| Backgrounds and Objectives Inflammatory bowel diseases (IBDs), consisting of Crohn's disease (CD) and ulcerative colitis (UC), are chronic debilitating disorders characterized by recurring episodes of relapsing and remitting inflammation of the gastrointestinal tract. IBDs belong to complex diseases which are very difficult to cure. With the rapid development of economy and society, the incidence of IBDs also increases gradually, so it has brought great burden to patients and their families, even to the society. How to resolve this problem has been a great challenge for the doctors and scientists. Although the precise etiology of IBD has not yet been elucidated, a complex interaction between predisposing genes, environmental factors, and dysregulated mucosal immune responses to the commensal gut microbiome has been thought to make a great contribution to the disease pathogenesis. Neuropeptide receptor 1 (NPSR1), previously called as G-protein-coupled receptor GPR154 or GPRA, is a member of G protein-coupled receptor family. NPSR1 polymorphisms have been reported to be associated with some autoimmune diseases such as asthma, rheumatoid arthritis and IBD, but the number of the relevant researches is very small. In addition, few studies have ever focused on the precise role of NPSR1 in the pathogenesis of these diseases. Until now, there are still no studies which have ever identify the associations of NPSR1 polymorphisms and IBD susceptibility in Asian populations, evaluate the mRNA and protein expressions of NPSR1 and NPS(the ligand of NPSR1) systematically (in the perapheal blood) and locally (in the intestine) in patients with IBD, and explore the possible mechanism in disease pathogenesis. It will be a great help to determine these issues for supporting important theoretical basis of whether NPSR1 can be a potential target for IBD therapy.Methods For genotyping and exploring the associations between genotype and clinical phenotypes,107 CD cases,710 UC cases and 605 healthy controls(HC) were included.2ml perapheal blood(PB) was obtained for each donor for extracting DNA. Six SNPs(rs323917, rs323922, rs324377, rs324384, rs324396 and rs740347) of NPSR1 were genotyped, and haplotype blocks were also constructed. For mRNA expression analysis in PB,37 CD cases,43 UC cases and 69 HCs were recruited, and 5ml PB was needed for each donor to extrat total RNA. Real-time PCR was used to detect the mRNA expressions of NPSR1-A, NPSR1-B, NPS, Tbet, GATA3, RORc and Foxp3. Bivariate correlation analysis was performed to confirm the asscotions between different indicators. For mRNA and protein expression analysis in intestinal biopsies,19 CD cases,17 UC cases and 69 HCs were recruited, and 3 to 5 pieces of biopsies were obtaind for each donor for total RNA and protein extraction. Real-time PCR and western blot were used to detect the mRNA and protein expressions of NPSR1-A, NPSR1-B, NPS, Tbet, GATA3, RORc and Foxp3. Bivariate correlation analysis was also perfonned to explore the asscotions between different indicators. For the immunofluorescence detection,10 CD cases,10 UC cases and 10 HCs were included, and 2 to 3 pieces of mucosal biopsies were obtained from intestine. The distribution of CD4, NPSR1-A and NPSR1-B in the intestinal mucosa was determined. A siRNA sequence which could effectively interfere the expressions of NPSR1 in vitro was identified. GO analysis based on detection of mRNA expression was performed to speculate the influence of downregulation of NPSR1 on the functions of CD4+ T cells from a holistic perspective. Flow cytometry, real-time PCR and CBA detection were used to observe the impact of downregulation of NPSR1 on the differentiation of CD4+ T cells and the immune responses of Th1, Th2, Th17 types. EdU incorporation was adopted to determine the effect of NPSR1 inhibition on the proliferation of CD4+ T cells. Flow cytometry was usded to evaluate the impact of NPSR1 inhibition on the apoptosis and the cell-cycles of CD4+ T cells. Real-time PCR was performed to detect the influence of downregulation of NPSR1 on the mRNA expressions of chemokines and their receptors. A transwell system was used to assess the impact of NPSR1 inhibition on the migration of CD4+ T cells towards some chemokines.Results The distribution of genotype and allelotype of NPSR1 rs324377 in CD was significantly different from that of HC(P=0.003 and 0.019, respectively). The difference of genotype frequencies of all the six SNPs between UC and HC didn't reach statistical significance, and the difference of allelotype frequencies of rs324384 and rs740347 between UC and HC was statistically different (P=0.026 and 0.047, respectively). The frequencies of haplotypes between CD or UC and HC also have statistical difference. Genotype and clinical phenotypes analysis revealed that the polymorphisms of rs324377 for CD and rs324384 and rs740347 for UC were statistically associated with gender, age at diagnosis, disease location (extent) and disease behavior. Compared with the controls, IBD cases had higher levels of mRNA and protein expressions of NPSR1. The mRNA and protein expressions of Tbet, GATA3, RORc and Foxp3 also increased or decreased to varying degrees in IBD PB or intestinal mucosa when compared with HC. Bivariate correlation analysis revealed that the mRNA and protein expressions of NPSR1 were consistent with that of Tbet and RORc. immunofluorescence detection indicated that the ratio of CD4+NPSR1+ cells was increased in IBD compared to HCs (P<0.05). GO analysis revealed that there were obvious difference about the functions of CD4+ T cells between the siRNA-101 group and siRNA-N group, including proliferation, apoptosis and immune process. Further studies found that siRNA-101 could reduce the mRNA expressions of Tbet, GATA3, RORc and Foxp3 (P<0.05), decrease the frequencies of CD4+Tbet+ cells and CD4+RORc+ cells (P<0.05), inhibit the mRNA and protein expressions of IL2, TNFa, IL4,IL6, IL10, IL17A (P<0.05), restrain the proliferation of CD4+ T cells (P<0.05), promote the apoptosis of CD4+ T cells (P<0.05), induce a G0/G1 arrest (P<0.05), and enhance the migration of CD4+ T cells towards to CCL2 (P<0.05).Conclusions 1) NPSR1 gene was a susceptibility locus for IBD in Chinese Han population. Its genotype polymorphisms were assciaoted with IBD clinical phenotype, which needs to be verified in a different population.2) The mRNA and protein expressions of NPSR1 were elevated in IBD patients both systematically and locally compared to HC, but more significantly in CD. Meanwhile the frequency of NPSR1+ cells was also increased in IBD intestinal mucosa, which increased our confidence that NPSR1 could be a potential target for IBD therapy.3) There were close connections between NPSR1 and CD4+ T cells.4) Downregulation of NPSR1 could inhibit the differentiations of Th1 cells and Th17 cells, restrain immune responses of Th1, Th2 and Th17 types. Downregulation of NPSR1 could depress the proliferation of CD4+ T cells, promote its apoptosis and induce a G0/G1 phase arrest, which leaded to the decrease of the number of CD4+ T cells. Downregulation of NPSR1 could also enhance the mRNA expression of CCL2 and boost the migration of CD4+ T cells to CCL2, which might be association with the ability of CCL2 to recruit Treg cells. Overall, downregulation of NPSR1 could inhibit CD4+T cells-mediated inflammatory responses significantly, which might be an important theoretical basis how NPSR1 take part in the pathogenesis of IBD. |
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