钙网蛋白参与缺氧预处理对氧化应激损伤大鼠成心肌H9c2细胞的保护作用

Calreticulin is involved in the protection of hypoxic preconditioning against cardiomyoblast H9c2 cell oxidative injury

目的:观察缺氧预处理(HPC)对氧化应激诱导大鼠成心肌H9c2细胞损伤的保护作用,探讨钙网蛋白(CRT)是否参与其保护效应及p38MAPK是否参与其信号转导过程。方法:H9c2细胞随机分为8组:氧化应激(H2O2)组、短暂缺氧(HPC)组、HPC+H2O2组、SB203580+HPC+H2O2组、反义干扰(AS)组、AS+H2O2组、AS+HPC+H2O2组和对照组。以细胞存活率、乳酸脱氢酶(LDH)活性及流式细胞术检测细胞损伤情况;采用RT-PCR和Westernblotting分别检测CRT表达和p38MAPK磷酸化水平。结果:(1)HPC可减轻氧化应激损伤,与H2O2组比较,HPC+H2O2组细胞凋亡率和LDH漏出分别降低13.4%和44.0%,存活率增高12.7%(均P<0.05);HPC前以特异性p38MAPK抑制剂SB203580预孵育消除HPC的保护作用,与HPC+H2O2组相比,细胞凋亡率和LDH漏出分别增高5.4%和45.0%,存活率降低5.0%(均P<0.05);(2)氧化应激明显上调CRT表达(较对照组高3.6倍)(P<0.05);单纯短暂缺氧可诱导CRT表达(较对照组高1.4倍,P<0.05),但上调程度较H2O2组低48%(P<0.05);HPC可降低CRT过表达程度(降低26%)(P<0.05);(3)反义寡核苷酸干扰CRT表达后HPC对氧化应激的保护作用降低,相关分析显示HPC诱导的CRT适度表达与细胞存活率正相关(r=0.8573,P<0.05);(4)HPC前应用p38MAPK抑制剂,抑制CRT表达上调(分别较HPC+H2O2组和HPC组低38%和23%)(均P<0.05)。结论:HPC可通过p38MAPK信号途径诱导CRT表达上调,减轻大鼠成心肌H9c2细胞氧化应激损伤。

AIM： To investigate whether hypoxic preconditioning （HPC） protects cardiomyoblast H9c2 cells against oxidative injury, and to discuss whether calreticulin （CRT） contribute to this protection through p38 MAPK signaling pathway. METHODS： Cardiomyoblast H9c2 cells were randomly divided into eight groups as follows： hydrogen peroxide stress （ H2O2 ） ; brief hypexic exposure of 20 min to simulate hypexic preconditioning （HPC） ; 20 rain of hypoxic exposure followed by 24 h of normoxic reoxygenation before hydrogen peroxide stress （ HPC ＋ H2O2 ）, SB203580 （ the specific inhibitors of p38 MAPK） ＋ HPC ＋ H2O2, antisense oligonucleotides transfection of calreticulin （ AS）, AS ＋ H2O2, AS ＋ HPC ＋ H2O2 and control. Morphological studies, estimation of lactate dehydrogenase （LDH） leakage and flow cytometry were employed to assess the cell apeptosis and necrosis. RT - PCR and Western blotting analysis was used to detect calreticulin expression and phosphorylation of p38 MAPK. RESULTS： The results obtained are as follows： （ 1 ） HPC relieved cell injury caused by H2O2. Compared with those in H2O2 group, apoptosis rate and LDH leakage in culture medium in HPC ＋ H2O2 group decreased 13.4% and 44.0%, respectively （ P 〈 0. 05 ）, and cell survive rate increased 12. 7% （ P 〈 0. 05 ）. SB203580, a selective p38 MAPK inhibitor presented before HPC, eliminated the eytoproteetion of HPC. Compared with HPC ＋ H2O2 group, apoptosis rate and LDH leakage increased 5.4% and 45.0%, respectively （P 〈0. 05）, and cell survive rate decreased 5.0% （ P 〈 0. 05 ）. （2） Brief hypoxia intimating HPC resulted in mild CRT up - regulation （ 1.4 - fold increased vs control group, P 〈 0. 05 ） , but this up - regulation was lower than that of 3.6 - fold increase induced by oxidative stress. HPC relieved the over - expression of CRT induced by H202（26% decreased vs H202 group, P 〈0. 05）. （3） Transfection of antisense oligonucleotides of CRT before HPC reduced cytoprotection against oxidative stress. Correlative analysis indicated that mild up - regulation of CRT induced by HPC was positively correlated with survive rate （ r = 0. 8573, P 〈 0. 05 ）. （4） SB203580 suppressed CRT up-regulation （the expression of CRT decreased 38% or 23% , vs HPC ＋ H2O2 group or HPC group, respectively）. CONCLUSION： These results suggest that hypoxic preconditioning up- regulates calreticulin expression through p38 MAPK signaling pathway and protects cardiomyoblast H9c2 cells against oxidative injury.