微囊蛋白-1调节铁死亡介导的线粒体稳态改善2型糖尿病伴高血压的肾功能损伤

Caveolin-1 ameliorates renal impairment in type 2 diabetes with hypertension regulating iron death-mediated mitochondrial homeostasis

目的探究微囊蛋白-1(caveolin-1,Cav-1)通过调节铁死亡介导的线粒体稳态来对2型糖尿病(T2DM)伴发高血压大鼠肾功能损伤的改善作用。方法于2021年5月至2022年5月采用自发性高血压大鼠(SHR)建立T2DM大鼠模型,将大鼠按随机数字表法分为对照组、SHR+T2DM组、pCDNA3.1(+)-NC组和pCDNA3.1(+)-Cav-1组,每组10只。测定大鼠收缩压、空腹血糖值(FBG)、空腹胰岛素(FINS)、糖化血红蛋白(GHbA1C)浓度、评价胰岛素抵抗指数(HOMA-IR)、血肌酐、尿素氮含量,并计算尿清蛋白与肌酐比值(UACR)HE染色检测肾组织病理学变化;检测肾组织中亚铁离子(Fe^(2+))、丙二醛含量及超氧化物歧化酶(SOD)活性;二氢乙锭(DHE)荧光染色法检测肾组织中活性氧水平;线粒体膜电位荧光探针(JC-1法)检测肾组织中线粒体膜电位(MMP)水平;蛋白质印迹法检测肾组织中谷胱甘肽过氧化酶4(GPX4)、x-CT蛋白表达。结果SHR+T2DM组收缩压(189.46±7.51)mmHg、FBG(23.46±1.28)mmol/L、FINS(1.62±0.15)mU/L、HOME-IR(1.52±0.13)、GHbA1C(689.31±45.81)nmol/L、血肌酐(83.69±4.25)mmol/L、尿素氮(19.32±2.54)mmol/L、UACR(76.51±8.09)、Fe^(2+)(0.45±0.05)mg/g、丙二醛(58.32±3.26)nmol/L、活性氧含量23.46±1.84均高于对照组[(110.34±5.26)mmHg、(5.12±0.94)mmol/L、(0.68±0.07)mU/L、0.31±0.04、(310.48±23.26)nmol/L、(32.08±3.61)mmol/L、(6.89±1.86)mmol/L、8.31±0.94、(0.13±0.02)mg/g、(15.68±2.15)nmol/L、1.31±0.23],SOD活性(11.56±1.62)U/mg、MMP水平0.25±0.03、GPX4(0.35±0.04)和x-CT蛋白0.51±0.06表达均低于对照组[(35.61±2.17)U/mg、0.62±0.08、1.00±0.10、1.00±0.09](P<0.05);和SHR+T2DM组相比,pCDNA3.1(+)-Cav-1组收缩压(136.27±6.09)mmHg、FBG(10.37±1.05)mmol/L、FINS(0.91±0.10)mU/L、HOME-IR(0.64±0.08)、GHbA1C(426.17±25.94)nmol/L、血肌酐(51.36±3.87)mmol/L、尿素氮(10.71±2.18)mmol/L、UACR(38.62±4.18)、Fe^(2+)(0.18±0.03)mg/g、丙二醛(21.39±2.64)nmol/L、活性氧含量5.47±1.06均明显降低,SOD活性(29.83±1.94)U/mg、MMP水平0.51±0.06、GPX4(0.86±0.09)和x-CT蛋白0.91±0.08表达升高(P<0.05)。结论Cav-1可通过抑制铁死亡来维持线粒体功能的稳态,进而对T2DM合并SHR大鼠的肾组织发挥保护作用。

Objective To investigate the ameliorative effect of Caveolin-1(Cav-1)on renal impairment in type 2 diabetes mellitus(T2DM)with hypertension by regulating iron death-mediated mitochondrial homeostasis.Methods A T2DM rat model was established from May 2021 to May 2022 using spontaneously hypertensive rats(SHR),and the rats were randomly numbered and tabulated into the control group,SHR+T2DM group,pCDNA3.1(+)-NC group,and pCDNA3.1(+)-Cav-1 group,with 10 rats in each group.Systolic blood pressure(SBP),fasting blood glucose value(FBG),fasting insulin(FINS),glycated hemoglobin(GHbA1C)concentration,homeostasis model assessment of insulin resistance(HOMA-IR),blood creatinine(Cr),and blood urea nitrogen(BUN)levels were measured in the rats,and the urinary ascorbic protein/creatinine ratio(UACR)was calculated.HE staining was used to detect the renal histopathological changes.Kidney tissue was tested for Fe^(2+),MDA content and SOD activity.Dihydroethidium(DHE)fluorescence staining was used to detect reactive oxygen species(ROS)level in renal tissue,JC-1 method to detect mitochondrial membrane potential(MMP)level in renal tissue,and protein blotting to detect glutathione peroxidase 4(GPX4)and x-CT protein expressions in renal tissue.Results SBP,FBG,FINS,HOME-IR,GHbA1C,Cr,BUN,UACR,Fe^(2+),MDA,and ROS levels in the SHR+T2DM group were higher than those in the control group[(189.46±7.51)mmHg vs.(110.34±5.26)mmHg,(23.46±1.28)mmol/L vs.(5.12±0.94)mmol/L,(1.62±0.15)mU/L vs.(0.68±0.07)mU/L,1.52±0.13 vs.0.31±0.04,(689.31±45.81)nmol/L vs.(310.48±23.26)nmol/L,(83.69±4.25)mmol/L vs.(32.08±3.61)mmol/L,(19.32±2.54)mmol/L vs.(6.89±1.86)mmol/L,76.51±8.09 vs.8.31±0.94,(0.45±0.05)mg/g vs.(0.13±0.02)mg/g,(58.32±3.26)nmol/L vs.(15.68±2.15)nmol/L,23.46±1.84 vs.1.31±0.23],while SOD activity,MMP levels,GPX 4 and x-CT protein in the SHR+T2DM group were lower than those in the control group[(11.56±1.62)U/mg vs.(35.61±2.17)U/mg,0.25±0.03 vs.0.62±0.08,0.35±0.04 vs.1.00±0.10,0.51±0.06 vs.1.00±0.09](P<0.05).Compared with the SHR+T2DM group,SBP,FBG,FINS,HOME-IR,GHbA1C,Cr,BUN,UACR,Fe^(2+),MDA,and ROS levels in the pCDNA3.1(+)-Cav-1 group were significantly decreased[(136.27±6.09)mmHg,(10.37±1.05)mmol/L,(0.91±0.10)mU/L,0.64±0.08,(426.17±25.94)nmol/L,(51.36±3.87)mmol/L,(10.71±2.18)mmol/L,38.62±4.18,(0.18±0.03)mg/g,(21.39±2.64)nmol/L,and 5.47±1.06,respectively],while SOD activity,MMP level,GPX 4,and x-CT protein expres‐sions were increased[(29.83±1.94)U/mg,0.51±0.06,0.86±0.09,and(0.91±0.08),respectively](P<0.05).Conclusion Caveolin-1 can maintain the homeostasis of mitochondrial function by inhibiting iron death,which in turn exerts a protective effect on renal tissue in T2DM combined with SHR rats.