[Protection of hepatocyte growth factor against hydrogen peroxide-induced mitochondria-mediated apoptosis in rat cortical neurons.].
Hepatocyte growth factor (HGF) pretreatment could protect multiple cell types from apoptosis induced by various damages including oxidative stress. The present study was designed to investigate the protective effect of HGF on rat cortical neurons against apoptosis induced by hydrogen peroxide (H2O2) in culture, and then to explore whether HGF could influence the mitochondrial pathway of apoptosis. Primary rat cortical neurons were isolated from Sprague-Dawley rats and cultured in serum free medium containing 2% B27 and Neurobasal-A. To mimic the oxidative stress damage, cortical neurons were exposed to 100 mumol/L H2O2 for 4 h. To explore the effects of HGF on the neurons subjected to H2O2 injury, cells were pretreated with HGF 15, 30, 60 ng/mL for 24 h, respectively, and then exposed to 100 mumol/L H2O2 for 4 h. The cell viability was measured by MTT colorimetric assay and cell injury was evaluated by lactate dehydrogenase (LDH) leakage rate. Apoptotic cells were detected by Hoechst 33258 staining and Annexin V-FITC/PI double labeled flow cytometry. The caspase-3 activity was assessed by colorimetry. The alteration of transmembrane potential of mitochondria was determined by confocal laser scanning microscopy. The expression of cytochrome C protein was measured by Western blot analysis. The results showed that H2O2 treatment significantly decreased the cell viability, increased LDH leakage rate and the percentage of apoptotic cells. Pretreatment of HGF at different concentrations (15-60 ng/mL) could remarkably increase the cell viability of neurons. Compared with that of H2O2 group (53.4%+/-7.4%), the cell viabilities of neurons treated with 15, 30, and 60 ng/mL HGF significantly increased to (69.3+/-6.4)%, (77.5+/-6.1)% and (82.9+/-9.3)% (P<0.05), respectively. HGF preincubation also evidently decreased the LDH leakage rate in cortical neurons damaged by H2O2. The results of Hoechst staining revealed that HGF pretreatment could significantly reduce the apoptotic rate of neurons. The apoptotic rate of H2O2 group was (62.8+/-7.1)%, while that of HGF groups decreased significantly to (34.8+/-8.4)%, (23.5+/-3.2)% and (18.6+/-4.5)% (P<0.05), respectively. The data from caspase-3 activity assay indicated that HGF preconditioning could also remarkably decrease the caspase-3 activity of neurons. In addition, in the presence of various concentrations of HGF, the decrease of transmembrane potential of mitochondria in neurons caused by H2O2 injury could be reversed. Moreover, as detected by Western blot analysis, HGF downregulated the expression of cytochrome C protein in neurons. These results suggest that HGF has a protective effect on rat cortical neurons against apoptosis induced by H2O2, which might be related to the inhibition of the mitochondrial apoptotic pathway and the suppression of the caspase-3 activity.
Hu
Zhi-Xing
ZX
Department of Pharmacology, Kunming Medical University, Kunming, China. xingzhihu@yahoo.com.cn
Geng
Ju-Min
JM
Liang
Dao-Ming
DM
Zhou
Yi-Ping
YP
Luo
Min
M
D000818:Animals; D017209:Apoptosis; D001921:Brain / Q000166:cytology; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008928:Mitochondria / Q000502:physiology*; D009474:Neurons / Q000166:cytology / Q000187:drug effects*; D018384:Oxidative Stress; D051381:Rats; D017207:Rats, Sprague-Dawley
D017228:Hepatocyte Growth Factor; D045304:Cytochromes c; D006861:Hydrogen Peroxide; C505410:Casp3 protein, rat; D053148:Caspase 3