右美托咪定对大鼠脑缺血再灌注损伤的保护作用及对铁死亡通路蛋白的影响

Protective effect of dexmedetomidine on cerebral ischemia-reperfusion injury in rats and effect on ferroptosis pathway proteins

目的通过动物模型来探讨外源性注射右美托咪定在大鼠脑缺血再灌注损伤过程中的保护作用,以及对铁死亡通路蛋白的影响。方法选择8周龄健康SPF级雄性SD大鼠45只,体质量约220 g。随机分为假手术组、模型组和右美托咪定组。改良Longa线栓法构建模型。24 h后取血肿周围组织原位末端标记法(TUNEL)染色检测神经元形态和凋亡率,氯代三苯基四氮唑(TTC)染色测定脑梗死体积,普鲁士蓝染色检测铁沉积量,酶联免疫吸附分析(ELISA)法检测丙二醛(MDA)、谷胱甘肽(GSH)和谷胱甘肽还原酶4(GPX4),Western blot法检测环氧合酶-2(COX-2)、血红素转运蛋白(HPC1)、转铁蛋白受体(TfR)和铁输出蛋白(FPN1)。结果与假手术组相比,模型组神经元凋亡率[(18.6±1.6)%vs(2.3±0.4)%]、脑梗死体积[(42.7±5.7)%vs(3.1±0.5)%]、铁沉积量[(35.1±4.4)%vs(2.6±0.3)%]、血清MDA水平[(42.52±6.63)mmol/L vs(15.63±3.26)mmol/L]、组织COX-2(0.79±0.13 vs 0.41±0.06)、HPC1(0.62±0.08 vs 0.34±0.03)和TfR蛋白(0.53±0.07 vs 0.24±0.03)相对表达量均明显升高,而血清GSH[(18.65±3.52)μmol/L vs(30.21±5.62)μmol/L]和GPX4水平[(24.52±4.65)μmol/L vs(39.63±6.03)μmol/L]及组织FPN1蛋白(0.36±0.03 vs 0.54±0.05)相对表达量明显降低(P<0.05)。与模型组相比,右美托咪定组神经元凋亡率[(9.1±1.1)%vs(18.6±2.3)%]、脑梗死体积[(22.3±3.2)%vs(42.7±6.1)%]、铁沉积量[(12.6±2.1)%vs(35.1±4.3)%]、血清MDA水平[(28.65±4.21)mmol/L vs(42.52±6.63)mmol/L]、组织COX-2(0.60±0.08 vs 0.79±0.13)、HPC1(0.55±0.06 vs 0.62±0.08)和TfR蛋白(0.41±0.05 vs 0.53±0.07)相对表达量明显降低,而血清GSH[(25.52±3.69)μmol/L vs(18.65±3.52)μmol/L]和GPX4水平[(33.26±5.25)μmol/L vs(24.52±4.65)μmol/L]及组织FPN1蛋白(0.42±0.04 vs 0.36±0.03)表达量明显升高(P<0.05)。结论右美托咪定可能通过影响铁死亡通路蛋白表达,进而参与大鼠脑缺血再灌注损伤的保护作用。

Objective To study protective effect of exogenous injection of dexmedetomidine on cerebral ischemia-reperfusion injury in rats and its effect on ferroptosis pathway proteins via animal models.Methods A total of 45 healthy SPF male SD rats aged 8-week-old with body mass about 220 g were sacrificed,and were randomly divided into sham operation group,model group and dexmedetomidine group.The modified Longa suture method was used to construct the model.After 24 hours,the morphology and apoptosis rate of neurons were detected by TdT-mediated dUTP nick end labeling(TUNEL)staining,the volume of cerebral infarction was measured by 2,3,5-triphenyltetrazolium chloride(TTC)staining,the amount of ferric deposition was detected by Prussian blue staining.The malondialdehyde(MDA),glutathione(GSH)and glutathione reductase 4(GPX4)were detected by enzyme-linked immunosorbent assay(ELISA).The cyclooxygenase-2(COX-2),heme transporter(HPC1),transferrin receptor(TfR)and ferric pump protein(FPN1)were detected by Western blot.Results The neuronal apoptosis rate[(18.6±1.6)%vs(2.3±0.4)%],cerebral infarction volume[(42.7±5.7)%vs(3.1±0.5)%],amount of ferric deposition[(35.1±4.4)%vs(2.6±0.3)%],serum MDA level[(42.52±6.63)mmol/L vs(15.63±3.26)mmol/L],tissue COX-2(0.79±0.13 vs 0.41±0.06),HPC1(0.62±0.08 vs 0.34±0.03)and relative expression of TfR protein(0.53±0.07 vs 0.24±0.03)in model group were significantly higher than those in sham operation group,while relative expression levels of serum GSH[(18.65±3.52)μmol/L vs(30.21±5.62)μmol/L]and GPX4[(24.52±4.65)μmol/L vs(39.63±6.03)μmol/L]and tissue FPN1 protein(0.36±0.03 vs 0.54±0.05)in model group were significantly lower than those in sham operation group(P<0.05).The neuronal apoptosis rate[(9.1±1.1)%vs(18.6±2.3)%],cerebral infarction volume[(22.3±3.2)%vs(42.7±6.1)%],ferric deposition[(12.6±2.1)%vs(35.1±4.3)%],serum MDA level[(28.65±4.21)mmol/L vs(42.52±6.63)mmol/L],tissue COX-2(0.60±0.08 vs 0.79±0.13),HPC1(0.55±0.06 vs 0.62±0.08)and relative expression of TfR protein(0.41±0.05 vs 0.53±0.07)in dexmedetomidine group were significantly lower than those in model group,while levels of serum GSH[(25.52±3.69)μmol/L vs(18.65±3.52)μmol/L]and GPX4[(33.26±5.25)μmol/L vs(24.52±4.65)μmol/L]and tissue FPN1 protein(0.42±0.04 vs 0.36±0.03)in dexmedetomidine group were significantly higher than those in model group(P<0.05).Conclusion It is demonstrated that dexmedetomidine may affect the expression of ferroptosis pathway protein,and then participate protective process of cerebral ischemia-reperfusion injury in rats.