Hydrogen Sulfide Suppresses The Increase In Reactive Oxygen Species In Medullary Neurons Induced By Angiotensin â…¡

Like nitric oxide (NO) and carbon monoxide (CO), H2S has been shown to be the third gaseous transimitter, which is a toxic gas with the smell of rotten eggs and is found in multitudes of systems. Evidences show that H2S protects neurons from oxidative stress by increasing the reducibility of neurons. Our previous morphological experiments demonstrated that H2S in the rostral ventrolateral medulla (RVLM) lowered blood pressure and heart rate by inhibiting the activity of NADPH oxidase in the SHR, thus reducing the production of reactive oxygen species (ROS). In the present study, we use molecular biological techniques to explore the underlying mechanism how H2S protects neurons from oxidative stress induced by increased ROS level of medullary neurons.Primary cultured medullary neurons were used in all experiments. To detect whether CBS expressed in primary cultured medullary neurons or not, immunohistochemical method with fluorescence microscope was used.The result displayed that CBS could express in these nurons, providing the structural basis for studying the physiological effects of the central H2S. To investigate the effect of Ang Ⅱ (angiotensin Ⅱ) on the ROS level in medullary neurons, Dihydroethidium (DHE) staining was used and the activity of neurons was detected by CCK-8assay. The results showed that Ang Ⅱ (100nmol/L) significantly increased the level of ROS in medullary neurons(P<0.01), but had no effect on the activity of neuvons(P>0.05), suggesting that Ang Ⅱ had no toxical effect on the medullary neurons. However,the effect of Ang Ⅱ could be significantly inhibited by NaHS (50μmol/L,100μmol/L、200μmol/L), especially100μmol/L NaHS (P<0.05), while NaHS alone did not change the level of ROS in these neurons. Furthermore, to clarify the molecular mechanisms that hydrogen sulfide suppresses the increase in ROS in medullary neurons induced by Ang Ⅱ, western blotting was used to detect the changes of phosphorylation of MAPK family(p-ERK1/2、p-p38、p-JNK).100umol/L NaHS was added to medullary neurons followed by Ang Ⅱ to observe whether100umol/L NaHS could reduce the high expression of p-ERK1/2on neurons induced by Ang Ⅱ. Our finding indicated that Ang Ⅱ(100nmol/L) induced a significant increase in the phosphorylation of p-ERK1/2at5min,10min,15min,30min,until to1h. At5min, it showed the greateast increase in the phosphorylation of p-ERK1/2on neurons (P<0.01) Besides, we also found that pretreatment of neurons by100μmol/L NaHS significantly supressed the high expression of p-ERK1/2induced by Ang Ⅱ at the5min time point(P<0.05). PD98059, which is the inhibitor of p-ERK1/2, was used to make further confirmation. The results showed that PD98059also decreased the increased ROS induced by Ang Ⅱ in medullary neurons(P<0.01). Thus, we concluded that H2S significantly reduces ROS increased by Ang Ⅱ in medullary neurons via activation of the expression of p-ERK1/2.Furthernore, the expression of p-p38of MAPK family also be detected in these neurons. The result showed that the pretreatment of neurons by100μmol/L NaHS significantly supressed the high expression of p-p38induced by Ang Ⅱ at the30min time point(P<0.01). However, Ang11(100nmol/L) did not induce a significant increase in the phosphorylation of p-JNK at5min,15min,30min,60min. From the above results, we could conclude that H2S significantly reduces ROS increased by Ang Ⅱ in medullary neurons via activation of the expression of p-p38, but not p-JNK.In order to elucidate whether the effect of H2S was related to the NADPH oxidase or not, Real time PCR was used to detect the changes of the mRNA level of p47phox and p67phox,which are the subunits of NADPH oxidase. The results showed that H2S downregulated the increase of mRNA level of p47phox and p67phox of NADPH oxidase in medullary neuons induced by Ang Ⅱ(P<0.05). In conclusion, the results of present study suggest that H2S could significantly supress the increasing ROS level of medullary neurons induced by Ang Ⅱ via two mechanisms:(1) activation of MAPK family signaling pathways, mainly through p-ERK1/2and p-p38, to rescue neurons from oxidative stress;(2) may decreasing the mRNA level of the subunits of NADPH oxidase:p47phox and p67phox, thus inhibiting the activity of NADPH oxidase to suppress ROS increase induced by Ang Ⅱ in medullary neurons.