Effects Of HGF On Neurons Subjected To Hypoxia/Reoxygenation And The Molecular Mechanisms Mediated The Effects

Objective:To investigate the effects of hepatocyte growth factor (HGF)on cerebral cortical neurons subjected to hypoxia/reoxygenation (H/R)and explore the molecular mechanisms mediated the effects.Methods:Primary cultured cerebral cortical neurons were prepared from Sprague-Dawley rats.Cell injury was evaluated by lactate dehydrogenase(LDH)release rate,cell viability was assessed by MTT assay and percentage of apoptotic cells was analyzed by Flow Cytometry The effects of hypoxia,low(no)glucose and hypoxia / reoxygenation on cultured cerebral cortical neurons were observed to establish a hypoxia/reoxygenation model in vitro.We studied the effects of HGF on cortical neurons by the model.The relationship between the influence of HGF on neurons exposed to H/R with MEK/ERK1/2 pathway,PI-3K/Akt pathway and apoptosis-related molecules(Bcl-2,Bcl-xL,Bax)was evaluated by MTT assay,Flow Cytometry,Hoechst 33258 staining,semiquantitative RT-PCR and Western blotting analysis.Then,we observed the alteration of HGF on the expression,of hypoxia signaling pathway by gene array technology.Real-time quantitative PCR assay was conducted to examine the level of Myb12 mRNA,Cygb mRNA,Birc5 mRNA,Cdc42 rnRNA,IL1βmRNA,NOS2 mRNA which had been affected by HGF.Western blotting analysis was performed to detect the expression of Cygb protein and NOS2 protein. Results:1.Effects of hypoxia,low glucose,glucose-free and hypoxia /reoxygenation on cortical neurons(1)MTT assay showed that the cell viability was decreased 1 h after hypoxia,and the LDH release rate was increased 4 h after hypoxia.As the treatment time went on,the cell viability was on the decline and the LDH release rate was on the rise.(2)Either low glucose(2.5mmol/L)or glucose-free induced cell injury.Compared with low glucose group,the cell viability of glucose-free group was lower and the LDH release rate was higher.(3)The tolerance to hypoxia in various group followed this rule: normal glucose＞low glucose＞glucose-free.(4)Hypoxia/reoxygenation decreased the cell viability,increased the LDH release rate and the apoptotic rate of cortical neurons exposed to hypoxia.Along with the duration of reoxygenation(0～24h),the cell viability was on the decline,the LDH release rate and the apoptotic rate were on the rise.2.Effects of HGF on cortical neurons subjected to hypoxia /reoxygenation(1)c-Met mRNA and c-Met protein were present in both the cultured cortical neurons at 10 days in vitro(DIV10)and the cerebral cortical tissue from post-natal day 10 rat detected by RT-PCR and Western blotting analysis.(2)Effects of HGF on cortical neurons subjected to hypoxia 8 h/reoxygenation 12 h(H₈/R₁₂):The,decrease of LDH release rate by HGF was observed at 20 ng/ml,and the maximum effect was at 60ng/ml; The increase of cell viability and the anti-apoptotic effect by HGF were observed at 10 ng/ml,and the maximum effect was at 60ng/ml. Furthermore,administration of HGF(60ng/ml)at 12 h before hypoxia,1 h before hypoxia,the same time of hypoxia or the same time of reoxygenation decreased the LDH release,rate,increased the cell viability and decreased the apoptotic rate.And the most potent effect of HGF mediated cell survival was observed,at 1 h before hypoxia.Application of c-Met inhibitor SUl1274(5μmol/L)significantly abolished the HGF mediated protection in cortical neurons subjected,to H₈/R₁₂.(3)Effects of HGF on cortical neurons exposed to oxygen-glucose deprivation 2 h/reperfusion 24 h(OGD₂/R₂₄):The decrease of LDH release rate,the increase of cell viability and the decrease of apoptotic rate by HGF were observed at 20 ng/ml,and the maximum effect was at 80ng/ml.Moreover,administration of HGF(80ng/ml)at 12 h before hypoxia,2 h before hypoxia,the same time of hypoxia or the same time of reoxygenation increased the cell viability and decreased the apoptotic rate.Treatment with HGF(80ng/ml)at the same time of reoxygenation had no significant effect on the LDH release rate.The most potent effect of HGF mediated cell survival and decrease of LDH release rate were observed at 2 h before hypoxia.Treatment with c-Met inhibitor SU11274 (5μmol/L)remarkably eliminated the neuroprotection by HGF.3.Relationship between the influence of HGF on neurons exposed to hypoxia/reoxygenation with MEK/ERK1/2 pathway, PI-3K/Akt pathway and apoptosis-related molecules(1)As detected by Western blotting analysis,HGF activated both MEK/ERK1/2 and PI3-K/Akt pathways in cerebral cortical neurons.(2)The presence of U0126(500 nmol/L),a potent inhibitor of MEK, significantly eliminated HGF mediated cell survival and anti-apoptotic effects on neurons subjected to H₈R₁₂.Inhibition of Akt activation with LY294002(10μmol/L)reduced the HGF mediated cell survival and anti-apoptotic effects.Neither inhibitor at the concentration used in this work,in the absence of HGF,significantly altered cell viability and the amount of apoptosis in cultures not exposed to H₈/R₁₂or H₈/R₁₂-treated culttires.(3)As determined by semi-quantitative RT-PCR and Western blotting analysis,the level of Bcl-2 mRNA,Bcl-xL mRNA,Bcl-2 protein and Bcl-xL protein in cortical neurons exposed to H₈/R₁₂were significantly decreased.The expression of Bax mRNA in cortical neurons exposed to H₈/R₁₂was remarkably increased,whereas no detectable change in Bax protein was observed.Pre-treatment with HGF(60 ng/ml) remarkably attenuated H₈/R₁₂induced decrease in the level of Bcl-2 mRNA,Bcl-xL mRNA,Bcl-2 protein and Bcl-xL protein.The expression of Bax mRNA and-Bax protein in cortical neurons exposed to H₈/R₁₂was not affected by the application of HGF(60 ng/ml).4.Influence of HGF on the expression of hypoxia signaling pathway in cortical neurons subjected to hypoxia/reoxygenation(1)Functional clustering analysis of the genes affected by HGF indicated that these genes were involved in metabolism,signal transduction,cell growth,transcription,cytoskeleton,apoptosis and so on.(2)As assessed by real-time quantitative PCR assay,H₈/R₁₂treatment decreased the level of Myb12 mRNA,Cygb mRNA,Birc5 mRNA,and increased the expression of Cdc42 mRNA,NOS2 mRNA,I11b mRNA in cortical neurons;Pre-treatment with HGF(60 ng/ml)remarkably inhibited H₈/R₁₂induced decrease in the level of Myb12 mRNA,Cygb mRNA, Birc5 mRNA and increase in the expression of Cdc42 mRNA,NOS2 mRNA,I11b mRNA.(3)As detected by Western blotting analysis,the level of Cygb protein in corticat neurons exposed to H₈/R₁₂was significantly decreased,and the expression of NOS2 protein was remarkably increased.HGF(60 ng/ml) significantly attenuated H₈/R₁₂induced decrease in the level of Cygb protein and increase of the expression of NOS2 protein. Conclusions:1.Hypoxia or low(no)glucose can induce injury in cortical neurons, which is closely related to sustained time,and the cortical neurons are very sensitive to hypoxia or low(no)glucose.2.Low glucose or glucose-free can aggravate the injury in cortical neurons resulting from hypoxia in a time-dependent manner,and the tolerance to hypoxia under glucose-free condition is lower than normal or low glucose.3.Hypoxia/reoxygenation can aggravate hypoxia induced injury and apoptosis of cortical neurons in a time-dependent manner.4.c-Met receptor is expressed in both the cerebral cortical tissue and the cultured cortical neurons.5.HGF has a direct protection on cultured cerebral cortical neurons subjected to hypoxia/reoxygenation in a dose-and time-dependent manner.6.The neuroprotection of HGF depend on ERK1/2 pathway,and,to a lesser extent,PI-3K/Akt pathway.7.Bcl-2 and Bcl-xL appear to be involved in the neuroprotection of HGF.8.The hypoxia signaling pathway related genes,which are influnced by hypoxia/reoxygenation are achieved.9.The hypoxia signaling pathway related genes in cortical neurons exposed to H/R,which are affected by HGF are clarified.10.The protection of HGF on cortical neurons subjected to H/R may be related to inhibition of decrease of the level of Myb12,Cygb,Birc5 and the increase of the expression of Cdc42,NOS2,IL1β.