The Neuroprotective Effect Of Paeonol In The Focal Cerebral Ischemia: Up-regulated PAkt, NRF2and HO-1Expression

Objective: Ischemic stroke with a high incidence is a leading cause ofdeath and permanent disability in adult worldwide. However, the treatmentremains obscure and chiefly empirical because the pathophysiology involvedwherein is complex and not yet well understood. During ischemia, oxidativestress induced by enormously reactive oxygen species (ROS) that promotesdamage to lipid, carbohydrates, proteins and DNA. Brain as the body’s highoxygen demand organ, contains large amounts of unsaturated lipids withrelatively deficient in anti-oxidative defense mechanism, is the mostsusceptible organ to oxidative damage. Recently, the close relationshipbetween oxidative stress and cerebral ischemia has generated considerableinterest in developing antioxidant therapies to combat ischemia-induceddamage.Several publications have demonstrated the protective role of Aktphosphorylation in preventing the pathogenesis of ischemic injury. Amongcell survival pathways, the protein kinase B (PKB)/Akt, as a family ofserine/threonine protein kinases, plays crucial roles in a diverse numbers ofprocesses including cell survival, gene expression, apoptosis, protein synthesis,energy metabolism by regulating fundamental transcriptional factors, suchas Nrf-2.Nuclear factor erythroid2-related fator2(Nrf2), which could combinewith the promotor sequence termed the antioxidant response element (ARE)and exert its protective effect. In addition, it controls the coordinatedexpression of important antioxidant and detoxification genes (Phase II genes).Among which, HO-1attracts more and more attention and becomes a spotlightin rencent researches. HO-1, a ubiquitous and redox-sensitive inducible stressprotein, can exert potent anti-oxidative function by degrading heme to CO, iron, and biliverdin. Based on the previous findings, it is now widely acceptedthat induction of HO-1expression represents an adaptive response thatincreases cell resistance to oxidative injury. It has proved that the increasingactivity and expression of Nrf2and HO-1could attenuate oxidative injuryinduced by brain ischemia. Moreover, Nrf2-/-mice have been demonstratedmore sensitive to oxidative stressors. Therefore the activation of Nrf2mayconfer protection to acute stroke.Paeonol (20-hydroxy-40-methoxyacetophenone), an active componentisolated from Cortex Moutan radix, has been reported the potential fortherapeutic use in the treatment of Alzheimer’s disease (AD), diabetes mellitus,cancer and Atherosclerosis resided in its anti-inflammatory and anti-oxidantproperties. It has also reported that paeonol could attenuate neurotoxicity andameliorate cognitive impairment induced by D-galactose, which generatessuperoxide anion and oxygen-derived free radicals. Furthermore, studies haveshown that PN exerts a protective effect on several models of cerebralischemic injury and myocardium injury induced by ischemia/reperfusion.However, the mechanism underlying have not been fully studied. Therefore, inaddition to its intrinsic antioxidant nature, we pursued to investigate thepossible involvement of Nrf2, HO-1in the neuroprotection of cerebralischemia by Paeonol as well as the role of the PKB/Akt pathway in thisprocess in permanent middle cerebral artery occlusion (pMCAO) brain.Methods: This study was consisted with two sections: Experiment1wasused to detect paeonol’s neuroprotective effect in focal cerebral ischemia.Experiment2was conducted to evaluate paeonol’s effect on the expression ofphospho-Akt, Nrf2, HO-1, Claudin-5.Experiment1, sixty male CD-1(ICR) mice were divided randomly andequally into five groups (12mice in each group): Sham operated group(Sham); pMCAO group (pMCAO); Vehicle group (Vehicle); Low dose group(PN-L); and High dose group (PN-H). Vehicle and pMCAO groups weresubjected to permanent focal cerebral ischemia by right MCAO occlusion.Neurological deficit was evaluated on a six point scale at24h after pMCAO; brain water content was measured; infarct size was analyzed with2,3,5-triphenyltetrazolium chloride (TTC).Experiment2, ninety mice were randomly and equally divided into fivegroups:(18mice in each group): Sham operated group (Sham); pMCAOgroup (pMCAO); Vehicle group (Vehicle); low dose group (PN-L); and highdose group (PN-H). PN solution (30mg/kg or60mg/kg) was intragastricadministration. Western blotting was used to analyse the expression ofphospho-Akt, Nrf2, HO-1, Claudin-5. Meanwhile the indexes were alsodetected by qR-T PCR. Activitity of superoxide dismutase (SOD) wasmeasured with a standard kit by spectrophotometer.Results:1Mann–Whitney U test analysis was conducted to detect the scores ofneurologic deficit on a6-point scale at24h after pMCAO. The scores weresignificantly reduced in PN-H group comparing with Vehicle group(P <0.05).PN-L reduced the scores, but did not reach a significant level.2In Sham operated group, ipsilateral brain water content was79.08±0.78%.In Vehicle group, the water content increased to84.61%±0.61%at24h after occlusion. Pre-treating with paeonol decreased brain watercontent in ipsilateral hemispheres. Water content reduced to82.16±1.02%inPN-L group and81.68±1.23%in PN-H group markedly comparing withvehicle group(P <0.05). However no statistical difference was observedbetween these two groups.(n=6in each group)3In Sham group, no infarction area was observed. Pre-treating withpaeonol significantly reduced the infarct volume comparing with Vehiclegroup,but only PN-H group reach a significant level.(P <0.05).4PN’s role in the expression of pAkt, Nrf2, HO-1: We analyzed theexpression of pAkt, HO-1and nuclear Nrf2at protein level after pre-treatingwith PN in ischemia brain by Western blot. It showed a significant increase ofpAkt, HO-1in PN-L and PN-H group (n=6in each group) vs. Vehiclegroup(P <0.05). PN high dose group significantly increased the expression ofnuclear Nrf2(P <0.05). Furthermore, the qR-T PCR indicated the expression of Nrf2and HO-1was upregulated significantly at mRNA level in PN-Hgroup (P <0.05). But total protein of Akt were not affected in mRNA level(P>0.05).5The western blot results showed Vehicle group induced sharplyreduction of Claudin-5comparing with sham operated group (P <0.05). Afterpre-treating with peaonol, the protein expression of Claudin-5in ischemiabrain was up-regulated (P <0.05). However the mRNA level was onlyincreased statistically in PN-H group(P <0.05).6Paeonol increased the activities of SOD significantly compared withVehicle group and Sham group (P <0.05).Conclusions: Taking together, our findings showed that systemicadministration of peaonol has ability of reducing neurological deficits, brainedema and infarct volumes in cerebral ischemia; the up-regulation of pAkt,Nrf2, HO-1, SOD activities and sustaining the tight junction of BBB furtherdemonstrated the potential neuroprotection of paeonol. Thus, a corollary ofthis study is that paeonol may represent a potentially beneficial therapeuticavenue to harness the neuroprotective Akt/Nrf2/HO-1axis for focal cerebralischemic stroke treatment.