Therapeutic Effect Of CP-25 On Sj?gren's Syndrome Model Induced By Antigen In C57BL/6 Mice And Effect Of CP-25 On BAFF-BAFFR-NF-?B Signaling Pathway

Primary Sj?gren's syndrome(pSS)is a chronic inflammatory autoimmune illness of the moisture-producing glands such as salivary glands that is characterized by variousimmune abnormalities.It is characterized by various clinical and laboratoryexamination,such as xerostomia and xerophthalmia,focal lymphocytic infiltration of exocrine glands,hypergammaglobulinemia,and the presence of autoantibodies which display multiple specificities.B cell activation factor is a member of the tumor necrosis factor ligand superfamily.It is mainly derived from monocytes,dendritic cells,macrophages,neutrophils and so on.It plays a vital role in the growth,maturation and homeostasis of B lymphocytes.BAFF combines with B cell maturation antigen(BCMA),transmembrane activator and calcium modulator and cyclophilin ligandinteractor(TACI)and B cell activation factor recptor(BAFFR).BAFFR is the specific high affinity receptor of BAFF,which can bind to BAFF specifically and has positiveregulation on the proliferation,differentiation and maturation of B cells.It has been proved that BAFF and BAFFR are involved in the development of many autoimmune diseases such as SLE,RA,pSS.A curative agent for pSS is lacking now.Suppression of an excessive abnormal immune response is critical to attenuate the symptoms of pSS patients,and glucocorticoids orimmunosuppressive therapy is therefore frequently utilized.When used for treatment,these drugs also have many adverse effects.Thus,effective and safe therapeutic agents that protect against pSS are urgently needed.TGP has been used in the treatment of SS,but the effect is slow and the curative effect is weak,which may be related to its low bioavailability.CP-25(Phenylsulfonylpaeoniflorin,code of CP-25)that has betterlipid-soluble and obvious effect of anti-inflammatory immunomodulatory is a single component of Paeoniflorin(Pae)modified by esterification.However,the efficacy of CP-25 in the treatment of pSS is not clear.The regulation mechanism of CP-25 about BAFF and BAFFR on pSS has not been reported.In this research,we established the model of antigen-induced C57BL/6 mice,and discussed the therapeutic effects in the model of pSS and its possible mechanism.the activation of B lymphocytes in the spleen and submandibular gland of Sj?gren'ssyndrome model mice induced by C57BL/6 antigen was measured by CP-25.The expression of BAFF and BAFFR in subsets and the regulation of the expression of TRAF2,P50,NIK and P100 in NF-?B signaling pathway in mouse submandibular gland tissue and in vitro cell experiment.The effect of CP-25 on the coexpression of TRAF2 and GRK2 in B lymphocytes stimulated by BAFF was detected byco-immunoprecipitation assay.The effect of CP-25 on the level of BAFF in thesupernanant of HSG stimulated by IFNa was detected by ELISA.We further applied MTT,flow cytometry,immunofluorescence,western blot method to study BAFF,BAFFR,TRAF2 and NF-?B signaling pathway in B lymphocytes and the mechanism of CP-25,which may provide more experiment basis for explorationBAFF-BAFFR-NF-?B signaling pathway participating the pathomechanism of abnormal in flammatory immune response in animal model of primary Sj?gren's syndrome in antigen-induced C57BL/6 mice and the target of CP-25.Objective:To establish an animal model of primary Sj?gren's syndrome in antigen-inducedC57BL/6 mice and to clarify the therapeutic effects in the model of pSS and its possible mechanism animal models.Further reveal the role of BAFF-BAFFR-NF-?B signaling pathway in induced Sj?gren's syndrome animal model and the effect of CP-25.Methods:1.80 female C57BL/6 mice under specific pathogen-free conditions were divided intoeight groups,namely control group,model group,CP-25(17.5mg/kg)group,CP-25(35mg/kg)group,CP-25(70mg/kg)group,Pae group(70mg/kg),TGP group(70mg/kg)and HCQ group(80mg/kg).SS was initiated in the C57BL/6 mice exceptcontrol group via intradermal immunization in the back.The day of the firstimmunization,the day of the second immunization and the day of the thirdimmunization were defined as day 0,day7 and day14.Adjuvant-injected mice weredivided into seven groups,in which SS mice were intragastrically administeredCP-25(17.5,35 and 70mg/kg/day),TGP(70 mg/kg/day),Pae(70 mg/kg/day)andHCQ(80mg/kg/day)from week 7 to week 8.Pilocarpine was used to stimulate thesecretion of saliva for 10min on week 6,week 7 and week 8;Blood was taken fromthe eyeballs of the mice and the mice were killed at the 9th week.The weight,thymus,spleen and submandibular gland index were assessed;The pathologicalchanges of submandibular gland in each group were observed under lightmicroscope and was assessed submandibular gland histopathological score;Thymocyte and splenocyte viability in the spleen of each group was detected byCCK-8 assay;Flow cytometry was used to detect the subsets of T?B cells and theexpression of DC on the surface of mice in each group.The levels of cytokines inserum of each group were detected by CBA kit.The concentrations of antiSSA/Ro,anti-SSB/La and IgG antibodies in serum were measured using ELISA kits.Theexpression of Th1 and Th2 cells in the salivary glands of mice were detected viaconfocal laser scanning microscopy.The level of ALT in serum of mice in eachgroup was detected by ALT-kit.2.The activation and subsets of B lymphocytes in peripheral blood and submandibulargland of Sj?gren's syndrome model mice induced by C57BL/6 antigen weredetermined by flow cytometry in vivo.The expression of BAFF and BAFFR inperipheral blood and submandibular gland was analyzed by ELISA andimmunohistochemical method.The expression of NF-?B related proteins in NF-?Bsignaling pathway was detected by Western blot.The effect of CP-25 on thecoexpression of TRAF2 and GRK2 in submandibular gland of mice was detected byimmunofluorescence.3.The level of BAFF in the supernatant of human submandibular gland cell line HSGstimulated by IFNa was detected by ELISA.The proliferation of B lymphocytes wasdetected by MTT,the subsets of B lymphocytes were detected by flow cytometry.Western blot was used to detect the effect of BAFF on BAFFR,downstreamjunction protein TRAF2 and NF-?B nonclassical signal pathway protein in Blymphocytes and the effect of CP-25.4.Flow cytometry and salivary amylase activity kit were used to detect the effect ofabnormal activated B lymphocytes on the function of HSG cells and the effect ofCP-25.Results:1.The model of Sj?gren's syndrome induced by antigen in C57BL/6 mice wassuccessfully constructed.CP-25 has therapeutic effect on Sj?gren's syndrome model induced by C57BL/6 mice antigen.Compared with normal group,the saliva in the model group decreased significantly.The mice in model group developed tissue destruction with lymphocytic infiltration insalivary glands.The mice in model group developed tissue destruction with lymphocytic infiltration in salivary glands.The concentrations of anti-SSA/Ro,anti-SSB/La and IgG antibody increased significantly in the model group.The results suggest that the model of Sj?gren's syndrome induced by antigen in C57BL/6 mice was successfully constructed.CP-25 can improve the general condition of pSS mice,increase the amount of saliva and improve the pathological changes of submandibular gland in pSS mice.CP-25could inhibit the submandibular gland index and the proliferation of T lymphocytes and B lymphocytes in pSS mice.CP-25 significantly decreased the proportion of Th17 cells and CD19~+PDCA1~+plasma cells,inhibited the expression of CD80 and MHC-?ofDCs in pSS mice.CP-25 increased the proportion of Treg cells and CD19~+CD27~+memory B cells in pSS mice.CP-25 could significantly decreased the levels of IL-4,IL-6,IL-17A and TNF in the sera and the expression of Th1 and Th2 in thesubmandibular gland in pSS mice.The therapeutic effect of CP-25 on Sj?gren's syndrome model induced by C57BL/6antigen was superior to that of TGP,Pae,HCQ,in submandibular gland index,saliva content,Treg cells,DCs and other immune cell subsets and cytokines,anti-ssa antibody level and so on.2.NF-?B signaling pathways mediated by BAFF and its receptor BAFFR involved in B cell activation in Sj?gren's syndrome model mice induced by C57BL/6 antigen.CP-25 can regulate NF-?B signaling pathways.The levels of BAFF in peripheral blood and submandibular gland of Sj?gren'ssyndrome model mice induced by C57BL/6 antigen were higher than that in the control group.The expression of BAFFR in submandibular gland of Sj?gren's syndrome model mice induced by C57BL/6 antigen were elevated.In the model group,the infiltratinglymphocytes in the submandibular gland were mainly B lymphocytes,and thepercentage of plasma cells and memory B cells increased significantly.The expression of TRAF2?GRK2?NIK and p52 in the submandibular gland of the model mice was higher than that in the control group.The co-expression of TRAF2 and GRK2 was enhanced in submandibular gland.CP-25 decreased the expression of BAFF and BAFFR in submandibular gland,anddecreased the percentage of B lymphocyte and its subgroup memory B cell and plasma cell in submandibular gland.The expression of TRAF2,GRK2,NIK and p52 in thesignal pathway mediated by BAFF and BAFFR decreased,while the coexpression of TRAF2 and GRK2 decreased.Expression of signal molecules in BAFF and BAFFR mediated signaling pathway in the B cells of the model mice.3.Human submandibular gland cell line HSG stimulated by IFNa could secrete BAFF,and culture human B cells with its supernatant.The percentage of plasmacyte andmemory B cells increased,the expression of BAFFR,TRAF2,GRK2,NIK and p52increased.CP-25 inhibited the proliferation of B cells,decreased the percentage ofmemory B cells and plasma cells,and decreased the expression of TRAF2,GRK2,NIK and p52.4.The abnormal activation of B lymphocytes affected the function of HSG cells,increased the apoptosis rate of HSG cells,and decreased the activity of amylase in the supernatant of HSG cells.The content of TNF-?in the supernatant of HSG cells was increased.CP-25 can restore the function of HSG cells by regulating the function of B cells.Conclusion1.Antigen-induced Sj?gren's syndrome model in C57BL/6 mice was successful set up.CP-25 has therapeutic effect on antigen-induced Sj?gren's syndrome model in C57BL/6mice.The therapeutic effect of CP-25 on antigen-induced Sj?gren's syndrome model in C57BL/6 mice is closely related to the regulation of immune cell function and cytokine level.The therapeutic effect of CP-25 on the induced Sj?gren's syndrome model wasbetter than that of TGP,Pae,and the adverse reaction was less than HCQ.2.High levels of BAFF and BAFFR involved in the activation of B cells through NF-?B signaling pathways in Sj?gren's syndrome model mice induced by C57BL/6antigen.CP-25 can regulate NF-?B signaling pathways3.The regulation of CP-25 on the function of B cells may be related to the regulation of NF-?B signaling pathway.By regulating the function of B cells,the function of HSG cells can be restored.4.CP-25 may be a promising anti-inflammatory and immunomodulatory softmodulator.It may play a role in the treatment of Sj?gren's syndrome model induced by C57BL/6 antigens through BAFF and its receptor mediated NF-?B nonclassicalsignaling pathway.