Hydrogen Sulfide Protects Blood-brain-barrier Integrity Following Cerebral Ischemia

Part Ⅰ Protective effects of hydrogen sulfide onblood-brain-barrier following cerebral ischemia in miceObjective: To investigate whether H2S protects the blood-brain-barrier integrityfollowing cerebral ischemia/reperfusion injury.Methods: ICR male mice were subjected to tMCAO (transient middle cerebral arteryocclusion). At3hours after reperfusion, mice were randomly divided to receive vehicle,the H2S dornor ADT（50mg/kg/day）or NaHS (25μmol/kg/day) intraperitoneally. The serumlevels of H2S were measured with methylene blue method at45min and3hours afterinjection. By using2,3,5-triphenyltetrazolium chloride (TTC) staining, we measured theinfarction sizes of each group. The post-MCAO permeability of BBB was assessed bymeasuring Evans blue leakage into the ischemic brain. The levels of tight junction proteinsZO-1and occludin were measured with western blotting.Results: Compared to the vehicle control, NaHS significantly elevated serum H2S levels at45min, but not at3h after injection. In contrast, ADT elevated serum levels of H2S both at45min and3h after injection. Compared to the vehicle group, ADT-treated mice displayedsignificantly reduced the infarction volumes and improved neurological deficits (Corticalinfarction of vehicle-treated mice vs. ADT-treated mice:66.0±10.5%vs.42.7±6.8%;hemispheric infarction of vehicle-treated mice vs. ADT-treated mice:66.0±25.5%vs.54.8±18.5%) at48h after MCAO. At48h after tMCAO, compared to vehicle, ADT andNaHS also significantly attenuated EB extravasation into the ipsilateral cortex and striatum. Compared to sham-operated mice, vehicle-treated mice displayed significantly decreasedlevels of two tight junction proteins, occludin and ZO-1, in the ipsilateral cortex at48hafter tMCAO. The MCAO-induced decrease in occludin and ZO-1in the ipsilateral cortexwas attenuated in ADT-treated mice.Conclusions: Using two structurally-unrelated H2S donors, we showed that H2Sprotected against BBB disruption following ischemia/reperfusion injury. Part II the machenism underlyingADT protection against BBBdisruption following cerebral ischemia/reperfusion injuryObjective: To investigate whether ADT protects the BBB integrity following cerebralischemia by acting through of NF-κB inhibition to supress post-ischemic inflammationand NADPH oxidase-mediated production of reactive oxygen species.Method: The mice receiving MCAO were divided into three groups: Sham, Veh groupand Veh+ADT group. The cortical samples were collected at24h or48h after the MCAO.Real-time PCR、ELISA、MPO kit、NOX kit were applied into determining expression ofpro-and anti-inflammation factors and the activity of MPO and NOX. Zymography andwestern blotting were used to measure the activy of MMP-9and the nuclear translocationof the NF-κB subunit p65, an indicator of NF-κB activationResults: ADT remarkably attenuated MCAO-induced mRNA expression of iNOS andIL-1β and enhanced mRNA expression of the anti-inflammatory marker arginase1in theipsilateral cortex compared to vehicle. ELISA results indicated that protein levels of IL-10were also higher in the ipsilateral cortexes of ADT-treated mice than in the ipsilateralcortexes of vehicle-treated mice. Compared to the vehicle group, ADT treatment blockedischemia-induced MPO activity in the ipsilateral cortex. Gelatin zymography assaysshowed that ADT attenuated ischemia-enhanced MMP-9activity in the ipsilateral cortex.Post-ischemic induction of NOX activity in the ipsilateral cortex was remarkablyattenuated by ADT. ADT treatment attenuated ischemia-induced nuclear translocation of p65in the ipsilateral cortex, suggesting that ADT inhibited post-ischemic NF-κBactivation.Conclusion: The slow-releasing donor ADT protected post-ischemic BBB integrity viasuppressing post-ischemic inflammation and NOX-derived ROS in the ischemic brainpossibly by acting through NF-κB inhibition.