The Neuroprotective Effect And Underlying Mechanism Of Rosmarinic Acid On Focal Cerebral Ischemia Reperfusion Injury In Mice

With the rapid development of the national economy and the arrival of anaging population, cerebrovascular disease(CVD) has become the main diseaseendangering people’s health and life. Ischemic stroke has a high incidence,mortality, morbidity and high recurrence rate, which results in heavy socialand economic burden.Brain is an organ most sensitive to hypoxia and brain weight accounts foronly2%of body weight. The brain oxygen consumption accounts for20%oftotal body oxygen consumption. When the ischemic stroke occurs, the keytreatment is to recover the blood supply timely and effectively. Recombinanttissue plasminogen activator (tPA), targeting the occlusion and inducingreperfusion, is the only FDA-approved stroke medication currently. However,recanalization following ischemia paradoxically causes severe cerebralischemia-reperfusion (I/R) injury, which always plagues clinical treatment andprognosis. Cerebral I/R injury is a complex pathophysiologic process and isthought to be a complex regulatory networks formed by the interaction of avariety of mechanisms, including inflammatory damage, free radical damage,oxidative stress, excitatory amino acid toxicity, intracellular calcium overload,etc. Cerebral I/R injury directly or indirectly leads to neuronal apoptosis/death,BBB damage, eventually leading to neurological deficit, and even endangerthe lives of patients. Therefore, seeking effective neuroprotective agent toischemic stroke has been the focus of the study. Nowadays, the use ofmedicinal plants is becoming an increasingly attractive approach as acomplement and an alternative for treating acute stroke.Rosmarinic acid (RA), is a naturally occurring hydroxylated polyphenoliccompound, and it is commonly found in various plant families. It has been reported that RA elicits multiple promising biological activities such asanti-oxidative, anti-inflammatory, antiviral and anticancer abilities. Abundantstudies have demonstrated the beneficial effects of RA in various diseases.However, whether RA could confer the neurorescue effects in ischemic strokein vivo has not been well investigated. Therefore, we designed this study toevaluate the role and possible mechanisms of RA on I/R brain injury.Male, healthy CD1mice were used and subjected to focal cerebralischemia/reperfusion by right MCA occlusion as described by Longapreviously. RA was used to treat ischemic stroke and its neuroprotective effectwas analyzed. Neurological deficits, brain water content and infarct volumewere evaluated. TUNEL staining was used to detect apoptosis. The expressionof HO-1, Nrf2, Bcl-2, Bax, MMP-9and claudin-5were detected. Furthermore,ZnPPIX (a common used HO-1activity inhibitor) and LY294002(a typicalPI3K/Akt pathway inhibitor) were used to explore the important role ofNrf2/HO-1pathway in the neuroprotection of RA against I/R brain injury.PartⅠ Anti-oxidative and Anti-apoptotic effects of RA against cerebralI/R injuryObjective：The aim of this study was to evaluate the neuroprotectiveeffects of RA against focal cerebral ischemia reperfusion. Neurologicaldeficits, infarct volume, the expression of HO-1, Nrf2, Bcl-2and Bax weremeasured.Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R). Experiment1was used to analyze RA’sneuroprotective effect. Neurological deficits, and infarct volume wereevaluated at24h after reperfusion. Mice were randomly assigned to fivegroups: Sham-operated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; and RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with low doseof RA10mg/kg (RA-L), middle dose of RA20mg/kg (RA-M) and high doseof RA40mg/kg (RA-H). RA was administered immediately at the onset of reperfusion. Experiment2was used to analyze RA’s anti-oxidative andanti-apoptotic effects. TUNEL staining was used to detect apoptosis. Theexpression of HO-1, Nrf2, Bcl-2and Bax were detected at24h afterreperfusion using western blot and RT-PCR. Mice were randomly assigned tofour groups: Sham-operated group (Sham): animals received sham operationand equal volume of saline; MCAO/R group: animals received transcientmiddle cerebral artery occlusion and equal volume of saline; and RA groups:animals received transcient middle cerebral artery occlusion and treated withmiddle dose of RA20mg/kg (RA-M) and high dose of RA40mg/kg (RA-H).RA was administered immediately at the onset of reperfusion.Results：1Mice in Sham group had no palsy and score was zero. Mice inMCAO/R group, low dose group, middle dose group and high dose groupperformed significant neurological deficits. Compared with MCAO/R group,there was a significant improvement in neurological deficit scores in the RA-Hgroup and RA-M group at24h after reperfusion (P <0.05). By contrast, therewas no significant effect in RA-L group compared with MCAO/R group (P>0.05).2Compared with MCAO/R group, RA-H and RA-M group reduced theinfarct volume significantly at24h after reperfusion(P <0.05). RA-L grouphad no effect on infarct volume (P>0.05). Based on the results above, wedemonstrated that RA delivered at middle dose (20mg/kg) and high dose (40mg/kg) have a better therapeutic effect after reperfusion, and therefore wefocused on the RA treatment at middle dose (RA-M) and high dose (RA-H)for subsequent molecular and biochemical analysis.3After reperfusion, TUNEL-positive cells were numerous in theMCAO/R group. Treatment with RA, the number of TUNEL-positive cellswas significantly decreased and the effect was more obvious in the RA-Hgroup(P <0.05).4Compared with MCAO/R group, RA-H group significantlyup-regulated the expression of Nrf2, HO-1, Bcl-2and down-regulated the expression of Bax at24h after reperfusion in protein and gene levels (P <0.05). A similar but less marked effect was observed in the RA-M groupcompared with the RA-H group (P <0.05).Conclusions：Systemic administration of RA is effective which candecrease the infarct volume, ameliorate the neurological deficits, and reducedTUNEL-positive cells. RA significantly increased the expression of Nrf2andHO-1, which suggested the Nrf2/HO-1pathway was activated and activationof Nrf2with RA results in upregulation of HO-1. Meanwhile, RAup-regulated the expression of Bcl-2and down-regulated the expression ofBax. Taken together, the expression changes of Nrf2, HO-1, Bcl-2and Baxwere involved in the underlying mechanism of anti-oxidantive andanti-apoptotic effects of RA against cerebral I/R injury.PartⅡ Multiple neuroprotection of RA against cerebral I/R Injury isattibutable to upregulation of HO-1Objective：This study is to evaluate the upregulation of HO-1after RAtreatment in multiple neuroprotection against cerebral I/R Injury by using acommonly used specific HO-1inhibitor ZnPPIX.Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R).Mice were randomly assigned to fivegroups: Sham-oprated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with RA (40mg/kg); RA+ZnPPIX group: Mice were pretreated with ZnPPIX(10mg/Kg)via intraperitoneal injection24h before operation and treated with RA at doseof40mg/kg after reperfusion. ZnPPIX group: Mice were pretreated withZnPPIX (10mg/Kg) via intraperitoneal injection24h before operation.Neurological deficits, brain water content and infarct volume were evaluatedat24h after reperfusion. The expressions of Bax, Bcl-2, MMP-9andclaudin-5were detected to evaluated the important role of HO-1upregulatedwith RA treatment on alleviating apoptosis and blood-brain barrier(BBB) permeability.Results：1Compared with MCAO/R group, RA decreased the neurological deficitscore, infarct volume and brain water content significantly at24h afterreperfusion (P <0.05). These protective role of RA was partly reversed withZnppIX adminiatration (P <0.05).2Compared with MCAO/R group, RA significantly up-regulated theexpression of claudin-5, Bcl-2and down-regulated the expression of MMP-9,Bax at24h after reperfusion in protein and mRNA levels(P <0.05). Theregulation of claudin5, MMP-9, Bcl-2and Bax expression was partly inhibitedwith ZnppIX applyment and there was a significant difference between RAgroup and ZnppIX co-treatment group.Conclusions：RA treatment reduced brain edema, increased the claudin-5expression and decreased the MMP-9expression, which suggested that RAalleviated BBB permeability. But the positive effect was reversed by ZnppIX.Futhermore, ZnppIX co-treatment partly abolished the regulation of Bcl-2andBax by RA. Taken together, the above results showed that the anti-apoptosisand alleviating BBB permeability abilities of RA were attributable toupregulation of HO-1.PartⅢ RA induces upregulation of Nrf2and HO-1expression involvingPI3K/Akt signal pathway in a mice model of focal cerebralischemia reperfusionObjective: This study is to evaluate the involvement of PI3K/Akt signalpathway in upregulating Nrf2and HO-1expression induced by RA after focalcerebral ischemia reperfusion.Methods：Male, healthy CD1mice were subjected to transcient middlecerebral artery occlusion (MCAO/R).Mice were randomly assigned to fivegroups: Sham-oprated group (Sham): animals received sham operation andequal volume of saline; MCAO/R group: animals received transcient middlecerebral artery occlusion and equal volume of saline; RA groups: animalsreceived transcient middle cerebral artery occlusion and treated with RA (40 mg/kg); RA+LY294002group: Mice were treated with LY294002viaintracerebroventricular injection15min before ischemia and treated with RAat dose of40mg/kg after reperfusion. DMSO group: Mice were treated withequal volume of DMSO via intracerebroventricular injection15min beforeischemia. Neurological deficits and infarct volume were evaluated at24hafter reperfusion. The expressions of p-Akt, Akt, HO-1and Nrf2weredetected to evaluated the important role of PI3K/Akt signal pathway inup-regulating Nrf2and HO-1expression.Results：1Compared with MCAO/R group, RA increased the expression of p-Aktin ischemic cortex significantly at24h after reperfusion (P <0.05).Intracerebroventricular injection of LY294002reversed the positive effectpartly and there was a great significance between RA group and LY294002co-treatment group (P <0.05). By contrast, there was no significant change inexpression of p-Akt between MCAO/R group and DMSO group(P>0.05).2The expression of HO-1and Nrf2was increased with RA treatment (P<0.05). Treated with LY294002via intracerebroventricular injection15minbefore ischemia partly abolished the induction of HO-1and Nrf2expression.There was a great significance between RA group and LY294002co-treatmentgroup (P <0.05). Meanwhile, there was no significant change in expression ofHO-1and Nrf2between MCAO/R group and DMSO group (P>0.05).Conclusions：RA increased the expression of p-Akt in ischemic cortexsignificantly after reperfusion and this positive effect was reversed byLY294002, which suggested that PI3K/Akt signal pathway was activated withRA treatment after focal cerebral ischemia reperfusion. The up-regulatedexpression of HO-1and Nrf2after RA administration was inhibited partly byLY294002, which suggested that the regulation of HO-1and Nrf2expressioninvolved in PI3K/Akt signal pathway.