The Effect Of A Caspase-1 Inhibitor CZL-80 Against Neurological Injury After Ischemic Stroke

Ischemic stroke is a neurological disease which is caused by temporary or permanent insufficient cerebral blood supply due to stenosis or occlusion of blood vessels.Given the complex pathological mechanisms of ischemic stroke,there is limited approaches for stroke therapy.Therefore,the therapeutic drugs for stroke treatment are urgently needed.Neuroinflammation is an important pathological process after ischemic stroke.Damaged associated molecular patterns?DAMPs?released by necrotic cells activate inflammasome,which mediates inflammatory process after cerebral ischemia,leading to neuronal damage.Caspase-1 activation is involved in the process and mediates the activation of inflammasome,suggesting Caspase-1 is a potential therapeutic target for the intervention of cerebral ischemia.We previously designed and screened a novel Caspase-1 inhibitor CZL-80.However,it is unclear whether CZL-80 is neuroprotective against cerebral ischemia.The study is aimed to identify the pharmacological effects of CZL-80 against ischemic stroke and to explore its mechanisms.Male C57/B6 mice and Caspase-1^-/-mice were subjected to photothrombosis?PT?-induced cerebral ischemia.CZL-80 was intraperitoneally injected daily in 1-7 d,1-4 d,4-7 d after cerebral ischemia.We used the grid-walking task and the cylinder task to determine the motor function of mice at indicated time points after cerebral ischemia.Immunoblotting and immunofluorescence were used to detect the expression of Caspaase-1 and GSDMD and their cellular localization in the peri-infarct brain cortex after ischemic stroke.The activation of microglia and neuron morphology in the peri-infarct brain cortex after ischemic stroke were also observed by immunofluorescence assay.The results showed neurological dysfunction in mice with different extension along with the recovery phase after ischemia.We documented the primary and the secondary neurological dysfunction at 1 and 5-7 d after PT onset.The upregulation of Caspase-1 peaked at 5-7 d after ischemic stroke.The immunofluorescence results showed that Caspase-1 was mainly derived from activated microglia rather than neurons.It is likely that Caspase-1 in microglia may be involved in secondary neurological dysfunction after ischemic stroke.Daily administration of CZL-80?30mg/kg?in 1-7 d significantly reduced foot faults of left forelimb in the grid-walking task and forelimb symmetry in the cylinder task,indicating improved motor function by CZL-80.Daily administration of CZL-80?30 mg/kg?in 1-4 d could not improve motor while daily administration in 4-7 d after PT onset significantly reduced foot faults and forelimb symmetry.Remarkably,CZL-80 administration during 4-7 d showed no significant difference in efficacy compared with the its administration during 1-7 d,which indicated a key therapeutic time window.Moreover,the neuroprotective effect of administration of CZL-80 in 4-7 d was available 43 d after ischemic stroke,indicating CZL-80 can improve the long-term neurological function after cerebral ischemia.Daily administration of CZL-80?30 mg/kg?during 4-7 d after PT onset in Caspase-1^-/-mice failed to improve the neurological function,which suggested that the neuroprotective effect of CZL-80 was Caspase-1-dependent.The results showed that GSDMD was transiently up-regulated 1 d after ischemia in neurons and CZL-80 did not inhibit the expression of GSDMD and failed to reduce neuronal loss by ischemia.These results indicated the effect of CZL-80 was not attributable to inhibited pyroptosis.We further found that CZL-80 significantly reduced the number of activated microglia in the peri-infarct brain cortex after ischemic stroke,which might be involved in its neuroprotective effect.In summary,the present study identified that CZL-80,a novel Caspase-1 inhibitor,improved neurological function after ischemic stroke in mice.The effective therapeutic window of CZL-80 would be 4-7 days after ischemia,when the secondary neurological dysfunction occurred.inhibiting microglia activation,but not neuronal pyroptosis was involved in the efficacy of CZL-80.