Exploring The Efficacy And Possible Mechanism Of Tofacitinib In The Treatment Of Primary Sj(?)gren’s Syndrome Based On Transcriptome Data Mining

Primary Sj(?)gren’s syndrome(pSS)is a polygenic,multipathogenic autoimmune disease characterized by exocrine glandular lymphocyte infiltration,and there is no specific treatment for pSS.Objective:1.Using the integrated analysis of multiple microarray transcriptome datasets,multiple databases,and various biological bioinformatics methods,we will analyze differentially expressed key genes,signaling pathways,and potential therapeutic drugs in pSS.2.We will provide a reference of small molecule drug targeted therapy by constructing an animal treatment model of pSS and evaluating the efficacy of tofacitinib for pSS.3.From the perspective of immunology and molecular biology,we will preliminarily explore the possible mechanism of tofacitinib in the treatment of pSS.Methods:1.We screened the pSS transcriptome microarray datasets suitable for analysis in the GEO database,respectively GSE23117,GSE40611,GSE80805,and GSE127952.We used the linear models for microarray data(LIMMA)package of R to filter differentially expressed genes(DEGs)of each dataset.We used robust rank aggregation(RRA)to integrate the processing results of the four data and filtered out the robust DEGs.We used Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)database to perform functional annotation and pathway enrichment analysis of the screened DEGs.We used the STRING database to construct a protein-protein interaction network(PPI).We used the Fun Rich software to perform protein domain enrichment analysis of the screened key genes.The transcriptome microarray dataset was adopted in immune infiltration analysis to target immune cells involved in the development of pSS.2.We selected NOD/ShiLtj mice as the subjects of tofacitinib treatment.Body weight,daily water intake,and fasting blood sugar of NOD/ShiLtj mice were monitored.Saliva secretion,tear secretion,corneal fluorescence staining,tear film breakup time,and organ index of NOD/ShiLtj mice were detected.HE staining was used to observe the pathological changes of the submandibular glands,corneas,and lacrimal glands of NOD/ShiLtj mice.AB-PAS staining,Masson trichrome staining,and Sirius red staining were used to observe the pathological changes of the submandibular glands of NOD/ShiLtj mice.ELISA was used to detect the anti-SSA and anti-SSB antibody levels in NOD/ShiLtj mice’s plasma.3.The spleen lymphocytes of NOD/ShiLtj mice were used as the flow cytometry samples.We analyzed the changes of Th1,Th2,Th17,and Treg subsets’ proportion of T cells in tofacitinib-treated NOD/ShiLtj mice’s spleens.The plasma of NOD/ShiLtj mice was selected as the ELISA samples.We analyzed the changes of IFN-γ,IL-4,IL-17,IL-10,and TGF-β levels in tofacitinib-treated NOD/ShiLtj mice’s plasma.Submandibular gland sections of NOD/ShiLtj mice were selected as immunohistochemical samples.We analyzed the JAK-STAT pathway-related protein expression changes in the submandibular gland of tofacitinib-treated NOD/ShiLtj mice.Results:1.RRA analysis identified 294 DEGs in pSS compared with the control group,of which in pSS 241 were significantly up-regulated,and 53 were significantly down-regulated.Among them,CXCL9 is the most aberrantly up-regulated significant gene.GO enrichment analysis identified that immune response,integral component of membrane,and protein binding terms were significantly involved in the pathogenesis of pSS.KEGG enrichment analysis identified that many genes involved in cytokine-cytokine receptor interaction and chemokine signaling pathway terms participated in the pathogenesis of pSS.PPI network analysis identified that PTPRC,CD86,LCP2,CCR5,CXCL10,STAT1,IRF8,CD19,SELL,and CD2 were key pathogenic genes of pSS.Protein domain enrichment analysis identified STAT as the potential protein target for pSS therapy.2.Tofacitinib can appropriately increase NOD/ShiLtj mice’s body weight,reduce NOD/ShiLtj mice’s daily water intake,and have no therapeutic effect on diabetes.Tofacitinib can improve salivary secretion of NOD/ShiLtj mice.Tofacitinib can improve tear secretion function of NOD/ShiLtj mice,decrease their corneal fluorescein staining score,and prolong their tear break-up time.Tofacitinib can reduce tissue swelling in the salivary glands,lungs,and spleen of NOD/ShiLtj mice.Tofacitinib can relieve lymphocyte infiltration in salivary glands of NOD/ShiLtj mice.Tofacitinib can increase the amount of protein secreted by salivary glands.Tofacitinib can relieve the surrounding infiltration foci and change the degree of fibrosis and collagen type.Tofacitinib can improve collagenous fiber arrangement in NOD/ShiLtj mice’s corneal stroma.Tofacitinib can attenuate lymphocyte infiltration in NOD/ShiLtj mice’s lacrimal glands.Tofacitinib can reduce anti-SSA and anti-SSB antibody levels in NOD/ShiLtj mice’s plasma.3.Twelve weeks of tofacitinib administration can reduce the proportion of Th1,Th2,and Th17 cells in spleen CD3+ T cells of NOD/ShiLtj mice and increase the proportion of Treg cells in spleen CD4+ T cells.With the increase in the dosing cycle,tofacitinib can gradually reduce plasma concentrations of IFN-γ,IL-4,and IL-17 in NOD/ShiLtj mice,while increasing plasma concentrations of IL-10 and TGF-β.Tofacitinib can reduce JAK1,JAK3,STAT1,STAT3,and STAT6 expression and increase STAT5 expression in NOD/ShiLtj mice’s submandibular glands.Conclusion:1.The enrichment analysis and functional annotation of DEGs indicate that T cells are involved in the development of pSS.The JAK-STAT signaling pathway is a potential pathway for the treatment of pSS.2.The results of tofacitinib-treated pSS mice indicate that tofacitinib can improve the exocrine function of pSS mice,reduce the lymphocyte infiltration of exocrine glands,improve local inflammation level,and reduce autoantibody levels.3.The results of tofacitinib-treated pSS mice indicate that tofacitinib restores the immune balance of T cell subsets in pSS mice.Tofacitinib’s therapeutic effect on pSS may be achieved by changing the proportion of lymphocyte populations.Tofacitinib reduces pro-inflammatory factor levels of pSS mice,and increases anti-inflammatory factor levels of pSS mice,which may act through the JAK-STAT signaling pathway.