摘要
目的探讨七氟烷对大鼠肾缺血再灌注损伤的影响及其作用机制。方法选取6~8周龄、体质量(250±30)g雄性SD大鼠40只,按数字表法随机分成假手术组(Sham组)、肾缺血再灌注组(IR组)、七氟烷预处理组(Sev组)、七氟烷预处理+沉默信号调节因子1(SIRT1)抑制剂组(EX组)4组,每组10只。肾缺血再灌注损伤模型制备前2天和术前10 min,Sham组、IR组、Sev组大鼠腹腔注射1%二甲基亚砜(DMSO)溶液(5 mL/kg),EX组腹腔注射含抑制剂EX 527(1 mg/mL)的1%DMSO溶液(5 mL/kg)。Sham组:切除右侧肾脏,暴露左侧肾脏但不夹闭肾蒂;IR组:切除右侧肾脏,制备左侧肾脏缺血再灌注模型;Sev组、EX组:先吸入七氟烷45 min后,再按IR组方法制备缺血再灌注模型。于制备缺血再灌注模型术后3 h,收集各组大鼠肾脏组织与左心室血液,取材后以颈椎脱位法处死大鼠。观察项目:(1)检测各组大鼠左心室血液血清肌酐(Scr)、尿素氮(BUN)。(2)制备各组大鼠肾脏组织病理切片,光镜下观察肾组织病理变化,对肾小管按Paller评分标准进行评分,评估肾脏损伤情况。(3)采用原位末端脱氧核苷酸转移酶介导的dUTP切口末端标记技术(TUNEL)检测各组大鼠肾脏组织细胞凋亡情况。(4)取各组大鼠肾脏组织病理切片,在透射电子显微镜下观察肾脏组织自噬情况。(5)采用蛋白质印迹法(Western blotting)检测各组大鼠肾脏组织SIRT1、叉头盒转录因子O3(FoxO3)蛋白,以及凋亡蛋白(BAX/Bcl 2)、自噬相关蛋白(Beclin1、LC3A/B)的表达水平。结果(1)Sham组大鼠血清Scr和BUN分别为(50.74±5.91)μmol/L和(10.11±0.80)mmol/L,IR组分别为(90.18±11.22)μmol/L和(53.39±6.29)mmol/L,Sev组分别为(63.70±8.69)μmol/L和(27.68±3.41)mmol/L,EX组分别为(80.18±9.15)μmol/L和(33.20±3.57)mmol/L。与Sham组相比,IR组、Sev组、EX组血清Scr、BUN水平均升高;与IR组相比,Sev组、EX组血清Scr、BUN水平均下降;与Sev组相比,EX组血清Scr、BUN水平均升高:差异均有统计学意义(P值均<0.05)。(2)Sham组肾小球和肾小管形态结构基本正常;IR组肾小管上皮组织肿胀明显,肾小管细胞排列紊乱,大量细胞核固缩,大量中性粒细胞浸润;Sev组:肾小管上皮细胞轻中度肿胀,少见核固缩坏死,中性粒细胞浸润明显减少;EX组:肾小管上皮组织肿胀明显,细胞核固缩和溶解增多,中性粒细胞浸润较多。Sham组、IR组、Sev组、EX组肾小管Paller评分分别为(0.61±0.15)、(5.05±0.55)、(2.68±0.33)、(4.51±0.49)分。与Sham组比较,IR组、Sev组、EX组Paller评分均升高;与IR组比较,Sev组、EX组Paller评分均下降;与Sev组相比,EX组Paller评分升高:差异均有统计学意义(P值均<0.05)。(3)Sham组、IR组、Sev组、EX组细胞凋亡率分别为8.71%±0.68%、38.15%±2.23%、14.51%±2.02%、32.55%±2.89%。与Sham组比较,IR组、Sev组、EX组细胞凋亡率均升高;与IR组比较,Sev组、EX组细胞凋亡率下降;与Sev组比较,EX组细胞凋亡率升高:差异均有统计学意义(P值均<0.05)。(4)与Sham比较,IR组、Sev组、EX组大鼠肾组织自噬小体数量均增加;与IR组比较,Sev组、EX组自噬小体数量增加且体积增大;与Sev组比较,EX组自噬小体数量明显减少:差异均有统计学意义(P值均<0.05)。(5)与Sham组比较,IR组和EX组大鼠肾脏组织FoxO3、Beclin1、LC3A/B蛋白、BAX/Bcl 2蛋白的相对表达量均升高,SIRT1的相对表达量均降低;Sev组SIRT1、FoxO3、Beclin1、LC3A/B、BAX/Bcl 2蛋白的相对表达量均升高:差异均有统计学意义(P值均<0.05)。与IR组比较,Sev组SIRT1、Beclin1、LC3A/B蛋白的相对表达量均升高,FoxO3、BAX/Bcl-2蛋白的相对表达量均降低;EX组SIRT1蛋白的相对表达量升高,FoxO3、BAX/Bcl 2蛋白的相对表达量均降低:差异均有统计学意义(P值均<0.05)。与Sev组比较,EX组SIRT1、Beclin1、LC3A/B蛋白的相对表达量均降低,FoxO3、BAX/Bcl 2蛋白的相对表达量均升高,差异均有统计学意义(P值均<0.05)。结论七氟烷可通过促进自噬,抑制细胞凋亡,减轻大鼠肾缺血再灌注损伤,其机制可能与激活SIRT1/FoxO3信号通路有关。
Objective This study aimed to investigate the effect of sevoflurane on renal ischemia‑reperfusion injury in rats and its mechanism.Methods Forty male SD rats,aged 6‑8 weeks old with a body weight of(250±30)g,were randomly divided into four groups:the sham surgery group(Sham group),renal ischemia‑reperfusion group(IR group),sevoflurane pretreatment group(Sev group),and sevoflurane pretreatment+silencing signal regulator 1(SIRT1)inhibitor group(EX group),with 10 rats in each group.Two days before the preparation of the renal ischemia‑reperfusion injury model and 10 min before surgery,the rats in the Sham,IR,and Sev groups were intraperitoneally injected with 1%dimethyl sulfoxide(DMSO)solution(5 mL/kg),and the EX group was intraperitoneally injected with 1%DMSO solution(5 mL/kg)containing the EX-527 inhibitor(1 mg/mL).In the Sham group,the right kidney was removed,and the left kidney was exposed but not clamped.In the IR group,the right kidney was resected,and an left renal ischemia‑reperfusion model was established.In the Sev group and EX group,sevoflurane was inhaled for 45 min,and then an ischemia‑perfusion model was prepared in accordance with the method used in the IR group.Three hours after the preparation of the ischemia‑reperfusion model,renal tissue and left ventricular blood of each group were collected,and the rats were sacrificed by cervical dislocation after the material was collected.Observation was performed as follows:(1)Serum creatinine(Scr)and urea nitrogen(BUN)were detected in the left ventricle of rats in each group.(2)Pathological sections of kidney histopathology of each group of rats were prepared;pathological changes in kidney tissue were observed under light microscopy,and renal tubules were scored in accordance with the Paller scoring standard to evaluate kidney damage.(3)In-situ terminal deoxynucleotidyl transferase-mediated dUTP incision-end labeling technology was used to detect apoptosis in kidney tissue cells of rats.(4)The pathological sections of kidney tissue in each group were taken,and the autophagy of kidney tissue was observed under transmission electron microscopy.(5)Western blotting was used to detect the expression level of SIRT1,forkhead box transcription factor O3(FoxO3),and apoptotic(BAX/Bcl-2)and autophagy-related(Beclin1,LC3A/B)proteins in rat kidney tissue.Results(1)Scr and BUN detection results in each group were shown as follows:(50.74±5.91)μmol/L and(10.11±0.80)mmol/L in the Sham group,(90.18±11.22)μmol/L and(53.39±6.29)mmol/L in the IR group,(63.70±8.69)μmol/L and(27.68±3.41)mmol/L in the Sev group,and(80.18±9.15)μmol/L and(33.20±3.57)mmol/L in the EX group.Compared with the Sham group,serum Scr and BUN levels were increased in the IR group,Sev group,and EX group.Compared with the IR group,serum Scr and BUN levels decreased in the Sev group and EX group.Compared with the Sev group,serum Scr and BUN levels were increased in the EX group.The differences were statistically significant(all P values<0.05).(2)Renal histopathological changes in each group:the morphological structure of glomeruli and renal tubules in the Sham group was basically normal;in the IR group,the tubular epithelial tissue was swollen;the tubular cells were arranged disorderly;a large number of nuclei were solidified,and a large number of neutrophils were infiltrated;in the Sev group,tubular epithelial cells swell mildly,nuclear shrinkage necrosis occurred rarely,and neutrophil infiltration significantly reduced;in the EX group,renal tubular epithelial tissue swelling was evident,nucleus contraction and lysis were increased,and neutrophil infiltration is high.The tubular Paller scores of the Sham group,IR group,Sev group,and EX group accounted for(0.61±0.15),(5.05±0.55),(2.68±0.33),(4.51±0.49).Compared with the Sham group,the Paller scores were higher in the IR group,Sev group,and EX group.Compared with the IR group,the Paller scores of the Sev group and EX group decreased.Compared with the Sev group,the Paller score was increased in the EX group,and the difference was statistically significant(all P value<0.05).(3)Apoptosis of renal histocytes in each group:the apoptotic rates in the Sham group,IR group,Sev group,and EX group accounted for 8.71%±0.68%,38.15%±2.23%,14.51%±2.02%,and 32.55%±2.89%,respectively.Compared with the Sham group,the apoptotic rate of cells in the IR group,Sev group,and EX group was increased.Compared with the IR group,the apoptotic rate of cells in the Sev group and EX group decreased.Compared with the Sev group,the apoptotic rate of cells in the EX group was increased,and the difference was statistically significant(all P value<0.05).(4)Autophagy in renal tissues of each rat group was compared under electron microscopy.Compared with the Sham group,the number of autophagies in the IR group,Sev group,and EX group increased.Compared with the IR group,the number and volume of autophagies increased in the Sev group and EX group.Compared with the Sev group,the number of autophagies in the EX group was significantly reduced.The difference was statistically significant(all P value<0.05).(5)The expression level of SIRT1,FoxO3,and apoptotic(BAX/Bcl-2)and autophagy-related(Beclin1 and LC3A/B)proteins was compared in renal tissues of rats.Compared with the Sham group,the relative expression level of FoxO3,Beclin1,LC3A/B protein,and BAX/Bcl-2 protein in the IR and EX groups was increased,whereas that of of SIRT1 was decreased.In addition,the relative expression level of SIRT1,FoxO3,Beclin1,LC3A/B,and BAX/Bcl-2 proteins in the Sev group was all increased,with statistical significance(all P values<0.05).Compared with the IR group,the relative expression level of SIRT1,Beclin1,and LC3A/B proteins was increased in the Sev group,whereas the relative expression level of FoxO3 and BAX/Bcl-2 proteins was decreased.The relative expression level of SIRT1 protein was increased in the EX group,whereas the relative expression level of FoxO3 and BAX/Bcl-2 proteins was decreased.The differences were statistically significant(all P values<0.05).Compared with the Sev group,the relative expression level of SIRT1,Beclin1,and LC3A/B proteins in the EX group was decreased,whereas the relative expression level of FoxO3 and BAX/Bcl-2 proteins was increased:the differences were statistically significant(all P values<0.05).Conclusion Sevoflurane treatment can protect rats from renal ischemia‑reperfusion injury by promoting autophagy and inhibiting apoptosis by an underlying mechanism related to the activation of the SIRT1-FoxO3 signaling pathway.
作者
杨传铭
易铭
韩冰
张一粟
李晓红
Yang Chuanming;Yi Ming;Han Bing;Zhang Yisu;Li Xiaohong(Department of Anaesthesiology,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233004,China;Graduate School of Bengbu Medical College,Bengbu 233030,China)
出处
《中华解剖与临床杂志》
2023年第11期749-757,共9页
Chinese Journal of Anatomy and Clinics
基金
安徽省高校省级自然科学研究重点项目(KJ2017A246,KJ2020A0581)
蚌埠医学院研究生科研创新计划项目(Byycxz20019)。
关键词
再灌注损伤
七氟烷
肾损伤
大鼠
模型
动物
自噬
沉默信号调节因子1
叉头盒转录因子O3
Reperfusion injury
Sevoflurane
Kidney injury
Rats
Models,animal
Autophagy
Silent information regulator sirtuin 1
Forkhead transcription factor O3
