The Effects And Mechanism Of Hydrogen Sulfide On The Extracellular ATPconcentration In PC12Cells With Oxygen-glucose Deprivation

Background Recent studies have found that H2S has neuroprotective effects on cerebral ischemic-hypoxic injury. Our previous research also found that H2S levels increased significantly in ischemic area after cerebral ischemia. Futhermore, it was reportedin the literature that extracellular ATP concentration increased rapidly after cerebral ischemia and hypoxia or in-vitro oxygen glucose deprivation (OGD), which led to serious secondary damage. So extracellular high concentrations of ATP are thought to be "death factors" and P2X7receptors are "death receptors". the degradation and inactivation of extracellular ATP are mainly relied on membrane adenosine diphosphate triphosphate hydrolase (NTPDASE-1/CD39).Now the expression and activity of CD39is considered to determine extracellular ATP concentration. Whether. H2S protects the neurons from OGD injury by regulating the expression and activity of adenosine diphosphate triphosphate hydrolase and extracellular ATP concentration is still unkown.Objectives To investigate the effects of exogenous and endogenous H2S on extracellular ATP levels and the expression of ATP hydrolase CD39to explore the regulatory mechanism of H2S on signalling molecular ATP induced by OGD.Methods C12cells were induced by nerve growth factor to differentiate into neurons. Then the cells were divided into normal control, NaHS (NaHS), amino hydroxy acetic acid (AOAA) and AOAA+NaHS, oxygen-glucose deprivation (OGD), NaHS+OGD and AOAA OGD, AOAA+NaHS+OGD8groups. The cell viability was assessed by XTT assay. The concentration of ATP in the culture supernatant was detected with the firefly luciferase ATP bioluminescent method. The distribution and the expression of adenosine diphosphate triphosphate hydrolase (NTPDASE-1/CD39) protein were examined by immunofluorescence staining and western blotting respectively. Data were analyzed by using SPSS13.0statistical software.Results1. The effect of H2S on PC12cell vitality after OGDCompared with control group, the cell viability of NaHS group, AOAA group and NaHS+AOAA group was no significantly different (P>0.05). The cell viability of OGD group was significantly lower than that of control group (P<0.01) and there was a further decrease of the cell viability in AOAA+OGD group(P<0.01), which shows that the inhibition of production of endogenous H2S can aggravate OGD injury. After NaHS(a exogenous H2S donor) treatment, the cell viability of NaHS+OGD group and NaHS+AOAA+OGD group increased significantly compared with that of OGD group (P<0.05) and the cell viability of NaHS+OGD was higher than that of NaHS+AOAA+OGD group (P<0.05).2. The effect of H2S on ATP content in the medium after OGDThe concentrations of ATP in the medium had no significant difference amony control group, NaHS group, AOAA group and NaHS+AOAA group(P>0.05). ATP content in the medium of OGD group increased obviously compared with that of control group (P<0.01), and ATP content in the medium of AOAA+OGD group was higher than that of OGD group (P<0.01), suggesting that inhibition of endogenous H2S can cause the increase of extracellular ATP. ATP content in the medium of NaHS+OGD group and NaHS+AOAA+OGD significantly decreased compared with that of OGD group(P<0.05), and ATP content in the midium of NaHS+OGD group was lower (P<0.05).3. The effect of H2S on the distribution of adenosine diphosphate triphosphate hydrolase(NTPDASE-1/CD39) after OGDThe results of immunofluorescence staining showed that the distribution of CD39did not change in cells of all groups and CD39protein was distributed mainly in the cytoplasm and cell membrane. Cells in control group,NaHS group and NaHS+AOAA group spread out, and the cell density and fluorescence intensity were similar between the3groups. After OGD treatment, cell shrinkage and the decrease of cell density wereobserved. Cell fluorescence intensity of OGD group and AOAA+OGD group was significantly decreased compared with that of control group. But the cell fluorescence intensity in NAHS+OGD group and NaHS+AOAA+OGD group was stronger than that of OGD group and AOAA+OGD group after NAHS treatment. So it proves that OGD treatment inhibit CD39expression and H2S can increase CD39expression in cells with OGD treatment.4.The effect of H2S on the expression of adenosine diphosphate triphosphate hydrolase(NTPDASE-1/CD39) after OGDNo significant differences of CD39expression were found in control group,NaHS group and NaHS+AOAA group (P>0.05). After OGD treatment, CD39expression decreased significantly(P<0.01), while CD39expression of AOAA+OGD group was further reduced compared with that of OGD group (P<0.05). So it is considered that the expression of CD39can be inhibited by blocking the generation of endogenous H2S. CD39expression in NAHS+OGD group and NAHS+AOAA+OGD group increased obviously compared with the OGD grou(P<0.05)p.Furthermore the CD39protein expression of NaHS+OGD group was higher than that of NASH+AOAA+OGD group(P <0.05). All of these shows that H2S can protect against OGD injury by increasing the expression of CD39which promotes the hydrolysis of extracellular ATP.ConclusionsH2S can protect PC12cells against injuries caused by OGD. The mechanism is probobly related to up-regulating of CD39expression which promotes the hydrolysis of extracellular ATP. So H2S can regulate ATP signalling molecules induced by OGD by increasing the expression of CD39to promote extracellular ATP hydrolysis.