The Protective Effects And Underlying Mechanism Of The PDE4 Inhibitor Roflumilast Against Blood-brain Barrier Disruption In Ischemic Stroke

Objective:The disruption of the blood-brain barrier(BBB)is one of the main pathological features of ischemic stroke.The decrease in junction proteins and structural changes lead to the destruction of BBB and the increase in vascular permeability,which subsequently causes the flow of toxic substances to flow into the brain and aggravates brain damage.Studies have shown that the expression of phosphodiesterase 4(PDE 4)is increased following ischemic stroke,and inhibition of PDE4 activity exerts protective effects against ischemic stroke.However,the underlying mechanism needs to be further studied.By employing human brain microvascular endothelial cells(HBMEC)and SD(Sprague-Dawley)rats,the present study aimed to explore whether inhibition of PDE4 by Roflumilast(Roflu)would attenuate BBB damage caused by ischemic stroke.We further aimed to investigate the possible mechanism.Methods:(1)HBMEC cells were subjected to oxygen-glucose deprivation and re-oxygenation(OGD/R)for various times to determine the optimal conditions of the BBB injury model in vitro.PDE4B plasmid,PDE4B siRNA and Hesl siRNA were transfected into cells with lipofectamine 2000.Western blot was used to detect the expression of junction proteins(Occludin,VE-Cadherin,ZO-1),apoptosis-related proteins(cleaved-caspase3,Bax,Bcl-xl,Bcl-2),and Notch signaling pathway-related proteins(Notch1,Hes1).The ratio of apoptotic cells was detected by MTT assay and Calcein/propidium iodide staining.(2)The rat focal cerebral ischemia model was established by Middle cerebral artery occlusion(MCAO).Neurological deficit score was used to evaluate the motor function of rats subjected to MCAO/R.The integrity of BBB of MCAO rats was detected by Evans blue staining.Western blot was used to detect the expression of junction proteins and Notch signaling pathway-related proteins in the ischemic penumbra of MCAO rats,and immunofluorescence was used to observe the vascular morphology and tight junction protein expression in the ischemic penumbra.Results:(1)The levels of junction proteins were decreased in a time-dependent manner in cells treated with OGD/R.(2)Overexpression of PDE4B significantly decreased the level of intracellular junction proteins.Consistently,knocking down of PDE4B significantly increased the expression of junction proteins,while the levels of anti-apoptosis related proteins were increased.(3)The PDE4 inhibitor Roflu reversed the loss of junction proteins,and the pro-apoptosis related proteins were decreased as well following Roflu treatment.(4)The PKA inhibitor H89 blocked the protective effects of Roflu,reduced the levels of junction proteins,and blocked the activation of the Notch signaling pathway in cells treated with Roflu.(5)Both the γ-secretase inhibitor DAPT and Hes1 siRNA blocked the inhibitory effect of Roflu on the reduction of junction proteins and apoptosis.(6)Inhibition of PDE4 by Roflu reduced the neurological deficit score in rats following ischemic stroke and reperfusion.Roflu also decreased the permeability of BBB,reduced the loss of junction proteins in the ischemic penumbra,enhanced the fluorescence intensity of Occludin and ZO-1,and maintained the integrity of microvascular morphology in the brains of MCAO rats.Conclusion:(1)Roflu,an inhibitor of PDE4,could antagonize OGD/R-induced BBB damage.The mechanism may be that Roflu activates PKA/Notchl/Hesl,and subsequently reduces the apoptosis of endothelial cells and increases the expression of junction proteins.(2)Treatment with Roflu attenuated the neurological deficit in MCAO rats,reduced BBB permeability,reversed the decrease in junction proteins induced by ischemia-reperfusion,and thus maintained the integrity of brain microvascular morphology.The mechanism may be related to the activation of the Notchl/Hesl signaling pathway by Roflu.