Protection Of A Cathepsin L Inhibitor Clik148on Focal Crerebral Ischemic Injury In Rats And Its Mechanism Of Inhibition Of Astrocytic Cell Death

Aim:To investigate the protective effect of a novel cathepsin inhibitor Clik148on focal cerebral ischemic injury in rats and its mechanism of inhibition of astrocytic cell death.Methods:In this study, we established two cerebral ischemia-hypoxia models in vivo and in vitro. Permanent middle cerebral artery occlusion (pMCAO) model was induced by using intraluminal filament technique in rats. Primary astrocytes was exposed to a paradigm of ischemic insult by using an oxygen-glucose deprivation (OGD) device. Clikl48was injected intracerebroventricularly (icv.)10min before the onset of ischemia or6h after ischemia. Neurological deficits were analyzed by scoring neurological deficits, grip force and right forelimb use and brain infarction volume was assessed with2,3,5-triphenyl tetrazolium chloride (TTC). The injury of astrocytes induced by OGD was detected with LDH test and the expression of GFAP by Western Blot analysis. The signaling pathway of cathepsins-caspases related protein cathepsin L, tBid, Cyt-c and Caspase-3were assessed with Western Blot analysis. Double immunofluorescence was employed to determine the activation of cathepsin L and caspase-3in GFAP-positive cell.Results:Clik148reduced the infarct volume, improved the neurological deficits and increased the expression of MAP2and GFAP at24h after ischemia. In OGD-induced primary cultured astrocytes injury, Clik148significantly decreased the LDH leakage and increased the number of astrocytes in a dose-dependent fasion, and also increased the expression of GFAP. Clikl48has no markedly effects on non-OGD astrocytes. In ischemic cortex, pMCAO induced an increase in activated cathepsin L, tBid and Caspase-3, reduced Cyt-c in mitochondria and increased Cyt-c in cytoplasm. Clikl48reversed pMCAO-induced increases in activated cathepsin L, tBid and Caspase-3, and a reduction in mitochondria Cyt-c and an increase in cytoplasm Cyt-c. Immunofluorescence showed that the dots distribution of cathepsinL was seen in astrocytes in the ischemic cortex of sham. while the diffussion distribution of cathepsin L was seen in astrocytes in the ischemic cortex of ischemic control rats at3h after ischemia, suggesting the release of cathepsin L from lysosome to cytosol after ischemia and activation of cathepsin L. pMCAO also induced the activation of caspase-3in ischemic astrocytes at12h after iscemia. Clik148reversed pMCAO-induced activation of cathepsinL and caspase-3in ischemic astrocytes. Western Blot analysis showed that OGD induced an increase in activated cathepsin L, tBid and Caspase-3, reduced Cyt-c in mitochondria and increased Cyt-c in cytoplasm.Clikl48blocked OGD-induced an increase in activated cathepsin L, tBid and Caspase-3, and a reduction in mitochondria Cyt-c and an increase in cytoplasm Cyt-c.Conclusion:The current findings provide the first evidences that Clikl48has a neuroprotective activity on cerebral ischemic brain injury and astrocytes injury, and this effect may be associated with its blocking the activation of cathepsin L-caspase apoptotic signaling pathway in astrocytes.