蛋白酶体抑制剂对心肌缺血再灌注大鼠CHIP、HSP70的影响

Proteasome inhibitor improves expressions of CHIP and HSP70 in ischemia reperfusion rats

目的观察蛋白酶体抑制剂MG-132(N-benz0y1 oxycarbonyl(Z)-Leu-Leuleucina1)对急性缺血再灌注大鼠心肌(carboxyl terminus of the Hsc70-interacting protein,CHIP)、HSP70的影响以探讨MG-132的心肌保护机制。方法选择SD大鼠结扎左冠状动脉前降支30min制作心肌缺血再灌注模型,54只大鼠按随机数字表法分入以下各组:治疗(I/R+T)组于再灌注前5min静脉注射MG-132(0.75mg/kg),对照(I/R)组及假手术(Sham)组注射生理盐水,并将I/R组和I/R+T组分为再灌注2、24h及7d亚组(各亚组各8只大鼠),分别用荧光定量PCR法及Western blot法检测CHIP及HSP70的mRNA及蛋白质表达,并进行相关性分析。结果与I/R各时相组相比,I/R+T各时相组CHIP及HSP70的mRNA水平显著升高[(0.87±0.10)vs(0.50±0.06),(0.57±0.07)vs(0.33±0.04),(0.47±0.06)vs(0.19±0.03),P<0.01]。与I/R2、24h组相比I/R+T组CHIP及mRNA水平显著升高[(1.15±0.12)vs(0.57±0.07),(0.81±0.09)vs(0.44±0.06),P<0.01];MG-132上调了I/R+T各时相组HSP70的蛋白质表达,与I/R各组相比,差异均有统计学意义[(1.28±0.16)vs(0.79±0.08),(0.88±0.12)vs(0.58±0.12),(0.9±0.17)vs(0.50±0.09),P<0.01];MG-132上调了I/R+T2、24h组CHIP的蛋白质表达,与I/R2、24h组相比,差异有统计学意义[(1.43±0.15)vs(1.04±0.11),P<0.01;(1.16±0.11)vs(0.96±0.13)],P<0.05)。结论心肌缺血再灌注前适量静注MG-132能显著刺激缺血再灌注大鼠心脏CHIP表达,从而进一步上调HSP70水平,从而保护心肌。

Objective To explore the influence of proteasome inhibitor, MG-132 （N-benzOyloxycarbonyl （Z）-Leu-Leuleucinal） on the expressions of carboxyl terminus of the Hsc70-interacting protein （CHIP） and heat shock protein 70 （HSP70） in ischemia reperfusion （I/R） rats and investigate the underlying mechanism of myocardial protection by the inhibitor. Methods Totally 54 adult SD rats were randomly divided into 3 groups, sham group （n =6）, I/R group （n =24） and treatment group （I/R ＋T, n =24）. The later 2 groups were further equally divided into 3 subgroups according different time of reperfusion. Left anterior descending （LAD） coronary artery was ligated for 30 rain and then released for 2 h or 24 h or 7 d in different subgroups. Five minutes before reperfusion, MG-132 at dose of 0.75 mg/kg was given intravenously in I/R ＋ T group, but I/R group and sham group were given the same volume of normal saline. The levels of CHIP and HSP70 at mRNA and protein level were detected by real-time PCR and Western blotting respectively. The correlation between CHIP and HSF70 expression was analyzed. Results Compared with I/R groups, the mRNA levels of CHIP and HSPTO were significantly increased in I/R ＋ T groups （P 〈0. 01 ）. Accordingly, the protein expression of them in I/R ＋ T groups were also notably raised compared with I/R groups （P 〈0.01 ）. MG-132 exerted its strongest effect on upregulating the levels of CHIP and HSP70 in mRNA and protein in reperfusion for 2 h. Furthermore, the mRNA and protein levels of HSP70 had significant positive correlation with the expression of CHIP in all groups （r = 0. 94, P 〈 0. 01; r =0. 89, P 〈0.05）. CONClUSiON MG-132 may play a role in myocardial protection by upregulating the expression of CHIP in ischemia reperfusion rats, which then further increases the level of HSP70.