人尿激肽原酶通过抑制细胞凋亡保护小鼠脑缺血再灌注损伤

Hnnan urinary kallidinogenase protects against cerebral ischemia reperfusion injury in mice

目的探讨人尿激肽原酶（human urinary kallidinogenase, HUK）对小鼠脑缺血再灌注损伤的影响。方法110只雄性ICR小鼠随机分为假手术组、对照组和HUK组，采用短暂性大脑中动脉闭塞法制作脑缺血再灌注模型。利用氯化三苯四氮唑染色检测梗死体积，蛋白质印迹法检测缺血皮质Bcl-2、Bax、胱天蛋白酶-3蛋白表达量，免疫组化染色检测缺血侧海马CA1区Bcl-2和Bax阳性细胞，TUNEL染色法检测缺血皮质细胞凋亡。结果假手术组未见梗死灶和神经功能缺损。缺血再灌注24 h时，HUK组梗死体积（P〈0.01）和神经功能缺损评分（P＝0.02）均显著低于对照组；缺血再灌注72 h时，HUK组梗死体积（P〈0.01）和神经功能缺损评分（P＝0.03）同样显著低于对照组。蛋白质印迹分析显示，HUK组缺血皮质Bcl-2表达显著高于对照组（P〈0.001），而胱天蛋白酶-3（P〈0.001）和Bax（P〈0.001）表达均显著低于对照组。假手术组小鼠未见凋亡细胞，HUK组缺血侧海马CA1区凋亡细胞数量（P〈0.01）和Bax阳性细胞数量（P〈0.01）显著少于对照组，而Bcl-2阳性细胞数量显著多于对照组（P〈0.01）。结论HUK对小鼠缺血再灌注损伤具有一定的保护作用，其机制可能与上调Bcl-2蛋白表达以及下调胱天蛋白酶-3和Bax蛋白表达从而抑制细胞凋亡有关。

ObjectiveTo investigate the effect of human urinary kallidinogenase （HUK） on cerebral ischemia reperfusion injury in mice.MethodsOne hundred and ten male ICR mice were randomly divided into sham operation, control and HUK groups. A cerebral ischemia-reperfusion model was induced by transient middle cerebral artery occlusion. The infarct volume was detected by triphenyltetrazolium chloride staining. Bcl-2, Bax, and caspase-3 expression levels in the ischemic cortex were detected by Western blot. Bcl-2 and Bax positive cells in the hippocampal CA1 area on the ischemic side were detected using Immunohistochemical staining. Apoptotic cells in the ischemic cortex were detected by TUNEL staining.ResultsNo infarction and neurological deficits were found in the sham operation group. At 24 h after ischemia-reperfusion, the infarction volume （P〈0.01） and neurological deficit score （P=0.02） in the HUK group were significantly lower than those in the control group; at 72 h after ischemia-reperfusion, the infarction volume （P〈0.01） and neurologic deficit score （P=0.03） in the HUK group were also significantly lower than those in the control group. Western blot analysis showed that the expression level of Bcl-2 in the ischemic cortex in the HUK group was significantly higher than that in the control group （P〈0.001）, and the expression levels of caspase-3 （P〈0.001） and Bax （P〈0.001） in the cerebral cortex in the HUK group were significantly lower than those in the control group. No apoptotic cells were found in the sham operation group. The number of apoptotic cells in hippocampal CA1 area （P〈0.01） and the number of Bax positive cells （P〈0.01） in the HUK group were significantly less than those in the control group, while the number of Bcl-2 positive cells was significantly more in the control group （P〈0.01）.ConclusionsHUK has a certain protective effect on ischemia-reperfusion injury in mice, its mechanism may be associated with the upregulation of Bcl-2 protein expression and downregulation of caspase-3 and Bax protein expression, thus inhibiting cell apoptosis.