The effect of IRE1-XBP1 pathway on regulation of polarization in activated Kupffer cells / 重庆医学

ABSTRACT

Objective To isolate and culture rats liver KCS ,and to explore the effect of IRE1-XBP1 pathway on regulation of polarization in activated Kupffer cells (KCs) .Methods (1)Rat KCs were isolated by Ⅳ type collagenase digestion and gradient cen-trifugation methods .(2)KCs were transfected and randomly divided into four groupsXBP1-shRNA group ,Ctrl-shRNA group , AdV-XBP1 group and Ctrl-AdV group .(3)The transfection level of KCs XBP1 ,IL-6 ,IFN-γ ,TNF-α and IL-17 were detected by RT-PCR ;the protein expression level of JAK1 ,JAK2 ,STAT1 and STAT3 were evaluated by Western blot .(4)The changes of KCs expression type in each group were detected by flow cytometry (FCM ) and the laser confocal .(5)T cells derived from rat spleen cells were co-cultured within the 4 groups of KCs mentioned above ;T cells proliferation was measured by Brdu labeling assay .(6)T cells apoptosis was determined by Annexin V /PI FCM analysis .(7)The density of IL-6 ,IFN-γ ,TNF-α ,IL-17 and IL-10 in the su-pernatant of co culture was assessed by ELISA .Results (1)The mRNA and protein level of XBP1 were measured by RT-PCR and western blot ,those in XBP1-shRNA group were significantly reduced compared with those in the other three groups ,while in AdV-XBP1 groups ,results demonstrated entirely the opposite tendency (P< 0 .05) .(2)The expression of marker molecules on the sur-face of KCs such as M HC Ⅱ ,CD86 and CD40 in XBP1-shRNA group were significantly lower (P< 0 .05) ,but CD204 and CD206 expression were much higher compared with the other three (P< 0 .05) .However the expression tendency of these surface markers were shown the opposite results in AdV-XBP1 group (P < 0 .05) .(3)Western blot revealed the XBP1-shRNA could statistically suppress the protein levels and phosphorylation of JAK 1 ,JAK2 ,STAT1 and STAT3 ,which involved in the pro inflammatory cyto-kines regulation and KCs polarization (P< 0 .05) .But in AdV-XBP1 group ,these protein and its phosphorylation were markedly promoted (P< 0 .05) .ELISA results collaborated with Western blot .(4)3 d after co cultured with KCs transfected with XBP1-shR-NA ,the levels of T lymphocyte proliferation and pro inflammatory cytokines secretion were significantly reduced ,but the levels of T lymphocyte apoptosis and anti inflammatory cytokines secretion were remarkably enhanced (P< 0 .05) .Conclusion Blockage of IRE1-XBP1 activation could alter the phenotype of active KCs to M 2 like type and attenuated the capacity of antigen present of KCs ,while up regulated the expression of IRE1-XBP1 pathway could change the phenotype of KCs to M 1 type plus the promotion of antigen present capacity .