Experimental Study On Mast Cell Activation Of Silence Effect Of PDIA3 In Dendritic Cell-line JAWSⅡ

Background and objective Intestinal mucosal immune dysfunction plays an important role in the pathogenesis of irritable bowel syndrome(IBS).The phenotype and function of intestinal lamina propria dendritic cells(LPDCs)changed in the development of PI-IBS,which induced the maintenance of intestinal mucosal immune activation and visceral hypersensitivity.The infiltration and activation of mast cells(MCs)in colonic mucosa is characteristics of IBS.Our previous study identified PDIA3 is differentially expressed in the IBS rat model and diarrhea-predominant irritable bowel syndrome(IBS-D).However,whether PDIA3 can cause abnormal immune response and then activate the MC is unknown.This study is to observe the silence effect of PDIA3 in dendritic cell-line JAWSII on mast cell activation.Methods PDIA3 in mice immature dendritic cell-line JAWSII was knocked down by lentiviral vector-mediated siRNA(LV-PDIA3-RNAi)(sh-PDIA3 group).JAWSII transfected with negative control viruses(sh-NC group)or without transfection(normal group)were regarded as the control group.The JAWSII of three groups were stimulated with LPS at 1μg/ml for 24 h,and were respectively incubated in vitro with spleen lymphocytes to perform mixed lymphocyte reaction(MLR).The supernatant of MLR was used on MCs.PDIA3 gene and protein expression in 3 groups were tested with q-PCR and Western Blot.Surface markers of JAWSII were analyzed by flow cytometry.The proliferation of CD4+T/CD8+T cells were examined by Cell Trace CFSE kits.Cytokine IL-4 and IL-9 were determined by ELISA.The vitality of MC was examined by CCK8 kits.The apoptosis of MC was detected by V/PI kits.Tryptase was determined by ELISA.The degranulation was observed by transmission electron microscope(TEM).Results Sh-PDIA3 group in compared with normal group and sh-NC group,the amount of PDIA3 gene expression(0.28±0.06 vs 1.00±0.07,0.91±0.03;P<0.01)and the amount of protein expression(0.39±0.20 vs 0.91±0.07,1.02±0.10;P<0.05)decreased.After LPS stimulation,the expression level of CD86 and MHCII raised.The secretion of IL-4 in MLR of sh-PDIA3 group decreased compared with normal group and sh-NC group(1.38±0.21 vs 2.59±0.39,2.44±0.77;P<0.05).No obvious differences was seen in the secretion of IL-9(57.89±9.32 vs 53.24±7.35,61.47±9.15;P>0.05),neither nor the MFI of CD4+T cells(26526.33±149.95 vs 26993.50±582.67,26554.67±474.09;P>0.05)and CD8+T cell(23949.67±311.51 vs 22831.67±430.00,23189.17±688.95;P>0.05).Sh-PDIA3 group in compared with normal group and sh-NC group,the vitality of MCs(0.31±0.01 vs 0.36±0.01.0.37±0.02;P<0.05)decreased,the tryptase concentration(408.46±35.32 vs 503.67±7.51,492.33±21.89;P<0.01)decreased and the early apoptosis rate(3.96%±0.27%vs 3.03%±0.19%,3.59%±0.04%;P<0.05)increased.Conclusion Silence effect of PDIA3 in dendritic cell-line JAWSII may contribute to the abnormal immune response,which reduce the secretion of IL-4 and activation of MCs and promote the apoptosis of MCs.PDIA3 may be the therapeutic target of IBS.