CathepsinB-Caspase Signaling Pathway Participates In Astrocytic Cell Death Induced By Cerebral Ischemia

Aim: To investigate the role of CathepsinB-Caspase pathway in astrocytic celldeath induced by cerebral ischemia and the effects of CathepsinB specific inhibitorCA-074Me.Methods: Permanent middle cerebral artery occlusion (pMCAO) model wasinduced by using intraluminal filament technique in rats. CA-074Me was injectedintracerebroventricularly (icv)10min before or6h after the onset of ischemia. Theneuroprotection of CA-074Me was analyzed by assessing brain infarction volume,behavioral symptoms and the expression of MAP2protein after ischemia. Theeffect of CA-074Me on cerebral blood flow was observed with relative cerebral bloodflow (rCBF). Time course changes of CathepsinB-Caspase signaling pathway relatedprotein and the effects of CA-074Me on the proteins expression in the ischemiccortex were assessed with Western Blot and immunohistochemical assays. Primaryastrocyte was exposed to a paradigm of ischemic insult by using oxygen-glucosedeprivation (OGD). LDH detected LDH leakage alternation in astrocytes afterCA-074Me administrated. Western Blot was employed to determine time coursechanges of CathepsinB and the effects of CA-074Me on the CathepsinB-Caspasepathway related proteins expression in the ischemic astrocytes induced by OGD.Results: In pMCAO rats, when administrated10min before the onset of ischemia,CA-074Me (0.65-5.2μg/μl) significantly diminished the infarction volume (P0.01);both0.65and1.3μg/μl of CA-074Me significantly ameliorated behavioral symptoms,and the expression of MAP2in the ischemic cortex (P0.05, P0.01). CA-074Me1.3μg/μl icv treatment at6h after pMCAO also notably reduced the infarctionvolume (P0.01) and ameliorated behavioral symptoms (P0.05, P0.01).CA-074Me0.65and1.3μg/μl had no effect on rCBF. Western Blot analysis revealedthat CathepsinB was significantly activated at6h after pMCAO in the ischemic cortex(P0.01); activation of Bid began at6h after pMCAO (P0.05) and peaked at24h(P<0.01); the level of Caspase3was increased at3h(P0.05) and peaked at24h (P<0.01). CA-074Me treatment markedly decreased the activation of CathepsinB, Bidand Caspase3(P0.01) and the translocation of Cyt-c from mitochondrial to cytoplasm(P0.01), suggesting that CA-074Me effectively inhibited the CathepsinB-Caspasepathway related protein in the ischemic cortex. Further evidence byimmunohistochemical analysis confirmed that CathepsinB and Caspase3in astrocytes inthe ischemic cortex were markedly activated at6h and12h post-ischemia respectively,and CA-074Me treatment downregulated the activation of CathepsinB-Caspase pathwayin the ischemic astrocytes. LDH showed that CA-074Me (5,10,20μM) reduced LDHleakage in a dose-dependent manner at12h after OGD (P0.01). Western Blot analysisdemonstrated that the expression of active-CathepsinB protein was significantlyincreased at6h after OGD(P0.01); CA-074Me treatment decreased the expression ofactive-CathepsinB, tBid、and active-Caspase3(P0.01) and the translocation of Cyt-cfrom mitochondrial to cytoplasm (P0.01), suggesting that CA-074Me effectivelyinhibited the CathepsinB-Caspase pathway related protein in the ischemic astrocytesinduced by OGD.Conclusion:1.CathepsinB specific inhibitor CA-074Me has neuroprotectiveeffects on pMCAO induced brain injury.2.CathepsinB specific inhibitor CA-074Me inhibits CathepsinB-Caspase pathwayin the ischemic cortex in pMCAO rats.3.CathepsinB specific inhibtor CA-074Me exerts protective effects onischemic/hypoxic astrocytes in vivo and in vitro and its mechanism is associated withinhibiting CathepsinB-caspase pathway.