| Background and Objective:SIGIRR, a novel member of the TLR/IL-1R superfamilies, can negatively regulate the TLR/IL-1 signaling pathway, and mainly in gastrointestinal inflammation. IL-1F5 is a new member of IL-1 family. Research has found that interaction of IL-1F5 and SIGIRR can inhibit LPS-induced inflammation. Both are the TLR/IL-1 signaling pathway negative regulators, to provide a new way of treatment of IBD. Therefore, through this study we investigate the role of TLR/IL-1 signaling pathway and its inhibitors SIGIRR and IL-1F5 in intestinal epithelial cells stimulated by LPS, the effect of exogenous SIGIRR and IL-1F5 on the TLR/IL-1 signaling pathway, and the mechanism of its negative regulatory, to provide experimental evidence for SIGIRR and IL-1F5 used for the treatment of IBD.Methods:1. Experimental groups as follows:①control group 1: T84 cell;②control group 2: T84 cell transfected with pUNO(blank plasmid);③control group 3: T84 cell transfected with pUNO-hSIGIRR;④experimental group 1: T84 cell + 1μg/ml LPS;⑤experimental group 2: T84 cell transfected with pUNO(blank plasmid) + 1μg/ml LPS;⑥experimental group 3: T84 cell transfected with pUNO-hSIGIRR + 1μg/ml LPS.Every group encluding 5ng/ml IL-1F5 pretreatment and non-treatment.2. Intestinal epithelial cell strain T84 was pretreated with IL-1F5 and transfected with pUNO-hSIGIRR respectively, LPS groups were incubated with 1μg/ml LPS medium for 12 hours after pretreatment and transfection.3. The mRNA expression of SIGIRR, TLR2, TLR4 and MyD88 were detected by RT-PCR.4. The inhibiting rate of cell growth were determined by MTT assay. Results:1. The mRNA expression of SIGIRR in T84 cells:①The mRNA expression of SIGIRR were significantly higher in the cells transfected with SIGIRR than those in non-transfected with SIGIRR and transfected with blank plasmid(P<0.001);②The mRNA expression of SIGIRR were significantly lower in the cells of experimental groups than those of control groups(P<0.05);③The mRNA expression of SIGIRR in the cells had no change after pretreated with IL-1F5(P>0.05).2. The mRNA expression of TLR2 in T84 cells: The mRNA expression of TLR2 in the cells were not affected with LPS, SIGIRR and IL-1F5(P>0.05).3. The mRNA expression of TLR4 in T84 cells:①The mRNA expression of TLR4 in the cells had no change after transfected with SIGIRR(P>0.05);②The mRNA expression of TLR4 were significantly higher in the cells of experimental groups than those of control groups(P<0.001), In experimental groups, the mRNA expression of TLR4 in cells pretreated with IL-1F5 were significantly lower than those in cells non-treatment(P<0.05), Whereas in control groups, the mRNA expression of TLR4 were not affected with IL-1F5(P>0.05);③In experimental groups, the mRNA expression of TLR4 were significantly lower in the cells single pretreated with IL-1F5 than those in the cells single transfected with SIGIRR(P<0.05), there is no significant difference between group of single pretreatment with IL-1F5 and group of pretreatment with IL-1F5 add transfection with SIGIRR(P>0.05).4. The mRNA expression of MyD88 in T84 cells:①Whether or not IL-1F5 pretreatment, in experimental groups, the mRNA expression of MyD88 were significantly lower in the cells transfected with SIGIRR than those in non-transfected with SIGIRR and transfected with blank plasmid (P<0.001), whereas in control groups, the mRNA expression of MyD88 were not affected with SIGIRR(P>0.05); ②The mRNA expression of MyD88 were significantly higher in the cells of experimental groups than those of control groups(P<0.05), In experimental groups, the mRNA expression of MyD88 in cells pretreated with IL-1F5 were significantly lower than those in cells non-treatment(P<0.05), Whereas in control groups, the mRNA expression of MyD88 were not affected with IL-1F5(P>0.05);③In experimental groups, the mRNA expression of MyD88 were significantly lower in group of pretreatment with IL-1F5 add transfection with SIGIRR than group of single transfection with SIGIRR(P<0.05), there is no significant difference between group of single pretreatment with IL-1F5 and group of single transfection with SIGIRR(P>0.05).5. The effect of IL-1F5 and LPS on vitality of T84 cell after stimulated by IL-1F5 and LPS:The growth inhibition rates were significantly higher in all experimental groups than those in control groups significantly(P<0.001), and were significantly higher in the cells stimulated with LPS than those in cells pretreated with IL-1F5(P<0.001). The growth inhibition rates were higher in the cells stimulated by IL-1F5 and LPS than those in cells single stimulated by IL-1F5 and single stimulated by LPS significantly(P<0.001).Conclusions:1. LPS could inhibit the mRNA expression of SIGIRR in T84 cells, while increase the mRNA expression of TLR4 and the downstream molecule(MyD88) of signal pathway.2. The exogenous SIGIRR gene can block the activation on the TLR/IL-1 signaling pathway stimulated by LPS, and downregulate the mRNA expression of MyD88, which may be one of the mechanisms of its negative regulatory to the TLR/IL-1 signaling pathway.3. The exogenous IL-1F5 can block the activation on the TLR/IL-1 signaling pathway stimulated by LPS, and downregulate the mRNA expression of TLR4 and MyD88, which may be one of the mechanisms of its negative regulatory to the TLR/IL-1 signaling pathway.4. The exogenous SIGIRR and IL-1F5 together could more availably block the activation on the TLR/IL-1 signaling pathway stimulated by LPS than SIGIRR alone and IL-1F5 alone, they are receptor/ligand each other, negatively regulate the TLR/IL-1 signaling pathway, inhibit inflammation.
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