内质网应激介导的细胞凋亡在新生大鼠缺氧缺血性脑损伤中的作用和机制研究

Effects and mechanisms of endoplasmic reticulum stress mediated apoptosis on hypoxic-ischemic brain damagein in neonatal rats

目的探讨内质网应激介导的细胞凋亡途径在新生大鼠缺氧缺血性脑损伤中的作用和机制。方法将7日龄新生SD大鼠72只按随机数字表法随机分为对照组、缺氧缺血组及抑制剂组,每组各24只。缺氧缺血组模型的制备参照Rice法进行,抑制剂组在造模成功后侧脑室注射Caspase-12特异性抑制剂Z-ATAD-FMK,对照组不缺血不缺氧。72h后取脑组织标本,HE染色光镜下观察脑组织病理变化;RT-PCR和Western Blot法检测各组大脑皮质区Caspase-12 mRNA和活化蛋白的表达变化;原位末端标记法(TUNEL)检测细胞凋亡情况。28日龄时采用Morris水迷宫实验检测各组大鼠学习记忆能力。结果HE染色提示,缺氧缺血组出现明显脑损伤,抑制剂组脑损伤病理改变较缺氧缺血组改善。RT-PCR结果表明,缺氧缺血组Caspase-12 mRNA相对表达量(0.567±0.063)高于对照组(0.318±0.046),抑制剂组Caspase-12 mRNA的相对表达量(0.455±0.030)低于缺氧缺血组(0.567±0.063),差异有统计学意义(F=53.482,P<0.05)。Western Blot结果表明,缺氧缺血组Cleaved Caspase-12蛋白的相对表达量(0.509±0.035)高于对照组(0.218±0.021),抑制剂组Cleaved Caspase-12蛋白的相对表达量(0.355±0.023)低于缺氧缺血组(0.509±0.035),差异有统计学意义(F=234.715,P<0.05)。TUNEL法检测结果提示,缺氧缺血组凋亡细胞率[(75.148±6.279)%]高于对照组[(3.468±0.994)%],抑制剂组凋亡细胞率[(47.680±8.786)%]低于缺氧缺血组[(75.148±6.279)%],差异有统计学意义(F=266.906,P<0.05)。缺氧缺血组大鼠穿越平台次数[(3.138±0.746)次]低于对照组[(6.225±0.962)次],抑制剂组大鼠穿越平台次数[(4.450±0.952)次]高于缺氧缺血组,差异有统计学意义(F=24.134,P<0.05)。结论内质网应激介导的细胞凋亡在新生大鼠缺氧缺血性脑损伤中起重要作用,抑制内质网应激介导的凋亡反应可以减轻新生大鼠缺氧缺血性脑损伤,改善认知功能。

Objective To investigate the effects and mechanisms of endoplasmic reticulum stress mediated apoptosis on hypoxic-ischemic brain damage in neonatal rats.Methods Seventy-two 7-day-old newborn SD rats were randomly divided into control group,hypoxicischemic group and inhibitor group(n=24).The model of hypoxic-ischemic brain damage was established by Rice method.Caspase-12 specific inhibitor Z-ATAD-FMK was injected into lateral ventricle after successful modeling in inhibitor group.RT-PCR and Western-blot methods were used to detect the expression of Caspase-12 in cortex after 72h of hypoxia and ischemia.TUNEL method was used to detect apoptotic morphology.H-E staining was used to observe the pathological changes of cerebral cortex under light microscope.The learning and memory abilities of rats in each group were measured by Morris test at 28 days after birth.Results RT-PCR:the expression of Caspase-12 mRNA in hypoxic-ischemic group(0.567±0.063)higher than those in control group(0.318±0.046),the expression of Caspase-12 mRNA in inhibitor group(0.455±0.030)lower than those in hypoxic-ischemic group(0.567±0.063),the differences werestatistically significant(F=53.482,P<0.05).Western Blot:the expression of Cleaved Caspase-12 in hypoxic-ischemic group(0.509±0.035)higher than those in control group(0.218±0.021),the expression of Cleaved Caspase-12 in inhibitor group(0.355±0.023)lower than those hypoxic-ischemic group(0.509±0.035),the differences werestatistically significant(F=234.715,P<0.05).The apoptosis rate in hypoxic-ischemic group[(75.148±6.279)%]was higher than that in control group[(3.468±0.994)%],the apoptosis rate in inhibitor group[(47.680±8.786)%]was lower than that in hypoxic-ischemic group[(75.148±6.279)%],the differenceswere statistically significant(F=266.906,P<0.05).Morris tests:the number of times crossing the virtual platform in hypoxic-ischemic group(3.138±0.746)were less than those in control group(6.225±0.962),the number of times crossing the virtual platform in inhibitor group(4.450±0.952)were more than those in hypoxic-ischemic group(3.138±0.746),the differenceswere statistically significant(F=24.134,P<0.05).Conclusion Endoplasmic reticulum stress mediated apoptosis plays an important role in hypoxic-ischemic brain damage in neonatal rats.Inhibit apoptosis mediated by endoplasmic reticulum stress can decrease hypoxic-ischemic brain damage and improve cognitive function.