The Mechanism Of Extracellular Matrix Decorin In The Pathogenesis Of Primary Sjogren’s Syndrome And Gut Microbiota Aberration In Patients Of Systemic Sclerosis And Bleomycin-induced Mice Model

Backgrounds:Primary sjogren’s syndrome(pSS)is a chronic inflammatory autoimmune disease that mainly involves the exocrine glands.In addition to infiltrating immune cells and cytokines,extracellular matrixs(ECMs)and matrix metalloproteinase(MMP)also play a key role in gland damage and inflammation in pSS patients.Previous studies have shown that MMP-9is highly expressed in pSS patients’ serum and tissue,and is closely related to the degree of pathological injury and the occurrence of disease.However,the downstream pathogenesis caused by MMP-9 activation remains unclear.Decorin is a proteoglycan found primarily in connective tissue that widely participates in a few of biological processes.Decorin is also an endogenous ligand of Toll-like receptors(TLRs),which has been shown to act as a damageassociated molecular pattern(DAMP)molecule to mediate aseptic inflammatory responses.As one of the DAMP molecules in tissues,its release depends on the proteolytic enzyme shear in the matrix under the condition of damage.Moreover,the decorin receptors TLR2 and TLR4 were also increasingly expressed in labial glandular tissues of pSS patients compared with healthy controls.Whether and how decorin plays a role in the pathogenesis of pSS remains to be further investigated.Objective:To explore the pathogenic mechanism and clinical significance of extracellular matrix decorin in the pathogenesis of pSS.Methods:The expression values of DCN gene and related clinical data from three different GEO database were extracted.The correlation between DCN expression and clinical parameters or other genes was analyzed.The deconvolution algorithm of CIBERSORT was used to analyze the infiltration of immune cells in parotid glands.Enzyme-linked immuno sorbent assay(ELISA)was used to determine the expression level of decorin in the peripheral blood of the included subjects,and the correlation with the clinical parameters was analysed.Hematoxylin-eosin staining(HE),immunohistochemical staining,and immunofluorescence staining were performed on the obtained labial glandular tissues.Salivary duct tumor cell line A253 and THP-1 cell line were cultured in vitro,and exogenous decorin stimulation at different concentrations was given.Cell morphology was observed.Cell apoptosis was determined by flow cytometry(FACS)and cytokine expression was detected by Reverse Transcription-Polymerase Chain Reaction(RT-PCR).Results:The expression of DCN as detected by the the transcriptome of pSS blood sample was significantly lower than that of healthy controls.The expression of DCN in parotid tissue RNA-seq was significantly higher than that in healthy controls.There was no correlation between DCN expression and the disease activity score.In the parotid tissue RNA-seq,DCN was positively correlated with the expression of apoptosis gene CASP3,and negatively correlated with the expression of proliferation gene MKI67.DCN was positively correlated with the expression of inflammatory factor IL1 B.And the DCN was positively correlated with the expression of TLR2 and My D88.The expression of decorin in the serum of pSS patients was significantly lower than that of healthy controls,and had no correlation with clinical parameters,including ESSDAI score,Focus score,Ig A,Ig M and Ig G.Immunohistochemical staining of labial glandular tissues showed that the expression of decorin and its receptor TLR4 were significantly increased in labial glandular tissues of moderate and severe pSS patients compared with SSICA patients.In addition,immunofluorescence co-localization staining indicated that decorin and TLR4 were colocalized around the duct of labial glandular tissue in pSS patients,and their expression was up-regulated compared with that in the SSCIA group.The analysis of immune cell infiltration in parotid tissue RNA-seq data showed that M1 Macrophages,γδ-T cells and T/B subsets of adaptive immune cells were dominant in the salivary tissue of pSS patients.Meanwhile,M1 Macrophages in the labial gland tissue of pSS patients were significantly higher than that of SSCIA patients.Exogenous decorin stimulation induced apoptosis of salivary ductal epithelial cells in a concentration-and time-dependent manner.Moreover,it could induce the up-regulation of TNF-α transcription in salivary ductal epithelial cells and the expression of related transcription factors of M1 Macrophages.Conclusion:Decorin and its gene DCN were reduced in peripheral blood of pSS patients,and were not correlated with disease activity,so they could not be used as biomarkers reflecting disease activity.Decorin is highly expressed in labial glandular tissues of pSS patients,and can act as a DAMP molecule to induce apoptosis of salivary ductal epithelium and the production of inflammatory factors.It can also act on Macrophages,promoting the macrophages polarization from M0 to M1,and secreting related inflammatory factors。Our exploratory study provides a scientific experimental basis for seeking new therapeutic targets for pSS chronic inflammation.Introduction Systemic sclerosis(SSc)is a systemic autoimmune disease of unknown etiology,which is mediated by immune system and has high morbidity and mortality.The main clinical manifestations of SSc patients include skin fibrosis(increased tension),visceral lesions(lung,kidney,gastrointestinal tract,etc.),vascular lesions,etc.Among the organs involved,the gastrointestinal tract(GIT)is the second most common one besides the skin.More than 90% of patients have GIT involvement,which is characterized by dysphagia,esophageal reflux,vomiting,constipation,and abdominal pains.There are currently very limited methods for the treatment.Unfortunately,there are still no ideal animal models that can fully replicate the four basic pathophysiological features of SSc,let alone animal models specifically designed to study gastrointestinal lesions.The bleomycin(BLM)-induced mouse model showed not only local skin sclerosis,but also systemic immune response,such as autoantibody production,which is a commonly used animal model of SSc.A few of studies have shown that the gut microbiota of SSc patients is disturbed and is involved in the occurrence and development of the disease.To verify whether BLM-induced mouse model could simulate the gut microbiota aberration of SSc,which can be further used to study the pathogenic mechanism of gut microbiota in SSc.Objective: To investigate the gut microbiota aberration in SSc patients and BLM-induced mouse models.Methods: Fecal samples were collected from 5 SSc patients and 10 healthy controls,as well as from mice.The 16 Sr RNA sequencing of the samples was commissioned by Shanghai Majorbio Biotech.After the sequencing data were corrected,the diversity of was analyzed on Majorbio Cloud platform.Results: In this study,it was found that gut microbiota aberration in BLM-induced mouse models had the similar changes as that in SSc patients.The Alpha diversity analysis showed that BLM-induced mice and SSc patients showed the same trend in species diversity index.And the microbiota composition analysis showed that,compared with the control group,the Firmicutes significantly enriched in the feces of BLM-induced mouse mice and SSc patients,while Bacteroidetes decreased.At the genus level,Lactobacillus increased in the feces of BLM-induced mice and SSc patients.PCo A and Lefse software analysis showed that Lactobacillus＿salivarius in the feces of SSC patients was significantly higher than that of healthy controls,with statistical significance.And unclassified＿g＿lactobacillus and Lactobacillus＿reuteri were also significantly increased in BLM-induced mice.Conclusion: BLM-induced mice model can likely mimic the pathological changes of gut microbiota in patients with SSc.In the future,this may offer an important potential platform for the in-depth understanding of gut microbiota aberration in patients with SSc and to devise potential disease-modifying treatments.