高血压大鼠心肌内质网应激时GRP78和CHOP的表达及其与心肌细胞凋亡的关系

Expressions of GRP78 and CHOP in endoplasmic reticulum stress of rat heart after aortic constriction and their relationship with cardiac myocyte apoptosis

目的:探讨心肌组织内质网应激相关因子葡萄糖调节蛋白78(glucose-regulated protein78,GRP78)和C/EBP环磷酸腺苷反应元件结合转录因子同源蛋白(C/EBP homologous protein,CHOP)在高血压大鼠心肌中的表达及其与心肌细胞凋亡的关系。方法:将48只SD大鼠随机等分为两个组,一组采用腹主动脉缩窄术(TAC)建立高血压模型,称为TAC组;另一组只进行相应的假手术,称为Sham组。按术后饲养时间(3 d、7 d、14 d和28 d)的不同,TAC组又分为4个组,即TAC3d组、TAC7d组、TAC14d组和TAC28d组,每组6只(n=6);Sham组也又分为4个组,即Sham3d组、Sham7d组、Sham14d组和Sham28d组,每组6例(n=6)。用颈动脉插管法测定各组大鼠的平均动脉压(MAP);用Western blot法检测GRP78蛋白和CHOP蛋白表达的水平;用TUNNEL法检测心肌细胞的凋亡。结果:①TAC3d组、TAC7d组、TAC14d组和TAC28d组的MAP均显著高于相应的各Sham组(P<0.05),且TAC各组的MAP随着术后时间的延长,血压值呈逐渐递增的趋势。②GRP78蛋白在TAC术后3 d即发现表达(0.20±0.02),明显高于Sham3d组(0.10±0.02)(P<0.05);但明显低于其他各TAC组(P<0.05)。随着术后时间的延长,TAC7d组GRP78蛋白的表达达到最高(0.94±0.03),其后各组呈递减趋势;但TAC7d组、TAC14d组和TAC28d组GRP78蛋白表达的水平均明显高于相应的各Sham组(P<0.05)。③随着TAC术后时间的延长,心肌组织中CHOP蛋白的表达呈递増趋势;TAC7d组、TAC14d组和TAC28d组CHOP蛋白表达的水平,分别为(0.17±0.01)、(0.36±0.03)和(0.61±0.02),均明显高于相应的各Sham组(P<0.05)。TAC3d组CHOP蛋白的表达与Sham3d组比较,无明显差异。④Sham3d组和TAC3d组中仅有极少数凋亡细胞;Sham7d组和TAC7d组可见较多的凋亡细胞,且随着时间的延长细胞凋亡率逐渐增加,TAC28d组的凋亡率达最高(30.23±0.17)%;TAC7d组、TAC14d组和TAC28d组细胞凋亡率显著高于相应的各Sham组(P<0.05)。⑤相关性分析显示,从TAC7d组、14d组及28d组的MAP与心肌组织中GRP78蛋白的表达呈显著的负相关(r=-0.882,P<0.01),与CHOP蛋白的表达呈显著正相关(r=0.933,P<0.01);CHOP蛋白的表达与GRP78蛋白的表达呈显著负相关(r=-0.980,P<0.01),与心肌细胞的凋亡率呈显著的正相关(r=0.997,P<0.01)。结论:高血压诱导心肌组织内质网应激反应在血压升高早期以GRP78的表达占优势,随着血压持续性升高可导致CHOP在心肌中超表达,参与心肌细胞凋亡的病理过程,并使心肌内质网应激稳态调节发生失衡。

AIM： To study the expression of glucose-regulated protein 78 （GRP78） and C/EBP homologous protein （CHOP） in rat heart after aortic constriction and their relationship with cardiac myocyte apoptosis. METHODS： Healthy male Sprague Dawley rats were randomly divided into transverse aomc constriction （TAC） group and sham group. The rats in TAC group received abdominal TAC. Blood pressures were measured on the 3rd, 7th, 14th and 28th day after surgery. Western blot was used to assess the protein level of GRP78 and CHOP and TUNEL was used to detect the cardiac myocyte apoptosis. RESULTS： Compared with those in sham group, the mean arterial pressure （MAP） of rats in TAC group increased significantly on the third day after TAC and increased progressively during the experiment （ P 〈 0.05 ）. On day 3 after TAC, the protein level of GRP78 in TAC group increased compared with that in control group （ P 〈 0. 05）, but its expression was lower than that on days 7, 14 and 28 （ P 〈 0.05 ）. On day 7 after TAC, the protein level in the TAC group was the highest and although it decreased on days 14 and 28, high levels sustained compared with sham group （P 〈0.05 ）. After TAC, the protein levels of CHOP in TAC groups increased progressively and, except on day 3 （ P 〉 0. 05 ）, they were higher than those in sham group （ P 〈 0.05 ）. On day 7 after surgery, cardiac myocyte apoptosis in both TAC group and sham group could be detected by TUNEL, and the incidence of cardiac myocyte apoptosis increased progressively. The incidence of cardiac myocyte apoptosis in TAC group on day 28 after TAC was （30.23 ±0. 17）% , which was the highest. Correlation analysis indicated a negative correlation between MAP and GRP78 protein expression in TAC group from day 7 after TAC （r= 0. 882, P 〈 0. 01 ） and a posi- tive correlation between the MAP and CHOP protein expression （r = 0. 933, P 〈 0. 01 ）. The analysis also indicated a negative correlation between CHOP protein expression and GRP78 protein expression （r = -0. 980, P 〈0. 01 ）, and a positive correlation between CHOP protein expression and the incidence of cardiac myocyte apoptosis （r = 0. 997, P 〈 0.01 ）. CONCLUSION： TAC-caused hypertension induces endoplasmic reticulum stress. At the early stage of hypertension, the expression of GRP78 has the highest status. Although prolonged endoplasmic reticulum stress activated by hypertension induces a significant upregulation in CHOP, CHOP may participate in myocyte apoptosis in hypertension and cause the regulatory disequilibrium of endoplasmic reticulum stress.