基于HSF1高表达H9c2细胞的参芎葡萄糖注射液抗氧化损伤作用探讨

Anti-oxidative Activity of Shenxiong Glucose Injection in H9c2 Cells with High Expression of HSF1

目的：建立热休克转录因子1（HSF1）高表达的H9c2细胞系,考察在HSF1高表达的情况下参芎葡萄糖注射液（SGI）对过氧化氢（H2O2）诱导的H9c2细胞氧化损伤的保护作用。方法：构建GV1422-HSF1的重组质粒,并利用Fu GENE 6转染试剂将重组质粒转染至H9c2细胞中,用遗传霉素（G418）来筛选稳定转染细胞株,并用实时荧光定量聚合酶链式反应法（Real-time PCR）和蛋白免疫印迹法（Western blot）检测HSF1和热休克蛋白70（HSP70）mRNA和蛋白的表达情况,来鉴定HSF1高表达的H9c2细胞系（H9c2-HSF1）是否建立成功。用H2O2（300μmol·L-1）处理H9c2-HSF1细胞0.5 h构建氧化损伤模型,考察HSF1高表达的情况下参芎葡萄糖注射液预处理对细胞存活率、乳酸脱氢酶（LDH）和肌酸激酶（CK）漏出量、丙二醛（MDA）和活性氧（ROS）含量、谷胱甘肽过氧化物酶（GSH-Px）和超氧化物歧化酶（SOD）活性的影响,同时Western blot检测HSF1和HSP70蛋白表达情况。结果：Real-time PCR和Western blot实验结果显示：筛选出来的稳定转染细胞的HSF1和HSP70蛋白表达显著上调（P〈0.01）,表明H9c2-HSF1细胞系构建成功。经参芎葡萄糖注射液预处理6 h,H2O2损伤0.5 h后,与H9c2＋H2O2组和H9c2-HSF1＋H2O2组比较,对应给药组细胞的HSF1和HSP70蛋白表达显著上调（P〈0.01）,细胞存活率显著增高（P〈0.01）,LDH,CK释放量和MDA含量显著减少（P〈0.01）;ROS含量显著降低,GSH-Px和SOD活性明显升高。结论：本研究成功构建了HSF1高表达H9c2细胞系,并发现HSF1高表达能明显增强参芎葡萄糖注射液抗氧化损伤作用,其机制可能不在于增加细胞清除ROS能力,但可能与上调HSP70表达相关,需要进一步研究。

Objective： To construct H9 c2 cell line with high expression of heat shock transcription factor1（HSF1）,in order to investigate the effect of Shenxiong glucose injection（SGI） in protecting H2 O2-induced H9 c2 cellular oxidation injury with a high expression of HSF1. Method： The recombinant plasmid of GV142-HSF1 was constructed and transfected into H9 c2 cells with Fu GENE 6 transfection reagent,and G418 was used to obtain stably transfected cell line. The expressions of HSF1 and heat shock protein 70（HSP70） were detected with Realtime RT-PCR and Western blot to confirm the establishment of H9 c2 cell line with high expression of HSF1（H9 c2-HSF1）. The H9 c2-HSF cells were pretreated with SGI for 24 h,and then treated with H2 O2（300 μmol·L-1）for 0. 5 h. After that,cell viability,contents of malondialdehyde（MDA） and reactive oxygen species（ROS）,releases of lactate dehydrogenase（LDH） and creatine kinase（CK）,activities of superoxide dismutase（SOD）and Glutathione peroxidase（GSH-Px）,and expressions of HSF1 and HSP70 were examined. Result： The results of Real-time PCR and Western blot assays showed that the mRNA and protein expressions of HSF1 and HSP70 were significantly up-regulated（P〈0. 01） compared with the untransfected cells,which verified the establishment of H9 c2-HSF1 cell line. Compared with H9 c2 cells ＋ H2 O2 and H9 c2-HSF1 cells ＋ H2 O2, corresponding administration unit showed an up-regulated expressions of HSF1 and HSP70（P〈0. 01）,an increased cell survival rate（P〈0. 01）,and decreased releases of LDH and CK and MDA generation（P〈0. 01） after 6 h pretreatment with SGI and 0. 5 h treatment with H2 O2,dereased content of ROS and increased the activities of SOD and GSHPx. Conclusion： H9 c2 cell line with high expression of HSF1 was constructed successfully. It is found that high expression of HSF1 can significantly enhance the anti-oxidative damage effect of SGI; the mechanism may not be associated with the enhancement of the cell＇s capability of clearing ROS,but may be correlated with the upregulation of HSP70 expression,which needs further study.