| Primary Sjogren’s syndrome（p SS）is a systemic autoimmune disease typically characterized by plasma-lymphocytic infiltration of the salivary and lacrimal glands.Dry mouth is a common presenting complaint in adults,while parotid swelling may be a more frequently encountered in children.While inflammation is primarily directed toward the exocrine glands,extraglandular manifestations can include arthritis,Raynaud’s phenomenon,purpura,pulmonary disease,renal disease,and neurological involvement.It most often affects middle-aged females with a prevalence of 0.1–3%and incidence of 3.9–5.3 yearly per 100,000 in the adult population.Genetic,hormonal,and environmental factors contribute to the development of the disease.Overactivity of B cells,the production of autoantibodies and the formation of ectopic germinal centers are the main features of p SS.In autoimmune process key cytokines can be detected which involved In the salivary glands and lacrimal gland,CXCL13 expressed by the epithelial cells is a kind of chemokines which can regulate B cell movement,higher expressed in several kinds of autoimmune diseases and related to severity of the disease.in addition,a expression of CXCL13 increased is mainly composed of B cells of ectopic germinal center,the formation of CXCL13 may affect B cells into the pathological changes of salivary glands.In the immune pathogenesis of p SS,IFN-αwhich is mainly synthesized by plasmodendritic cells has also attracted more and more attention.It plays an important role in rheumatoid arthritis,systemic lupus erythematosus,inflammatory bowel disease and other autoimmune diseases by activating the JAK-STAT signaling pathway.Currently,there is a lack of safe and effective drugs for the treatment of p SS.CP-25（Phenylsulfonylpaeoniflorin,termed CP-25）is a single component modified by esterification on the basis of paeoniflorin,and its lipid solubility and oral bioavailability have been significantly optimized.In the early stage of the study,we through the establishment of submandibular gland（SG）protein-immunised experimental Sj?gren’s syndrome（ESS）mice,confirms the CP-25 mouse model has certain curative effect of ESS,and may be through influencing CXCR5-GRK2-MAPK signaling pathways in B cells.In our study,we focus on under the stimulus IFN-α,whether the secretion of the inflammatory factor CXCL13 can be increased by activating the JAK-STAT signaling pathway on the submandibular gland epithelial cells,so as to bind to CXCR5,the specific receptor of CXCL13 on the B cells,and affect the migration of B cells to the inflammatory tissues.Whether CP-25 can achieve the treatment goal by regulating the binding of GRK2 and JAK1 and weakening the activation of this signaling pathway is the main content of this study.Objective:1. Establishing ESS mouse model,to determine the therapeutic effect of CP-25 on ESS mice model.To determine the regulatory effect of CP-25 on the B cell subsets in ESS mice.To determine whether CP-25 affects B cell function by regulating the JAK1-STAT1/2-CXCL13 signaling pathway in submandibular tissues.2. To observe the expression of IFN-α-JAK1-STAT1/2-CXCL13 signaling pathway and B cells subsets in labial glandular tissues and blood samples of p SS patients.3.To clarify the effect of CP-25 on JAK1-STAT1/2-CXCL13 signaling pathway in in human salivary gland epithelial cells（HSGECs）and its effect on the migration function of B cells,and to reveal whether CP-25 can regulate this signaling pathway by affecting the binding of GRK2-JAK1 on HSGECs,thereby improving the function of B cells.Method:In vivo,we established ESS mouse model induced by SG protein,and divided each group of 10 mice into normal group,model group,CP-25（35mg/kg、70mg/kg）group and hydroxychloroquine（HCQ 80mg/kg）group.During the modeling process,the systemic manifestation of mice were observed,and the weekly body weight of mice was recorded.Mouse saliva flow was measured.The infiltration of lymphocyte in submandibular gland was observed by H&E staining.Spleen,thymus and salivary gland were collected to detect organ index.T lymphocytes of thymus and B lymphocytes of spleen were extracted and cell proliferation was detected by CCK8 method.The changes of B-cell subsets in peripheral blood and salivary gland were detected by flow cytometry.Protein expression of JAK1-STAT1/2 signaling pathway in salivary gland was detected by western blot.The expression of CXCL13 in mouse serum was detected by ELISA.The expression of CXCL13 in mouse salivary gland was detected by Laser scanning confocal microscopy（LSCM）.We collected samples of human peripheral blood and labial gland tissues in clinical,The changes of B-cell subsets in peripheral blood were detected by flow cytometry.LSCM assay was used to detect the expression of p-JAK1,p-STAT1,CXCL13 and IFN-αprotein in human labial gland tissue.In vitro,HSGECs were selected as models for the study of p SS,and the expression of CXCL13 was detected by immunofluorescence,ELISA and RT-PCR under IFN-αstimulation.The expression of JAK1-STAT1/2 signaling pathway protein was detected by western blot.Salivary gland epithelial cells were co-cultured with B lymphocytes,and the migration ability of B cells was detected by Transwell.The co-expression of GRK2 and JAK1 was detected by CO-IP and LSCM assay.Results:In vivo,in ESS model group,the saliva flow decreased,the lymphocyte infiltration degree in salivary gland tissues increased,the salivary gland indexes and spleen indexes increased,and the splenic B lymphocytes viability increased significantly.The CP-25（35,70 mg/kg）-treated group and the HCQ（80mg/kg）-treated group increased the saliva flow,reduced the degree of lymphocyte infiltration,and reduced the proliferation activity of spleen B lymphocytes.In the CP-25（70mg/kg）group,spleen index and salivary gland index were decreased,while HCQ（80mg/kg）group only decreased salivary gland index.In ESS model mice’s peripheral blood,the percentage of total B cells（CD19+）and CXCR5+B cells increased,while the percentage of memory B cells（CD27+CD19+）decreased.CP-25（35,70 mg/kg）group and HCQ（80mg/kg）group reduced the proportion of total B cells（CD19+）and CXCR5+B cells in mice’s peripheral blood,and increased the proportion of memory B cells（CD27+CD19+）in mice’s peripheral blood.In the salivary gland of the mice,the percentage of total B cells（CD19+）,CXCR5+B cells and memory B cells（CD27+CD19+）in the ESS model group increased,CP-25（35,70 mg/kg）group and the HCQ（80mg/kg）group decreased the proportion of total B cells（CD19+）,memory B cells（CD27+CD19+）and CXCR5+B cells in the salivary gland.The expressions of p-JAK1,p-STAT1,p-STAT2 and CXCL13 increased in the salivary glands of ESS mice,while the expressions of CXCL13 in the serum of ESS mice were increased.CP-25（70mg/kg）group and the HCQ（80mg/kg）group decreased the expressions of p-Jak1,p-Stat1 and p-Stat2 in the salivary glands,and CP-25（35,70mg/kg）treatment group decreased the expressions of CXCL13 in the mice’s salivary glands and serum.In clinical,collected samples of human peripheral blood and labial gland tissues.Compared with normal people there were significant lymphocytic infiltration and lymphocytic foci in the labial glands of p SS patients.The expression of CXCL13 in HSGECs（labeled HSGECs byα-amylase）,IFN-α,p-JAK1 and p-STAT1 were markedly increased in p SS patients.In human peripheral blood,flow cytometry results showed that compared with normal people,the percentage of total B cells（CD19+）and CXCR5+B cells in the peripheral blood of p SS patients increased,while the percentage of memory B cells（CD27+CD19+）decreased.In vitro,in HSGECs,the IFN-α（10ng/ml）group increased the expression of CXCL13,p-JAK1,p-STAT1 and p-STAT2 proteins,CP-25(10-7mol/l,10-6mol/l,10-5mol/l)groups reduced the expression of CXCL13.CP-25(10-5mol/l)group and TOF（0.1μM,1μM,10μM）groups reduced the expression of p-JAK1,p-STAT1,p-STAT2protein.We stimulated HSGECs with IFN-α,then co-culturing HSGECs with B cells,the IFN-α（10ng/ml）group increased the migration of B cells.CP-25(10-6mol/l,10-5mol/l)groups,Tofacitinib（TOF,10μM）group and CXCL13 inhibitor group reduced the migration of B cells.LASM and CO-IP results showed that co-expression of GRK2and JAK1 existed in HSGECs cells.After IFN-αstimulation,co-expression of GRK2and JAK1 decreased,and CP-25(10-5mol/l)increased the co-expression of GRK2 and JAK1.Conclusion:1.CP-25 may play a therapeutic role in SG protein-immunised ESS mice model by regulating B cell subsets and B cell viability.2. The proportion of B cell subsets in peripheral blood of p SS patients was abnormal,and the expression of IFN-α,CXCL13 increased and the JAK-STAT pathway was activated in human labial glands.3. CP-25 may inhibit the activation of JAK1-STAT1/2-CXCL13 signaling pathway by increasing the binding of JAK1 and GRK2,reduce the expression of CXCL13 and reduce B cells migration,achieving the purpose of treating pSS. |
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