冷诱导RNA结合蛋白在心肌缺血再灌注小鼠急性肾损伤中的作用:与NF-κB信号通路的关系

Role of cold-inducible RNA-binding protein in acute renal injury in a mouse model of myocardial ischemia-reperfusion:relationship with NF-κB signaling pathway

目的评价冷诱导RNA结合蛋白(CIRP)在心肌缺血再灌注小鼠急性肾损伤中的作用及其与NF-κB信号通路的关系。方法SPF级健康雄性C57BL/6小鼠24只,6~8周龄,体质量24~28 g,采用随机数字表法分为3组(n=8):假手术组(Sham组)、心肌缺血再灌注组(I/R组)和心肌缺血再灌注+CIRP衍生肽C23组(I/R+C23组)。采用结扎左冠状动脉前降支30 min,再灌注120 min的方法制备心肌缺血再灌注小鼠急性肾损伤模型,I/R+C23组分别于心肌缺血前和再灌注前腹腔注射CIRP衍生肽C238 mg/kg,Sham组小鼠只穿线不结扎。于再灌注120 min时分离右侧颈内动脉取血样,采用ELISA法检测血清CK-MB、LDH、Cr和BUN水平;取部分肾组织,观察病理学结果,进行肾小管损伤评分;采用Western blot法检测NF-κB、p-NF-κB、NOD样受体蛋白3(NLRP3)、IL-1β和IL-18的表达;RT-PCR法检测Toll样受体4(TLR4)、NLRP3、IL-1β、TNF-α及IL-6的mRNA表达。结果与Sham组比较,I/R组血清CK-MB、LDH、Cr、BUN水平和肾小管损伤评分升高,p-NF-κB、NLRP3、IL-1β和IL-18表达上调,TLR4、NLRP3、IL-1β、TNF-α和IL-6的mRNA表达上调(P<0.05),肾脏组织病理学损伤加重;与I/R组比较,I/R+C23组血清CK-MB、LDH、Cr、BUN水平和肾小管损伤评分降低,p-NF-κB、NLRP3、IL-1β和IL-18表达下调,TLR4、NLRP3、IL-1β、TNF-α和IL-6的mRNA表达下调(P<0.05),肾脏组织病理学损伤减轻。结论CIRP参与了心肌缺血再灌注小鼠急性肾损伤的过程,与激活NF-κB信号通路,促进炎症反应有关。

Objective To evaluate the role of cold-inducible RNA-binding protein(CIRP)in acute renal injury in a mouse model of myocardial ischemia-reperfusion(I/R)and the relationship with nuclear factor kappa B(NF-κB)signaling pathway.Methods Twenty-four SPF-grade healthy male C57BL/6 mice,aged 6-8 weeks,with body mass index of 24-28 g,were divided into 3 groups(n=8 each)using a random number table method:sham operation group(Sham group),myocardial I/R group(I/R group)and myocardial I/R+CIRP-derived peptide C23 group(I/R+C23 group).The model of myocardial I/R was developed by ligation of the left anterior descending coronary artery for 30 min followed by 120-min reperfusion in anesthetized animals.CIRP-derived peptide C238 mg/kg was intraperitoneally injected before myocardial ischemia and reperfusion in I/R+C23 group,while Sham group was only threaded without ligation.Blood samples were collected from the right internal carotid artery at 120 min of reperfusion for determination of the serum creatine kinase isoenzymes(CK-MB),lactic dehydrogenase(LDH),creatinine(Cr)and blood urea nitrogen(BUN)concentrations.Renal tissues were obtained for examination of the pathological changes,and the tubular injury score was assessed.The expression of NF-κB,phosphorylated NF-κB(p-NF-κB),Nod-like receptor protein 3(NLRP3),interleukin-1beta(IL-1β)and IL-18 in renal tissues was detected by Western blot.The expression of Toll-like receptor 4(TLR4),NLRP3,IL-1β,TNF-αand IL-6 mRNA was determined by real-time polymerase chain reaction.Results Compared with Sham group,the levels of serum CK-MB,LDH,Cr and BUN and renal tubule injury score were significantly increased,the expression of p-NF-κB,NLRP3,IL-1βand IL-18 was up-regulated,the expression of TLR4,NLRP3,IL-1β,TNF-αand IL-6 mRNA was up-regulated(P<0.05),and the pathological injury to renal tissues was aggravated in I/R group.Compared with I/R group,the serum CK-MB,LDH,Cr,BUN and renal tubular injury score were significantly decreased,and the expression of p-NF-κB,NLRP3,IL-1βand and IL-18 was down-regulated,the expression of TLR4,NLRP3,IL-1β,TNF-αand IL-6 mRNA was down-regulated(P<0.05),and the pathological injury to renal tissues was alleviated in I/R+C23 group.Conclusions CIRP is involved in the process of acute renal injury in a mouse model of myocardial I/R,which is associated with activation of NF-κB signaling pathway and promotion of inflammatory responses.