Study On The Roles Of HIFs And Their Target Genes In Chemical Hypoxia Preconditioning Protection

Hypoxia is a common injurious factor in many clinical diseases and some specific environments such as high altitude, aviation and diving. Although the hypoxic reactions vary from cell to cell, as hypoxia injury aggravates and exposure time prolongs, metabolic block and cellular malfunction happen, even cell death occurs. Neuronal systems are extremely dependent on aerobic oxidation to supply energy,therefore, neurons are very sensitive to hypoxia, and easy to be hurt by hypoxia. It is very important to look for the approaches to enhance hypoxia tolerance of neurons, and to look for new strategies to prevent and treat clinical hypoxic diseases. Recent studies have showed that hypoxia preconditioning can protect neurons from damages caused by severe hypoxia or otherwise lethal injury. This neuroprotective effect has gained more and more attentions from researchers. By now a number of mechanisms and molecules have been linked with neuroprotective effects of hypoxia preconditioning. Unfortunately, this array of different molecules has not yielded a clear systematic approach to understanding mechanisms of hypoxia preconditioning. Hypoxia-inducible factors, regulated by multiple injury modalities, can affect many signal pathways, and are master regulators of cellular adaptive responses to hypoxia by mediating hypoxic signal transduction and activateing transcription of hypoxia related genes. Hypoxia-inducible factors are involved in the adapting of cells to changes in the O2 level, which is essential for the survival of cells under low oxygen tension. Recently the roles of HIFs and HIFs target genes arouse many researchers'concern. There are many reports about the roles of HIFs and HIFs target genes/proteins in hypoxia preconditioning. But the results are very controversial. For example, some studies suggest that up-regulation of HIF-1 appears to be protective, while some suggest that down-regulation of HIF-1 is likely to be the case, and still some reports indicate that the protection of hypoxia preconditioning is independent of HIF-1 signaling cascade. Whether HIFs and HIFs target genes/proteins correlate with protection afforded by hypoxia preconditioning is still underwater and needs further investigation. CoCl2 can mimic hypoxia, and this chemical also can mimic the effect of hypoxia preconditioning in the brain. In this study, we established an experiment model of CoCl2 chemical hypoxia preconditioning by using differentiated neuronal SH-SY5Y cells to investigate the effects of chemical hypoxia preconditioning on hypoxia-induced cell injury and the roles of HIF-1α,HIF-2αand their target genes (VEGF,GLUT-1,EPO,LDH-A) in chemical hypoxia preconditioning. Further evaluation of the potential neuroprotective effect of VEGF was done by investigating the effect of either recombinant human VEGF or neutralizing anti-VEGF antibody on subsequent hypoxia injury. The main methods, results and conclusions are as following:Methods1. The human neuroblastoma cell line SY-SY5Y cells were cultured in MEM/F12 medium supplemented with 15% (v/v) foetal calf serum, following 3 to 7 days differentation in the 10μM retinoic acid.2. Differentiated SH-SY5Y cells were randomly divided into control group, chemical hypoxic preconditioning group (50μMCoCl2, 3 h, following 1h normal culture, 2% O2, 28 h) and hypoxia group (2% O2, 28 h). Or differentiated SH-SY5Y cells were randomly divided into control group, chemical hypoxia group (250 MCoCl2, 24 h) and chemical hypoxic preconditioning group (75 MCoCl2, 1.5 h, following 3h normal culture, 250MCoCl2, 24 h). Cell viability was evaluated by measuring lactate dehydrogenase (LDH) release and MTT assay to determine the neuroprotective effects of chemical hypoxia preconditioning.3. RT-PCR was used to examine the expressions of VEGF,GLUT-1,EPO,LDH-A mRNA.4. Western Blotting was used to examine the expressions of VEGF proteins.5. Differentiated SH-SY5Y cells were randomly divided into control group, chemical hypoxic preconditioning group (50μMCoCl2, 3 h, following 1 h normal culture, 2% O2, 28 h), chemical hypoxic preconditioning plus VEGF antibody group (50μMCoCl2+40μg/ml VEGF antibody, 3 h, following 1h normal culture, 2% O2+40μg/ml VEGF antibody, 28 h), hypoxia group (2% O2, 28 h) and hypoxia plus VEGF group (2% O2+100 ng/ml VEGF, 28 h). 6. Western Blotting was used to examine the expressions of HIF-1α, HIF-2αproteins.Results1. The preconditioned neuronal cells had a higher survival rate (P<0.05) and a lower lactate dehydrogenase leakage (P<0.05). (In the experiment model of CoCl2 preconditioning against CoCl2 chemical hypoxia injury )2. The preconditioned neuronal SH-SY5Y cells had a higher survival rate (P<0.05) and a lower lactate dehydrogenase leakage (P<0.01). (In the experiment model of CoCl2 preconditioning against normal baric hypoxia injury )3. The levels of GLUT-1,EPO mRNA increased in the preconditioning group compared with those in the hypoxia group (P<0.05), so is the case with VEGF mRNA expression (P<0.01), but the LDH-A mRNA expression had no apparent difference between the two gropes.4. The expression of VEGF protein increased in the preconditioning group compared with that in the hypoxia group (P<0.01).5. The effect of precondition was reduced by using a neutralizing anti-VEGF antibody (40μg/ml) to block VEGF, and was emulated with the application of recombinant human VEGF (100 ng/ml) based on MTT assay.6. The expression of HIF-2αprotein increased in the preconditioning group compared with that in the hypoxia group (P<0.01), while the expression of HIF-1αprotein had no difference between the two gropes.Conclusions1. Based on lactate dehydrogenase (LDH) release and MTT assay, an experiment model is established which proves that CoCl2 preconditioning can enhance chemical hypoxia tolerance of neuronal SH-SY5Y.2. Based on lactate dehydrogenase (LDH) release and MTT assay, an experiment model was established which proves that CoCl2 preconditioning can protect neuronal SH-SY5Y cells from hypoxia (2% O2) injury.3. The preconditioning effects of CoCl2 against normal baric hypoxia injury are greater than against CoCl2 chemical hypoxia injury.4. The enhanced GLUT-1, EPO, VEGF mRNA lever might be involved in the observed chemical hypoxia preconditioning effects. 5. VEGF plays an impotant role in the neuroprotection effects of chemical hypoxia preconditioning.6. HIF-2, rather than HIF-1 is involved in the observed preconditioning effects, by enhancing the hypoxia-regulated genes including VEGF.