Study On Neuronprotective Effect And Mechanism Of Sonic Hedgehog Signaling On Cortical Neurons Against Oxidative Stress

PartⅠThe study on the relationship between Sonic hedgehog signaling and cortical neurons under oxidative stressObjective:To investigate the change of endogenous Sonic hedgehog signaling in cortical neurons under oxidative stress.Methods:The primary cultured neurons were treated with 100μmol/l H2O2 for 3h, 6h,24h respectively. The expression of SHH, PTCH1, GLI1, which were key moleculars belong to Sonic hedgehog (SHH)signaling, were detected by Quantitative realtime RCR and Western blot at different time points. The primary cortical neurons were doubled marked by NSE and SHH fluorescent antibodies and the expression and location of SHH protein in neurons were observed with confocal microscope.Results:The expression of SHH, PTCH1, GLI1 were up regulated after the primary cortical neurons treated with 100μmol/l H2O2 for 3h. The peak of mRNA level expression appeared after 100μmol/l H2O2 treatment for 6h while the climax for protein expression appeared after 100μmol/l H2O2 treatment 24h later.The distributions of SHH protein from different treatments were located mainly in cytoplasm of neurons.Conclusions:Endogenous Sonic hedgehog signaling is activated in cortical neurons under oxidative stress. The activation is time dependent. SHH protein is distributed in neuronal cytoplasm. Part II The significance of the activation of Sonic hedgehog signaling in cortical neurons under oxidative stressObjective:To study the effects of the activation of Sonic hedgehog signaling on the survival of cortical neurons under oxidative stress.Methods:Cyclopamine and 5E1 were chose to block Sonic hedgehog signaling selectively. The primary cortical neurons were divided into five different groups depending on the different treatments:control (CTRL),100μmol/l H2O2 (H202),20μmol/l cyclopamine (Cyc),100μmol/l H202+20μmol/l cyclopamine(H202+ Cyc),100μmol/l H202+10μmol/l 5E1 (H2O2+5E1). After 24h, MTT was used to detect the cell survival rate. Exogenous SHH protein was used to activate Sonic hedgehog signaling while cyclopamine treatment blocked it. The different treatments on primary cortical neurons were divided into following groups: control (CTRL),100μmol/l H2O2 (H202),100μmol/l H2O2+3μg/ml SHH(H202+SHH), 100μmol/l H2O2+3μg/ml SHH+20μmol/1 cyclopamine(H202+SHH+Cyc). MTT was used to detect the cell survival rate after 24h. The neurons apoptosis was measured by Flow cytometry and observed by Hoechst staining.Results:Cyclopamine or 5E1 treatment separately induced the decreased survival rate on neurons under oxidative stress significantly (p<0.05). Exogenous SHH protein activated Sonic hedgehog signaling, enhanced the cell survival, decreased the cell apoptosis, which were significantly different from the 100μmol/l H202 alone group (p<0.05). The effects were partly reversed by cyclopamine treatment.Conclusions:The activation of Sonic hedgehog signaling plays a protective role on neurons under oxidative stress PartⅢThe study on the protective factors involved in the neuroprotective effects of Sonic hedgehog signaling under oxidative stressObjective:To evaluate the changes of the factors involved in the neuroprotective effect of Sonic hedgehog signaling under oxidative stressMethods:The primary cortical neurons were treated after culture for 10 days and divided into four different groups:control (CTRL),100μmol/l H202 (H202), 100μmol/l H2O2+3μg/ml SHH (H202+SHH),100μmol/l H2O2+3μg/ml SHH+20μmol/l cyclopamine(H202+SHH+Cyc). The levels of ROS in neurons were detected by DHE probe. The production of SOD, MDA, GSH-Px, LDH from cells or supplement culture medium were examined by the kits according to the instructions. Western blot and Quantitative realtime PCR were used to measure the expression of Bcl-2 and Bax. The expression of VEGF and BDNF were detected by ELISA and Quantitative realtime PCRResults:The levels of ROS induced by neurons under oxidative stress decreased after the activation of Sonic hedgehog signaling. Sonic hedgehog signaling promoted the production of SOD and GSH-Px and decreased the production of MDA and LDH.It also up regulated the expression of Bcl-2 and down regulated the expression of Bax. The expression levels of VEGF and BDNF were increased when Sonic hedgehog signaling was activated. Cyclopamine treatment attenuated these effects (p<0.05).Conclusions:Activation of Sonic hedgehog signaling on neurons under oxidative stress mediates the neuroprotective effects by multiple factors, which include regulating the oxidation-reduction system by increasing anti-oxidant capability and reducing the toxic products, up-regulating the anti-apoptosis factor/apoptosis factor ratio, increasing the neuroprotecrive factors. Part IV The study on the pathways involved in the neuroprotective effects of Sonic hedgehog signaling under oxidative stressObjective:To explore the underlying pathways involved in the neuroprotective effects of Sonic hedgehog signaling under oxidative stress.Methods:The primary cortical neurons were divided into four different groups: control(CTRL),100μmol/l H2O2, 100μmol/1 H2O2+3ug/mlSHH(H2O2+SHH),100μmol/l H2O2+3μg/ml SHH 20μmol/1 cyclopamine(H2O2+ SHH+Cyc). Western blot was used to detect the expressions of p-ERK, p-Akt, p-p38 protein. LY294002 (20μmol/l) was used to selectively block the PI3K/Akt signaling. The primary cortical neurons were then divided into five different groups:100μmol/l H2O2(H2O2),100μmol/l H2O2+3μg/ml SHH(H2O2 +SHH),100μmol/lH2O2+3μg/mlSHH+20μmol/lcyclopamine(H2O2+SHH+Cyc),100μmol/l H2O2+3μg/mlSHH+20μmol/l LY294002(H2O2+SHH+LY294002). MTT was used to measure the cell survival. The cells apoptosis were analyzed by FCAS. Western blot was used to measure the expression of Bcl-2 and Bax protein.Results:Exogenous SHH treatment enhanced the expression of p-Akt protein and attenuated the expression of p-ERK protein significantly (p<0.05), but not p38 proteins (p>0.05). All the effects could be partially reversed by cyclopamine. While LY294002 was added to block PI3K/Akt pathway, the effects induced by SHH,including enhanced cell survival, the decreased cell apoptosis and the up regulation of Bcl-2/Bax were reversed significantly (p<0.05).Conclusions:Inhibition of ERK pathway and activation of PI3K/Akt pathway are involved in the neuroprotective effects of SHH. PI3K/Akt pathway is required for SHH to decrease cell apoptosis and enhance cell survival. SHH/Akt/Bcl-2 pathway may be the underlying mechanism to protect neurons against H2O2-induced apoptosis.