| Objective To evaluate the prevention of DSS-induced acute ulcerative colitis(UC) in mice with TLR2 and TLR4 monoantibodies.Methods Fifty male BALB/c mice were randomly assigned into five groups from A to E:the control group(group A), the UC model group (group B), the TLR2mAb intervention group(group C), the TLR4mAb intervention group(group D), the TLR2mAb and TLR4mAb intervention group (group E). Group B to group E were given 5.0% DSS solution as drinking water for days 1-7 to induce acute intestinal inflammation, while group A drank distilled water freely. Groups C, D and E received TLRmAb injection intraperitoneally once on days 1,3,5 and 7 with 10μg TLR2mAb each time for the group C, with 10μg TLR4mAb each time for group D, with 10μg TLR2mAb and 10μg TLR4mAb each time for group E. Group A and B received a normal sodium injection intraperitoneally as control. Daily disease activity index (DAI) and histopathological score (HS) were evaluated. The quantities of mRNA for TLR2, TLR4, IFN-γ, IL-4 and IL-17 were detected by Realtime-PCR, the protein expression of P-P38αMAPK, c-jun, c-fos and P-IKK were examined by Western Blotting assay, the activity of NF-κB was measured by EMSA.ResultsPart 1 TLR2mAb and TLR4mAb improve the symptoms and histopathological change in murine DSS-induced acute colitisMice with DSS-induced colitis(model group) showed typical symptoms (like diarrhoea,hematochezia and bodyweight loss) of acute ulcearative colitis. The DAI in group A to group E were 0.50±0.707,9.70±2.406,6.75±3.495,7.75±3.919,5.70±3.498 respectively. The DAI in the UC model group were much higher than those in the control group (P<0.01); the DAI in group E were much lower than those in group B(P<0.05) and higher than group A(P<0.01); The DAI in group C and group D had no statistical significance with group B(P>0.05). The HS in group A to group E were 6.40±3.273,16.50±2.991,13.25±3.576,14.50±3.625,9.50±3.308 respectively. The HS in the UC model group were much higher than those in the control group (P<0.01); the HS in group E were much lower than those in group B(P<0.01) and group D(P<0.05), and had no statistical significance with group A(P>0.05); the HS in group C and group D had no statistical significance with group B(P>0.05).Part 2 TLR2mAb and TLR4mAb suppress the expression of cytokine in murine DSS-induced acute ColitisThe mucosal TLR2mRNA expression in group A to group E were 0.26±0.397,1.96±1.558,0.64±1.030,0.39±0.594,0.42±0.461 respectively. The TLR2mRNA expression in the UC model group were much higher than those in the control group (P<0.01); the TLR2mRNA expression in group C, Dand E were much lower than those in group B(P<0.05), and had no statistical significance with group A(P>0.05).The mucosal TLR4mRNA expression in group A to group E were 0.52±0.414,1.16±0.568,0.45±0.346,0.51±0.374,0.44±0.335 respectively. The TLR4mRNA expression in the UC model group were much higher than those in the control group (P<0.01); the TLR4mRNA expression in group C, Dand E were much lower than those in group B(P<0.01, P<0.05, P<0.01), and had no statistical significance with group A(P>0.05).The levels of IFN-γmRNA in group A to group E were 0.80±0.802,2.40±1.089,0.95±1.075,0.97±0.716,0.93±0.661 respectively. The levels of IFN-γmRNA in the UC model group were markedly higher than those of the control group(P<0.01); the levels of IFN-γmRNA in group C, Dand E were much lower than those in group B(P<0.05, P<0.05, P<0.01), and had no statistical significance with group A(P>0.05).The levels of IL-4mRNA in group A to group E were 0.12±0.107,0.86±1.185,0.02±0.025,0.02±0.025,0.03±0.024 respectively. The levels of IL-4mRNA in the UC model group were markedly higher than those of the control group(P<0.05); the levels of IL-4mRNA in group C, D and E were much lower than those in group B and group A(P<0.01).The levels of IL-17mRNA in group A to group E were 0.18±0.230,0.61±0.296,0.18±0.236,0.17±0.197,0.06±0.039 respectively. The levels of IL-17mRNA in the UC model group were markedly higher than those of the control group(P<0.01); the levels of IL-17mRNA in group C, Dand E were much lower than those in group B(P<0.01), and had no statistical significance with group A(P>0.05).Part 3 TLR2mAb and TLR4mAb suppress the TLR2 and TLR4 signaling pathway in murine DSS-induced acute colitisThe levels of P-P38aMAPK in group A to group E were 0.45±0.037,1.42±0.131,1.57±0.068,0.89±0.033,0.75±0.024 respectively. The levels of P-P38aMAPK in group B, C and D were markedly higher than those of group A (P<0.01); the levels of P-P38αMAPK in group D and group E were much lower than those in group B(P<0.01); those in group C had no statistical significance with group B(P>0.05); those in group E had no statistical significance with group A(P>0.05).The levels of c-jun in group A to group E were 0.28±0.060,1.17±0.074,1.00±0.078,0.75±0.183,0.42±0.062 respectively. The levels of c-jun in group B, C and D were markedly higher than those of group A (P<0.01); the levels of c-jun in group D and group E were much lower than those in group B(P<0.01); those in group C had no statistical significance with group B(P>0.05); those in group E had no statistical significance with group A(P>0.05).The levels of c-fos in group A to group E were 0.46±0.013,0.48±0.010,0.46±0.017,0.45±0.012,0.45±0.015 respectively. The level of c-fos was similar in group A to E(P>0.05).The levels of P-IKK in group A to group E were 0.11±0.022,0.66±0.013,0.34±0.014,0.18±0.010,0.14±0.013 respectively. The levels of P-IKK in group B, C and D were markedly higher than those of group A (P<0.01); the levels of P-IKK in group C, D and E were much lower than those in group B(P<0.01); the levels of P-IKK in group D and group E were much lower than those in group C (P<0.01); those in group E had no statistical significance with group A(P>0.05).The activity of NF-κB in group A to group E were 5.18±0.685,21.24±1.150,18.13±1.45,6.15±0.429,6.12±0.575 respectively. The activity of NF-κB in group B and group C were markedly higher than those of group A (P<0.01); the activity of NF-κB in group C, D and E were much lower than those in group B(P<0.01); the activity of NF-κB in group D and group E were much lower than those in group C (P<0.01); those among group D, E and A had no statistical significance (P>0.05).Conclusions Expression of TLR2 and TLR4 increased in the colonic mucosa of mice with DSS-induced colitis. TLR4mAb can prevent the development of DSS-induced colitis through the TLR2 and TLR4 mediated P-P38αMAPK-c-jun pathway and NF-κB pathway, which can decrease the protein expression of P-P38αMAPK, c-jun, p-IKK and the activity of NF-κB, While TLR2mAb can prevent the development of DSS-induced colitis through the TLR2 and TLR4 mediated NF-κB pathway, which can decrease the protein expression of p-IKK and the activity of NF-κB, and down-regulate the mRNA expression of cytokines (IFN-γ,IL-4,IL-17), relieve symptoms (like diarrhoea,hematochezia and bodyweight loss) and colonic mucosa inflammatory reaction. TLR2mAb and TLR4mAb have similar prevention and obvious synergy in DSS-induced colitis.
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