姜黄素对小鼠肾小管上皮细胞体外再灌注损伤的保护作用及机制

Protective effect of curcumin against reperfusion injury of mouse renal tubular epithelial cells in vitro

目的研究姜黄素对小鼠肾小管上皮细胞体外再灌注损伤的保护作用及机制。方法采用Percoll溶液密度梯度离心法,分离并培养小鼠肾小管上皮细胞。培养的细胞分为空白对照组、缺氧再灌注组、姜黄素低剂量组(5μmol/L)及高剂量组(25μmol/L)。流式细胞仪分析细胞凋亡率;荧光定量PCR及蛋白质印迹法检测细胞内TLR4表达,蛋白质印迹法检测NF-κB p65磷酸化;荧光定量PCR检测各组细胞内IL-6、IL-1β、TNF-α和MCP-1的mRNA水平,ELISA法检测培养液上清内上述因子的含量。结果分离的肾小管上皮细胞纯度可以达到95%以上,活性高于93%。与缺氧再灌注组相比,姜黄素低剂量及高剂量组细胞凋亡率降低(P<0.01),细胞内TLR4表达降低(P<0.01),p65磷酸化受到抑制,细胞内IL-6、IL-1β、TNF-α、MCP-1的mRNA水平以及培养液上清内上述因子含量降低(P<0.05或P<0.01)。结论姜黄素对小鼠肾小管上皮细胞体外再灌注损伤具有保护作用,其机制可能与抑制细胞内TLR4表达并减轻TLR4所激发的炎性损伤有关。

Objective To evaluate the protective effect of curcumin against reperfusion injury of mouse renal tubular epithelial cells in vitro and the related mechanisms.Methods The renal tubular epithelial cells（TECs） were isolated by an improved Percoll density gradient centrifugation method.The cultured TECs were divided into blank group,hypoxia reperfusion group,low-concentration curcumin（5 μmol/L） and high-concentration curcumin（25 μmol/L） groups.Apoptotic rate of TECs was examined with FACS,and the mRNA and protein levels of TLR4 were detected with RT-PCR and Western blotting analysis,respectively.Meanwhile,expression of IL-6,IL-1β,TNF-α and MCP-1 mRNA in TECs was examined by RT-PCR,and their contents in the supernatants of TECs were measured by ELISA.Results The purity of the isolated TECs was higher than 95%,and the cell viability was higher than 93%.Compared with hypoxia reperfusion group,the two curcumin groups exhibited significantly lower apoptotic rates（P0.01） and lower levels of TLR4 mRNA and protein（P0.01）,inhibited P65 phosphorylation,and significantly decreased levels of IL-6,IL-1β,TNF-α and MCP-1 in the TECs（at both mRNA and protein levels） and their supernatant（P0.05 or P0.01）.Conclusion Curcumin can protect TECs from reperfusion injury in vitro,which might be associated with the inhibition of TLR4 expression and the consequent attenuation of TLR4-trigered inflammatory damages.