Toll样受体4抑制剂对大鼠肾缺血再灌注损伤的保护作用

The protective effect of TLR4 inhibitor on renal ischemia reperfusion injury in rat

目的探讨Toll样受体4（TLR4）小分子抑制剂（TK242）对实验性大鼠肾缺血再灌注损伤的保护作用。方法36只成年健康雄性SD大鼠,体重200~250g,随机分为3组：假手术组（Sham组）,肾缺血再灌注组（IR组）和TAK242治疗组（IR＋TAK242组）。Sham组切除右肾后仅分离左肾动脉而不夹闭,而IR组与TAK242治疗组则于右肾切除后分离左肾动脉,利用无损伤血管夹夹闭左肾动脉45分钟后恢复灌注。TAK242治疗组术后立即腹腔注射给予TAK242（3mg/kg,溶于含1%DMSO双蒸水）,Sham组及IR组则均于同期给予相同剂量的含1%DMSO双蒸水。3组均于术后48小时处死,取血及右肾待测。利用专用试剂盒检测各组血肌酐及尿素氮变化,评估肾功能改变。再利用RT-PCR检测比较3组肾组织中肿瘤坏死因子（TNF）-α、白介素（IL）-1β、细胞间粘附因子（ICAM）-1水平变化,同时利用western印迹法评估TLR4、pIκBα、IκBα、p-NF-κB p65、NF-κB p65蛋白表达。结果应用TAK242治疗后,因肾缺血再灌注损伤导致的血肌酐（Cr）及尿素氮（BUN）升高得到明显改善（P〈0.05）,而缺血再灌注后肾组织中升高的TNF-α、IL-1β、ICAM-1 m RNA表达则明显下降（P〈0.05）。TAK242治疗还可以显著缓解肾缺血再灌注损伤诱导的肾组织TLR-4蛋白表达及IκBα和NF-κB p65磷酸化水平升高（P〈0.05）。结论 TLR4小分子抑制剂（TK242）可以减轻肾缺血再灌注损伤而起保护作用,其作用与TLR4/NF-κB信号通路介导的炎症反应相关。

Objective To explore the protective effect of Toll-like receptor（TLR） 4 inhibitor（TAK242） on renal ischemia reperfusion injury（IR） in rat. Methods 36 SD rats were divided to three groups,as Sham group,IR group,and TAK242 treatment group. IR injury was induced by clamping left renal artery for 45 min,and right kidney was resected. The rats in TAK242 group were given TAK 242 solution（3mg/kg,dissolved in double distilled water contains 1%DMSO） by intraperitoneal injection immediately after surgery,while other groups accept similar dose of double distilled water contains 1%DMSO. After 48 hours from operations,all rats were sacrificed,and blood creatinine（Cr） and usea nitrogen（BUN） were tested by ELISA kit. In addition,kidney TNF-α,IL-1β,ICAM-1 m RNA expression were analyzed by RT-PCR,while kidney protein expressions of TLR4 、p-IκBα、IκBα、p-NF-κB p65、NF-κB p65 were detected by western blot. Results The appliance of TAK242 ameliorated the increase of Serum Cr and BUN induced by IR（P〈0.05）. It also gave rise to decreases in m RNA expressions of kidney TNF- α,IL-1β,ICAM-1,as well as the increased TLR4 protein expression and IκBα and NF-κB p65 phosphorylation（P〈0.05）. Conclusion TLR4 inhibitor（TAK242） can protect renal IR via attenuating inflammation regulated by TLR4/NF-κB pathway.