Protect Effect And Mechanism Of Anethole On The Blood-brain Barrier Permeability On Mice With Focal Cerebral Ischemia Injury

Objective: Stroke is one of the leading causes of death and disability worldwide,especially ischemic stroke.At the acute phase of cerebral ischemia,secondary brain injury includes: calcium overload,inflammation,apoptosis,oxidative stress,excitotoxicity.Oxidative stress and inflammation are closely related to the blood-brain barrier(BBB)damage,and the damage of BBB furtherly aggravates brain injury.The process is a vicious cycle,so it is important to reduce the secondary brain damage and protect the BBB for the treatment of cerebral ischemia.Therefore,new drugs to protect the blood brain barrier are expected to be an effective strategy of ischemic stroke.Anethole(referred to as “AN”),originates from umbelliferae plant fennel,osmorhiza aristata,illicium verum,cupressaceae plants,ocimum basilicum,agastache rugosus and aster.Anethole had been reported to exert anti-inflammatory effect on cancer,skin lesions,liver damage and so on.The experiment is made to replicate the permanent middle cerebral artery embolism model(permanent middle cerebr alanery occlusion,pMCAO).After anethole intervention,it would been evaluated to anti-inflammatory effect and protective effect on BBB,aiming to prove the brain protection and discuss the possible mechanism of anethole.Methods:Male and healthy CD-1 mice were subjected to pMCAO,as described by Longa previously.1.Experiment 1 was conducted to to detect neuroprotection of anethole.Seventy-two mice were randomly assigned to four groups: Sham operated group(Sham+10% Tween 80-saline),p MCAO group(pMCAO + 10% Tween 80-saline),AN low-dose group(pMCAO + AN 40mg/kg),AN high-dose group(pMCAO + AN 80mg/kg).AN was administrated via intraperitoneal injection immediately after brain ischemia happened.In the cases of pMCAO and Sham group,equal volume of 10% Tween 80-saline was administered in the same manner.Six mice of each group were conducted to evaluate the neuroprotective effect:neurological deficit(24h),brain water content(24h),infarct size(24h).2.Experiment 2 was to investigate possible mechanisms of AN on ischemia brain and BBB.Six mice of each group were used to examine the inflammatory cytokines(including TNF-α、IL-1β and IL-6)and the indicators of BBB(including Claudin-5、occludin and JAM-1)detected by western blot(6 mice of each group)at 24 h after ischemia.Results:1 Anethole reduced neurological deficits.All mice were detected the scores of neurologic deficit at 24 h after ischemia.Mann–Whitney U test analysis was used to do statistical analysis.Mice in pMCAO group,AN low-dose group and AN high-dose group performed a leftpalsy.Compared with pMCAO group,the scores in AN low-dose group and AN high-dose group were lowered significantly(P<0.05).2 Anethole reduced the infarct volume.There was extensive lesion in both striatum and lateral cortex in pMCAO group.In AN low-dose group and AN high-dose group,the infarct volume were significantly reduced after p MCAO(pMCAO vs.L-AN,H-AN:73.62% ± 4.83% vs.38.36% ± 1.16%,43.19%± 1.09%,P < 0.05,).Although the infarct volume of AN low-dose group is smaller than AN high-dose group,no significant difference was found in infarct volume between AN low-dose group and AN high-dose group.3 Anethole ameliorated the brain edema at 24 h after ischemia.In Sham operated group,the brain water content of ipsilateral hemisphere was 82.92% ± 1.11%.In p MCAO group,the water content increased to 87.81% ± 0.82% and at 24 h after occlusion.In AN low-dose group,the brain water content was reduced significantly compared with pMCAO group at 24 h after occlusion(86.29% ± 1.31%,P<0.05).4 Anethole ameliorated BBB permeability at 24 h after ischemia..The protective effect of anethole was observed by examination of EB content at 24 h.Compared with the p MCAO,significantly lower EB extravasation was observed in AN low-dose group(P<0.05).5 Anethole improved the ability of resistance to inflammation.In this experiment,compared with sham group,the inflammatory cytokines(TNF-α、IL-1β、IL-6)of pMCAO group in ipsilateral hemisphere homogenate were significantly improved at 24 h after ischemia.The inflammatory cytokines of AN low-dose group decreased.The difference was statistically significant(P < 0.05).6 Anethole affected the protein expression of claudin-5、occludin and JAM-1.It was showed that the expression of claudin-5 and cccludin and JAM-1 decreased after 24 h in pMCAO group.After applying anethole,the protein expression of claudin-5,JAM-1 and cccludin were up-regulated.They all had significant differences compared with pMCAO group(P<0.05).Conclusions: Anethole has protective effect on ischemic brain,composed of reducing the acute neurologic deficits,diminishing infarction volume,and relieving cerebral edema.Additionally,anethole can improve the permeability of blood brain barrier,up-regulating the expression of Claudin-5,Occludin and JAM-1 quantity,while down-regulating the inflammatory cytokines(TNF-α、 IL-1 β、 IL-6).We conclude that anethole has neuroprotective effect on ischemic stroke,and the study provides a strong basis to develop new therapeutic agents in the treatment of cerebral ischemia.