Role Of The Central PGE2-mediated Oxidative Stress In Regulation Of Cardiovascular Function

Hypertension is a kind of chronic diseases with high morbidity,leading to a variety of serious complications.The etiology of more than 90% of hypertensive patients is unknown.Although there are a variety of treatments to decrease blood pressure,the complications such as heart failure and stroke are still high.Therefore,there are great theoretical and practical significance to study the occurrence and development of hypertension.There are two main aspects involoved in regulating blood pressure,including humoral and neural mechanisms which includes sympathetic and vagal nerves.The key sympathetic center lies in the rostral ventrolateral medulla(RVLM),which receives and integrates brainstem and central nuclei [such as the nucleus of the solitary tract(NTS),paraventricular nucleus(PVN)],and peripheral baro/chemoreceptor afferent nerve impulses,connects with sympathetic preganglionic neurons in the intermediolateral column of spinal cord.Therefore,RVLM is the pivotal region in the central nervous system to regulate sympathetic outflow and cardiovascular function.It has been shown that the microinjection of ?-aminobutyric acid into the RVLM can effectively inhibit the sympathetic function and reduce blood pressure;and neurons by current stimulation in the RVLM can make the sympathetic function hyperactivity and blood pressure high.The increasing excitability of the presympathetic neurons of RVLM is the main reason of abnormal sympathetic hyperactivity,which plays an important role in the pathogenesis of hypertension.It has been documented that sympathetic hyperactivity is closely related to the development and prognosis of cardiovascular disease.Therefore,it is of great significance for the prevention and treatment of hypertension to elucidate the mechanism of RVLM and to explore the effective methods to attenuate sympathetic hyperactivity.The development of hypertension and the damage of the terminal organs are closely related to the oxidative stress caused by the immune system disorders and chronic low-grade inflammation.Once the production of reactive oxygen species(ROS)are increased or the ability of scavenging is weakened,which leads to the excessive accumulation of ROS,breaking the balance between the generation and degradation of ROS,and results in oxidative stress.Oxidative stress contributes to the development of high blood pressure,in turns,the sustained high blood pressure can also promote the increased level of oxidative stress,which establishes a vicious cycle.The production of ROS is mainly charged of nicotinamide adenine dinucleotide phosphate(NADPH)oxidase,mitochondrial respiration,and nitric oxide synthase pathway,among which NADPH oxidase is the main pathway.Superoxide dismutase(SOD)is a major component of the endogenous antioxidant defense mechanism and participates in the blockade of ROS on cell damage and repairing cell damage caused by ROS.The level of oxidation stress in the RVLM of spontaneously hypertensive rats(SHR)and angiotensin II(AngII)-induced hypertensive rats is increased and causes enhancement in activity of neurons,resulting in sympathetic hyperactivity.Furthermore,microinjection of the SOD mimic Tempol into RVLM can decrease the level of ROS and effectively reduce blood pressure.The inflammatory response of the immune system is one of the important resources to generate ROS.Moreover,the chronic continuous low-grade inflammatory reaction leads to ROS production,contributing to the development of hypertension and end organ damage.In central nervous system,cerebral vascular secretion of Prostaglandin E2(PGE2)is an important inflammatory factor through autocrine and paracrine effects of neurons.Prostaglandins(Prostaglandins,PGs)have a wide range of biological effects,playing an important role in regulating cardiovascular function.The cyclooxygenase(Cyclooxygenase,COX)is the rate limiting enzyme for generation of PGEs.The previous study suggests that the COX/PGEs axis exerts an important role in the development of hypertension and hypertension-associated with end organ damage.In rats,ventricles(Intracerebroventricular,ICV)infusion of prostaglandin E2(Prostaglandin E2,PGE2)or microinjection of PGE2 into the paraventricular nucleus(Paraventricular nucleus,PVN)enhances renal sympathetic nerve activity(RSNA)and blood pressure.However,other kinds of PGs probably be not involved in the central regulatory mechanism of cardiovascular activity.However,the mechanism of central PGE2 inducing oxidative stress is not clear.Although RVLM is an important region of the central nervous system to regulate sympathetic outflow and cardiovascular function,whether PGE2 is involved in the regulation of cardiovascular function in the RVLM has not been reported.Therefore,it is very important to explore the effects of PGE2 in the RVLM on cardiovascular function,and to clarify its underlying regulatory mechanism.The hypothesis of this study is that PGE2 may increase the production of ROS through up-regulating the protein expression of NOX and down-regulating the protein expression of SOD,which results in the excessive accumulation of ROS and the increase of oxidative stress.[Methods]The experimental animals are male SHR and WKY rats of 16-week old and Sprague-Dawley(SD)rats of 280-320 g with intraventricular(ICV)perfusion of AngII(24 g/d)for 7 days via femoral arterial cannulation to measure mean arterial pressure(MAP)and heart rate(HR),Enzyme linked immunosorbent assay(ELISA)to measure PGE2 content in the RVLM and Western Blot to detect the expression of the type of COX protein in the RVLM.The animal in vivo experiment of SD rats is used to observe the microinjection of PGE2 on the effect of MAP,HR,and RSNA and to determine the cardiovascular effects of PGE2 after the pretreatment with Tempol in the RVLM.On each side of the RVLM received by 3 sites of PGE2 was used to detect ROS content by DHE and the expression of NOX and SOD protein in the RVLM Western Blot.[Result]1.PGE2 content in the RVLM was increased in hypertensive rats1.1 PGE2 content in the RVLM was increased in hypertensive ratsThe PGE2 content in the RVLM in SHR was significantly higher than in WKY rats(0.25 ± 0.01 vs.0.17 ± 0.01 ?g/mg,p<0.05,n=5).Likewise,the PGE2 content in the RVLM in the Ang II-induced hypertensive rats was also significantly higher than in aCSF infused control rats(0.26 ± 0.01 vs.0.15 ± 0.01 ?g/mg,p<0.05,n=4).These results showed that the PGE2 content in the RVLM was increased in hypertenion.1.2 Microinjection of PGE2 into the RVLM increased blood pressure and sympathetic activityEscalating doses of PGE2 range from 0.3 pmol to 30 pmol were unilaterally microinjected into the RVLM in normotensive rats.Compared with microinjection of aCSF(BP: 2.25 ± 0.49 mmHg;0.3 ± 1.7 bpm;RSNA: 0.4 ± 1.0 %),0.3 pmol(BP: 11.75±1.93 mmHg;RSNA: 12.75 ±2.29 %),3 pmol(BP: 24.25±3.43 mmHg;RSNA: 24.25 ±3.43 %)and 30 pmol(BP: 13.75±1.38 mmHg;RSNA: 14.25 ±1.89 %)of PGE2 microinjection significantly increased BP and RSNA.These results showed that microinjection of PGE2 into the RVLM resulted in an increase in RSNA and BP.The effects of 3 pmol PGE2 on cardiovascular function is most significant,therefore,this dosage was selected in the subsequent experiments.1.3 COX-2 protein expression in the RVLM was increased in hypertenisive ratsThe protein expression of COX-2 in the RVLM in SHR group was significantly higher than WKY group(1.80 ± 0.11 vs.1.00 ± 0.16,n=4).There was no significant difference in COX-1 protein expression between SHR group and WKY group(1.17 ± 0.38 vs.1.00 ± 0.19,n=4).The protein expression of COX-2 in Ang II group was significantly higher than aCSF group(2.86 ± 0.46 vs.1.00 ± 0.16,n=5).There was no significant difference in COX-1 protein expression between AngII group and aCSF group(1.16 ± 0.31 vs.1.00 ± 0.32,n=5).These results suggested that the increased PGE2 in the RVLM may be mediated by COX-2 in hypertension2.Microinjection of PGE2 into the RVLM induced oxidative stress2.1 Microinjection of PGE2 into the RVLM increased production of ROSThe production of superoxide anion in the RVLM in PGE2 group was significantly increased,compared with aCSF group(1.50 ± 0.05 vs.1.00 ± 0.08,p<0.05,n=5).2.2 Microinjection of PGE2 into the RVLM decreased the protein expression of SOD1 and SOD2The level of SOD1 expression in the RVLM in PGE2 group was significantly decreased compared with aCSF group(0.16 ± 0.03 vs.1.00 ± 0.19,p<0.05,n=5).Moreover,the level of SOD1 expression in the RVLM in PGE2 group was also significantly down-regulated compared with aCSF group(0.68 ± 0.05 vs.1.00 ± 0.11,p<0.05,n=5).These results indicated that PGE2 inhibited the degradation of ROS in the RVLM.2.3 Microinjection of PGE2 into the RVLM increased the protein expression of NOX2 and NOX4Compared with aCSF group,the expression of NOX2 protein in the RVLM in PGE2 group was significantly increased(1.00 ± 0.17 vs.2.75 ± 0.62,p<0.05,n=5).Likewise,the expression of NOX4 protein in PGE2 group was also significantly higher than that in aCSF group(3.65 ± 0.35 vs.1.00 ± 0.24,p<0.05,n=5).These results suggested that PGE2 increased the production of ROS in the RVLM.3.Microinjection of Tempol into the RVLM attenuated the PGE2-induced cardiovascular effectsMicroinjection of PGE2 into the RVLM increased blood pressure and sympathetic activity,which was attenuated by pretreatment with Tempol(MAP: 21.00 ± 3.27 vs.7.20 ± 2.76 mmHg,n=5;RSNA: 21.00 ± 3.27 vs.7.20 ± 2.76 %,n=5).These results indicated that Tempol attenuates the PGE2-induced increase in sympathetic activity and blood pressure.ConclusionThe present study shows that PGE2 derived from COX-2 participates in the regulation of cardiovascular function in the RVLM.PGE2 up-regulated the level of NOX2/4 expression and down-regulated the level of SOD1 and SOD2 expression,which leads to an increase in ROS in the RVLM.Furthermore,cardiovascular dysfunction induced by PGE2 was attenuated by Tempol pretreatment.Taken together,the central PGE2-mediated oxidative stress participates in regulation of cardiovascular function in hypertension.