| Backgrounds and ObjectiveEssential hypertension is a common chronic disease of human beings.It is a serious disease characterized by high blood pressure with the serious blood vessel and heart complication.According to the Guideline for Hypertension Prevention in China?Revised Edition 2017?,the number of hypertensive patients in China is estimated to be 270 million,while the control rate of hypertension in China is only 17%.Therefore,the study of the pathogenesis of hypertension and the search for more efficient treatment strategies are still a focal point in the biomedical field.In addition to a series of characteristics of the peripheral cardiovascular system in hypertension,the hyperactivity of sympathetic tone in the central nervous system is also an important feature of hypertension.In many central nuclei,the rostral ventrolateral medulla?RVLM?is a key center for regulating sympathetic output.Dysfunction of RVLM neurons is one of the mechanisms of increased blood pressure and sympathetic excitability.Angiotensin converting enzyme 2?ACE2?is a member of RAS family,Angiotensin I,?Ang I?and Angiotensin II?Ang II?can be converted into Angiotensin-1-7[Ang-?1-7?]by ACE2.ACE2 plays compensatory function by antagonizing the Ang II pressor effects,and the balance between RAS and ACE2 associated compensatory axis regulation is very important for maintaining normal blood pressure and regulation of central autonomic nerve function.Previous studies have confirmed that the level of RVLM-ACE2 in rats with essential hypertension is significantly decreased,while overexpression of ACE2 in this area obviously inhibits the tonically active input of glutamatergic into RVLM and decreases blood pressure and renal sympathetic activity.At the same time,there is also evidence showing that exercise not only reduces the blood pressure and renal sympathetic nerve activity in hypertensive rats,but also inhibit production of angiotensin II and expression of angiotensin II type 1 receptor?AT1R?and increase the expression level of ACE2 and MasR in SHR RVLM.Therefore,the role of ACE2 in the central regulation of cardiovascular function is very significant,and it is also a potent target for the study of new strategies for the prevention and treatment of hypertension.The function of ACE2 is widely studied at present,but the mechanism of its upstream regulation is still not clear.ACE2 is a type I transmembrane protein,with an extracellular N-terminal domain containing the active site and a short intracellular C-terminal tail.A soluble form of ACE2,lacking its cytosolic and transmembrane domains.Studies have shown that A Disintegrin and Metalloproteases 17?ADAM17?can regulate ACE2 activity,ADAM17 is a kind of metalloproteinase and Disintegrin embedded on the surface of cell membrane.It can cut a variety of protein on the surface of the cell and play a variety of biological functions.It is expressed in many organs in the body and participates in some physiological and pathological processes.On the cell membrane surface,ADAM17 can hydrolyze the ACE2 by proteolysis,resulting in the sheding of ACE2 ectodomain which can enter the extracellular fluid,finally reduce activity of cellular ACE2.In type 1 diabetes mellitus,the expression of ADAM17 is increased in renal tubules of Akita mice,which can cut ACE2 on cell membrane and increase ACE2 segment?70 KDa?in urine,but insulin treatment can reverse this change.Previous studies have confirmed that Ang II perfusion increases the activity of ADAM17and decreases the content and activity of ACE2 in the heart,eventually causes cardiac hypertrophy and fibrosis.Tissue inhibitor of matrix metalloproteinase 3?TIMP3?is a natural endogenous inhibitor of ADAM17,which can inhibit ADAM17-mediated shedding of substrate.It has been reported that Ang II can increase the expression of TIMP3 in myocardium,while Ang?perfusion can not make cardiac hypertrophy in TIMP3-/-mice.TIMP3 is involved in the process of myocardial hypertrophy induced by Ang II.ADAM17is expressed in brain.It is not clear whether it is involved in the regulation of ACE2 in the central nervous system,especially in the sympathetic center RVLM.Resveratrol?RSV?is a small phenolic compound.RSV exerts multiple protective effects of cardiovascular regulation.Resveratrol can increase the expression and activity of ACE2 in many pathological and physiological processes and improve the state of disease.It is reported that resveratrol could inhibit ApoE-/-mice abdominal aortic aneurysm growth by increasing the expression and activity of the abdominal aorta and renal aorta ACE2.In addition,the intake of RSV could increase the expression of ACE2 in the fat tissue of high fat diet mice and reduce the expression of ACE.Previously,our team found that the perfusion of RSV into fourth ventricle could produce an effect of antihypertension.The change of ACE2 in the RVLM could directly affect the blood pressure and sympathetic output.Therefore,the goal of this study is:?1?to determine the role of central RAS system on the activity of ACE2in RVLM,Werther antihypertensive effect of resveratrol in the centre is associated with regulating the expression and activity of ACE2 in the central RVLM,Werther TIMP3/ADAM17 pathway is related to the abnormal ACE2 activity in RVLM of hypertensive rats and is involved in the regulation of the ACE2 activity by resveratrol.?2?to determine whether the decrease of ACE2 activity in the RVLM of hypertensive rats is related to ADAM17,whether ADAM17 is involved in the regulation of RVLM-ACE2 activity by RSV and in the regulation of ACE2 activity by RAS system.?3?to determine whether TIMP3/ADAM17 pathway resveratrol is involved in the expression and activity of ACE2.MethodThe experimental animals were male WKY rats and spontaneous hypertensive rats?SHR?,and the body weight was between 250g-330g.The cell model was PC-12 cell line.The central drug delivery was accomplished through perfusion of RSV into fourth ventricle on rats,blood pressure in rats was detected by the non-invasive tail artery monitor method;cerebrospinal fluid was collected by microdialysis in the RVLM;the effect of RAS to ACE2 and ADAM17in the RVLM is observed by perfusion of Ang II into fourth ventricle in WKY,the ACE2activity of brain tissue and cerebrospinal fluid was detected by using quenched fluorescent substrate;l of PC-12 cell was imitated by Ang?stimulating.TIMP3-siRNA interference was used to determine whether RSV regulates ACE2 through ADAM17/TIMP3 pathway.Western blot was used to detect the expression changes of the RVLM and intracellular protein.Result1.The expression level of ADAM17 on RVLM presympathetic neurons of SHR was increasedImmunofluorescent assay showed that ADAM17 was expressed on presympathetic neurons of rat RVLM;Western Blot detection showed that the ADAM17 expression level of SHR group was significantly higher than that of WKY in the RVLM?P<0.05,n=5?.2.Ang II downregulated the function of ACE2 within the RVLM through TIMP3/ADAM17pathway?1?Ang II attenuated the ACE2 function in the RVLM tissue while enhanced the ACE2activity of RVLM CSF.The fourth ventricle in WKY was perfused with aCSF and Ang II for a week respectively.Compared with aCSF perfusion,perfusion of Ang II for a week increased significantly,the blood pressure and heart rate?MAP:113±4 vs.151±4 mmHg;HR:360±5 vs.395±6 beats/min,P<0.05,n=5?.The RVLM protein extracting solution and microdialysis solution was detected with ACE2 Activity Kit showing that:ACE2 activity in RVLM tissue of WKY+Ang II group was lower than that of WKY+aCSF group?454±44.7vs.180.5±73.2 AFU/ug/min,P<0.05,n=5?,While ACE2activity in CSF was higher than that of WKY+aCSF group?1370±470 vs.0,AFU/ml/min,P<0.05,n=5?.Western Blot detection showed that the expression of ACE2 in the RVLM tissue of WKY+Ang II group was lower than that of the control group.?2?Ang II reduced the expression of TIMP3 in RVLM tissues and increased the expression of ADAM17.Western Blot detection showed that the expression level of ADAM17 in WKY-RVLM of central perfusion of Ang II was higher than that of the control group,while the expression level of TIMP3 was lower than that of the control group.Therefore,Ang II is involved in the regulation of ACE2 through the TIMP3/ADAM17 pathway.3.Resveratrol upregulated ACE2 expression in the RVLM neurons in hypertensive rats through the TIMP3/ADAM17 pathwayRats for the fourth ventricle perfusion were divided into four groups:WKY+aCSF,WKY+RSV,SHR+aCSF and SHR+RSV.?1?Central RSV decreased blood pressure of hypertensive rats.After perfusion of RSV into SHR fourth ventricle?SHR+RSV?,the blood pressure began to decrease on the fifth day,and it became stable on seventh days.Compared with SHR+aCSF group,blood pressure of the SHR+RSV group was decreased significantly?161±2 vs.143±2 mmHg,P<0.05,n=5?.The heart rate of WKY was not significantly changed compared with that before injection?P>0.05,n=5?.?2?Central perfusion of resveratrol increased the ACE2 activity of RVLM tissue and reduced the ACE2 activity in the cerebrospinal fluid?CSF?in the RVLM in hypertensive rats.ACE2activity of brain tissue and cerebrospinal fluid was detected by ACE2 activity kit,showing that ACE2 activity in RVLM extracellular fluid?CSF?of SHR+aCSF group was significantly higher than that of WKY+aCSF?1455±287 vs.0,AFU/ml/min,P<0.05,n=8 and RVLM?,while ACE2 activity in the tissue cell is significantly lower than that of WKY+aCSF group?291.4±13.2 vs.464.0±48.5 AFU/?g/min,P<0.05,n=8?;ACE2 activity in RVLM CSF of SHR+RSV group was significantly lower than that of control group?SHR+aCSF??607.5±48.3 vs.1455.0±287.0 AFU/ml/min,P<0.05,n=8?,while the ACE2 activity in RVLM tissue was significantly increased compared with control group?399±12.8 vs.291.4±13.2,AFU/?g/min,P<0.05,n=8?.?3?Central perfusion of resveratrol could significantly increase the transformation of Ang II into Ang?1-7?and increase the activity of ACE2 in SHR-RVLM.The content of Ang II and Ang?1-7?in the RVLM was tested by the Elisa kit,showing that the content of Ang II in SHR+aCSF group was significantly higher than that of WKY+aCSF group?19.6±1.73 vs.9.26±0.72 pg/?g,P<0.05,n=6?,and Ang?1-7?was significantly lower than that of WKY+aCSF group?2.25±0.185 vs.4.48±0.44 pg/?g P<0.05,n=6?;Ang II content in RVLM of hypertensive rats perfused resveratrol group?SHR+RSV?was significantly lower than the control group?SHR+aCSF??13.7±1.38 vs.19.6±1.73 pg/?g,P<0.05,n=6?,and Ang?1-7?was significantly higher than the control group?3.95±0.172 vs.2.25±0.18 pg/?g,P<0.05,n=6?.The ratio of Ang?1-7?/Ang II in SHR+aCSF group was significantly lower than that in WKY+aCSF?P<0.05?,and it was significantly higher in group SHR+RSV than that in SHR+aCSF?P<0.05?.?4?Central perfusion of resveratrol significantly increased the protein expression of ACE2 in the RVLM of hypertensive rats.After a week of perfusion,the expression level of ACE2 in SHR+aCSF group was significantly higher than that in WKY+aCSF group,while the expression level of ACE2 in the group of hypertensive rats after perfusion of resveratrol?SHR+RSV?was significantly higher than that in control group?SHR+aCSF?.Central perfusion of resveratrol could significantly increase the level of ACE2 content in the RVLM of hypertensive rats.?5?Resveratrol increased the expression of TIMP3 in SHR-RVLM and downregulated the expression of ADAM17.Western Blot test showed that:the expression level of ADAM17protein in RVLM of SHR+aCSF group was significantly higher than that of WKY+aCSF group,while the level of TIMP3 expression in RVLM was significantly lower than that of WKY+aCSF group.The expression level of ADAM17 in the RVLM of SHR+RSV group was significantly lower than that of the control group?SHR+aCSF?,while the expression level of TIMP3 was increased significantly.The expression of TIMP3 and ADAM17 protein in the RVLM between two WKY groups was not changed significantly.4.Central resveratrol upregulated ACE2 content through TIMP3/ADAM17 pathway in PC-12cells?1?Ang II increased the level of ADAM17 expression in PC-12 cells and reduced the expression level of ACE2 and TIMP3.The rat pheochromocytoma cell line?PC-12?,under the action of beta-NGF?50ng/ml?for 5-7 days,the cells differentiated completely into dendrites and developed long axons.Differentiated PC-12 cells treated with Ang II?100 nM and 1000nM?for 24 hours,was performed for extraction of cell protein.Western Blot detection showed that:Compared with the control group,the expression of ACE2 and ADAM17 in 100 nM and1000nM Ang II groups were significantly reduced,but there was no significant difference between 100 nM and 1000 nM Ang?groups.?2?Resveratrol treatment can inhibit the effect of Ang-II to PC12 cells,increase the expression of ACE2 and TIMP3,and reduce the expression of ADAM17.The experiments were divided into four groups,Control+DMSO,Control+RSV,Ang II and Ang II+DMSO+RSV,cells protein was extracted,the expression of ACE2 and TIMP3 of Ang II+RSV group were significantly higher than that of Ang II+DMSO group,while the expression of ADAM17 was lower than that of Ang II+DMSO group,but there was no significant difference between Control+RSV and Control+DMSO group in protein expression.?3?Interference of TIMP3 expression reduced the effect of resveratrol on the expression of ACE2 in PC-12 cells with Ang?stimulation.The experiments were divided into three groups,PC-12 cells were stimulated with Ang II,treated negative control group?Negative,Control,NC?,the second group was the control group treated with RSV?NC+RSV?,the third group was RSV treatment group withTIMP3-siRNA interference?siRNA+RSV?.It was showed that:compared with NC group,the expression level of TIMP3 and ACE2 of NC+RSV group was significantly increased,while the expression level of ADAM17 was decreased;Compared with NC+RSV group,TIMP3 and ACE2 expression of siRNA+RSV significantly decreased,while the expression of ADAM17 was significantly increased.ConclusionThis study suggestes that central RAS is an important factor to activate ADAM17 and subsequently downregulates ACE2 in the RVLM.In addition,it also confirmed that resveratrol decreases blood pressure of SHR by upregulating ACE2 activity in the RVLM,and RSV upregulates ACE2 activity through TIMP3/ADAM17 pathway.This study revealed the new mechanism of central ACE2 regulation and provided new ideas for prevention and treatment of hypertension. |
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