Study On Anti-inflammatory And Blood-brain Barrier Protection Effect Of A Polyphenol Complex In Transient Middle Cerebral Artery Occlusion Mice Model

Background and purposeCerebral infarction is mainly caused by thrombotic,atherosclerotic embolism or cerebral arterial occlusion that caused by microarterial occlusion,and usually induces pathological reactions such as severe inflammatory reactions.Neumentix-a dietary supplement which are rich in multiple polyphenols,contains 14.9%rosmarinic acid and 29.9%total phenol content,and polyphenols have proven beneficial for a variety of pathological processes.Therefore,we investigated the effect of Neumentix on post-ischemia intracranial inflammation and blood-brain barrier（BBB）destruction of transient middle cerebral artery occlusion（t MCAO）model mice.MethodsWe used adult male C57BL/6J mice（23～27g,8 weeks old）in this experiment.The mice in the control group（including the sham operation group.That is,the sham operation+solvent group n=3,t MCAO+solvent group n=7,all treated with solvent 10 m L/kg body weight/d）or the experimental group（including the sham operation group.That is,sham operation+solvent+Neumentix group n=3,t MCAO+solvent+Neumentix group n=12,all administered with Neumentix 134mg/kg body weight/d which containing rosmarinic acid 20 mg/kg body weight/d,dissolved in 10 m L/kg body weight/d of solvent,intraperitoneal injection）.After 14days of pretreatment,experimental mice underwent transient middle cerebral artery occlusion（t MCAO）for 60 minutes and then reperfused,or underwent sham surgery.And from the day of t MCAO or sham surgery onset and 5 days after that,they continued to receive the solvent or solvent+Neumentix daily as before.The neurological assessment of corner tests and Bederson scores were conducted at 1 day before,1st,3rd,and 5th days after t MCAO or sham surgery day,and then evaluated cerebral infarct volume by Nissl staining,after sacrifice.Either immunohistochemistry or immunofluorescence was adopted to estimate the level of microglia-specific biomarker Iba-1,proinflammatory markers TNF-α,MCP-1,and the destruction of BBB（assessed by observing dissociation of astrocytes and vascular basement membranes,and by evaluating level of extravascular Ig G leakage）in brain tissue sections of each group.Results（1）In either of the control group（solvent solely）or solvent+Neumentix treated group,the mice undergoing t MCAO for 60 minutes exerted significant larger cerebral infarction volume compared with the sham-operated mice,by Nissl stain result（control group 22.27±6.02 mm³vs.0 mm³;solvent+Neumentix treated group 15.48±8.46 mm³vs.0 mm³）,moreover,the infarct volume of the t MCAO+solvent+Neumentix group was significantly lower than that of the t MCAO+solvent group（15.48±8.46 mm³vs.22.27±6.02 mm³,#P<0.05）;（2）In mice underwent t MCAO,the solvent+Neumentix treated group significantly improved corner test result on the 3rd day（7.25±2.14 times vs.9.57±0.79 times,*P<0.05）and the 5th day（6.42±2.39 times vs.9.57±0.79 times,**P<0.01）from the surgery day onset,in comparison with the control group（solvent treated solely）.Although Bederson scores showed a trend of improvement,no significant difference was observed(on the 1^st,3^rdand the 5^thday after t MCAO,P>0.05).And solvent+Neumentix treatment（vs.solvent treatment solely）also inhibited the activation of microglial（by evaluating the level of Iba-1,59.62±13.06 vs.148.10±13.34positive cells/0.15mm²,§§§§P<0.0001）,suppressed the expression of TNF-α（102.30±7.03 vs.150.30±13.10 positive cells/0.15mm²,####P<0.0001）and MCP-1（109.10±6.16 vs.149.10±17.49 positive cells/0.15mm²,###P<0.001）,and ameliorated the BBB destruction after t MCAO（AOD value of Ig G DAB staining,0.20±0.01 vs.0.21±0.02/0.15mm²,§P<0.05）.ConclusionsThe dietary supplement that is rich in polyphenols,could reduce BBB destruction and inhibited pro-inflammatory factor levels in peri-ischemic zone in t MCAO model mice with middle cerebral artery occluded for 60 minutes,suggesting that this kind of supplement might have a potential neuroprotective effect on cerebral infarction.