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Protection of hepatocyte growth factor on neurons subjected to oxygen--glucose deprivation/reperfusion

He Fang, Wu Lixiang, Liu Fayi, Yang Lijuan, Zhang Yan, Zhang Haifu, Zhou Ju, Huang Baisheng, Deng Xiaolu

Department of Physiology, Xiangya School of Medicine, Central South University.Changsha 410008,Hunan

Abstract

The present study was conducted to investigate the effect of hepatocyte growth factor (HGF) on cortical neurons exposedto oxygen-glucose deprivation/reperfusion (OGD/R). Primary cultured cerebral cortical neurons were prepared from Sprague-Dawleyrats. The cells were used for experiments after culture for 12 d in vitro. To initiate OGD/R, the culture medium was replaced by glucose-free medium, and cells were transferred to a humidified incubation chamber flushed by a gas mixture of 95% N_(2) and 5% CO_(2) at 37 ℃for 2 h. Following this treatment, neurons were fed with glucose-supplemented (25 mmol/L) medium, and returned to the incubatorunder normoxic condition for 0-24 h. The cell viability was assessed by MTT assay, and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. The percentage of apoptotic cells was analyzed by flow cytometry and Hoechst 33258 staining.The expressions of c-Met mRNA and protein were detected by RT-PCR and Western blot analysis, respectively. Oxygen-glucosedeprivation for 2 h decreased the cell viability and increased LDH leakage rate in cultured cerebral cortical neurons. The cell viabilitydeclined and LDH leakage rate increased with the reperfusion time going on (0-24 h). To explore the influence of HGF on neurons underoxygen-glucose deprivation for 2 h/reperfusion for 24 h (OGD_(2)/R_(24)) condition, the cultures were pretreated with HGF at differentconcentrations (5-120 ng/mL) 2 h prior to OGD_(2)/R_(24) The results showed that OGD_(2)/R_(24) treatment significantly decreased the cellviability, increased LDH leakage rate and the percentage of apopototic cells. Pretreatment with HGF at 5 ng/mL and 10 ng/mL did notaffect the decrease in cell viability resulting from OGD_(2)/R_(24) In the presence of 20 nedmL HGF,the increase in cell viability in corticalneurons exposed to OGD_(2)/R_(24) began to appear,and 80 ng/mL of HGF exhibited the maximal effect.HGF at 5,10 and 20 ne/mL did notaffect the increase in LDH leakage rate in cortical neurons exposed to OGD_(2)/R_(24) In the presence of 40 nedmL HGF,the decrease in LDH leakage rate in cortical neurons subjected to OGD_(2)/R_(24) began to appear,and 80 ng/mL of HGF displayed the maximal effect. In addition, HGF at 80 ng/mL significantly attenuated cell apoptosis resulting from OGD_(2)/R_(24). As detected by semi-quantitative RT-PCR and Western blot analysis.c-Met mRNA and protein were expressed in cerebral cortical neurons cultured for 12 d in vitro.c-Met mRNA and protein expressions in cortical neurons exposed to OGD_(2)/R_(24) were significantly upregulated and were not affected by pretreatmentof HGF at 80 ne/mL.Treatment with c-Met inhibitor SU11274 (5 #mu#mol/L)completely eliminated HGF-mediated protection of corticalneurons subjected to OGD_(2)/R_(24). The results suggest that HGF directly protects cortical neurons against OGD/R-induced cell injury in a dose-dependent manner,and HGD has a potent anti-apoptotic action on neurons exposed to OGD/R.

Key words: hepatocyte growth factor;Neurons;oxygen;glucose;reperfusion

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Citing This Article：

He Fang, Wu Lixiang, Liu Fayi, Yang Lijuan, Zhang Yan, Zhang Haifu, Zhou Ju, Huang Baisheng, Deng Xiaolu. Protection of hepatocyte growth factor on neurons subjected to oxygen--glucose deprivation/reperfusion. Acta Physiol Sin 2008; 60 (2): 235-242 (in Chinese with English abstract).