半胱氨酸蛋白酶抑制剂C对脑缺血再灌注大鼠Bcl-2、Bax和Beclin-1的表达及细胞凋亡的影响

目的:应用不同浓度的半胱氨酸蛋白酶抑制剂C(cystatin C,Cys C)干预脑缺血再灌注大鼠,检测Bcl-2、Bax、Beclin-1阳性细胞的表达,探讨自噬蛋白Beclin-1与凋亡之间的关系。方法:成年雄性SD大鼠随机分成假手术组(Sham组),缺血再灌注组(I/R组),Cys C低、中、高浓度组。用线栓法制备大鼠右侧大脑中动脉栓塞(MCAO)模型,缺血2 h再灌注24 h。采用免疫印迹半定量检测损伤中心脑皮质组织凋亡相关蛋白Bcl-2、Bax和Beclin-1的表达;免疫组织化学检测Bcl-2、Bax和Beclin-1阳性细胞数;TUNEL染色法检测脑组织细胞凋亡。结果:与I/R组相比,Cys C低、中浓度组Bcl-2的表达有不同程度的升高,Bax的表达降低,细胞凋亡减少;而Cys C高浓度组Bcl-2的表达明显降低,Bax的表达显著上升,细胞凋亡增加;Cys C各浓度组Beclin-1的表达都有不同程度的升高。凋亡细胞与Beclin-1表达的相关性分析显示,Cys低、中浓度组细胞的自噬和凋亡呈负相关;Cys C高浓度组细胞的自噬和凋亡呈正相关。结论:Cys C在一定浓度范围内,随自噬蛋白Beclin-1表达的升高可抑制细胞的凋亡,对缺血再灌注损伤脑组织具有保护作用。其作用机制和Bcl-2的表达上调,Bax的表达下调有关;而Cys C较高浓度则无上述作用。

Effects of cystatin C pretreatment on protein expression of Bcl-2, Bax,Beclin-1 and apoptosis in rats with cerebral ischemia reperfusion injury

Objective: To study the effect of different concentrations of cystatin C (Cys C) pretreatment on proteins expression of Bcl-2,Bax and Beclin-1 in rats following focal cerebral ischemia reperfusion injury.Methods: Sixty male SD rats were randomly divided into five groups: the sham operation group (Sham group),the cerebral ischemia reperfusion injury group (I/R group) and low concentration Cys C (2mg/l) group (Cys C low group),middle concentration Cys C (5mg/l) group (Cys C middle group) and high-concentration Cys C (10mg/l) group (Cys C high group) (n=12).The right middle cerebral artery occlusion (MCAO) for 2h was induced by thread embolism and the reperfusion lasted for 24 hours.Western blotting method was used to detect apoptosis related proteins (the expression of Bcl-2,Bax and Beclin-1) in the injured center area of the cerebral cortex.Bcl-2,Bax,Beclin-1 positive cells were marked by immunohistochemistry method.TUNEL staining method was used to detect brain cell apoptosis.Results: Compared with I/R group,the expression of Bcl-2 had different degree of increase in the Cys C low and middle group,the expression of Bax decreased and apoptosis positive cells decreased significantly.The expression of Bax and apoptosis positive cells in the Cys C high group was higher than that in the Cys C low and middle group,and the Bcl-2 expression was obviously reduced;the expression of Beclin-1 protein in each concentration Cys C group increased gradually.The correlation analysis between apoptotic cells and Beclin-1 protein expression showed that in the Cys C low and middle groups,autophagy and apoptosis were negatively related;while in the Cys C high group,autophagy and apoptosis were positively correlated.Conclusion: Within a certain concentration range,cystatin C can inhibit apoptosis and increase the expression of autophagy Beclin-1.It has a protective effect on cerebral ischemia reperfusion model rats.Its possible mechanism is related to up-regulation of Bcl-2 and down-regulation of Bax expression.However,the higher concentration of cystatin C does not have the same effect.