p-BRAF在心肌缺血再灌注损伤中的作用和机制研究

The Role and Mechanism of p-BRAF in Myocardial Ischemia Reperfusion Injury

为探究p-BRAF通过影响线粒体功能参与心肌缺血再灌注损伤(MIRI)的分子机制,构建心肌缺血再灌注(MIR)模型。采用钙离子荧光探针(Rhod-2/AM)和钙黄绿素乙酰氧基甲酯(Calcein AM)染色,观察心肌细胞线粒体Ca^(2+)、线粒体通透性转换孔(mPTP)水平。碘化丙啶染色观察经环孢素A(CsA)和Ru360处理后细胞死亡情况。蛋白免疫印迹检测p-BRAF表达,并在干扰/过表达p-BRAF后,观察细胞死亡情况及线粒体Ca^(2+)和mPTP水平。实验结果显示,与常氧组相比,缺氧复氧组心肌细胞Rhod-2/AM荧光强度增加,Calcein AM荧光强度降低,细胞死亡增加(P均<0.05);CsA和Ru360处理后,细胞死亡减少(P<0.05)。MIR中p-BRAF表达明显升高(P<0.05)。敲低BRAF后,MIR造成的细胞死亡减少,Rhod-2/AM荧光强度增加得到恢复(P均<0.05)。过表达p-BRAF后,细胞死亡增加,Rhod-2/AM荧光强度增加,Calcein AM荧光强度降低(P均<0.05)。因此,p-BRAF在MIR中上调并可通过介导线粒体Ca^(2+)超载和mPTP开放促进心肌细胞死亡,其可能成为防治MIRI的重要靶标。

P-BRAF participates in myocardial ischemia-reperfusion injury(MIRI)by influencing mitochondrial function.A myocardial ischemia reperfusion(MIR)model was constructed to explore its mechanism.Calcium fluorescence probe(Rhod-2/AM)and calcein acetyloxymethyl ester(Calcein AM)staining were used to observe the levels of mitochondrial Ca^(2+) and mitochondrial permeability transition pore(mPTP)in cardiomyocytes.The cell death after cyclosporine A(CsA)and Ru360 treatment was observed by propidium iodide staining.The expression of p-BRAF was detected by western blot.Cell death and mitochondrial Ca^(2+) and mPTP levels were observed after interfering/overexpressing p-BRAF.The results show that compared with normoxia group,the fluorescence intensity of Rhod-2/AM increases,the fluorescence intensity of Calcein AM decreases and the cell death increases in hypoxia reoxygenation group(all P<0.05).Cell death is reduced after CsA and Ru360 treatment(P<0.05).The expression of p-BRAF increases significantly in MIR(P<0.05).The increase of cell death and Rhod-2/AM fluorescence intensity caused by MIR are recovered after knocking down BRAF(all P<0.05).After overexpression of p-BRAF,cell death increases,Rhod-2/AM fluorescence intensity increases,and Calcein AM fluorescence intensity decreases(all P<0.05).Therefore,p-BRAF is up-regulated in MIR and can promote cardiomyocyte death by mediating mitochondrial Ca^(2+) overload and mPTP opening,which may be an important target for the treatment of MIRI.