氢气对大鼠局灶性脑缺血再灌注损伤内质网应激及神经细胞凋亡的影响

Effect of hydrogen gas on endoplasmic reticulum stress and neural cell apoptosis in rats with cerebral ischemia-reperfusion injury

目的探讨吸入高浓度氢气(H2)对大鼠局灶性脑缺血再灌注损伤(IRI)内质网应激(ERS)相关蛋白葡萄糖调节蛋白78(GRP78)、半胱氨酸蛋白酶12(Caspase-12)及脑皮质神经细胞凋亡和凋亡相关蛋白Bcl-2、Bax表达的影响。方法选取72只健康成年SPF级雄性SD大鼠,将其分为对照组(Ⅰ组:不作任何处理)、假手术组(Ⅱ组)、脑IRI组(Ⅲ组)、氢气治疗组(Ⅳ组),每组18只。采用线栓法建立大鼠局灶性脑IRI模型。于再灌注24h后行神经功能缺损评分(NDS),采用2,3,5-氯化三苯基四氮唑(TTC)染色观察大鼠脑缺血梗死程度并计算脑梗死面积,原位末端标记法(TUNEL)技术检测各组大鼠脑皮质神经细胞凋亡并计算凋亡指数(AI),Western blot法和免疫组织化学法检测大鼠脑皮质GRP78、Caspase-12、Bcl-2及Bax蛋白表达。结果与Ⅰ、Ⅱ组比较,Ⅲ、Ⅳ组大鼠脑IRI后NDS、脑梗死面积、AI均明显增加,GRP78、Caspase-12、Bax蛋白表达明显升高,Bcl-2蛋白表达明显下降(P<0.05);与Ⅲ组比较,Ⅳ组大鼠NDS、脑梗死面积、AI及Caspase-12、Bax蛋白表达明显减少,GRP78、Bcl-2表达明显增加(P<0.05)。结论再灌注同时吸入高浓度H2可通过增加脑IRI后内质网GRP78蛋白表达,并抑制Caspase-12的激活进而抑制ERS并促进内质网功能修复,对大鼠脑IRI起到一定的保护作用。

Objective To investigate the effects of inhaling high concentration of hydrogen gas on the expressions of endoplasmic reticulum stress（ERS）related protein glucose regulated protein 78（GRP78）,Caspase-12 and the neural cell apoptosis and related proteins Bcl-2and Bax in the rats with focal cerebral ischemia reperfusion（I/R）injury.Methods Seventy-two healthy SPF male Sprague-Dawley rats were selected and then randomly divided into the control group（Ⅰ：without any treatment）,sham operation group（Ⅱ）,cerebral IRI group（Ⅲ）and hydrogen gas treatment group（Ⅳ）,18 cases in each group.Focal cerebral ischemia reperfusion injury（IRI）model was induced by using the suture-occluded method.The neurological deficits score（NDS）was assessed at 24 hafter cerebral reperfusion in four groups.The cerebral infarction severity and size were detected by TTC staining and neuronal apoptosis of brain cortex were tested by TUNEL technique.The apoptosis index（AI）was calculated.Then the expressions of GRP78,Caspase-12,Bcl-2and Bax were assessed by Western blot and immunohistochemistry.Results As compared with the group Ⅰand Ⅱ,NDS score,cerebral infarction size,AI and the expressions of GRP78,Caspase-12 and Bax in cerebral cortex in the group Ⅲ and Ⅳ were significantly increased,while the expression of Bcl-2in cerebral cortex was markedly decreased（P〈0.05）;compared with the groupⅢ,NDS score,brain infarction size,AI and the expression of Caspase-12 and Bax in cerebral cortex in the group Ⅳ were markedly decreased,while the expressions of GRP78 and Bcl-2were dramatically increased（P〈0.05）.Conclusion Inhaling high concentration of hydrogen gas has a certain protective effect on cerebral IRI in rats through increasing endoplasmic reticulum GRP78 protein expression after IRI and inhibiting Caspase-12 activation,thus inhibiting ERS and promoting the repair function of endoplasmic reticulum.