Protective Effects And Mechanisms Of Hydrogen Sulfide Against Oxidative-stress Damage In PC12 Cells

Objective: Using H₂O₂-induced PC12 cells damage as the model of nerve cells damage induced by oxidative stress, to investigate the protective effects and mechanisms of hydrogen sulfide against oxidative-stress damage in neurocyte.Methods: The change of cell shape and nuclear morphology were observed by light microscope and nuclear staining with Hoechst 33258 after H₂O₂-induced for 24 hours. The proliferation of PC12 cells was detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazol -ium bromide (MTT) assay. The apoptosis of PC12 cells was detected by flow cytometry (FCM) with propidium iodide (PI) stain and DNA Ladder assay. To study the change of reactive oxygen species (ROS) content in PC12 cells, the Dihydrohodamine123 (DHR123) fluorescence intensity of DHR123-stained PC12 cells was observed by fluorescence microscope and detected by FCM. To investigate the chang of mitochondrial membrane potential (△Ψm), the rhodamine123 (Rh123) fluorescence intensity of PC12 cells with Rh123 stain was observed by fluorescence microscope and detected by FCM. Western Blot was used to detect protein expression of Bcl-2 and Caspase-3 in the PC12 cells, respectively. Results:1. H₂O₂ induced PC12 cells oxidative stress damageâ‘ After PC12 cells incubated with different dose of H₂O₂ (50,100,200,400,600μmol/L) for 24 hours, the survival rate of cells was reduced in dose-dependent manner by MTT assay.â‘¡While PC12 cells were treated with H₂O₂ (200 and 400μmol/L) for 24h, the PC12 cells were evidently showed: round, decreased, refraction attenuate and adherence nature degrade, compared with the normal cells.â‘¢As shown in the nuclear staining assay using Hoechst 33258, the cells exposed to H₂O₂ (200 and 400μmol/L) for 24h appeared typical characteristics of apoptosis, including apoptotic nuclear condensation.â‘£Exposure to 200 and 400μmol/L H₂O₂ for 24h, the apoptosis proportion of PC12 cells measured by FCM was increased from 3.2% to 28.4% and 45.1%, respectively.2. The protective effects of H₂S on PC12 cells damage induced by H₂O₂2.1 H₂S decreased the inhibitory action of PC12 cells activity induced by H₂O₂â‘ In the presence of NaHS (400 and 800μmol/L), the inhibitory rates of PC12 cells induced by H₂O₂ (200 and 400μmol/L) was significantly decreased (P<0.05).â‘¡As shown in light microscope, the H₂O₂ (200μmol/L)-induced damage to the PC12 cells was evidently decreased in the presence of NaHS (400μmol/L).2.2 H₂S inhibited the apoptosis of PC12 cells induced by H₂O₂â‘ 400μmol/L NaHS rescuesed DNA fragmentation of the PC12 cells induced by 200μmol/L H₂O₂.â‘¡As shown in Hoechst 33258 staining assay, the number of cells with nuclear condensation induced by 200μmol/L H₂O₂ was significantly reduced in the presence of 400μmol/L NaHS.â‘¢As shown in FCM assay, the apoptosis proportion of the PC12 cells induced by H₂O₂ (200 and 400μmol/L) was significantly decreased in the presence of NaHS (400 and 800μmol/L) (P<0.05).3. The mechanisms of hydrogen sulfide against oxidative-stress damage in PC12 cellsâ‘ Compared with non-treated control cells, the DHR123 fluorescence and MFI in PC12 cells treated with 200μmol/L H₂O₂ for 3h were increased. However, the increase in DHR123 fluorescence and MFI induced by 200μmol/L H₂O₂ was significantly inhibited by 400μmol/L NaHS, suggesting that H₂S can inhibit the increase of ROS induced by H₂O₂ in PC12 cells.â‘¡The Rh123 fluorescence and MFI in PC12 cells treated with 200μmol/L H₂O₂ for 6h were significantly decreased. However, the decrease in Rh123 fluorescence and MFI induced by 200μmol/L H₂O₂ was prevented by 400μmol/L NaHS, suggesting that H₂S can inhibit the decrease of△Ψm induced by H₂O₂ in PC12 cells.â‘¢Expression of Bcl-2 was substantially decreased after 200μmol/L H₂O₂ for 24h but prevented by 400μmol/L NaHS.â‘£Expression of Caspase-3 was substantially increased after 200μmol/L H₂O₂ for 24h but prevented by 400μmol/L NaHS.Conclusions:1) H₂S has neuroprotective effect against oxidative stress-induced damage in PC12 cells;2) The neuroprotective effects of H₂S against oxidative stress might be related to attenuate the H₂O₂-induced intracellular ROS generation, preserve△Ψm, and prevent the H₂O₂-induced down-regulation of Bcl-2 and up-regulation of Caspase-3.