to explore the inducing effect of LUT on regulatory B cells and its intrinsic mechanism through cell experiments and to explore the preventive effect of diet rich in LUT on rheumatoid arthritis through animal experiments in order to provide theoretical basis for the prevention and nursing of rheumatoid arthritis.Method(1)To reveal the role and mechanism of LUT-induced regulatory B cells through in vitro experiments.Five drugs were screened from the literature,namely:LUT,kaempferol,chlorogenic acid(CGA),isoliquiritigenin(ISL)and sesamin.Flow cytometry was used to compare the induction of regulatory B cells by five drug treatments to determine the research drug for this topic.RT-q PCR,ELISA and Western Blot were used to detect the ratio of cytokines in the cells and supernatant,the transcription level of transcription factors,the protein and protein phosphorylation level after LUT treatment of regulatory B cells,and revealed that LUT promotes regulatory Pathway of B cell differentiation.(2)To reveal the effect of LUT on rheumatoid arthritis through in vivo experiments.Male DBA/1 mice from 6 to 8 weeks old were divided into control model group(CIA group),LUT group,CGA group and ISL group.The number of mice was 5,8,10,10 and 10,respectively The animal model of collagen-induced arthritis was established by immunizing mice with bovine typeⅡcollagen emulsion.Two mice in CIA group died because of intolerance to collagen emulsion,and finally eight mice were included in the model.Mice in LUT group,CGA group and ISL group were fed with 6g/kg LUT feed,4 m M/L CGA drinking water and 3 g/kg ISL feed for 46 days.The preventive effect of LUT-rich diet on rheumatoid arthritis was evaluated by mouse body weight arthritis index score flow cytometry and micro-CT.Results(1)LUT may promote IL-10 in regulatory B cells by activating p38MAPK pathway and inhibiting histone deacetylase activity.To observe the promotion of IL-10 and the inhibition of IL-6 in regulatory B cells treated with five drugs,LUT was selected as the research subject.The drug concentration was 8μM LUT up-regulated IL-10(P<0.05)and down-regulated IL-6 and IL-1β(P<0.05);The expression of IL-6 in cell supernatant was inhibited(P<0.01).In MAPK pathway,the phosphorylation level of p38 protein kinase increased after LUT treatment of regulatory B cells(P<0.05);The phosphorylation level of P38 protein kinase decreased(P<0.05)and the ability of regulatory B cell differentiation decreased(P<0.05)after inhibition of p38 branch.ELISA showed that LUT inhibited histone deacetylase activity in regulatory B cells(P<0.01).(2)LUT-rich diet reduced arthritis symptoms and joint damage in mice.Compared with CIA group,LUT group had no significant change in body weight(P>0.05),CGA group and ISL group had a lower weight increase(P<0.05);The three drug groups alleviated the redness and swelling symptoms of mouse claws to a certain extent;The score of arthritis index of mice in LUT group was lower than that in CGA group and ISL group,and the score of arthritis index of mice in CGA group and ISL group was higher;The proportion of CD4~+T cells increased but there was no significant difference in IL-10 and IL-6 of regulatory B cells in each drug group(P>0.05);Articular bone erosion in CGA group and more severe LUT and ISL group.Conclusion(1)LUT may induce regulatory B cell differentiation by activating p38 MAPK pathway and inhibiting histone deacetylase activity.(2)LUT-rich diet in CIA animal model can protect the joints of mice with rheumatoid arthritis and delay the progression of the disease.