右美托咪定通过α_(2)受体/HSP70/NLRP3通路减轻肾缺血再灌注损伤细胞焦亡的机制研究  

Dexmedetomidine protects against renal ischemia reperfusion injury and cell pyroptosis via theα_(2)-receptor/heat shock pro-tein 70/NOD-like receptor-related protein 3 pathway

作　　者：陈歆予 张冕 王宜蒇 彭科[1] 孟晓文[1] 单希胜[1] 嵇富海[1] Chen Xinyu;Zhang Mian;Wang Yichan;Peng Ke;Meng Xiaowen;Shan Xisheng;Ji Fuhai(Department of Anesthesiology,the First Affiliated Hospital of Soochow University,Institute of Anesthesiology,Soochow University,Suzhou Key Laboratory of Anesthesiology,Suzhou 215006,China)

机构地区：[1]苏州大学附属第一医院麻醉科,苏州大学麻醉研究所,苏州市麻醉学重点实验室,苏州215006

出　　处：《国际麻醉学与复苏杂志》2024年第8期785-792,共8页International Journal of Anesthesiology and Resuscitation

基　　金：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)。

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α_(2)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 50μg/kg,IR+Dex+ATI组在给予Dex的同时腹腔注射ATI 250μg/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,HSP70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、cObjective To observe the protective effect of dexmedetomidine(Dex)on renal ischemia reperfusion(IR)and to in-vestigate whether the mechanism is through activation ofα_(2)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,50μg/kg of Dex was intraperitoneally injected 30 min before clamping the renal clitoris and immediately after releasing the clamp,while 250μg/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-1βand IL-18,the prote

关 键 词：肾脏 缺血再灌注损伤 热休克蛋白 细胞焦亡 右美托咪定 

分 类 号：R614[医药卫生—麻醉学]