| Objective:To investigate the effect of retinoic acid receptor??RAR??agonist Am80 on cerebral ischemia reperfusion injury and its possible mechanisms.Methods:Adult male Sprague-Dawley?SD?rats,weighing 260-280 g.Rats were subjected to middle cerebral artery occlusion?MCAO?for 90 min followed by 24 h reperfusion,were the I/R models.Experiment 1:SD rats were randomly divided into 2 groups:sham operation group?Sham?;ischemia-reperfusion group?I/R?;24 h after operation,the neurological function was evaluated by using double blind method before being killed;TTC staining was used to determine the volume of cerebral infarction;the expressions of RAR?,Bcl-2,Bax and Akt protein and the mRNA level of RAR?,Bcl-2,Bax,Akt,Caspase-3 in brain tissues were detected by Western blot and Real-time quantitative PCR,respectively.Experiment 2:SD rats were randomly divided into 4 groups:ischemia-reperfusion group?I/R?;tamibarotene 2 mg·kg-1 group?Am80-L?;tamibarotene 6 mg·kg-1 group?Am80-M?;tamibarotene 10 mg·kg-1 group?Am80-H?.24 h after operation,the neurological function was evaluated by using double blind method before being killed;TTC staining was used to determine the volume of cerebral infarction;apoptosis changes of neurons were observed with TUNEL staining and transmission electron microscopy?TEM?to observe the pathological changes of neurons;the expressions of Bcl-2,Bax and Akt protein and the mRNA level of Bcl-2,Bax,Akt,Caspase-3 in brain tissues were detected by Western blot and Real-Time Quantitative Polymerase Chain Reaction?RT-PCR?,respectively.Experiment 3:SD rats were randomly divided into 5 groups:sham operation group?Sham?;ischemia-reperfusion group?I/R?;tamibarotene 6mg·kg-1 group?Am80-M?;tamibarotene 6 mg·kg-1+Wortmannin group?Am80+W?;ischemia-reperfusion+Wortmannin group?W?.24 h after operation,the neurological function was evaluated by using double blind method before being killed;TTC staining was used to determine the volume of cerebral infarction;the expressions of Bcl-2,Bax and Akt protein and the mRNA level of RAR?,Bcl-2,Bax,Akt,Caspase-3 in brain tissues were detected by Western blot and RT-PCR,respectively.Results:?1?The middle cerebral artery occlusion can cause neurological dysfunction,cerebral infarction volume increased,the expression of RAR?was significantly decreased in hippocampus after ischemia reperfusion.?2?RAR?agonist Am80 has protective effects on cerebral ischemia reperfusion injury.Compared with I/R group,the neurological scores were significantly reduced and the volume of cerebral infarction was effectively reduced in Am80 groups,Am80 significantly reversed these I/R-induced the apoptosis and pathological damage of neurons.Western blot results showed that the expression of RAR?,Bcl-2 and p-Akt were significantly decreased in I/R group compared with sham group,and the expression of Bax was significantly increased?P<0.01?.Compared with I/R group,the expression of Bcl-2 and p-Akt were significantly increased in Am80-M and Am80-H groups,but the expression of Bax was significantly reduced?P<0.05,P<0.01?,there was no significant difference between in Am80-L group and I/R group.The RT-PCR results showed that Bcl-2,Bax,Caspase-3 mRNA increased in I/R group compared with in Sham group?P<0.05,P<0.01?;RAR?mRNA expression decreased.Bax,Caspase-3 mRNA expression of Am80-L group,Am80-M group and Am80-H group was lower than that of I/R group?P<0.05,P<0.01?;while Bcl-2 mRNA expression increased significantly in Am80-M group and Am80-H group?P<0.05,P<0.01?;There was no significant difference of Bcl-2 mRNA expression between Am80-L group and I/R group?P>0.05?.The results of the two methods showed that the expression of Akt in each experimental group,there was no significant difference among all groups?P>0.05?.?3?Western blot and RT-PCR results showed that the expression of apoptosis related proteins and genes were observed in all groups,PI3K inhibitor Wortmannin eliminated the neuroprotective effect of Am80.Conclusion:The expression of RAR?and the level of p-Akt in brain tissue decreased after I/R,indicating that RAR?and Akt phosphorylation may play an important role in I/R injury.Am80 intervention can improve neurological deficit after I/R,reduce the volume of cerebral infarction,inhibition of apoptosis,Am80 has a protective effect on cerebral I/R.Am80can increase the expression of Bcl-2 protein,decrease the expression of Bax protein,promote the phosphorylation of Akt,the total amount of Akt does not change,Am80 through anti-apoptotic effect on cerebral ischemia reperfusion injury.The use of Wortmannin can inhibit the effect of Am80,said that Am80 protection mechanism may be through the regulation PI3K/Akt signaling pathway to achieved. |
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