Role Of The Central SIRT1-mediated Anti-oxidative Stress In Improving Cardiovascular Function In Hypertension

IntroductionEssential Hypertension is a common cardiovascular disease,and its complications such as heart failure and stroke have a serious threat to human health.In addition to increased blood pressure(BP),sympathetic nerve overactivity is another major characteristic of hypertension,which is closely related to its development and prognosis.The key regulating center of sympathetic nerve activity is located in the rostral ventrolateral medulla(RVLM)of the brainstem.It has been widely accepted that the increased excitability of neurons in the RVLM contributes to sympathetic overactivity and high BP.Furthermore,inhibition in the activity of RVLM neurons can decrease the BP and sympathetic outflow in hypertention.SIRT1(silent information regulator-2 related enzyme 1)is a kind of nicotinamide adenine dinucleotide(NAD+)-dependent protein deacetylase,which is the closest mammalian homologue of yeast silent information regulator-2(Sir2).SIRT1 deacetylates several lysine residues of histones and downstream transcription factors(such as p53,NF?B,FOXOs and PPAR-?),and then regulates the transcription of genes expression,and plays an important role in the metabolism,aging,cancer,circadian rhythm,and other physiological or pathological activities.In recent years,studies in the periphery cardiovascular system have confirmed that SIRT1 has cardiovascular protection functions through enhancing vascular endothelial function,improving myocardial ischemia,and reducing the incidence of atherosclerosis.SIRT1 also exists in the central nervous system and has biological effects.However,there is no evidence showing the effect and significance of the central deacetylase SIRT1 in mediating cardiovascular regulation.The enhanced oxidative stress is one of the important mechanisms responsible to the pathological process of cardiovascular diseases,and it has been recognized as a key contributor to sympathetic overactivity under the pathophysiological conditions such as chronic heart failure and hypertension.It has been documented that oxidative stress is dependent on increased generation of reactive oxygen species(ROS).Superoxide-anion(O2-),a major component of ROS,was produced by oxygen reacting with electron.It is suggested that the increased formation of O2-in the RVLM is the main mechanism for sympathetic overactivity,and scavenging ROS in the RVLM by the superoxide dismutase(SOD)genes overexpression lowers high BP.A number of studies suggest that SIRT1 is closely related to oxidative stress in the heart and blood vessels.However,the role and significance of this relationship in the RVLM needs to be determined.Therefore,the aim of this study was to determine the role and underlying mechanism of the central deacetylase SIRT1 in mediating regulation of cardiovascular activity.The data obtained from this study would provide the theory basis for the significance of the central SIRT1 in mediating abnormal sympathatic nerve regulation in cardiovascular diseases,and would be helpful to our understanding of generation and protective strategy for hypertension.MethodsThe 18-week old male Wistar-Kyoko rats(WKY)and Spontaneously Hypertensive Rats(SHR),weighting from 280-350 g,were used in this study.First,the SIRT1 agonist resveratrol(RSV)was injected into the fourth ventricle of animal using micro-osmotic pumps,the artificial cerebrospinal fluid(aCSF)was used as vehicle groups.In order to determine the role of the central SIRT1 in mediating the regulation of cardiovascular activity,the level of BP and heart rate(HR)were observed continuously before and after the administration,and the renal sympathetic nerve activity(RSNA)was also recorded.Experimental animals were randomly divided into four groups: WKY with aCSF perfusion group(WKY+aCSF),WKY with RSV perfusion group(WKY+RSV),SHR with aCSF group(SHR+aCSF),and SHR with RSV perfusion group(SHR+RSV).In order to further determine the function and underlying mechanism of SIRT1 in the RVLM,injections of gene lentivirus was used to overexpression of SIRT1.BP and HR were continuously observed before and after RVLM gene transfer.Experimental animals were randomly divided into 4 groups: WKY with LenVi-GFP transfection group(WKY-GFP),WKY with LenVi-SIRT1 transfection group(WKY-SIRT1),SHR with LenVi-GFP transfection group(SHR-GFP),SHR with LenVi-SIRT1 transfection group(SHR-SIRT1).DHE fluorescent probe staining techniques were used to detect the ROS production in the RVLM.Western Blot analyses were used to detect the expression of the ROS-related protein NOX4 and antioxidant enzymes SOD1 in the RVLM.Results1.The cardiovascular effects of the central deacetylase SIRT1 on SHR1)The expression of SIRT1 in the RVLM of SHRImmunofluorescence co-localization analysis confirmed that SIRT1 was expressed in the presympathetic neurons of the RVLM.Western Blot analysis showed that the level of SIRT1 protein expression in the RVLM was obviously lower in SHR group than in WKY group(P<0.05,n=5).2)The central SIRT1 decreases BP and sympathetic outflow in SHRThe results of hemodynamic observation under awake and anesthetized conditions showed that the BP level of SHR-RSV group gradually decreased after perfusion with RSV compared with basal BP.Compared with SHR-aCSF group(162 ± 1.8 mmHg),BP levels were decreased in SHR-RSV group(145 ± 1.7 mmHg).And RSNA levels in SHR-RSV group(26.6 ± 1.0 %)were lower than that of SHR-aCSF group(38.1 ± 1.7 %)(P<0.05,n=5).BP and RSNA levels of WKY groups were no obvious difference before and after the administration(P>0.05,n=5).3)SIRT1 overexpression in the RVLM decreases BP in SHRThe results of hemodynamic observation under awake and anesthetized states showed that BP of SHR-SIRT1 group gradually decreased after SIRT1 overexpression in the RVLM compared with the basal BP.Compared with SHR-GFP group(168 ± 1.3 mm Hg),BP levels were decreased in SHR-SIRT1 group(143 ± 2.7 mmHg)(P<0.05,n=5).BP levels of WKY groups were no obvious difference before and after the gene transfer(P>0.05,n=5).2.The central SIRT1 inhibits oxidative stress in RVLM of SHRThe results of DHE fluorescent staining revealed that the fluorescence intensity in the RVLM was obviously higher in SHR groups than WKY groups.Compared with SHR-GFP group,the fluorescence intensity of SHR-SIRT1 group was reduced(P<0.05,n=5),suggesting that the ROS content in the RVLM of SHR was higher than that of WKY,and it was reduced after SIRT1 overexpression in SHR.3.The central SIRT1 downregulated the expression of NOX4 protein and upregulated the expression of SOD1 protein in the RVLM of SHR.Western Blot analysis demonstrated that expression level of NOX4 in the RVLM of SHR-GFP group was obviously higher than that of WKY-GFP group,whereas SOD1 expression level is lower than that of WKY-GFP group.Compared with SHR-GFP group,NOX4 protein expression in the RVLM of SHR-SIRT1 group was downregulated for about 50%,while the expression of SOD1 was upregulated for more than 1 times(P<0.05,n=4).ConclusionOur findings suggest that the central deacetylase SIRT1 in the RVLM decreases BP and sympathetic outflow in SHR,which is associated with anti-oxidative stress mechanism.