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

LIU Ting; SONG Fei; YANG Jian; LU Ding-yan; LIN Cun-yong; XUE Wei-na; SUN Jia

doi:10.13422/j.cnki.syfjx.20180822

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

更新时间：2024-01-04

- Anti-oxidative Activity of Shenxiong Glucose Injection in H9c2 Cells with High Expression of HSF1
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 24, Issue 8, Pages: 85-90(2018)
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- DOI：10.13422/j.cnki.syfjx.20180822
  CLC： R285
- Published：2018
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LIU Ting, SONG Fei, YANG Jian, et al. Anti-oxidative Activity of Shenxiong Glucose Injection in H9c2 Cells with High Expression of HSF1[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(8): 85-90.
DOI：

LIU Ting, SONG Fei, YANG Jian, et al. Anti-oxidative Activity of Shenxiong Glucose Injection in H9c2 Cells with High Expression of HSF1[J]. Chinese journal of experimental traditional medical formulae, 2018, 24(8): 85-90. DOI： 10.13422/j.cnki.syfjx.20180822.

摘要

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FuGENE 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（300 μmol·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。

Abstract

Objective: To construct H9c2 cell line with high expression of heat shock transcription factor 1(HSF1)

in order to investigate the effect of Shenxiong glucose injection (SGI) in protecting H2O2-induced H9c2 cellular oxidation injury with a high expression of HSF1. Method: The recombinant plasmid of GV142-HSF1 was constructed and transfected into H9c2 cells with FuGENE 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 Real\|time RT-PCR and Western blot to confirm the establishment of H9c2 cell line with high expression of HSF1 (H9c2-HSF1). The H9c2-HSF cells were pretreated with SGI for 24 h

and then treated with H2O2(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 H9c2-HSF1 cell line. Compared with H9c2 cells+H2O2 and H9c2-HSF1 cells+H2O2

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 H2O2

dereased content of ROS and increased the activities of SOD and GSH-Px. Conclusion: H9c2 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 up-regulation of HSP70 expression

which needs further study.

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