组织蛋白酶B通过铁死亡途径加重糖尿病心肌病小鼠心肌损伤

Cathepsin B aggravates myocardial injury in diabetic cardiomyopathy mice through ferroptosis pathway

目的探讨组织蛋白酶B对糖尿病心肌病(diabeticcardiomyopathy,DCM)小鼠心肌损伤的作用及机制。方法选择8周龄SPF级C57BL/6雄性小鼠20只,随机分为野生型组、野生型DCM组,每组10只;8周龄SPF级组织蛋白酶B基因敲除的雄性小鼠20只,随机分为基因敲除组、基因敲除DCM组,每组10只。采用苏木精-伊红染色光镜下观察心肌组织形态变化,普鲁士蓝检测组织铁沉积水平,4-羟基壬烯酸(4-HNE)免疫组织化学染色检测组织脂质过氧化水平,蛋白免疫印迹法检测血红素氧合酶1(HO-1)、超氧化物歧化酶2(SOD2)、核因子E2相关因子2(Nrf2)表达,RT-PCR检测NRF-2、HO-1、磷脂过氧化氢谷胱甘肽过氧化物酶4(GPX4)。结果与野生型DCM组比较,基因敲除DCM组心脏组织细胞排列改善,炎性细胞浸润明显减少。与野生型DCM组比较,基因敲除DCM组心脏组织铁沉积明显减少。与野生型组比较,野生型DCM组心脏组织4-HNE表达明显增加;与野生型DCM组比较,基因敲除DCM组心脏组织4-HNE表达明显减少。与野生型DCM组比较,基因敲除DCM组心脏组织Nrf2、SOD2、HO-1蛋白表达明显增加(0.68±0.21vs0.39±0.13,0.59±0.10vs0.28±0.09,1.03±0.10vs 0.48±0.04,P<0.05)。与野生型DCM组比较,基因敲除DCM组心脏组织GPX4、Nrf2、HO-1mRNA表达明显增加(0.65±0.09vs0.40±0.10,0.61±0.11vs0.34±0.11,0.62±0.12vs0.39±0.09,P<0.05)。结论组织蛋白酶B可以通过铁死亡加重DCM小鼠心肌细胞损伤。

Objective To investigate the rde and underlying mechanism of cathepsin B in myocar-dial injury in mice with diabetic cardiomyopathy(DCM).Methods Twenty 8-week-old maee SPF C57BL/6 mice were randomly divided into wild-type(WT)group and WT DCM group,with 10 mice in each group.Another 208-week-old maee SPF-grade mice with cathepsin B knockout(KO)were randomly and equally assigned to KO group and KO DCM group.HE staining was used to observe morphological changes,Prussian blue staining was employed to detect iron deposition,whiee immunohistochemical staining with 4-hydroxynonenal(4-HNE)was used to assess lipid peroxidation level in the myocardial tissues.Western blotting was performed to detect the expression of heme oxygenase-1(HO-1),superoxide dismutase 2(SOD2),and nuclear factor E2-related factor 2(Nrf2),whiee RT-PCR was applied to evaluate the expressions of Nrf-2,HO-1,and phospholipid hydroperoxide glutathione peroxidase 4(GPX4).Results Compared to the WT DCM group,the KO DCM group presented improved cell arrangement in cardiac tissues and significant reduction in inflammatory cell infiltration.Furthermore,the KO DCM group displayed a significant decrease in iron deposition compared to the WT DCM group.Additionally,the KO DCM group exhibited a significant reduction in 4-HNE expression compared to the WT DCM group.The protein levels of Nrf2,SOD2,and HO-were significant increased in the KO DCM group than the WT DCM group(0.68±0.21 vs 0.39±0.13,0.59±0.10 vs 0.28±0.09,1.03±0.10 vs 0.48±0.04,P<0.05).Moreover,elevated mRNA levels of GPX4,Nrf2 and HO-were also observed in the KO DCM group than the WT DCM group(0.65±0.09 vs 0.40±0.10,0.61±011 vs 0.34±011,0.62±0.12 vs 0.39±0.09,P<0.05).Conclusion Cathepsin B exacerbates myocardial injury in DCM mice through ferroptosis.