| Background and objective:Sepsis is a systemic inflammatory response syndrome caused by infection. It is one of serious complications in trauma, burns, shock, infection and other clinical acute and severe patients. Furthermore, it is the important reasons which can induce multiple organ disorder syndromes. The mortality rate of sepsis can be as high as 30% -50% in severe cases. TLRs (Toll like receptors) signal pathway plays a vital role in inflammatory response of sepsis. TLRs can sense a different PAMPs (pathogen associated molecular patterns), then it can cause a series of inflammatory response through MyD88 (myeloid differentiation factor 88), TRIF (TIR-domain-containing adapter-inducing interferon-β) and other downstream junction protein. Many studies show that, after pro-contacted with PAMPs, the body is exposed to the same high-dose ingredients or bacteria again, and the sensitive of response will be down-regulation, which is called immune tolerance. We can observe that it reduced the production of inflammatory cytokine, which would improve survival rate of animals. However, in clinical practice, it is difficult to induce tolerance by given small doses of pathogens in advance. Research has found that BLP (bacterial lipoprotein)-induced septic shock is associated with TLR2. In this study, we investigated whether the anti-TLR2 antibody can reduce the production of inflammatory factors induced by THP-1 cells attacked by BLP. We also investigated the change of the signal pathway of TLR2.Methods:1. The experiment was divided into 5 groups. The blank control group: Neither TLR2 antibody nor BLP were added into THP-1 cells; The isotypism control group: After adding homologous immunoglobulin of anti-TLR2 antibody (10μg/ml) the THP-1 cells were incubated at room temperature 0.5h, and then incubated in CO2 for 14h with BLP (1000ng/ml); The direct attack group: BLP (1000ng/ml)was added into THP-1 cells directly then they were incubated in CO2 for 14h; The A+B group: After adding anti-TLR2 antibody (10μg/ml) they were incubated at room temperature 0.5h, and then were incubated in CO2 for 14h with the BLP (1000ng/ml). The B+A group: After adding the BLP (1000ng/ml) they were incubated at room temperature 0.5h, and then were incubated in CO2 for 14h with anti-TLR2 antibody (10μg/ml).2. The concentrations of TNF-αand IL-6 were determined by ELISA.3. Western Blotting was used to test the expression of NF-κB in nuclear, TLR2 and MyD88 in cytoplasm.4.The effect of anti-TLR2 antibody on the nuclear translocation of P65 was examined by immunofluorescence.Results:1. The expressions of TNF-αin the blank control group, the isotypism control group, the direct attack group ,the A+B group and the B+A group were: 98.14±22.14 pg/ml,1423.18±363.14 pg/ml,1408.05±420.00 pg/ml,1026.59±286.76 pg/ml and 878.80±349.02 pg/ml ;Their expressions of IL-6 were: 0 pg/ml,140.69±59.79 pg/ml,155.44±73.64 pg/ml,70.60±16.19 pg/ml and 66.49±6.69 pg/ml ;The expressions of TNF-α,IL-6 in the isotypism control group, the direct attack group, the A+B group and the B+A group were significantly higher than the control group(P﹤0.05). The expressions of TNF-α,IL-6 in the A+B group and the B+A group were lower than the isotypism control group and the direct attack group, with significant difference(P﹤0.05).2. The expression of NF-κB in the isotypism control group, the direct attack group ,the A+B group and the B+A group were significantly higher than the control group(P﹤0.05). The expressions of NF-κB in the A+B group and the B+A group were lower than the isotypism control group and the direct attack group, with significant difference(P﹤0.05).3. Immunofluorescence showed that the NF-κB nuclear translocation of the A+B group and the B+A group were significantly lower than the isotypism control group and the direct attack group.4. There was no significant difference among each group in the expression of TLR2 and MyD88 (P> 0.05).Conclusions: The blocking TLR2 antibody decreased the expression of NF-κB and the release of TNF-αand IL-6 which was induced by the attacking of BLP to THP-1 cells significantly. This mechanism might provide protective effects on the body.
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