摘要
目的 观察右美托咪定(Dex)对肾脏缺血再灌注(IR)的保护作用,探讨其机制是否通过激活α2受体/热休克蛋白70(HSP70)从而减轻NOD样受体相关蛋白3(NLRP3)介导的细胞焦亡.方法 SPF级C57/BL6雄性小鼠64只,8～10周龄,体重24～26 g,采用随机数字表法分为4组(每组16只):假手术组(Sham组)、肾IR组(IR组)、IR+Dex组、IR+Dex+阿替美唑(ATI)组(IR+Dex+ATI组).除Sham组外,其余组均夹闭双侧肾蒂45 min后恢复血流灌注48 h,建立肾IR损伤模型.IR+Dex组在夹闭肾蒂前30 min和松开夹子即刻腹腔注射Dex 50 μg/kg,IR+Dex+ATI组在给予Dex的同时腹腔注射ATI 250 μg/kg,其余组则腹腔注射等量的无菌生理盐水.再灌注48 h后收集小鼠血液和肾组织,采用全自动生化分析仪检测血清肌酐(Cr)、尿素氮(BUN)水平以评估小鼠肾功能,苏木精-伊红染色(H-E染色)检测肾组织病理损伤及评分,免疫组化技术检测HSP70的表达,免疫印迹法(Western blot)检测HSP70、NLRP3、消皮素D(GSDMD)、胱天蛋白酶(caspase)-1、凋亡相关斑点样蛋白(ASC)、活化的胱天蛋白酶-1(cleaved caspase-1)、GSDMD蛋白N端片段(GSDMD-N)蛋白水平,实时荧光定量聚合酶链反应(FQ-PCR)法检测HSP70、NLRP3、GSDMD、caspase-1、ASC、白细胞介素(IL)-1β、IL-18的mRNA水平.结果 与Sham组比较:IR组血清Cr、BUN 水平,肾损伤评分,HSP7 0 阳性率,HSP70、NLRP3、GSDMD、caspase-1、ASC、IL-1β、IL-18 的 mRNA 水平,HSP70、NLRP3、GSDMD、caspase-1、GSDMD-N、cleaved caspase-1 及 ASC蛋白水平较高(均P＜0.05).与 IR组比较:IR+Dex组血清 Cr、BUN 水平,肾损伤评分,NLRP3、GSDMD、caspase-1、ASC、IL-1β、IL-18 的 mRNA 水平,NLRP3、GSDMD、caspase-1、GSDMD-N、cleaved caspase-1、ASC蛋白水平较低(均P＜0.05);HSP70阳性率,HSP70的mRNA水平,HSP70蛋白水平较高(均P＜0.05).与IR+Dex组比较:IR+Dex+ATI组血清 Cr、BUN水平,肾损伤评分,NLRP3、GSDMD、caspase-1、ASC、IL-1β、IL-18 的 mRNA水平,NLRP3、GSDMD、caspase-1、GSDMD-N、cleaved caspase-1、ASC 蛋白水平较高(均P＜0.05);HSP70 阳性率,HSP70 的 mRNA水平,HSP70蛋白水平较低(均P＜0.05).结论 Dex可减轻肾IR损伤,其机制可能与激活α2受体上调HSP70,从而抑制NLRP3炎症小体及焦亡级联反应相关.
Abstract
Objective To observe the protective effect of dexmedetomidine(Dex)on renal ischemia reperfusion(IR)and to in-vestigate whether the mechanism is through activation of α2 receptor/heat shock protein 70(HSP70)thereby attenuating NOD-like receptor-related protein 3(NLRP3)-mediated cellular pyroptosis.Methods Sixty-four SPF-grade C57/BL6 male mice,8-10 weeks old,weighing 24-26 g,were selected.According to the random number table method,they were divided into four groups(n=16):a sham-operated(Sham)group,a renal IR group,an IR+Dex group,and an IR+Dex+atipamezole(ATI)(IR+Dex+ATI)group.A renal IR injury model was established by clamping the bilateral renal clitoris for 45 min and then restoring blood perfusion for 48 h in all groups except the Sham group.In the IR+Dex group,50 μg/kg of Dex was intraperitoneally injected 30 min before clamping the renal clitoris and immediately after releasing the clamp,while 250 μg/kg of Dex was intraperitoneally injected at the same time in the IR+Dex+ATI group,and the other groups received the same amount of sterile normal saline.Then,blood samples and kidney tissues were collected from mice after 48 h of reperfusion.The serum creatinine(Cr)and urea nitrogen(BUN)levels were detected by an automatic biochemi-cal analyzer to evaluate the renal function of mice.The hematoxylin-eosin staining(H-E staining)was used to evaluate the renal patho-logical damage,the expression of HSP70 was detected by immunohistochemistry.The levels of HSP70,NLRP3,gasdermin D(GSDMD),caspase-1,apoptosis-associated speck-like protein(ASC),cleaved caspase-1,and the N terminal fragment of GSDMD protein(GSDMD-N)were detected by Western blot.The mRNA levels of HSP70,NLRP3,GSDMD,caspase-1,ASC,interleukin(IL)-1β,and IL-18 were detected by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).Results Compared with the Sham group,the IR group showed increases in serum Cr and BUN levels,renal injury scores,HSP70 positive rate,the mRNA levels of HSP70,NLRP3,GSDMD,caspase-1,ASC,IL-1β and IL-18,the protein levels of HSP70,NLRP3,GSDMD,caspase-1,GSDMD-N,cleaved caspase-1 and ASC(all P＜0.05).Compared with the IR group,the IR+Dex group presented decreases in serum Cr and BUN levels,renal injury scores,the mRNA levels of NLRP3,GSDMD,caspase-1,ASC,IL-1β and IL-18,the protein levels of NLRP3,GSDMD,caspase-1,GSDMD-N,cleaved caspase-1,and ASC(all P＜0.05),as well as increases in HSP70 protein levels(all P＜0.05).Compared with the IR+Dex group,the IR+Dex+ATI group showed increases in serum Cr and BUN levels,renal injury score,the mRNA levels of NLRP3,GSDMD,caspase-1,ASC,IL-1β and IL-18,the protein levels of NLRP3,GSDMD,caspase-1,GSDMD-N,cleaved caspase-1,and ASC(all P＜0.05);as well as decreases in HSP70 positive rate,and the mRNA and protein levels of HSP70(all P＜0.05).Conclusions Dex can alleviate renal IR injury,possibly by activating α2 receptors to upregulate HSP70,thereby inhibiting NLRP3 inflammasome activation and the cascade of pyroptosis-related reactions.
基金项目
国家自然科学基金(82072130)
江苏省医学科研重点项目(ZD2022021)
江苏省高等学校自然科学研究面上项目(22KJD320002)
苏州市麻醉学临床医学中心项目(Szlcyxzxj202102)
国家临床重点专科建设项目(苏财社[2021]143号)
苏州市医学创新应用研究(SKY2022138)
江苏省自然科学基金青年项目(BK20200195)
NETL
NSTL
万方数据