Effect Of CP-25 To Sj?gren’s Syndrome Model Induced By Antigen In C57BL/6 Mice And CXCL13-CXCR5-MAPK Signaling Pathway In B Lymphocyte Function

Sjogren syndrome（SS）is an autoimmune disease affected the exocrine glands,such as lacrimal gland,parotid gland and salivary gland etc.This disease mainly characterized by dry eyes,difficulty swallowing,and often involving the skin,liver,kidney and neurovascular structures etc.The lymphocytes infiltration in various organs is an important manifestation of Sjogren’s syndrome.Recent studies have found that B lymphocytes play a crucial role in the pathogenesis of Sjogren’s syndrome.The infiltration of B lymphocytes in the salivary glands usually means the bad prognosis in patients.At the same time,B lymphocytes can also secrete a variety of autoantibodies,such as Anti-SSA,Anti-SSB,which aggravate the development of the disease.on the early state,B lymphocytes can also secrete some cytokines to promote the formation of ectopic germinal centers.At present,there is no specific medicine for treating Sjogren’s syndrome in clinical.Alternative treatments such as artificial tears,pilocarpine,etc.and immunosuppressant is used in most patients.Immunotherapy and other treatment methods cannot fundamentally solve the patient’s pain,also can’t effectively stop the deterioration of the disease.There is an urgent need for a low-toxic and highly effective therapeutic drug for Sjogren’s syndrome.CP-25（Phenylsulfonylpaeoniflorin,code CP-25）is a single component obtained by Chemical modified of Paeoniflorin（Pae）.Its onset time and intensity are significantly better than total glucosides of paeony（TGP）and Pae.CP-25 can significantly inhibit the proliferation of T and B lymphocytes,reduce the levels of CD80,CD86,MHC-II,PGE2 and TNF-α,and increase the proportion of Treg cells.The previous research of our group showed that CP-25 have a significant effect on the mouse model of primary Sjogren’s syndrome,but the mechanism of CP-25 is still unclear.Based on our previous research,this study established an animal model of primary Sjogren’s syndrome in C57BL/6 mice.The effective dose of CP-25 was administered to observe the pathological changes of salivary gland tissue and the expression of CXCR5in the salivary gland.The relationship between CXCL13/CXCR5 and the change.The human salivary gland（HSG）cell line were co-culture with B lymphocytes by using IFN-αas stimulator,using Transwell,Co-immunoprecipitation and Western blot,to observe CXCR5 receptor signal transduction pathway and GRK2 Interactions,and the regulation of CP-25 on the interaction between CXCR5 and GRK2,elucidating the regulation of GRK2 on CXCR5 receptor signaling and the role of CP-25,providing a mechanism for revealing the anti-inflammatory and immunomodulatory effects of CP-25.OBJECTIVE:To establish a model of spontaneous Sjogren’s syndrome in C57BL/6mice and study the role of B lymphocytes in Sjogren’s syndrome.To study the role of CXCL13-CXCR5-MAPK signaling pathway in the Sjogren’s syndrome model of C57BL/6 mice.And the mechanism of CP-25 regulate the CXCL13-CXCR5-MAPK signaling pathway by GRK2.Method:Animal experiments:According to saliva and body weight,the C57BL/6 mice were randomly divided into 5 groups.That is:the normal group,the model group,CP-25 two different dosage groups（35mg/kg,75mg/kg）and HCQ group,each group have 8 mice.In the usual feeding,pay attention to observe the mouse hair color and other general conditions,every week have a weighing.The spleen,salivary gland and thymus index were assessed;pilocarpine stimulated salivary gland secretion 10min saliva;H&E staining and assessment of salivary gland histopathological score;CCK-8 method was used to detect T,B lymphocyte proliferation in the spleen of each group;the change of B cell subgroup in peripheral blood and salivary gland was detected by flow cytometry,and the expression of related protein in salivary gland of mice was detected by Western blot.Cell experiment:According to the Drug Administration,divided into five groups,that is,the normal group,the control group,IFN-αStimulation Group,IFN-α+CP-25(10^-5-10^-7mol/ml).The expression of CXCL13 after IFN-αstimulation was detected by Q-PCR,CCK-8 detect the viability of HSG cell and B cell after co-culture,Transwell detect the B cell migration after co-culture.The expression of B lymphocyte-related protein was detected by Western blot.Results:Therapeutic effect of CP-25 on Sjogren’s syndrome model of C57BL/6 mice1)Compared with the normal group,the hair color in the model group grew to dry,the weight was lower than the normal group,the phenomenon of scratching the lips and licking the tongue was reduced,and the symptoms of the CP-25 group were relieved.2)Compared with the normal group,the amount of saliva decreased significantly in the model group mice after modeling.After 1 weeks of administration,CP-25（70mg/kg）,HCQ（80mg/kg）significantly increased saliva volume,and 2 weeks after administration,the amount of saliva in CP-25（35,70mg/kg）and HCQ（80mg/kg）group were increased significantly.3)Compared with the normal group,the spleen Index,thymus index and salivary gland index of mice in the model group increased significantly.The CP-25 group can significantly reduce the spleen index,thymus index and salivary gland index and the HCQ group could reduce thymus index and salivary gland index.4)There were significant elevated levels of spleen T and B lymphocyte proliferation in mice in C57BL/6 model group.CP-25（35,70mg/kg）and HCQ（80mg/kg）inhibited the proliferation of T lymphocytes,and CP-25（70mg/kg）and HCQ（80mg/kg）significantly inhibited the proliferation of B lymphocytes.5)The H&E staining of C57BL/6 mice in the model group showed the infiltration of lymphocytes,while the degree of infiltration was serious,which was mainly manifested in the increase of the number of lymphocyte infiltration stoves and the enlargement of the area.CP-25（70mg/kg）and HCQ（80mg/kg）can significantly reduce thepathological classification of salivary gland.6)In the detection of B lymphocyte subsets in peripheral blood,the proportions of model plasma cells,memory B cells and immature B cells increased significantly,and the proportion of mature B cells decreased.CP-25 can significantly reduce theproportion of peripheral plasma cells,memory B cells and immature B lymphocytes,and there is no statistically different effect on the elevation of mature B lymphocytes.HCQ could increase the proportion of immature B lymphocytes,and there was nostatistically different effect on the proportion of plasma cells,memory B cells and mature B cells.In the detection of B lymphocyte subsets of salivary gland,theproportion of plasma cells increased significantly,CP-25 could reduce the proportion of plasma cells,and the effect of HCQ on the proportion of salivary gland pulp cells was not statistically different.7)CD19,CXCR5 double immunofluorescence staining was carried out on the salivary gland tissue and the fluorescence quantitative analysis of the tissues was carried.Thedetection of salivary gland tissue protein was carried out by Western blot method,and the expression of p-ERK and p-p38 was obviously increased.CP-25 group can reduce the expression of abnormal protein.8)After stimulation with IFN-αfor 48h,HSG cells could express CXCL13 highly,and there was no statistical difference between CP-25 group and model group.9)After co-culture with B lymphocytes,the viability of HSG cells is decreased,and CP-25 could increase viability of HSG cells after co-culture.10)After 24 hours of co-culture,B lymphocyte migration ability is increased,and CP-25 can decrease B lymphocyte migration ability.11)Expression of GRK2,CXCR5,ERK and p-ERK in B lymphocytes after co-culture with HSG cells by Western blotConclusion:1.CP-25 has therapeutic effect on antigen-induced Sjogren syndrome mouse model2.The therapeutic effect of CP-25 may be related to its regulation of B cell subset ratio and proliferation and migration.3.CP-25 regulates B lymphocyte function through regulating GRK2 to affecting CXCL13-CXCR5-MAPK signaling pathway.