摘要
目的探讨肌肽(CAR)对链脲佐菌素(STZ)诱导的糖尿病小鼠肾脏铁死亡和炎症的影响。方法以STZ诱导C57小鼠构建1型糖尿病小鼠模型(STZ模型),以正常C57小鼠作为正常对照。小鼠分为5组(每组6~8只):正常对照(NC)组、肌肽(CAR)组、STZ模型(STZ)组、STZ模型+肌肽(STZ+CAR)组、STZ模型+铁死亡抑制剂(STZ+Fer-1)组。喂养小鼠16周后,收集小鼠血清检测血肌酐(CRE)和尿素氮(BUN)水平,尿液检测小鼠24 h尿白蛋白水平。HE染色、PAS染色观察肾脏病理损伤程度。透射电镜观察肾脏线粒体的形态。PCR检测小鼠肾脏组织炎症因子白细胞介素(IL)-1β、IL-6、单核细胞趋化蛋白-1(MCP-1)和肿瘤坏死因子-α(TNF-α)的表达。免疫荧光法检测小鼠肾脏活性氧(ROS)的表达。Western blot法检测肾脏组织铁死亡指标谷胱甘肽过氧化物酶4(GPX 4)和长链脂酰辅酶A合成酶4(ACSL 4)蛋白表达水平的改变。对小鼠肾脏组织进行丙二醛(MDA)、谷胱甘肽(GSH)和Fe^(2+)含量的测定。结果与NC组相比,STZ组CRE和BUN水平升高(P<0.001);与STZ组相比,STZ+CAR组CRE和BUN水平降低(P<0.001,P<0.01)。肾脏组织病理学检查显示,与NC组相比,STZ组小鼠肾组织肾小管扩张明显、炎症细胞浸润和糖原沉积增加;与STZ组相比,STZ+CAR组肾小管扩张、炎症细胞浸润和糖原沉积减少。透射电镜检测结果显示STZ组小鼠肾脏线粒体肿胀,膜密度增加,线粒体脊减少或缺失,STZ+CAR组和STZ+Fer-1组肾小管扩张明显改善、炎症细胞浸润减少。Real-time PCR检测结果显示,与NC组相比,STZ组小鼠肾脏炎症因子(IL-1β、IL-6、MCP-1和TNF-α的mRNA表达水平升高(P<0.001或P<0.01);与STZ组相比,STZ+CAR组IL-1β、IL-6、MCP-1和TNF-α)的mRNA表达下降(P<0.01或P<0.05)。免疫荧光结果显示,与NC组相比,STZ组小鼠肾脏组织ROS水平升高(P<0.001);与STZ组相比,STZ+CAR组小鼠肾脏组织ROS水平表达下降(P<0.01)。与NC组相比,STZ组小鼠肾脏GPX4表达与GSH含量下降(P<0.001),ACSL4蛋白表达、MDA和Fe2+含量增加(P<0.01或P<0.001),STZ+CAR组GPX4表达与GSH含量增加(P<0.01),ACSL4蛋白表达、MDA和Fe2+含量下降(P<0.01或P<0.001)。结论CAR能够抑制STZ诱导的糖尿病小鼠的铁死亡和炎症,改善糖尿病小鼠肾脏病理损伤。
Objective To investigate the effects of carnosine(CAR)on streptozotocin(STZ)induced renal ferroptosis and inflammation in diabetic mice.Methods Type 1 diabetes mice model were induced by STZ,and normal C57 mice were used as normal control group.The C57 mice were divided into 5 groups(6-8 mice in each group):normal control group(NC),normal control+carnosine group(NC+CAR),STZ model group(STZ),STZ model+carnosine group(STZ+CAR),STZ model+ferroptosis inhibitor group(STZ+Fer-1).After feeding the mice for 16 weeks,serum samples were collected to detect blood creatinine(CRE)and urea nitrogen(BUN)levels.The urine of mice was collected to detect the 24-hour urinary albumin level.HE staining and PAS staining were performed to observe the degree of renal pathological injury.Real-time PCR was used to detect the expression of interleukin(IL)-1β,IL-6,monocyte chemotactic protein 1(MCP-1)and tumor necrosis factor-α(TNF-α)in mouse kidney tissue.The expression of reactive oxygen species(ROS)in mouse kidney was detected by immunofluorescence.Morphology of renal mitochondria was observed by transmission electron microscopy.The protein expression levels of glutathione peroxidase 4(GPX 4)and long-chain lipoacyl-CoA synthetase 4(ACSL4),which are ferroptosis indexes,were detected by Western blot.The contents of malondialdehyde(MDA),glutathione(GSH)and Fe~(2+)in mouse kidney tissue were determined.Results Compared with NC group,CRE and BUN levels in STZ group increased(P<0.001);and ompared with STZ group,these indexes decreased in STZ+CAR group(P<0.001,P<0.01).Renal histopathological examination showed that compared with NC group,renal tubule dilatation,inflammatory cell infiltration and glycogen deposition significantly increased in STZ group;and compared with STZ group,tubule dilatation,inflammatory cell infiltration and glycogen deposition decreased in STZ+CAR group.Electron microscope results showed that the renal mitochondria in STZ group were swollen,membrane density increased,mitochondrial ridges decreased or absent,renal tubule dilation was improved significantly in the STZ+CAR group and STZ+Fer-1 group,and inflammatory cell infiltration was reduced.Real-time PCR test results showed that compared with NC group,mRNA expression levels of inflammatory factor(IL-1β,IL-6,MCP-1 and TNF-α)increased in STZ group(P<0.001 or P<0.01);and mRNA expressions of IL-1β,IL-6,MCP-1 and TNF-αwere decreased in STZ+CAR group compared with STZ group(P<0.01 or P<0.05).Immunofluorescence results showed that compared with NC group,ROS level in kidney tissue of mice in STZ group increased(P<0.001);and compared with STZ group,the expression of ROS in kidney tissue of STZ+CAR group decreased while ROS expression in STZ+CAR group decreased(P<0.01).Compared with NC group,GPX4 expression and GSH content in kidney of STZ group decreased(P<0.001),and ACSL4 protein expression and MDA and Fe^(2+)contents increased(P<0.01 or P<0.001),GPX4 expression and GSH content increased(P<0.01),ACSL4 protein expression and MDA and Fe~(2+)content decreased in STZ+CAR group(P<0.01 or P<0.001).Conclusion CAR inhibits ferroptosis and inflammation in the kidney in diabetic mice induced by STZ,and improved renal pathological injury in diabetic mice.
作者
张崧
刘雪琪
姜玲
吴永贵
Zhang Song;Liu Xueqi;Jiang Ling;Wu Yonggui(Dept of Nephrology,The First Affliated Hospital of Anhui Medical University,Hefei 230022)
出处
《安徽医科大学学报》
CAS
北大核心
2023年第8期1322-1328,共7页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81761138042)。
关键词
糖尿病肾病
铁死亡
炎症
肌肽
diabetic nephropathy
ferroptosis
inflammation
carnosine
作者简介
张崧,女,硕士研究生;吴永贵,男,教授,博士生导师,责任作者,E-mail:wuyonggui@medmail.com.cn。
