| Objective:To investigate the inhibitory effect of icariside Ⅱ(ICS Ⅱ)on blood-brain barrier(BBB)dysfunction induced by cerebral ischemia reperfusion(I/R)in rats and explore its potential mechanism.Methods: Male Sprague-Dawley rats(250-280 g)were allowed to acclimatize to the experimental conditions for a week.Laboratory chow and tap water were allowed ad libitum,the cerebral I/R injury was induced by middle cerebral artery occlusion(MCAO).The rats were subjected to surgical procedure and divided into four groups: sham,model(MCAO),sham+ICS Ⅱ and MCAO+ICS Ⅱ.Sham+ICS Ⅱ and MCAO+ICS Ⅱ groups were treated with ICS Ⅱ(16 mg/kg)by gavage twice a day for 3 days,while animals in sham and MCAO groups received the same volume of normal saline.A laser-doppler flowmeter was used to monitor cortical blood flow(CBF).The neural deficit scores were observed using Longa 5 method;BBB disruption was quantitatively determined by Evans blue staining;Histopathological examination and neuronal loss were observed using Hematoxylin and eosin(H&E)and Nissl staining;the cellular apoptosis was evaluated by TUNEL staining.The expressions and position of matrix metalloproteinases 2/9(MMP 2/9)and tissue inhibitor of metalloproteinase 1(TIMP1)were detected by immunohistochemistry.The expressions of MMP2,MMP9,TIMP1,claudin 5,occludin,ZO 1,Bax,Bcl-2,and the cleaved-caspase 3 level of penumbra were determined using Western blot.Furthermore,molecular docking was used to evaluated the relationship between ICS Ⅱ and MMP2/9.Results: The CBF of rats was reduced to less than 20% of the base value after MCAO,and the CBF was restored to 80% of base value.The neurological scores were significantly increased,and the ischemic brain displayed a much severer leakage of Evans Blue dye in MCAO group.In addition,the neurons of CA1,CA3 and DG regions in hippocampus,cortex and striatum were disorderly arranged and lost,the nucleus were shrunk and contracted in MCAO group than those of sham group.The expressions of MMP2 and MMP9 were significantly increased and the expressions of TIMP1,claudin 5,occludin and ZO 1 were decreased in the cortex,striatum and penumbra.Furthermore,the apoptotic cells were remarkably augmented in hippocampus and cortex in MCAO group.The expressions of Bax and the level of cleaved-caspase 3 were significantly increased,and the expression of Bcl-2 were decreased in penumbra than those of sham group.Whereas,ICS Ⅱ effectively attenuated neurological scores and the leakage of Evans Blue dye.Neurons of CA1,CA3 and DG regions in hippocampus,cortex and striatum exhibited clear boundary and normal morphology,and the number of normal Nissl bodies were increased after treatment with ICS Ⅱ.Additionally,ICS Ⅱ not only reduced the apoptotic cells,but also decreased the expressions of MMP2,MMP9,Bax,the level of cleaved-caspase 3,and increased the expressions of,claudin 5,occludin,ZO 1,TIMP1,Bcl-2.ICS Ⅱ might be directly bind and inhibit MMP2/9.Conclusion: The current study reveals that ICS Ⅱ significantly ameliorates I/R-induced BBB disruption through regulating the balance of MMP2,MMP9/TIMP1,and inhibiting neuronal apoptosis via caspase 3-dependent apoptosis signaling pathway. |
