The Relations Between TLR/NF-κB Signal Pathway And IBD & Its Negative Factor SIGIRR

Objective:To discuss the role of TLR/NF-κB signal pathway in IBD and the mechanism of negative regulatory of its inhibitor SIGIRR on LPS/TLR inflammatory signal pathway. To provide new way of treatment of IBD.Methods:⑴The changes of TLR/NF-κB signal pathway in the intestinal mucosa of patients with ulcerative colitis and their relations: The protein expressions of TLR4 and NF-κB P65 in 68 biopsy intestinal mucosa samples were determined by immunohistochemistry, 68 biopsy samples including: UC 53 (according to Tmelove-Richards Classification: grades I: 9, grades II: 15, grades III: 19) and the control group 15.⑵The effect of SIGIRR on LPS/TLR signal pathway of macrophage cells:①Macrophage cells Raw264.7 non-transfected with SIGIRR was used as SIGIRR low expressing cell strain. Whereas macrophage cells Raw264.7 were transfected with pUNO-mSIGIRR using LipofectamineTM2000 to construct SIGIRR high expressing cell strain.②Groups: Control group 1:Raw264.7;Control group 2: Raw264.7 + pUNO ( blank plasmid); Control group 3: Raw264.7 cell + pUNO-mSIGIRR; LPS Group 1:Raw264.7 cell +LPS;LPS Group 2: Raw264.7 + pUNO(blank plasmid) + LPS;LPS Group 3: Raw264.7 cell + pUNO-mSIGIRR + LPS;LPS groups were incubated with 10 ng/ml LPS medium for 12 hours after transfection;③The mRNA expressions of SIGIRR, TLR2, TLR4, MyD88, IRAK-1 and TRAF6 were determined by RT-PCR.Results:⑴The changes of TLR/NF-κB signal pathway in the intestinal mucosa of patients with ulcerative colitis and their relations:①The expressions of TLR4 and NF-κB P65 in the intestinal mucosa of patients with ulcerative colitis were 50/53(94.3%) and 46/53(86.8%), respectively, which were higher than those in control groups 6/15(40%)和5/15(33.3%), respectively, (P<0.01). And the expressions were higher when the pathological changes were more serious;②The expressions of TLR4 and NF-κB P65 in the intestinal mucosa of patients with ulcerative colitis were significantly positive correlated (r =0.923,P<0.01).⑵The effect of SIGIRR on LPS/TLR signal pathway in macrophage cells:①The mRNA expression of SIGIRR in Raw264.7 cells transfected with SIGIRR was 0.470±0.045, which was significantly higher than those in cells non-transfected or transfected with pUNO (0.210±0.021 and 0.212±0.027,P<0.001).②In Raw264.7 cells transfected or non-transfected with SIGIRR, the mRNA expressions of SIGIRR in cells stimulated with LPS were significantly lower than those in control groups (0.114±0.052 vs 0.210±0.021, 0.109±0.047 vs 0.212±0.027, 0.302±0.036 vs 0.470±0.045)(P<0.001).③The mRNA expressions of TLR2 in Raw264.7 cells transfected or non-transfected were not affected with LPS (0.222±0.037 vs 0.218±0.036, 0.220±0.039 vs 0.215±0.035, 0.217±0.032 vs 0.205±0.029);and the mRNA expressions of TLR2 in Raw264.7 cells were not affected with SIGIRR (P>0.05).④In Raw264.7 cells transfected or non-transfected with SIGIRR, the mRNA expressions of TLR4 in cells stimulated with LPS were significantly higher than those in control groups (0.587±0.103 vs 0.309±0.046 , 0.564±0.092 vs 0.291±0.050, 0.558±0.085 vs 0.210±0.062)(P<0.001); the mRNA expressions of TLR4 of Raw264.7 cells were not affected with SIGIRR (P>0.05);⑤In Raw264.7 cells non-transfected with SIGIRR, the mRNA expression of MyD88 of cells stimulated with LPS were significantly higher than those in control groups (0.509±0.134 vs 0.890±0.144, 0.480±0.134 vs 0.974±0.166)(P<0.001), Whereas in Raw264.7 cells transfected with SIGIRR, the mRNA expressions of MyD88 were not significantly different in those two groups (0.478±0.151 vs 0.341±0.130)(P>0.05). In LPS groups, the mRNA expressions of MyD88 in Raw264.7 cells transfected with SIGIRR (0.341±0.130) were significantly lower than those in cells non-transfected (0.890±0.144) or transfected with pUNO (0.974±0.166) (P<0.001) Whereas in control groups, the mRNA expressions of MyD88 were not affected with SIGIRR (0.509±0.134, 0.480±0.134, 0.478±0.151) (P>0.05).⑥In Raw264.7 cells non-transfected with SIGIRR, the mRNA expressions of IRAK1 of cells stimulated with LPS were significantly higher than those in control groups (0.964±0.208 vs 0.522±0.096, 1.042±0.256 vs 0.491±0.121) (P<0.001),Whereas in Raw264.7 cells transfected with SIGIRR, the mRNA expressions of IRAK1 were not significantly different in those two groups (0.440±0.114 vs 0.530±0.134) (P>0.05); In LPS groups, the mRNA expressions of IRAK1 in Raw264.7 cells transfected with SIGIRR (0.440±0.114) were significantly lower than those in cells non-transfected (0.964±0.208) or transfected with pUNO (1.042±0.256) (P<0.001), Whereas in control groups, the mRNA expressions of IRAK1 were not affected with SIGIRR (0.522±0.096, 0.491±0.121, 0.530±0.134) (P>0.05).⑦In Raw264.7 cells transfected or non-transfected with SIGIRR, the mRNA expressions of TRAF-6 in cells stimulated with LPS were significantly higher than those in control groups (0.458±0.039 vs 0.263±0.051, 0.444±0.049 vs 0.268±0.043, 0.320±0.061 vs 0.247±0.034) (P<0.001 or P<0.01); In LPS groups, the mRNA expressions of TRAF-6 in Raw264.7 cells transfected with SIGIRR (0.320±0.061) were significantly lower than those in cells non-transfected (0.458±0.039) or transfected with pUNO (0.444±0.049) (P<0.001), Whereas in control groups, the mRNA expressions of TRAF-6 were not affected with SIGIRR (0.263±0.051, 0.268±0.043, 0.247±0.034) (P>0.05)Conclusion⑴The expressions of TLR4 and NF-κB p65 in the intestinal mucosa of patients with UC were significantly upregulated, which may increase the sensibilities of intestinal epithelial cells and lamina propria mononuclear cells to bacteria and endotoxin in intestine, and generate excessive immune inflammatory response, thus cause IBD.⑵The expressions of TLR4 and NF-κB P65 in the intestinal mucosa of patients with ulcerative colitis were higher when the pathological changes were more serious, which indicate that TLR4/ NF-κB signal pathway participate in the occurrence and development of UC.⑶LPS can significant inhibit the expression of SIGIRR in macrophage cells Raw 264.7, and upregulate the expressions of TLR4 and the downstream molecules (MyD88,IRAK-1 and TRAF6) of the signal pathway.⑷SIGIRR provided extrinsically can block the activation LPS/TLR signal pathway, and downregulate the expression of MyD88,IRAK-1 and TRAF6, which may be one of the mechanisms of negative regulatory to the TLR signal pathway. ⑸SIGIRR provided extrinsically has no effect on the TLR signal pathway of macrophage cells Raw264.7 not-stimulated with LPS, but can block the activation of LPS to the TLR signal pathway, thus inhibit the immune inflammatory response induced by LPS. Therefore, SIGIRR may be one of therapeutic targets of IBD and other inflammatory diseases.