title: Anti-hypoxia effect of adenovirus-mediated expression of heat shock protein 70 (HSP70) on primary cultured neurons.
abstract: Heat shock protein 70 (HSP70) has attracted great attention recently in hypoxia injury because of its close link to the recovery after hypoxic-ischemic damage in organs. However, the cellular mechanism underlying its protective roles remains unclear. In this study, we developed a recombinant adenovirus containing HSP70-GFP (vAd-HSP70-GFP) and studied the effect of virus-mediated expression of exogenous HSP70 gene on neurons in response to hypoxia-reoxygenation injury. Virus-mediated expression of HSP70 was detected as early as 24 hr and lasted until 10 days after infection. Neurons with 48 hr vAd-HSP70-GFP infection were exposed to 0, 0.5, 1, 2, 3, or 4 hr hypoxia followed by 1 hr reoxygenation. The mRNA and protein levels of HSP70 in neurons exposed to different lengths of hypoxia were compared by using RT-PCR and Western blotting (WB). The 1-hr hypoxia exposure showed the most significant increases in the HSP70 mRNA and protein level compared with other exposure durations. MTT assay showed that HSP70 overexpression significantly increased the neuronal viability, accompanied by decreased lactate dehydrogenase (LDH) activity in the culture medium after hypoxia-reoxygenation. Neurons with vAd-HSP70-GFP exhibited increased levels of mitochondrial cytochrome C (Cyt-C) and decreased levels of cytoplasmic Cyt-C compared with vAd-GFP-infected cells. These results suggest a neuroprotective role of exogenous HSP70 against hypoxia-reoxygenation injury, possibly via preventing initiation of mitochondrial apoptosis.
authors.affiliation: Department of Intensive Care Unit, Municipal Hospital of Medical College of Qingdao University, Qingdao, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.forename: Fuqiang
authors.initials: F
authors.lastname: Chen
authors.forename: Chun
authors.initials: C
authors.lastname: Guan
authors.forename: Fangfang
authors.initials: F
authors.lastname: Yang
authors.forename: Yan
authors.initials: Y
authors.lastname: Qu
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D015687:Cell Hypoxia / Q000502:physiology*; D049109:Cell Proliferation; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D045304:Cytochromes c / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism*; D006931:Hyperbaric Oxygenation; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D051379:Mice; D008815:Mice, Inbred Strains; D009474:Neurons; D012333:RNA, Messenger / Q000378:metabolism; D014161:Transduction, Genetic
chemical_list: D018840:HSP70 Heat-Shock Proteins; D012333:RNA, Messenger; D049452:Green Fluorescent Proteins; D045304:Cytochromes c; D007770:L-Lactate Dehydrogenase
keywords: adenovirus; heat shock protein 70 (HSP70); hypoxia; neurons; reoxygenation