Neuroprotective Effects Of Aliskiren On Focal Cerebral Ischemia/Reperfusion Injury In Mice And Its Mechanism Of Signal Transduction Pathway

Ischemic cerebrovascular disease is the most common type of disease,which is the first leading cause of death and the most frequent cause ofpermanent disability in adults. Ischemic stroke is one of the main diseasesendangering people’s health in our state, have a high incidence, mortality,morbidity, high recurrence rate, and the characteristics of the slow recovery.Brain tissue secondary injuries to ischemia often lead to an aggravated illnessafter cerebral ischemia/reperfusion. Cerebral ischemia/reperfusion injury isaslo a complex pathophysiologic process which is not been clarified now.With the rapid development of modern immunology and molecular biology,recent studies found that the injuries caused by cerebral blood flow cessationand reperfusion are a rapid cascade reaction which includes inflammatorydamage, oxidative stress, excitatory amino acid toxicity, apoptosis,intracellular calcium overload, free radical damage and so on. Thesepathophysiologic processes overlap and intercommunicate then form a viciouscycle which direct/indirect resulting in blood-brain barrier disruption and brainedema formation, neurological deficit, as well as cell apoptosis or necrosis.Nowadays, beyond only a restricted number of hospitalized patientsprofiting from thrombolytic therapy, the vast majority of patients sufferingfrom stroke resulted in disability even death owing to the limited therapies.Delaying the development of brain damage and protecting neuron areremained. There a large number of agents have been proved to displayanti-inflammation, anti-oxidation properties but never employed in treatingcerebral ischemia. It is a focus in neuroscience that looking for the idealneuroprotective agents that can block ischemic cascade reaction.Aliskiren, the first orally effective direct renin inhibitor, is an effectiveantihypertensive agent with distinctive properties including placebo-like tolerability, pharmacologic effects that persist after drug discontinuation, and aunique mechanism of action. When combined with agents that inhibit thereninangiotensin-aldosterone system (RAAS), such as angiotensin-convertingenzyme inhibitors, angiotensin receptor blockers, or β-blockers, additionalblood pressure reduction refl ects more complete RAAS blockade. Concernthat marked elevation in plasma renin concentration following aliskirenadministration might lead to RAAS-induced paradoxical blood pressureincreases appears unfounded, based upon analyses of patients participating inclinical trials. Studies in animals and humans indicate that aliskirenaccumulates in renal tissue, blocks the intrarenal RAAS, and interferes withdeleterious cellular effects of angiotensin II by mechanisms that may includeenzymatic blockade of renin and prorenin at the site of the (pro) renin receptor.Evidence has shown that aliskiren possesses a wide range of biological effects,such as antithrombotice, and anti-inflammatory activities to treat hypertension,progressive renal disease, atherosclerosis. However, the molecular targets andmechanisms underlying aliskiren are not completely characterized, and theeffect of the aliskiren in acute stroke is still unknown. In the present study, wedemonstrated the unexplored potential of aliskiren for the treatment ofcerebral ischemic damage and its potential mechanism.ICR mice were induced into focal cerebral ischemiaby transient middlecerebral artery occlusion (MCAO), and received aliskiren treatment beforeMCAO. The present study examined the ability of aliskiren, the first orallyeffective direct renin inhibitor, to induce expression of ERK, HO-1, HMGB1,TLR4, NF-κB, p85(PI3-kinase), AKT phospholation, Bcl-2and Baxanalyzed its signaling mechanism in mice brain. The neurological deficits,brain water content, infarct volume and the expression of ERK, HO-1,HMGB1, TLR4, NF-κB, p85(PI3-kinase), AKT phospholation, Bcl-2andBax were measured at24h and72h after cerebral ischemia/reperfusion. Theactivities of superoxide dismutase (SOD) and malondialdehyde (MDA)content in ischemic cortical tissue were aslo detected to examine the oxidativeresponse at24h and72h after ischemia. The study was divided into three part list as below.PartⅠ Anti-inflammatory effect of aliskiren on focal cerebralischemia/reperfusion injury in mice and its mechanism of signaltransduction pathwayObjective: This study is to evaluate the expression ofHMGB1/TLR4/MAPKs/NF-κB in experimental mouse after ischemic injuryat different time points and estimate the anti-inflammatory effects of aliskirenon focal cerebral ischemia/reperfusion injury in mice and explore its possiblemechanisms and related signal transduction pathways.Methods: C57/BL mice were subjected to transient focal cerebralischemia/reperfusion model was established by middle cerebral arteryocclusion using modified suture occlusion technique. Aliskiren protectedagainst damage from cerebral ischemia/reperfusion. Aliskiren was oraladministrated five days before MCAO, once daily for five consecutive days.Mice were reanesthetized and killed at24h and72h after MCAO. In this part,mice were individed into4groups randomly. Group1: Sham-operated group(Sham): animals received sham operation, group2: Vehicle controls (Vehicle):animals received transient MCAO and equal volume NaCl0.9%saline fivedays before MCAO, once daily for five consecutive days, group3: Aliskirenlow dose group (L-AS): animals received transient MCAO and10mg/kg ofAliskiren five days before MCAO, once daily for five consecutive days, group4: MCAO-Aliskiren high dose group (H-AS): animals received transientMCAO and25mg/kg of Aliskiren five days before MCAO, once daily for fiveconsecutive days. Brain water content was measured by wet-dry method,Infarct volume was analyzed with2,3,5-triphenyltetrazolium chloride (TTC)staining. Immunohistochemistry, RT-qPCR and Western blot were used toanalyse the expression of HMGB1, TLR4and NF-κ B.Results:1Mice in Sham group had no palsy and a neurological score of zero.Mice in Vehicle group, low dose group and high dose group performed a leftpalsy. Compared with Vehicle group, there was a significant improvement in neurological function scores in the H-S group both at24h and72h (P <0.05).By contrast, there was no significant effect in L-AS group compared with at24h and72h (P>0.05for all).2Compared with Vehicle group, there was a significant improvement inbrain water content in the H-S group both at24h and72h (P <0.05). Bycontrast, there was no significant effect in L-AS group compared with at24hand72h (P>0.05for all).3Compared with Vehicle group, there was a significant improvement ininfarct volume in the H-S group both at24h and72h (P <0.05). By contrast,there was no significant effect in L-AS group compared with at24h and72h(P>0.05for all).4In immunoreactive, compared with Sham group, HMGB1, TLR4andNF-κB were upregulated in ischemic brain (P <0.05). Compared with Vehiclegroup, aliskiren in high dose group dramatically decreased the positive cells ofHMGB1, TLR4and NF-κ B in the ischemic cortex at all time points (P <0.05), there was no significant difference between Vehicle group and L-ASgroup (P>0.05for all).5Compared with Vehicle group, aliskiren in high dose groupdramatically decreased the expression of HMGB1, TLR4and NF-κB in theischemic cortex at all time points in protein levels (P <0.05), there was nosignificant difference between Vehicle group and L-AS group (P>0.05forall).6In agreement with the results of immunohistochemistry and westernblotting, compared with Vehicle group, aliskiren in high dose groupdramatically decreased the mRNA expression of TLR4and NF-κ B in theischemic cortex at all time points in mRNA levels (P <0.05), there was nosignificant difference between Vehicle group and L-AS group (P>0.05forall). HMGB1expression was not affected by cerebral ischemia at the mRNAlevel.Conclusions: The expression of HMGB1, TLR4and NF-κB wereup-regulated after ischemia. Systemic administration of Aliskiren is effective which can decrease the expression of HMGB1, TLR4and NF-κB. Therefore,the excessive inflammation of the brain ischemia was alleviated. Aliskirenreduce cerebral infarct volume, brain water content and improve neurologicimpairment and protected the brain from damage caused by focal cerebralischemia/reperfusion injury. Thus, aliskiren may be useful as a therapeuticagent for the treatment of cerebral ischemic-associated disorders. Theneuroprotection of aliskiren was accomplished by antiinflammation.PartⅡ Anti-oxidative effect of aliskiren on focal cerebralischemia/reperfusion injury in mice and its mechanism of signaltransduction pathwayObjective: This study is to evaluate the expression ofand estimate theanti-inflammatory effects of aliskiren on and explore its possible mechanismsand related signal transduction pathways. The aim of this study was toevaluate the neuroprotective effects of aliskiren and the underlyingmechanisms in focal cerebral ischemia/reperfusion injury in mice. Brain watercontent, infarct volume, the expression of ERK, HO-1, activites of superoxidedismutase (SOD) and malondiadehyde (MDA) content were measured.Methods: C57/BL mice were subjected to transient focal cerebralischemia/reperfusion model was established by middle cerebral arteryocclusion using modified suture occlusion technique. Aliskiren protectedagainst damage from cerebral ischemia/reperfusion. Aliskiren was oraladministrated five days before MCAO, once daily for five consecutive days.Mice were reanesthetized and killed at24h and72h after MCAO. In this part,mice were individed into5groups randomly. Group1: Sham-operated group(Sham): animals received sham operation, group2: Vehicle controls (Vehicle):animals received transient MCAO and equal volume NaCl0.9%saline fivedays before MCAO, once daily for five consecutive days, group3: Aliskirenlow dose group (L-AS): animals received transient MCAO and10mg/kg ofAliskiren five days before MCAO, once daily for five consecutive days, group4: MCAO-Aliskiren high dose group (H-AS): animals received transientMCAO and25mg/kg of Aliskiren five days before MCAO, once daily for five consecutive days, group5: MCAO-Aliskiren high dose group (H-AS)+PD98059（AS+PD98059): animals received transient MCAO and25mg/kgof Aliskiren five days before MCAO, once daily for five consecutive days,PD98059immediately injected intraperitoneally at50mg/kg dissolved in1%DMSO diluted in PBS. Brain water content was measured by wet-dry method,Infarct volume was analyzed with2,3,5-triphenyltetrazolium chloride (TTC)staining. Immunohistochemistry, RT-qPCR and Western blot were used toanalyse the expression of ERK, HO-1.Results:1Mice in Sham group had no palsy and a neurological score of zero.Mice in Vehicle group, low dose group and high dose group performed a leftpalsy. Compared with Vehicle group, there was a significant improvement inneurological function scores in the H-S group both at24h and72h (P <0.05).By contrast, there was no significant effect in L-AS group compared with at24h and72h (P>0.05for all). And the effect of aliskiren on neurologicalfunction scores was antagonized by treatment with ERK inhibitors PD98059.2Compared with Vehicle group, there was a significant improvement inbrain water content in the H-S group both at24h and72h (P <0.05). Bycontrast, there was no significant effect in L-AS group compared with at24hand72h (P>0.05for all). And the effect of aliskiren on brain water contentwas antagonized by treatment with ERK inhibitors PD98059.3Compared with Vehicle group, there was a significant improvement ininfarct volume in the H-S group both at24h and72h (P <0.05). By contrast,there was no significant effect in L-AS group compared with at24h and72h(P>0.05for all). And the effect of aliskiren on infarct volume wasantagonized by treatment with ERK inhibitors PD98059.4In immunoreactive, compared with Vehicle group, aliskiren in highdose group dramatically upregulated the positive cells of p-ERK and HO-1inthe ischemic cortex at all time points (P <0.05), there was no significantdifference between Vehicle group and L-AS group (P>0.05for all). And theeffect of aliskiren was antagonized by treatment with ERK inhibitors PD98059.5Compared with Vehicle group, aliskiren in high dose groupdramatically decreased the expression of upregulated the positive cells ofp-ERK and HO-1in the ischemic cortex at all time points in protein levels (P<0.05), there was no significant difference between Vehicle group and L-ASgroup (P>0.05for all). And the effect of aliskiren was antagonized bytreatment with ERK inhibitors PD98059.6In agreement with the results of immunohistochemistry and westernblotting, compared with Vehicle group, aliskiren in high dose groupdramatically decreased the mRNA expression of ERK and HO-1in theischemic cortex at all time points in mRNA levels (P <0.05), there was nosignificant difference between Vehicle group and L-AS group (P>0.05forall). And the effect of aliskiren was antagonized by treatment with ERKinhibitors PD98059.7Compared with Vehicle group, aliskiren in high dose groupsignificantly increased the activities of SOD and decreased the levels of MDAat24h and72h (P <0.05), there was no significant difference betweenVehicle group and L-AS group (P>0.05for all). And the effect of aliskirenwas antagonized by treatment with ERK inhibitors PD98059.Conclusions: The expression of ERK and HO-1were up-regulated afterischemia. Systemic administration of aliskiren is effective which can decreasethe expression of TLR4, NF-κB and TNF-α. Therefore, the excessiveinflammation of the brain ischemia was alleviated.Systemic administration of aliskiren is effective which can ameliorate theneurological deficit, improve the brain edema, decrease the infarct size andup-regulate the expression of ERK and HO-1. Aliskiren significantlyincreased the activities of SOD, decreased the levels of MDA. The underlyingmechanism of this neuroprotection may be involved in increasing the activitiesof SOD, decreasing the levels of MDA and up-regulating ERK andHO-1expression. PartⅢ Anti-apoptosis effect of aliskiren on focal cerebralischemia/reperfusion injury in mice and its mechanism of signaltransduction pathwayObjective: This study is to evaluate the expression of PI3K/AKT/Bcl-2/Bax in experimental mice after ischemic injury at different time points.The aim of this study is to explore anti-apoptosis effect of aliskiren and theunderlying mechanism in rat experimental ischemic stroke.Methods: C57/BL mice were subjected to transient focal cerebralischemia/reperfusion model was established by middle cerebral arteryocclusion using modified suture occlusion technique. Aliskiren protectedagaist damag from cerebral ischemia/reperfusion. Aliskiren was oraladministrated five days before MCAO, once daily for five consecutive days.Mice were reanesthetized and killed at24h and72h after MCAO. In this part,mice were individed into5groups randomly. Group1: Sham-operated group(Sham): animals received sham operation, group2: Vehicle controls (Vehicle):animals received transient MCAO and equal volume NaCl0.9%saline fivedays before MCAO, once daily for five consecutive days, group3: Aliskirenlow dose group (L-AS): animals received transient MCAO and10mg/kg ofAliskiren five days before MCAO, once daily for five consecutive days, group4: MCAO-Aliskiren high dose group (H-AS): animals received transientMCAO and25mg/kg of Aliskiren five days before MCAO, once daily for fiveconsecutive days, group5: MCAO-Aliskiren high dose group (H-AS)+LY-294002（AS+LY-294002): animals received transient MCAO and25mg/kg of Aliskiren five days before MCAO, once daily for five consecutivedays and LY-294002(10μL10mM dissolved in3%DMSO). Brain watercontent was measured by wet-dry method, Infarct volume was analyzed with2,3,5-triphenyltetrazolium chloride (TTC) staining. Immunohistochemistry,RT-qPCR and Western blot were used to analyse the expression of PI3K, AKT,Bcl-2, Bax.Results:1Mice in Sham group had no palsy and a neurological score ofzero. Mice in Vehicle group, low dose group and high dose group performed a left palsy. Compared with Vehicle group, there was a significant improvementin neurological function scores in the H-S group both at24h and72h (P <0.05). By contrast, there was no significant effect in L-AS group comparedwith at24h and72h (P>0.05for all). And the effect of aliskiren onneurological function scores was antagonized by treatment with PI3Kinhibitors LY-294002.2Compared with Vehicle group, there was a significant improvement inbrain water content in the H-S group both at24h and72h (P <0.05). Bycontrast, there was no significant effect in L-AS group compared with at24hand72h (P>0.05for all). And the effect of aliskiren on brain water contentwas antagonized by treatment with PI3K inhibitors LY-294002.3Compared with Vehicle group, there was a significant improvement ininfarct volume in the H-S group both at24h and72h (P <0.05). By contrast,there was no significant effect in L-AS group compared with at24h and72h(P>0.05for all). And the effect of aliskiren on infarct volume wasantagonized by treatment with PI3K inhibitors LY-294002.4In immunoreactive, compared with Vehicle group, aliskiren in highdose group dramatically upregulated the positive cells of p-PI3K, p-AKT,Bcl-2, Bax in the ischemic cortex at all time points (P <0.05), there was nosignificant difference between Vehicle group and L-AS group (P>0.05forall). And the effect of aliskiren was antagonized by treatment with PI3Kinhibitors LY-294002.5Compared with Vehicle group, aliskiren in high dose groupdramatically decreased the expression of upregulated the expression of p-PI3K,p-AKT, Bcl-2, Bax in the ischemic cortex at all time points in protein levels (P<0.05), there was no significant difference between Vehicle group and L-ASgroup (P>0.05for all). And the effect of aliskiren was antagonized bytreatment with PI3K inhibitors LY-294002.6In agreement with the results of immunohistochemistry and westernblotting, compared with Vehicle group, aliskiren in high dose groupdramatically decreased the mRNA expression of Bcl-2and Bax in the ischemic cortex at all time points in mRNA levels (P <0.05), there was nosignificant difference between Vehicle group and L-AS group (P>0.05forall). And the effect of aliskiren was antagonized by treatment with PI3Kinhibitors LY-294002.Conclusions: The expressions of PI3K, AKT and Bcl-2weredown-regulated after ischemia, but the expression of Bax was up-regulatedafter ischemia. Systemic administration of aliskiren is effective which canincrease the expression of PI3K, AKT and Bcl-2and decrease the expressionof Bax. Therefore, the excessive apoptosis of the brain ischemia wasalleviated.