| Objective:Rheumatoid arthritis is an autoimmune disease characterized by changes in joint pathology and joint function.The treatment and rehabilitation of the damage caused by it is an important clinical problem that needs to be solved urgently.Among them,drugs and surgery are the main treatment methods for RA at present.However,these two methods can only relieve the symptoms of RA patients.Long-term use of drugs for the treatment of RA patients has large toxic and side effects,which brings a heavy burden to patients and society.Therefore,it is urgent for us to find RA prevention with high efficiency and low toxic and side effects.and therapeutic drugs.Flavonoids,the largest group of plant phenolic compounds that make up our diet,have anticancer and anti-inflammatory activities.In this study,the induction of Breg cells by apigenin in natural compounds was taken as the breakthrough point,the pharmacological activities of Chinese medicinal molecules were explored from the perspective of immune environment modification,the collagen-induced arthritis model was constructed,and the therapeutic effect of apigenin on rheumatoid arthritis was discussed.Its molecular mechanism aims to provide zoological and molecular biology basis for clinical nursing and rehabilitation guidance,provide a more scientific reference for dietary care of patients with clinical rheumatoid arthritis,and give full play to the advantages of traditional Chinese medicine dietary care in the prevention and treatment of chronic diseases.Method:1.The effect of apigenin on the differentiation and function of Breg cells.In this study,we first compared the induction of Breg cells by apigenin in vitro experiments,and treated Breg cells with apigenin.We observed the effect of apigenin on Breg cells from cell experiments and explored the role of apigenin in the differentiation process of Breg cells.In this study,LPS was used.In the induced inflammation model,mouse B cells were cultured in vitro to induce differentiation into Breg cells,and Breg cells were treated with apigenin.The optimal concentration of apigenin was screened based on drug toxicity and efficacy.Two methods of feeding were adopted:intraperitoneal injection and apigenin feed,and the expression levels of cytokines(IL-10,IL-6)were detected by flow cytometry,ELISA detection and real-time quantitative-q PCR(RT-q PCR).To determine the effect of apigenin on promoting IL-10 secretion and inhibiting inflammation.2.The preventive and therapeutic effects of apigenin on rheumatoid arthritis.In vivo experiments were conducted to investigate the apigenin treatment of rheumatoid arthritis mouse animal model,and to observe whether apigenin can improve the symptoms of arthritis in mice.In this study,bovine type II collagen was used to induce DBA/1 mice to establish autoimmunity.Arthritis(CIA)model,set up blank control group(WT),arthritis group(CIA),prevention group(CIA-Prevention)and treatment group(CIA-Therapy),Apigenin(2 g/kg)was incorporated into the animal feed,7 animals/group were fed with apigenin feed respectively,and different administration periods were designed,namely the prevention period and the treatment period.The disease process was observed by taking pictures,recording the changes in arthritis scores and body weight.After treatment,hematoxylin-eosin staining(HE)staining,flow cytometry,and high-resolution micro-computed tomography(Micro-CT)were used together.To evaluate the effect of apigenin in the prevention and treatment of CIA mice.3.The mechanism of apigenin on Breg cell differentiationRNA-seq technology was used to explore the effect of API on Breg cell signaling pathway,p38 inhibitor small molecule and apigenin were used to co-treat Breg cells,and Western blot technology was used to explore the mechanism of apigenin-induced Breg cell differentiation.Result:1.The effect of apigenin on the differentiation and function of Breg cellsThe results of flow cytometry,ELISA and RT-QPCR showed that apigenin at the concentration of 5μM had better cell activity,and was more conducive to promoting the secretion of anti-inflammatory IL-10 and inhibiting the expression of pro-inflammatory IL-6 in Breg cells.In the screening of better treatment and prevention methods,two methods were adopted in this study:intraperitoneal injection and apigenin feed feeding.The results showed that the apigenin feed feeding group could improve the proportion of IL-10 in blood of mice,while the expression of IL-10 in mice was not changed in the intraperitoneal injection group.2.The preventive and therapeutic effects of apigenin on rheumatoid arthritisThe results of flow cytometry,HE staining,and Micro-CT detection showed that apigenin had the best therapeutic effect in the mice in the prevention group,which could significantly improve the sudden weight loss and arthritis swelling of the mice.Joint HE staining showed that the infiltration of inflammatory cells was reduced,the damage of articular cartilage was repaired,the joint space was close to that of the blank control group,and the Micro-CT results were consistent with the pathological HE results.The spleen and blood flow detection results of the prevention group and the treatment group showed that apigenin relieved arthritis in mice by enhancing the differentiation function of Breg,decreasing the proportion of IL-17 cells in CD4~+,and increasing the expression of CD4~+T and CD8~+T cells.3.The mechanism of apigenin on Breg cell differentiationRNA-seq analysis showed that after apigenin treatment of Breg cells,KEGG-pathway enrichment analysis showed that MAPK signaling pathway was significantly enriched in all differential genes and up-regulated differential genes.Flow cytometry results showed that after treatment with p38 small molecule inhibitor,blocked apigenin-mediated secretion of IL-10 in Breg cells,and the Western blot results were consistent with the results of p38 small molecule inhibitor treatment.Apigenin regulated Breg cell differentiation and IL-10 expression by activating the p38 MAPK signaling pathway.Conclusion:1.Apigenin can induce the differentiation of mouse Breg cells,promote the expression of anti-inflammatory factor IL-10 in Breg cells,and reduce the secretion of pro-inflammatory factor IL-6.2.Apigenin can relieve the symptoms of arthritis in the CIA mouse model,slow down the progression of arthritis,and have preventive and therapeutic effects on arthritis.3.Apigenin participates in Breg cell differentiation by activating p38MAPK signaling pathway. |
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