| Sjogren's syndrome(SS)is an autoimmune disease charicatarized with salivary glands and lacrimal glands mainly involved.The manifestation of the disease is dry mouth and eyes.It can also affect other systems such as the skin and other organs.There are lots of lymphocytes infiltrating in the exocrine glands and a variety of autoantibodies can be detected in the serum,which often accompanied by high immunoglobulinmia.At present,there is no ideal drugs of SS in clinical treatment with only local alternative treatment and systemic immunotherapy.Alternative treatment cannot fundamentally treat SS and there are many serious adverse reactions during the treatment of SS.Therefore,a high efficiency and low toxicity drugs are needed clinically.CP-25(Phenylsulfonylpaeoniflorin,code of CP-25)is a single component of Paeoniflorin(Pae)modified by esterification.It was significantly better than the total total glucosides of paeony(TGP)and Pae in anti-inflammatory action and effect.CP-25can significantly inhibit the proliferation of T and B lymphocytes,reduce the levels of CD80,CD86,MHC-II,PGE2 and TNF-?and increase the percentage of Treg cells.However,the efficacy of CP-25 on the SS is unclear.SS is mainly the damage of exocrine glandular glands with the decrease of exocrine fluid and the decrease of mucin content leading to dry mouth and dry eyes.This is one of the typical features of the decrease of exocrine gland secretion.Human submandibular gland(HSG)cells are salivary gland cells that play a major role.Saliva is the main substance for maintaining oral hygiene and dental health.In addition to water and inorganic substances such as metal ions,there are many organic substances such as proteins that constitute the oral defense system,such as lysozyme,?-amylase,mucin,and peroxide.Enzymes,immunoglobulins,lectins,etc.Under physiological conditions,the secretion of saliva is controlled by the sympathetic and parasympathetic nerves(SNS).In addition,?adrenergic receptors(?AR)signaling may be involved in the regulation of inflammatory immune responses.Saliva secretion can also be regulated by the activation of SNS under physiological conditions.Therefore,changes in various components and levels of saliva may be one of the main features of SS disease.Non-obese diabetic(NOD)mice are typical type 1 diabetic mice characterized by spontaneous pancreatic injury.However,because of its characteristics of salivary glands and lacrimal gland injury after the 8th to 12th week-age,it has become a typical animal model for the study of self-sudden dry syndrome in recent years.Objective:To establish an animal model of spontaneous Sj?gren's syndrome in NOD/LtJ mice and to evaluate the corresponding indexes of the model.To clarify the therapeutic effect of CP-25 on spontaneous SS animal models and to clarify the function and related signal changes of submandibular gland cells.Methods:NOD/LtJ mice were randomly divided into 7 groups according to the weight of saliva and body,namely model group,CP-25 low,middle and high dosage groups(17.5,35 and 70mg/kg),Pae and HCQ group;ICR mice were used as Control group.The spleen,submandibular gland and thymus index were assessed;pilocarpine was used to stimulate the secretion of saliva for 10min;H&E staining and assessment of submandibular gland histopathological score;CCK-8 method was used to detect T,B lymphocyte proliferation in the spleen of each group;The expression of cytokines in serum of mice in each group were detected by CBA-Kit.Anti-SSA,anti-SSB antibody and IgG levels in serum were detected by laser scanning confocal microscopy.Th1 and Th2 subsets in submandibular gland cells were detected by confocal laser scanning microscopy.In vitro,HSG cells were divided into 4 groups,Control group,ISO group,ISO+CGP20712A group,ISO+ICI118551 group.In addition to the Control group.HSG cells were treated with a certain concentration of ISO(10~-66 mol/L),while the corresponding?-adrenergic receptor antagonists were administered to CGP20712A and ICI118551groups(both 10~-66 mol/L)after 48h continuous detection of the corresponding cell function and indicators.The morphology of HSG cells was observed under an upright microscope;the glycosaminoglycan sulfate(SGAG)was detected by DMMB;expressions of?-amylase in HSG cells were detected;the cells apoptosis was detected by the kit;Molecular expression level was measured by Western Blot.Results:1 Effects of CP-25 on NOD/LtJ mouse model of spontaneous Sjogren syndrome1.1 NOD/LtJ mice general changes in conditionHair of NOD/LtJ mice gradually turned yellow.Mice become thin and low in weight and scratch the lips and tongue.1.2 Effects of CP-25 NOD/LtJ mice organ indexThymus index of NOD/LtJ mice was significantly lower and submandibular gland index was significantly higher.CP-25(35 mg/kg),CP-25(70 mg/kg),TGP(70 mg/kg)and HCQ can improve theses indicators significantly.1.3 Effects of CP-25 on NOD/LtJ mice salivaThe saliva in the model group decreased significantly.CP-25(70 mg/kg),TGP(70mg/kg)and HCQ(80 mg/kg)significantly increased salivary volume after 1 week of treatment.The amount of saliva in HCQ(80mg/kg)group increased significantly.1.4 Effects of CP-25 NOD/LtJ mouse submandibular gland pathologyThe submandibular gland of mice in each group showed different extent of lymphocyte infiltration,which was mainly manifested by the increase of lymphocyte infiltration foci and the infiltration lesion area.CP-25(70mg/kg),HCQ(80mg/kg)can significantly reduce submandibular gland pathological grade.1.5 Effects of CP-25 NOD/LtJ miceon T,B lymphocyte proliferationNOD mouse spleen T,B lymphocyte proliferation was significantly increased.CP-25(70mg/kg),Pae(70mg/kg),TGP(70mg/kg)and HCQ(80mg/kg)all inhibited the proliferation of T lymphocytes;HCQ(80mg/kg)significantly inhibited the proliferation of B lymphocytes.1.6 Effects of CP-25 on NOD/LtJ mice Th17,Treg cell subsetsThe proportion of Th17 cells in NOD mice was significantly increased;the proportion of Treg cells was significantly decreased.CP-25(70mg/kg),TGP(70mg/kg),Pae(70mg/kg)and HCQ(80mg/kg)significantly decreased the proportion of Th17cells.1.7 Effects of CP-25 on NOD/LtJ mice serum cytokinesThe levels of IFN-?,IL-4,IL-6and IL-17A in peripheral blood of NOD/LtJ mice were significantly increased.The levels of IL-6,IL-17A and IFN-?in sera of NOD/LtJ mice were significantly decreased by CP-25(70mg/kg),TGP(70mg/kg),Pae(70mg/kg)and HCQ(80mg/kg);CP-25(35mg/kg)can significantly reduce the level of IFN-?.1.8 Effects of CP-25 on Anti-SSA Antibody and Anti-SSB Antibody in NOD/LtJ Mouse SerumLevels of anti-SSA antibody and anti-SSB antibody in the serum of NOD/LtJ mice increased significantly;CP-25(17.5,35,70mg/kg),TGP(70mg/kg),HCQ(80mg/kg)can significantly reduce NOD/LtJ mice serum anti-SSB antibody levels.1.9 Effects of CP-25 on NOD/LtJ mouse serum IgG levelsThe level of IgG in serum of NOD/LtJ mice increased significantly.Serum IgG levels of NOD/LtJ mice were significantly decreased by CP-25(70mg/kg),TGP(70mg/kg)and HCQ(80mg/kg).1.10 Effects of CP-25 on the mouse submandibular gland tissue Th1 and Th2 cell levelsThe expression of Th1 and Th2 cells in submandibular gland tissues of NOD/LtJ mice were significantly increased.In NOD/LtJ mice,CP-25(70 mg/kg)significantly reduced the expression of Th1 and Th2 cells.2 Effects of Isoprenaline on Function and Related Signaling Molecules of Submandibular Gland Cells2.1 HSG cells in the morphological changes under the expose of ISOThe Control group cell morphology showed elongated or fusiform morphology with clear cell boundaries.After 48 hours of ISO treatment,the cell morphology showed polygons or became shorter and the cell borders were not clear.2.2 HSG cells in the role of ISO under the action of mucopolysaccharide sulfate(SGAG)metabolic changesAfter 48h,the proportion of SGAG released by HSG cells increased significantly.The results showed that the sustained effect of ISO led to the disorder of glucagum metabolism in HSG cells.2.3 HSG cells under the action of ISO intracellular alpha-amylase expression levelsAfter 48 hours of ISO treatment,the expression of?-amylase in HSG cells was significantly down-regulated,suggesting that the sustained ISO effect may have some damaging effects on HSG cell function.2.4 HSG cells under the action of ISO changes in the proportion of apoptosisAfter 48 hours of ISO treatment,the apoptosis of HSG cells in ISO group increased significantly,suggesting that?2 adrenergic receptor may be involved in the apoptosis of HSG cells.2.5 HSG cells under the action of ISO?AR and related signaling moleculesThe results showed that after 48 hours of ISO treatment,both?1AR and?2AR were significantly down-regulated,while the corresponding?-adrenergic receptor antagonists antagonized this effect and up-regulated the expression of?1AR and?2AR.Compared with Control group,GRK2 in ISO group increased significantly and G?s level decreased significantly.Conclusion:1 NOD/LtJ model mice have the features of spontaneous Sjogren's syndrome2 CP-25 has a therapeutic effect on NOD/LtJ mice model of Sjogren's syndrome3 The therapeutic effect of CP-25 on NOD/LtJ mice syndrome model is closely related to the regulation of immune cell function and cytokine levels4 ISO on HSG cell function and related signaling molecules:4.1 HSG cells in the role of ISO under the morphological and functional changes4.2?AR and its signal molecules involved in the occurrence of HSG cell dysfunction... |
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