| Objective This study was designed to investigate the effect of TLR2monoclonal antibodies (Toll like receptor2monoclonal antibodies,TLR2mab) and TLR4monoclonal antibodies(Toll like receptor4monoclonal antibodies,TLR4mab) on the recipient immune regulation in the model of Wistar to SD rat cardiac allotransplantation. The mechanisms of rejection in allotransplantation will be explored in the model.Materials and methods Donor(Wistar rat) and recipient(SD rat) were randomly divided into four groups. Group A(no treatment): The heart from Wistar rat was transplanted into the abdomen of SD rat without any pre‐treatment; Group B(TLR2mAb Group): On the day before surgery,The recipients SD rats were injected with TLR2mab100ug/kg in abdomen and recieved TLR2mAb injection intraperitoneally every48hours after the success of transplantation. Group C(TLR4mAb Group): On the day before surgery,The recipients SD rats were injected with TLR4mab100ug/kg in abdomen and recieved TLR4mAb injection intraperitoneally every48hours after the success of transplantation. Group D(TLR2mAb and TLR4mab Group): On the day before surgery,The recipients SD rats were injected with TLR2mab100ug/kg plus TLR4mab100ug/kg in abdomen and recieved TLR2mAb plus TLR4mab injection intraperitoneally every48hours after the success of transplantation. Observations: The cardiac allograft mean survival time(MST), pathological changes； The proportion of peripheral blood CD4+CD25+Treg and the expressin of Foxp3protein in Tregs were detected by flow cytometry; the expression of TNF‐α,IFN‐γ,IL‐6,IL‐2,IL‐10,TGF‐β1in peripheral blood; The mRNA level of MyD88,IRF1,NF‐κB in mononuclear cells were detected by Semi‐quantitative RT‐PCR and Quantitative real‐time RT‐PCR.Results (1)MST:Group A:7.8±0.84d; Group B:25.8±2.48d; Group C:24.8±2.15d;GroupD:32.6±3.26d. Compare with Group A,B,C, The MST of Group D was significantly prolonger,P<0.05;Compare Group A, The MST of Group B,C were significantly prolonger,p<0.05.(2) The results of receptor CD4+CD25+Treg and the expression of Foxp3protein in the Treg cells in peripheral blood: Group A:2.5±0.47%,42.68±5.47%；Group B:7.98±1.33%,63.12±5.14%；Group C:8.98±1.48%,67.54±4.37%； Group D:19.08±1.46%,82.02±4.61%.The peripheral blood CD4+CD25+Treg of Group D was significantly higher compared with Group A,B,C,P<0.05(.3) The results of TNF‐α in the peripheral blood of recipient's(pg/ml): Group A538.59±13.7；Group B:271.19±44.07；Group C:268.77±29.06；Group D:157.18±26.77.The average serum levels of TNF‐α in Group B,C and D were significantly lower compared with Group A,P <0.05;and the lowest was found in group D,significantly different from the levels in group B and C,p<0.05.(4)The results of IFN‐γ in the peripheral blood of recipient's(pg/ml): Group A:527.67±62.13；Group B:176.83±17.35；Group C:154.25±18.44；Group D:104.99±14.39。The average serum levels of INF‐γ in Group B,C and D were significantly lower compared with Group A,P <0.05;and the lowest was found in group D,significantly different from the levels in group B and C,p<0.05.(5) The results of IL‐6in the peripheral blood of recipient's(pg/ml): Group A:302.21±20.76；Group B:113.59±15.07；Group C:100.07±13.98；Group D:64.75±13.98.The average serum levels of IL‐6in Group B,C and D were significantly lower compared with Group A,P <0.05;and the lowest was found in group D,significantly different from the levels in group B and C,p<0.05.(6)The results of IL‐2in peripheral blood of recipient's(pg/ml):Group A:5.87±0.22; Group B:2.94±0.13; Group C:3.6±0.14；Group D:2.11±0.14；The average serum levels of IL‐2in Group B,C and D were significantly lower compared with Group A,P <0.05;and the lowest was found in group D,significantly different from the levels in group B and C,p<0.05.(7)The results of IL‐10in the peripheral blood of recipient's(pg/ml): Group A:23.91±3.51；Group B:43.46±4.25；Group C:35.75±5.68；Group D:63.31±4.53;The serum levels of IL‐10in Group D is significantly higher compared with Group A,B,C,p<0.05; and the lowest was found in group D,significantly different from the levels in group B and C,p<0.05.(8) The results of TGF‐β1in the peripheral blood of recipient's(pg/ml):Group A:120.74±7.05;Group B:164.91±14.25;Group C:169.6±10.38;Group D:201.36±12.36; The serum levels of TGF‐β in Group D is significantly higher compared with Group A,B,C,p<0.05; and the highest was found in group D,significantly different from the levels in group B and C,p<0.05.(9) The mRNA level of MyD88,IRF1,NF‐κB:β‐actin is regarded as standard control (value=1), The mRNA levels of MyD88,IRF1,NF-κB in Group D were lower compared with Group A,B,C,p<0.05.Conclusions (1) The application of TLR2mab and TLR4mab can affect TLR2/4‐‐Myd88‐‐TRAF6‐NF‐kB signal path, lowered the mRNA level of MYD88,NF‐ΚB, reduced the related cytokines such as TNF‐α,IFN‐γ,IL‐6,IL‐2, increased IL‐10and TGF‐β1expression, so reduced acute rejection, and prolong the survival time of allograft.(2)TLR2mab and TLR4mab can blocking TLR2/4‐Myd88‐IFR1signal path,increase the proportion of peripheral blood CD4+CD25+Treg and the expression of Foxp3protein in Tregs, and significantly prolong the survival time of allograft.(3) We can observe that preventive effects of TLR2monoclonal antibodies and TLR4monoclonal antibodies can significantly reduce the acute vascular rejection (AVR) occurs, but can not completely overcome its occurrence. |
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