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Mechanisms of Central Cardiovascular Effects of H2S in SHR Rats: the Role of Reactive Oxygen Species

Author: YuHaiYun
Tutor: LuNing
School: Fudan University
Course: Physiology
Keywords: Hydrogen sulfide Reactive oxygen species Spontaneously hypertensive NADPH oxidase RVLM
CLC: R331
Type: Master's thesis
Year: 2011
Downloads: 58
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Abstract


Hydrogen sulfide (Hydrogen sulfide, H2S) is the the third class gasotransmitters found after the nitric oxide (NO) and carbon monoxide (CO). Despite a wide range of physiological role of H2S in the body, but its central cardiovascular effects and its regulatory mechanism is poorly understood. Studies have shown, H2S can reduce the generation of reactive oxygen species in the neurons. Reactive oxygen species (reactive oxygen species, ROS) in vivo activity oxygenates the general term, NADPH oxidase plays a major role in central catalytic generation of reactive oxygen. ROS in the central and peripheral cardiovascular system have an important impact the central ROS-mediated sympathetic activation induced cardiovascular effects. Based on the above theory, the issues raised central H2S may reduce the generation of reactive oxygen species by inhibiting the activity of NADPH oxidase, which in turn inhibits sympathetic activation and cardiovascular effects. This issue through the overall function of experimental methods and molecular experiments, expect more in-depth study of the central cardiovascular effects of H2S machine production. First, using immunohistochemical methods observed spontaneous hypertensive rats (SHR) and normotensive rats (WKY) the medulla oblongata generated H2S enzyme CBS expression explicitly to cardiovascular nuclei CBS expression characteristics. The results show that in the rat rostral ventrolateral (RVLM) and nucleus of solitary tract (NTS) are CBS expression of immunoreactive cells. Immunoblotting experiments (Western blot) for the determination of the level of expression of the SHR and WKY rats hub CBS quantitative analysis of the expression of CBS in the ventrolateral medulla and the hypothalamic paraventricular nucleus, the results show, CBS belly in SHR and WKY rat medullary lateral and paraventricular nucleus are expressed, and the expression level of 17 weeks SHR ventrolateral medulla CBS lower than WKY rats (P lt; 0.05), 8 week rats CBS were no significant differences in the expression of two parts . These results suggest that the decreased production of endogenous H2S may be related to the incidence of spontaneously hypertensive. CBS in the distribution of the cells of the central nervous system for observation, using double immunofluorescent labeling method combined with laser scanning confocal microscope observation of CBS expression in neurons and glial cells EVLM area, results showed: the RVLM area neurons CBS expression CBS expression of glial cell that CBS is mainly distributed in the neurons of the RVLM area through the the intracerebroventricular injection RVLM microinjection method we observed cardiovascular effects of H2S. In SHR and WKY rats, the intracerebroventricular injection CBS agonist SAM can cause a decrease in blood pressure and heart rate slows down (P lt; 0.05), low dose H2S donor NaHS (2nmol/10μ1 4nmol/10μI ) only in SHR rats produce cardiovascular effects of lowering blood pressure and slowing heart rate (P lt; 0.05), no significant role in WKY rats. High doses of NaHS (20nmol/10μ1) can cause SHR rats with high blood pressure and increased heart rate. Prompted low dose of H2S can produce cardiovascular effects of lowering blood pressure and heart rate in SHR rats. Based on the morphology of the experimental results, prompted central H2S in SHR and WKY effect differences may be related to the difference of two animals central endogenous H2S concentration. In order to further clarify the role of H2S as nuclei involved in cardiovascular, we observed the effect of the RVLM District microinjection of NaHS results showed: the SHR rat RVLM microinjection of NaHS (0.4nmol/0.1μ1) can cause a drop in blood pressure (P lt; 0.05 ), prompted the RVLM H2S produce central cardiovascular effects nuclei, and other brain nuclei injection NaHS cardiovascular effects may be integrated in the RVLM. In order to observe whether the cardiovascular effects of H2S caused by inhibiting the generation of reactive oxygen species, we use the the DHE fluorescence assay observed the level of reactive oxygen species in SHR and WKY rats RVLM. The results showed that: in WKY control group of red fluorescent cells more scattered, relatively dense red fluorescent cells in the SHR control group, SHR intracerebroventricular injection of low-dose NaHS (4nmol/10μ1) group of red fluorescent cells compared with SHR control group significantly reduce (P lt; 0.05) . To further verify the above results, we use the enzyme linked immunosorbent assay determination of the level of superoxide anion, results showed that the levels of reactive oxygen species in SHR control rats RVLM area than WKY rats (P lt; 0.05), intracerebroventricular injection of low dose NaHS of SHR rats ROS levels than SHR decrease in the control group (P lt; 0.05). The results suggest that the central H2S can cause the reduction of the level of ROS in SHR RVLM area. In order to observe the effect of H2S whether through inhibition of NADPH oxidase-mediated, we are using the ELISA method for the determination of the different groups of SHR and WKY rats the RVLM District NADPH oxidase activity: SHR control group rats NADPH oxidase activity than the WKY rats height (P lt; 0.05); intracerebroventricular injection the NaHS can inhibit NADPH oxidase activity (P lt; 0.05) produced similar results with the NADPH oxidase inhibitor Apocynin. These results also support our hypothesis that the central cardiovascular effects of H2S reduction of reactive oxygen species. For further observation of the relationship between the central cardiovascular effects of H2S with NADPH oxidase, we use the Real-Time PCR and Western-blot methods were used to detect the NADPH oxidase subunit gp91phox the P47phox mRNA levels and P47phox the, the level of protein phosphorylation. The results show that the intracerebroventricular injection of low-dose NaHS can reduce the ventrolateral medulla P47phox mRNA level and reduce the ratio of the phosphorylation of proteins relative to the total protein in P47phox gp91phox, mRNA levels had no significant change. These results suggest that of H2S can reduce RVLM area P47phox's level of phosphorylation, inhibition of NADPH oxidase activity, thereby reducing the generation of reactive oxygen species. In summary, our results suggest that the central cardiovascular effects of H2S in the spontaneously hypertensive rat (SHR) may be through the following channels mediated: H2S inhibition of the RVLM area NADPH oxidase activity, reducing the generation of reactive oxygen species such as superoxide anion, resulting in lower blood pressure cardiovascular effects and heart rate. Decreased production of endogenous H2S may be involved in the the spontaneously hypertensive pathological process.

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CLC: > Medicine, health > Basic Medical > Human Physiology > Blood and circulatory physiology
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