Mechanisms Of Central Cardiovascular Effects Of H₂S In SHR Rats: The Role Of Reactive Oxygen Species

H2S is considered to be a new gaseous molecular alongside with nitric oxide (NO) and carbon monoxide (CO). Lots of evidences indicate that H2S plays important roles in various systems such as nervous system, cardiovascular system, digestive system, et al. Recent study indicated that H2S could protect neurons from oxidative stress. Since accumulating evidence indicates that the level of reactive oxygen species (ROS) in RVLM could regulate sympathetic nervous system activity, we tested the hypothesis that H2S in CNS may inhibit NADPH oxidase and decrease the production of ROS, thus mediate cardiovascular effects. We use morphological and molecular biological techniques to explore the machanism of central cardiovascular effects of H2S.First of all, we used immunohistochemistry method and western-blot to observe the expression of CBS in CNS. In the medulla, neurons expressing CBS were located mainly in the rostral ventrolateral medulla (RVLM) and NTS in both WKY and SHR rats. Western-blot showed that the level of CBS in RVLM in SHR rats was lower compared with WKY rats (P<0.05), but there is no difference in paraventricular nucleus of the hypothalamus between the two groups. These findings indicated that the synthesis of H2S is probably decreased in SHR, which might be responsible for the different effects of H2S between SHR and WKY rats.Immunofluorescence double-labeling was also used in our experiment to identify the cellular distribution of CBS in RVLM. The results showed that CBS is mainly expressed in the neurons of RVLM, not the glia cells.To observe the central cardiovascular effects of H2S, intracerebroventricular injection (ICV) and microinjection were employed in SHR and WKY rats. we infused SAM (1nmol/10μl) or NaHS (4nmol/10μl) into the lateral ventricular of both SHR and WKY, and recorded mean blood pressure (MAP) and heart rate (HR). As shown, after the infusion of SAM, MAP and HR significantly decreased both in SHR and WKY rats compared with treatment with aCSF respectively. But infusion of NaHS only decreased MAP and HR in SHR rats (P<0.05). Microinjection of NaHS (400pmol/0.1μl) into the RVLM significantly decreased mean arterial pressure (P<0.05). These results indicate that NaHS in CNS may regulate cardiovascular effect in SHR rats and RVLM is possibly the main area of the central cardiovascular effects of H2S.In order to observe the distribution and level of ROS in RVLM in different groups, we used confocal analysis of DHE fluorescence and Lucigenin-enhanced Chemiluminescence to estimate. The level of ROS in RVLM of SHR control with LCV with aCSF was higher than WKY control (P<0.05). But after infusion of NaHS (4nmol/10μ1), the level of ROS decreased compared with SHR control (P<0.05). We also tested whether the decrease of ROS associated with the inhibition the activity of NADPH oxidase. By testing the level of ROS with NADPH or APO into the incubation medium, the activity of NADPH oxidase in RVLM of SHR control with LCV with aCSF was higher than WKY control (P<0.05). But after infusion of NaHS (4nmol/10μ1), the activity of NADPH oxidase decreased compared with SHR control (P<0.05), in the same change with APO, an inhibiter of NADPH oxidase. These results indicate that ROS produced by NADPH oxidase may mediate the central cardiovascular effects of H2S.Furthermore, we detected the mRNA level of gp91phox and p47phox, both of which are NADPH oxidase's subunits. By using Real-Time PCR, we also tested the level of p47phox and the phosphorylation of serine residues of p47phox after infusion of aCSF and NaHS into the lateral cerebroventricular. The results demonstrated that icv NaHS could significantly decreased p47phox mRNA level in the RVLM (P<0.05). Also, a significant decrease of phosporylated p47phox was detected 10 min after icv NaHS (P<0.05). Taking together, these results suggest that NaHS may reduce serine phosphorylation of p47phox, decrease the activity of NADPH oxidase, thus reduce the prodution of superoxide.In conclusion, the results of present study suggest that H2S in CNS may lower blood pressure and heart rate by inhibiting the activity of NADPH oxidase in the RVLM in SHR. Since the overproduction of ROS, mainly superoxide (O2-) in CNS by NADPH oxidase is involved in the etiology of hypertension, it can therefore to expect that inhibiting NADPH oxidase and O2- formation may become a therapeutic strategy in preventing hypertension.