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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 study the protective effect of curcumin on renal tubular epithelial cells in vitro and its mechanism. Methods Percoll solution density gradient centrifugation was used to separate and culture mouse renal tubular epithelial cells. The cultured cells were divided into blank control group, hypoxia-reperfusion group, curcumin low dose group (5μmol / L) and high dose group (25μmol / L). Flow cytometry was used to analyze the apoptotic rate. Fluorescent quantitative PCR and Western blotting were used to detect the intracellular TLR4 expression. The phosphorylation of NF-κB p65 was detected by Western blotting. The levels of IL-6, IL-1β, TNF-α and MCP-1 mRNA levels, ELISA assay of the supernatant content of the above-mentioned factors. Results The purity of isolated tubular epithelial cells can reach more than 95%, the activity is higher than 93%. Compared with hypoxia reperfusion group, the apoptotic rates of curcumin in low and high dose groups were decreased (P <0.01), TLR4 expression decreased (P <0.01), phosphorylation of p65 was inhibited, IL-6 , IL-1β, TNF-α, MCP-1 mRNA levels and the above-mentioned factors in the culture supernatant decreased (P <0.05 or P <0.01). CONCLUSION: Curcumin can protect renal tubular epithelial cells from reperfusion injury in vitro. The mechanism may be related to the inhibition of TLR4 expression and the alleviation of TLR4-induced inflammatory injury.