Calpain Participates In Neurovascular Unit Damage Caused By Focal Cerebral Ischemia Via PARP-NF-?B Inflammatory Signaling

Background and purpose:Calpain is a calcium-dependent neutral protease,which strictly regulated by calcium and calpastatin,an endogenous inhibitor.A great number of studies have determined the activation of calpain due to cerebral ischemia,subsequently,degrading intracellular target proteins,and leading to neuronal death.Various calpain inhibitors have been conformed to decrease the infarct focus in the rodent models of focal cerebral ischemia.Neurovascular unit(NVU)is a dynamic structure,which is composed of cerebral microvessels,extracellular matrix(ECM),astrocyte endfeet,pericytes,neurons and their axon,and other support cells.NVU is a basic functional unit to maintain normal function in the central nervous system.In recent years,the focus of the study of experimental stroke is gradually shifted to NVU protection from single neuronal protection.A growing number of data also confirms the key role of NVU in the mechanism and treatment of ischemic stroke.Calpain is an important regulator of inflammatory signalling pathway,which may participate in the regulation of inflammatory response after focal cerebral ischemia.At the same time,Calpain has important regulatory effects on PARP and NF-?B,and PARP-NF-?B inflammatory signaling are involved in secondary ischemic injury.However,the role of Calpain in NVU injury after focal cerebral ischemia and its role in regulating PARP-NF-?B inflammatory signaling are not clear.The purpose of this study was to determine the protective effects of Calpain inhibitor in NVU injury in the penumbra and core regions caused by focal cerebral ischemia,to confirm that Calpain is involved in NVU injury in the penumbra and core regions caused by focal cerebral ischemia,and to further determine whether Calpain participates in NVU injury after focal cerebral ischemia by regulating PARP-NF-?B inflammatory signaling,providing theoretical basis for the combined application of ischemic cerebral protective agents and NVU protection.Methods:1.The middle cerebral artery occlusion(MCAO)model of rat was prepared by intraluminal filament.Rats were subjected to 1.5 hours-ischemia,followed by 4.5 or 22.5 hours of reperfusion.MDL28170(MDL),a specific calpain inhibitor was used.The rats were randomly divided into 3 groups:Sham(solvent),vehicle(solvent),and calpain inhibitor(MDL).6 and 24 hours after focal cerebral ischemia,the modified neurological deficit score(mNSS score)was used to evaluate neurological deficits in rats(n=8 per group);After 24 hours of sham operation or onset of ischemia,the brain swelling and infarct volume were determined by triphenyl tetrazolium chloride(TTC)staining combined with image analysis;6 and 24 hours after focal cerebral ischemia or sham operation,the TUNEL staining was performed to evaluate the apoptosis of cells.2.6 and 24 hours after focal cerebral ischemia,the Evan's blue levels in penumbra and core were assayed by a multifunctional microplate reader,to evaluate the changes in BBB permeability(n=8 per group);The brain tissues of penumbra and core region were collected 6 and 24 hours after onset of ischemia,and the NVU ultra-structural changes were examined by transmission electron microscopy(n=5 per group).The tissues of the penumbra and core region were collected 6 and 24 hours after onset of ischemia,and the levels of extracellular matrix proteins such as Laminin and Collagen ? were determined by Western blot combined with gel image analyzer(n=5 per group),to evaluate the destruction of extracellular matrix in BBB.3.6 and 24 hours after focal cerebral ischemia,the MPO activity was assayed according to the instruction manual of MPO kit.And then,the protein levels of PARP and NF-?B p65 in cytosolic and nuclear fractions and the protein levels of I?B?,ICAM-1,TNF-?,IL-1?,MMP-2 and MMP-9 in cytosolic fractions were determined by Western blot,to understand the expression of PARP-NF-?B inflammation signaling pathway and related inflammatory mediator proteins.Results:1.We successfully established a rat MCAO model.Compared to Vehicle group,the Calpain inhibitor reduced the neurological deficits 6 and 24 hours after onset of ischemia,decreased the volumes of infarction and brain edema significantly,markedly reduced the TUNEL staining positive score in the penumbra and core 24 hours after induction of ischemia.2.Compared with Vehicle group,the Calpain inhibitor decreased the EB levels in the penumbra and core 6 and 24 hours after induction of ischemia,reduced the ultrastructural scores in the penumbra and core 6 and 24 hours after induction of ischemia,and lessened the perivascular edema in penumbra and core 24 hours after induction of ischemia.It also reduced the perivascular edema in penumbra or core 6 hours after induction of ischemia,but it did not reach statistical significance.And the Calpain inhibitor enhanced the level of extracellular matrix proteins such as Laminin and Collagen ? in penumbra and core at 6 and 24 hours after ischemia.3.Compared with the Vehicle group,the Calpain inhibitor markedly reduced the MPO activities in penumbra and core at 6 and 24 hours after induction of ischemia.Calpain inhibitor reduced the PARP protein levels in the cytosol and nucleus in penumbra and in the nucleus in core at 6 and 24 hours after induction of ischemia.Although it had no significant effects on the protein levels of NF-?B p65 in cyotsol in penumbra and core at 6 hours after ischemia,but it significantly reduces the protein levels of NF-?B p65 in the cytosol in penumbra and core at 24 hours after ischemia and that in the nucleus in penumbra and core at 6 and 24 hours after ischemia.Calpain inhibitor markedly enhanced the protein levels of I?B? in penumbra and core at 6 and 24 hours after ischemia.Although treatment with Calpain inhibitor had no significant effects on the protein levels of MMP-2 in penumbra at 6 and 24 hours after ischemia and TNF-? in penumbra at 6 hours after ischemia,it markedly reduced the levels of ICAM-1,IL-1? and MMP-9 in the penumbra and core at 6 and 24 hours after ischemia,and it markedly reduced the protein levels of MMP-2,TNF-? in core at 6 and 24 hours and TNF-? in penumbra at 24 hours after ischemia.Conclusions:1.Calpain inhibitor has clear protective effects on focal cerebral ischemia.2.Calpain is involved in the penumbra and core area NVU injury caused by focal cerebral ischemia.3.Calpain participate in NVU injury caused by focal cerebral ischemia by up-regulating PARP-NF-?B inflammatory signaling.4.Calpain participates in NVU damage mediated by inflammatory response after focal cerebral ischemia,which is possibly by up-regulating PARP-NF-?B inflammatory signaling in the penumbra and the core area.