摘要
目的探讨中性粒细胞胞外诱捕网(NETs)在肾缺血再灌注损伤中的作用,以及NETs与细胞焦亡之间的关系。方法将30只C57小鼠随机分为肾缺血再灌注损伤组(IRI组)、假手术组(Sham组)和脱氧核糖核酸酶Ⅰ组(DNaseⅠ组),每组10只。使用无创血管夹夹闭双侧肾蒂45 min、再灌注24 h的方法构建小鼠肾缺血再灌注模型;DNaseⅠ组小鼠术前1 h腹腔注射DNaseⅠ2.5 mg/kg。采用HE染色法评估各组小鼠肾脏损伤情况。检测各组小鼠外周血肌酐(Scr)、尿素氮(BUN)水平。采用免疫荧光双染检测各组肾组织中NETs标志物中性粒细胞髓过氧化物酶(MPO)与淋巴细胞抗原6G(LY6G)蛋白情况。采用蛋白质印迹法测定各组肾组织中瓜氨酸化组蛋白(Cit-H3)、核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、gasdermin D(GSDMD)、GSDMD-N端片段(N-GSDMD)水平。使用实时荧光定量反转录PCR检测肾组织中NLRP3、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)信使RNA(mRNA)水平。使用末端脱氧核苷酸转移酶dUTP缺口末端标记(TUNEL)染色法检测细胞焦亡情况。结果Sham组小鼠肾脏组织结构清晰,肾小球结构完整;IRI组部分肾小管上皮细胞空泡变性、胞质粉染,肾组织内MPO与LY6G免疫荧光强度表达增加;DNaseⅠ组肾小管上皮细胞空泡变性改善、胞核脱落减轻,肾组织内MPO与LY6G免疫荧光强度表达减弱。IRI组血清Scr、BUN水平[(292.400±20.340)μmol/L、(135.900±9.980)mmol/L]及肾组织中Cit-H3、NLRP3、GSDMD、N-GSDMD蛋白水平(1.100±0.107、3.001±0.092、1.120±0.089、1.194±0.122)高于Sham组[(25.880±7.144)μmol/L、(24.790±5.063)mmol/L、0.302±0.039、0.512±0.108、0.538±0.042、0.735±0.060],肾脏细胞焦亡指数[(71.920±12.890)%]高于Sham组[(1.000±0.433)%],肾组织中NLRP3、IL-1β、IL-18 mRNA表达水平(2.841±0.925、1.725±0.164、2.081±0.394)高于Sham组(1.035±0.057、1.008±0.008、1.007±0.011),差异均有统计学意义(P<0.05)。DNaseⅠ组血清Scr与BUN水平[(31.160±3.607)μmol/L、(22.310±1.897)mmol/L]及肾组织中Cit-H3、NLRP3、GSDMD、N-GSDMD蛋白表达水平(0.612±0.107、0.817±0.143、0.466±0.077、0.673±0.050)低于IRI组,肾脏细胞焦亡指数[(1.786±0.345)%]低于IRI组,肾组织中NLRP3、IL-1β、IL-18 mRNA表达水平(1.162±0.256、0.998±0.093、1.031±0.157)低于IRI组,差异均有统计学意义(P<0.05)。结论缺血再灌注损伤可导致NETs表达上调并促进细胞焦亡,抑制NETs后,可减轻缺血再灌注损伤诱导的肾损伤以及细胞焦亡。NETs可能是通过促进细胞焦亡加重肾缺血再灌注损伤。
Objective To investigate the role of neutrophil extracellular traps(NETs)in renal ischemia-reperfusion injury and the relationship between NETs and pyroptosis.Methods Thirty C57 mice were randomly divided into ischemia-reperfusion injury group(IRI group),sham-operated group(Sham group)and deoxyribonucleaseⅠgroup(DNaseⅠgroup),with 10 mice in each group.The IRI model was constructed by clamping the left and right nephrectomy for 45 min followed by 24 hours reperfusion.DNaseⅠgroup was injected DNaseⅠat a dose of 2.5 mg/kg 1 hour before ischemia.The renal damage of mice was in each group was evaluated by HE staining method.Peripheral blood levels of creatinine(Scr)and blood urea nitrogen(BUN)of mice were measured in each group.Immunofluorescence double staining was used to detect the expression of NETs markers in the renal tissues of each group,including myeloperoxidase(MPO)and lymphocyte antigen 6G(LY6G)protein.Western blotting was employed to determine the levels of citrullinated histone H3(Cit-H3),NLRP3,gasdermin D(GSDMD)and N-terminal fragment of GSDMD(N-GSDMD)in the renal tissues of each group.Real-time quantitative PCR was used to measure the mRNA levels of NLRP3,interleukin(IL)-1βand IL-18 in the renal tissues.Terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)staining was used to detect cell pyroptosis.Results The renal tissue structure in the Sham group was clear,with intact glomerular structure;in the IRI group,some renal tubular epithelial cells exhibited vacuolar degeneration and eosinophilic cytoplasm,increased immunofluorescence intensity of MPO and LY6G in renal tissues;in the DNaseⅠgroup,vacuolar degeneration of renal tubular epithelial cells was improved,nuclear shedding was reduced,and the immunofluorescence intensity of MPO and LY6G was decreased.The levels of serum Scr and BUN[(292.400±20.340)μmol/L,(135.900±9.980)mmol/L]and the protein expression levels of Cit-H3,NLRP3,GSDMD and N-GSDMD(1.100±0.107,3.001±0.092,1.120±0.089,1.194±0.122)in the renal tissue in the IRI group were higher than those in the Sham group[(25.880±7.144)μmol/L,(24.790±5.063)mmol/L,0.302±0.039,0.512±0.108,0.538±0.042,0.735±0.060],the pyroptosis index of renal cells in the IRI group[(71.920±12.890)%]was higher than that in the Sham group[(1.000±0.433)%],mRNA expression levels of NLRP3,IL-1 and IL-18(2.841±0.925,1.725±0.164,2.081±0.394)in the renal tissue were higher than those in the Sham group(1.035±0.057,1.008±0.008,1.007±0.011),with statistically significant differences(P<0.05).In the DNaseⅠgroup,the levels of serum Scr and BUN[(31.160±3.607)μmol/L,(22.310±1.897)mmol/L]and the protein expression levels of Cit-H3,NLRP3,GSDMD and N-GSDMD(0.612±0.107,0.817±0.143,0.466±0.077,0.673±0.050)in the renal tissue were lower than those in the IRI group,pyroptosis index of renal cells[(1.786±0.345)%]and the mRNA expression levels of NLRP3,IL-1βand IL-18(1.162±0.256,0.998±0.093,1.031±0.157)in the renal tissue were lower than those in the IRI group,with statistically significant differences(P<0.05).Conclusion Ischemia-reperfusion injury can lead to upregulation of NETs expression and promote cell pyroptosis,and inhibition of NETs can alleviate ischemia-reperfusion injury-induced renal injury and cell pyroptosis.NETs may exacerbate renal ischemia-reperfusion injury by promoting cell pyroptosis.
作者
张秋雯
朱丽容
虞燕青
陆兵
李海滨
孙煦勇
ZHANG Qiuwen;ZHU Lirong;YU Yanqing;LU Bing;LI Haibin;SUN Xuyong(Department of Immunology,School of Basic Medical Sciences at Guangxi Medical University,Nanning,Guangxi 530021,China;Institute of Transplantation Medicine,the Second Affiliated Hospital of Guangxi Medical University,Nanning,Guangxi 530007,China;Guangxi Clinical Research Center for Organ Transplantation,Nanning,Guangxi 530007,China;Guangxi Key Laboratory of Organ Donation and Transplantation,Nanning,Guangxi 530007,China)
出处
《检验医学与临床》
2025年第6期747-752,759,共7页
Laboratory Medicine and Clinic
基金
广西壮族自治区重点研发计划项目(桂科AB24010059)
广西壮族自治区自然科学基金区域高发疾病研究联合专项资助项目(2024GXNSFAA010050)。
