Study On Theoretical Basis And Mechanism Of Lung-large Intestinal Combination Therapy For Sjogren’s Syndrome

Primary Sjogren’s syndrome(SS)is an autoimmune disease whose prevalence is only to rheumatoid arthritis.SS involves targeting the exocrine glands causing dry mouth and dry eyes,frequently affecting multisystem damage.The direct and indirect health costs of SS patients are enormous due to relatively limited treatment and poor prognosis.Maidong Dishao Decoction(thereinafter referred to as MDSD)is founded by ’The lung stands in interior-exterior relationship with the large intestine’ based on Traditional Chinese medicine(TCM)in treating Sjogren’s Syndrome.According to previous studies and experimental results,MDSD might play a therapeutic role in regulating vasoactive intestinal peptide(VIP).Objective:To explore the theoretical basis of Traditional Chinese medicine(TCM)in treating Sjogren’s Syndrome by Maidong Dishao Decoction by The lung stands in interior-exterior relationship with the large intestine’.To study the therapeutic effect of Maidong Dishao Decoction,a representative prescription based on a therapeutic method of ’The lung stands in interior-exterior relationship with the large intestine’ for NOD mice and the mechanism of the vasoactive intestinal peptide on T cell subsets.Methods:1.Theoretical research:To summarize the etiology and pathogenesis of Sjogren’s syndrome of medical research progress recently;To summarize the treatment experience of Sjogren’s syndrome in Traditional Chinese medicin e;To overview the theoretical research of MDSD Decoction in treating Sjogren’s syndrome.2.Experimental study:2.1 The therapeutic effect of Maidong Dishao Decoction on NOD miceEight-week-old female C57/BL6 served as the normal group,and eight-week-old female NOD mice were randomly divided into four groups:the model group,the hydroxychloroquine(HCQ)group,MDSD decoction low dose group(thereinafter referred to as MDSD.L group),MDSD high dose group(thereinafter referred to as MDSD.H group).Water-consumption was measured as the weekly average.The salivary flow rate was measured before and after drug administration.After eight weeks,the submandibular glands(SMGs)of the mice were observed by hematoxylin and eosin(H&E)staining,and the expression of PTEN,PI3K,and p-PI3K in SMGs was evaluated by immunohistochemistry tests.The levels of VIP in the serum and SMGs,interleukin(IL)-2,and IL-10 in the spleen were assessed by enzyme-linked immunosorbent assay(ELISA).VIP,VPAC1,and VPAC2 gene expressions of the spleen were carried out using real-time quantitative polymerase chain reaction(Q-PCR)analyses.2.2 Regulation of VIP on PTEN and PI3K/AKT pathwayMonocytes were extracted from the spleen of 8 female NOD mice aged 17 weeks.Cell counting kit(CCK)-8 assay was used for the monocyte viability analysis of YIP affecting the splenic lymphocytes from NOD mice.Small interference RNA(siRNA)of PTEN was used to knock down the expression of PTEN in the splenic lymphocytes.The most efficient gene knockdown was selected by qPCR.Western blot was used to analyze the expression of PTEN,AKT,PI3K,and p-AKT,p-PI3K after the siRNA transfection,the Treg and Th17 cells from the splenic lymphocytes were determined using flow cytometry.2.3 Regulation of MDSD Decoction and VIP on PD-1/PD-L1 pathwayEight-week-old female NOD mice were randomly divided into six groups:control group,PD-L1 antagonist group,MDSD decoction+PD-L1 antagonist group,VIP+PD-L1 antagonist group,VIP medium-dose group,and VIP high-dose group.They were given orally and intraperitoneally from 10 weeks of age.Each group was treated for four weeks.After anesthesia,the SMGs were measured for H&E staining.Flow cytometry was used to detect the proportion of Treg cells in the spleen from each group.Results:1.Theoretical researchSjogren’s syndrome is a disease of abnormal body fluid metabolism.The functional coordination of ’the lung and large intestine’ is closely related to body fluid production and delivery.The imbalance of T cell subsets is one of the pathogenesis of Sjogren’s syndrome.VIP could regulate the immune balance and be associated with the combined treatment of_the lung and intestine.2.Experimental research2.1 The results showed that MDSD decoction could reduce the average water intake and increase the salivary flow rate of NOD mice.Submandibular-glands(SMGs)of mice in the model group were seen with severe lymphocyte infiltration,while the lymphocyte infiltration of the MDSD.H group and the normal group was not serious.MDSD decoction could increase the expression of VIP in serum and submandibular gland tissue,Treg-related cytokines IL-10 and IL-2 levels in the spleen.Moreover,MDSD decoction could increase the mRNA levels of VIP and VPAC1 in the spleen of NOD mice.In addition,MDSD decoction may increase the protein levels of PTEN and inhibit the expression of p-PI3K in NOD mice.2.2 The results showed that VIP could time-dependent increase the protein expression of phosphorylated PTEN after PTEN silencing.In addition,VIP could reduce the expression of the phosphorylated PI3K/AKT pathway and upregulate the proportion of Treg/Th17 cells through PTEN.2.3 The results showed that VIP could play a protective effect on lymphocytic infiltration of the submandibular gland in mice.After blocking PD-L1,both MDSD decoction and VIP could not modify the severity of sialadenitis,but the proportion of Treg cells was increased in the spleen in NOD mice.In addition,the regulation of Treg cells by VIP was affected by the microenvironment.Conclusion:The theoretical basis in TCM for treating Sjogren’s Syndrome by MDSD decoction is sufficient.MDSD decoction might alleviate lymphocyte infiltration and salivary gland dysfunction in SMGs of NOD mice,an experimental SS mouse model.It can also increase the level of VIP in serum and play the role of anti-inflammation.The mechanism may be related to the protective role of VIP in the pathological procession of pSS.Mechanistically,VIP could rescue the balance of the Treg/Th17 ratio,resulting in the amelioration of the dryness symptoms of SS mice.