WDR5在大鼠肾脏缺血再灌注损伤中的作用及机制研究

The Role and Mechanism of WDR5 in Rat Renal Ischemia Reperfusion Injury

目的:探讨WDR5基因在肾脏缺血再灌注损伤中的作用及机制。方法:成年雄性SD大鼠48只,按照随机数表法分为对照组、缺血再灌注损伤组(IR组)、药物溶剂组(DMSO组)、WDR5-0103低剂量组(1mg/kg)、WDR5-0103中剂量组(5mg/kg)、WDR5-0103高剂量组(25mg/kg)。对照组:仅切除大鼠右肾;IR组:切除大鼠右肾后用无创伤血管夹夹闭大鼠左肾肾蒂血管45min,松开血管夹恢复血流再灌注,模拟缺血再灌注过程;DMSO组:手术同IR组,在闭合左肾血管45min,松开血管夹后腹腔注射DMSO;不同浓度WDR5-0103组:腹腔注射不同浓度的WDR5-0103,余同IR组。检测各组大鼠血清尿素氮和肌酐水平,免疫组化染色检测WDR5蛋白表达变化,TUNEL染色检测细胞凋亡,qRT-PCR检测肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和细胞间黏附分子-1(ICAM-1)表达水平,Western Blot检测TLR4蛋白和核因子-κB(NF-κB)蛋白表达。结果:与对照组相比,IR组大鼠血Cr和BUN水平升高,肾组织WDR5蛋白水平升高,肾组织损伤明显,细胞凋亡率增加,TNF-α、IL-1β和ICAM-1的mRNA水平明显升高,TLR4/NF-κB蛋白表达水平明显升高(P均<0.01)。与IR组相比,不同浓度的WDR5-0103组血Cr和BUN水平显著降低,且25mg/kg组降低最明显;WDR5-0103高剂量组WDR5的蛋白表达降低;大鼠肾组织的病理学损害减轻,细胞凋亡率明显减少;TNF-α、IL-1β和ICAM-1mRNA水平明显降低;TLR4/NF-κB表达水平也明显降低(P均<0.01)。结论:WDR5-0103可减轻IR后的细胞凋亡与炎性反应,从而调控肾脏缺血再灌注损伤,其机制可能与下调TLR4/NF-κB通路有关。

Objective:To investigate the role and mechanism of WDR5 gene in renal ischemia reperfusion injury.Method:48 adult male SD rats were divided into control group,ischemia reperfusion injury group(IR group),medicine solvent group(DMSO group),WDR5-0103 low dose group(1mg/kg),WDR5-0103 medium dose group(5mg/kg),WDR5-0103 high dose group(25mg/kg).Control group:only the right kidney of the rat was removed;IR group:after the right kidney was removed,the left kidney renal pedicle vessel of the rat was clamped with a non-traumatic vascular clip for 45 minutes.The vascular clip was released to restore blood flow and reperfusion to simulate ischemia and reperfusion.DMSO group:the operation was the same as the I/R group,the left renal blood vessel was closed for 45 minutes,and DMSO was injected intraperitoneally after the vascular clamp was released;WDR5-0103 group:before the establishment of the IR model,different concentrations of WDR5-0103 were injected intraperitoneally.Serum urea nitrogen and creatinine levels in each group of rats were detected immunohistochemical staining was used to detect changes of WDR5 protein expression,TUNEL staining was used to detect cell apoptosis,Realtime-PCR was used to detect TNF-α,IL-1βand ICAM-1 expression levels,Western Blot was used to detect TLR4 protein and NF-κB protein expression.Results:Compared with the control group,the blood Cr and BUN levels of the rats in the IR group increased,the renal tissue WDR5 protein level increased,the renal tissue was significantly adjusted:the number of apoptotic cells increased,and the levels of TNF-α,IL-1βand ICAM-1 The mRNA level was significantly increased,and the TLR4/NF-κB protein expression level was significantly increased(all P<0.01);compared with the IR group,the blood Cr and BUN levels of the WDR5-0103 group at different concentrations were significantly reduced,and the 25mg/kg group was most obvious;WDR5-0103 high-dose group WDR5 protein expression decreased;the pathological damage of rat kidney tissue was reduced,and the number of apoptotic cells was significantly reduced;TNF-α,IL-1βand ICAM-1 mRNA levels were significantly decreased;TLR4/NF-κB The expression level of NF-κB was significantly reduced(all P<0.01).Conclusion:WDR5-0103 can alleviate cell apoptosis and inflammatory response after IR,thereby regulate renal ischemia-reperfusion injury.The mechanism may be related to down-regulation of TLR4/NF-κB pathway.