The Neuroprotective Role Of Ang-(1-7) In Hypertension And Ischemic Injury And Its Related Mechanisms

The neuroprotective role of Ang-(1-7) in hypertension andischemic injury and its related mechanismsStroke is one of the main causes of morbidity and mortality worldwidely andpresents as a great burden to family and society. As the most powerful risk factor,hypertension plays a vital role in stroke onset. It has been reported approximately54%of strokes can be attributed to hypertension. Apart from other risk factors, therelative risk of stroke increases by49%and46%when systolic and diastolic bloodpressure is increased by10mmHg and5mmHg, respectively. Therefore, it is of greatsignificance to strengthen hypertension prevention and explore mechanisms ofneuroprotection.The renin-angiotensin system (RAS) is an important circulative system inenvioromental homeostasis regulation. Classically, angiotensinogen (AGT) ishydrolyzed by renin to form the decapeptide Angiotensin I (Ang I), which is thenconverted by angiotensinconverting enzyme (ACE) into the biologically activepeptide Angiotensin I (Ang II). The Ang II receptor type (AT1R) is the primaryreceptor of Ang II and the ACE-Ang-II-AT1R axis has long been thought to be themain path in blood pressure control and renal sodium and water reabsorption.Overactivation of RAS is always closely associated with the development andmaintenance of hypertension, and the increase of Ang II has been found to be the keytarget of hypertension-related disasters. By modulating different redox signaling, AngII could elevate oxidative stress, promote inflammatory response, accelerate cellapoptosis, induce endothelial dysfunction and stimulate sympathetic tone, thusincreasing the risk of stroke onset. Angiotensin-(1-7)[Ang-(1-7)] is a newlyestablished bioactive fragment of RAS and generated predominately from Ang II byACE2. Through acting with the receptor Mas, the ACE2-Ang-(1-7)-Mas axiscounteract with ACE-Ang-II-AT1R axis and play a vital role in envioromentalhomeostasis. As a protective component of RAS, Ang-(1-7) is reported to oppose thedeleterious effects of Ang II, such as blood pressure downregulation, vasodilatory promotion, vascular smooth muscle cell growth inhibition, and provides beneficialeffects on hypertension-related end-organ damage. Moreover, in a previous studyfrom our group, central administration of Ang-(1-7) was found to ameliorateoxidative stress, minimize the size of cerebral infarction and improve neurologicalfunctions in a rat model of middle cerebral artery occlusion (MCAO). However, themechanisms of Ang-(1-7) on pathophysiology of hypertension and neuroprotection incerebral ischemia are still unknown.Therefore, in the first part of present study, we adopted spontaneouslyhypertensive rats (SHRs) and Wistar-Kyoto rats (WKYs), to investigate the effects ofAng-(1-7) on classic components of RAS in SHR brain, and to explore whetherAng-(1-7) attenuates brain oxidative stress, prevents neurons from apoptosis andinfluences autophagic activation in SHRs. In the second part, we employedpermanent middle cerebral artery occlusion model (pMCAO) to investigate whetherAng-(1-7) exerts anti-inflammatory effects and contributes to neuroprotectionfollowing cerebral ischaemia.In the present, blood pressure control has got more and more attention to theprevention and treatment of stroke. Meanwhile, timely thrombolysis has beenrecognized and neuroprotactants are also being constantly explored. In recent years,attention has been focused on the endogenous neuroprotectiond. Ischemicpreconditioning (IPC) is the phenomenon whereby a brief non-injurious episode ofischemic stress renders the organ resistant to a subsequent lethal ischemic insult.Although extensive research has demonstrated that IPC reduces cerebral ischemicdamage, it is clinically feasible only when the occurrence of stroke is predictable.Ischemic postconditioning (IPOC) is induced by a repetitive series of briefinterruptions of reperfusion, which is applied at the onset of reperfusion after aprolonged period of ischemia. It has translational relevance to reperfusion andthrombolitic therapy in acute cerebral ischemia, and represents a promising strategyto reduce ischemia/reperfusion injury. In the research field of myocardial ischemia,the intensive research of IPOC has even led to clinical trials. Moreover, theneuroprotective effects of IPOC also have been proved in animal studies in cerebralischemia. However, the mechanism has not been fully understood. Thus, in the third part, we intended to explore the mechanism of IPOC-induced tolerance to cerebralischemia, and to provide a novel therapeutic strategy for the treatment of stroke.Part I Angiotensin-(1-7) modulates renin-angiotensin systemassociated with reducing oxidative stress and attenuatingneuronal apoptosis in the brain of hypertensive ratsObjectives: To investigate the effects of Ang-(1-7) on the pathophysiologicchanges caused by hypertension in brain of spontaneously hypertensive rats SHRs.Methods: Sixteen-week-old male normotensive WKYs and SHRs were chosenin this study. Different doses of Ang-(1-7) or artificial CSF were continuousadministrated by implanted Alzet osmotic minipumps into lateral cerebral ventricle.After4weeks, brain tissues were collected and Ang II was detected by ELISA. Theexpressions of ACE, AT1R, AT2R, iNOS, gp91phox, and some others related toapoptosis and autophagy were detected by western blotting. The MDA content andSOD activity was determined by spectrophotometric assays. TUNEL staining wasperformed to detect the apoptotic neurons in frontal cortex and hippocampus. Inaddition, the immunofluorescence of LC3was analysed with a fluorescencemicroscopy and the morphological changes of neurons were observed under TEM.Results:(1) Compared with WKYs, the SHRs exhibited a significant increase inAng II, ACE, AT1R expression and a marked reduction in AT2R level. Medium-andhigh-dose of Ang-(1-7) significantly reduced the content of Ang II and expression ofAT1R in SHRs, but it didn’t affect the expressions of AT2R and ACE.(2) In the brainof SHRs, the level of MDA and total SOD activity increased, which is accompaniedby upregulations of NADPH oxidase subunit gp91phoxand iNOS. Medium-andhigh-dose of Ang-(1-7) inhibited the oxidative stress by downregulating MDAcontent and total SOD activity, and decreasing the expressions of gp91phoxand iNOS.(3) The hypertension-induced increase in the percentage of TUNEL-positive neuronsand Bax to Bcl-2ratio in SHRs brain was also attenuated by medium-and high-doseof Ang-(1-7).(4) Compared with WKYs, autophagy was activated with theupregulation of LC3/Beclin1and downregulation of p62in SHRs. Meanwhile, theimmunostaining of LC3increased and the cortical neurons were vacuolated with disrupted cell structure and double-membraned autophagosomes. Medium-andhigh-dose of Ang-(1-7) attenuated autophagy induction by decreasing the expressionsof LC3/Beclin1, increasing p62level and inhibiting autophagosomes formation.(5)All the aforementioned effects induced by Ang-(1-7) were abolished by Mas inhibitorA-779.Conclusion: Our findings demonstrated that treatment with Ang-(1-7) for4weeks modulated the component of RAS accompanied by reducing oxidative stress,inhibiting neuronal apoptosis and attenuating autophagic activation in the brain ofSHRs. These findings indicated that chronic treatment with Ang-(1-7) may bebeneficial to attenuate hypertension-induced physiopathologic changes in brain andprovide a novel therapeutic foundation to demonstrate the role of RAS inhypertension related disease.Part II Suppressing inflammation by inhibiting the NF-κBpathway contributes to the neuroprotective effect ofangiotensin-(1-7) in rats with permanent cerebral ischaemiaObjectives: To explore whether Ang-(1-7) exerts anti-inflammatory effect andcontributes to the neuroprotection in a rat model of permanent middle cerebral arteryocclusion.Methods: We infused aCSF, Ang-(1-7) or A-779into the right lateral ventricleof male Sprague-Dawley rats from48h before onset of pMCAO until the rats werekilled. Twenty-four hours after ischemia, the neuroprotective effect of Ang-(1-7) wasanalysed by evaluating infarct volume and neurological deficits. The MDA contentand SOD activity were determined by spectrophotometric assays. The expression ofNF-κB p65subunit in cell nuclei of the ischemic cortex were determined by westernblotting analysis and the spatial distribution of NF-κB p65subunit was detected byimmunohistochemical assay. The level of COX-2was tested by Western blotting andconcentrations of pro-inflammatory cytokines such as TNF-、IL-1β were measuredby ELISA.Results: Infusion of Ang-(1-7), i.c.v., significantly reduced infarct volume andimproved neurological deficits. It decreased the level of MDA, increased total SOD activity and suppressed NF-κB activity, which was accompanied by a reduction ofpro-inflammatory cytokines and COX-2in the peri-infarct regions. These effects ofAng-(1-7) were reversed by A-779. Additionally, infusion of A-779alone increasedoxidative stress levels and enhanced NF-κB activity, which was accompanied by anup-regulation of pro-inflammatory cytokines and COX-2.Conclusion: Our findings indicate that Ang-(1-7) significantly improvedneurological deficits, reduced infarct volume, inhibited oxidative stress andinflammatory response via Mas receptor. Suppressing NF-κB dependent pathwaymay represent one mechanism that contributes to the anti-inflammatory effects ofAng-(1-7) in rats with pMCAO, which offers a direct evidence to study the role ofRAS in stroke onset and exacerbation.Part III Inhibition of Autophagy Contributes to IschemicPostconditioning-Induced Neuroprotection AgainstFocal Cerebral Ischemia in RatsObjectives: To determine the role of autophagy in IPOC-inducedneuroprotection against focal cerebral ischemia in rats.Methods: A focal cerebral ischemic postconditiong model was established withpermanent MCA occlusion plus transient CCA occlusion and postconditioning wasperformed at the onset of reperfusion. Rapamycin and3-MA was infuced into the leftlateral ventricle of different group rats respectively. The expressions of LC3, Beclin1,and p62were evaluated by Western blotting. The immunofluorescence of LC3andBeclin1was analysed with a fluorescence microscopy and the morphologicalchanges of neurons were observed under TEM.Results: We found that autophagy was markedly induced with the upregulationof LC3/Beclin1and downregulation of p62in the penumbra at various time intervalsfollowing ischemia. IPOC reduced infarct size, attenuated brain edema, inhibited theupregulation of LC3/Beclin1, and reversed the reduction of p62simultaneously.Rapamycin, an inducer of autophagy, partially reversed all the aforementioned effectsinduced by IPOC. Conversely, autophagy inhibitor3-MA attenuated the ischemicinsults, inhibited the activation of autophagy, and elevated the expression of anti-apoptotic protein Bcl-2, to an extent comparable to IPOC.Conclusions: The present study suggests that autophagy is induced in cerebralischemia and varied with the exent and time. Inhibition of the autophagic pathwayplays a key role in IPOC-induced protection against focal cerebral ischemia, whichcould be simulated by3-MA. Thus, pharmacological inhibition of autophagy mayprovide a novel therapeutic strategy for the treatment of stroke.In summary, our present study has the following new concerns:1. Ang-(1-7) exerts multi-target neuroprotective effects on pathophysiologyof hypertension. By interacting with Mas receptor, chronic treatment with Ang-(1-7)remarkably reduced oxidative stress levels, attenuated neuronal apoptosis,downregulated autophagic induction, and modulated RAS activity in SHRs brain. Allof these findings provide a theoretical basis to further elucidate the importantance ofAng-(1-7) in hypertension and target RAS in clinical treatment ofhypertension-related end-organ damage.2. Ang-(1-7) participates in inflammatory response and contributes toneuroprotection after stroke. With an interaction with Mas receptor, Ang-(1-7)significantly reduced infarct volume and improved neurological deficits, which wasaccompanied by a reduction of oxidative stress and inflammation suppression. Thesefindings deepen our understanding of the role of Ang-(1-7) in stroke and may offerdirect evidence to recognize the mechanism of RAS in stroke onset and exacerbation.3. IPOC exerts endogenous neuroprotection and attenuatedischemia/reperfusion injury. IPOC inhibited neuronal apoptosis, downregulatedautophagic induction, attenuated ischemia/reperfusion injury, and then initiatedendogenous neuroprotection after stroke, which may provide a new perspective forthe development of novel options for stroke treatment.