The Preliminary Study For Toll-like Receptors Expression Influences Cardiomyocytes Apoptosis In Heart Xenotransplantation

Objective: After heart transplantation, acute vascular rejection induces infiltration in neutrophils, death in vascular endothelial cell, edema, hemorrhage and apoptosis in cardiomyocytes. We designed this study to explore the TLRs’ expression and the TLR-NF-κB signaling pathway in cardiomyocytes apoptosis.Methods: Cells culture:(1) Cardiomyocytes were isolated from C57BL6 mice.(2) Splenocytes were harvested from SD rats and BALB/C mice. Cells models: Heart allotransplantation and xenotransplantation cells models were structured between C57BL6 mice and BALB/C mice, C57BL6 mice and SD rats respectively. Cardiomyocytes from C57BL6 mice were divided into 6 groups:(1) A Group: with PBS;(2) B Group: with TNF-α;(3) C Group: with splenocytes from BALB/C mice;(4) D Group: with splenocytes and serum from BALB/C mice;(5) E Group: with splenocytes from SD rats;(6) F Group: with splenocytes and serum from SD rats. After a co-culture time of 108 h, the levels of apoptosis in cardiomyocytes were examined by Annexin-V/PI kit, and the differences between allotransplantation and xenotransplantation were achieved by comparision. The expressions of TLR-1, 2, 3, 4, 5, 7, 9 from cardiomyocytes were evaluated by RT-PCR in A Group, C Group, D Group, E Group, F Group. The most activated one had been found. After blocked the TLR pathway by antibody, the levels of apoptosis and NF-κB p65 expressions in cardiomyocytes were examined by Annexin-V/PI kit and Western-blot respectively.Results: Cardiomyocytes from C57BL6 mice were isolated and cultured by enzyme digestion successfully, so that the vitality and purity(80%-90%) were feasible. In the experiment of cardiomyocytes apoptosis, the levels of apoptosis in B, E and F groups were 71.31±7.71%, 70.05±5.73%, 68.61±4.71%. So they were decreased markedly compare with A, C and D groups(P<0.001). The levels of Toll-Like Receptors expression in untreated cardiomyocytes were low and unactivated. TLR-2 and TLR-3 expressions were promoted obviously in E group and F group among all of them(P<0.05), and TLR-3 was the higher(P<0.001). After blocked TLR-3 signal pathway in the cells model, the levels of apoptosis in cardiomyocytes were declined(P<0.05), and they were similar to NF-κB p65(P<0.05).Conclusion: 1. For the mouse-to-rat heart xenotransplantation in vitro, TLR-2 and TLR-3 were concerned in the process of cardiomyocytes apoptosis; in addition that TLR-3 played the most important role in all TLRs.2. The suppression of Toll-Like Receptor 3 expression, which was able to make an inhibition to NF-κB pathway, could attenuate the cardiomyocytes apoptosis.