CZL80,a Novel Caspase-1 Inhibitor,Reduces Microglial Activation In Ischemic Brain And Its Effect On Post-stroke Depression

Ischemic stroke is a neurological disorder with high morbidity,mortality and recurrence rate.Ischemic stroke not only causes defects in motor,sensory and other neurological function,but also closely relates with post-stroke depression（PSD）.Neuroinflammation plays an important role in the pathological process of ischemic stroke.Particularly,the activation of inflammasome in neurons,glial cells and vascular endothelial cells evoke neuroinflammation and aggravate the neurological dysfunction after stroke,which is a risky factor for the development of PSD.Caspase-1 is a key component of inflammasome.Inhibition of caspase-1 has been proposed as a potential strategy for treating ischemic stroke and related neurological disorders.We previously designed and found CZL80,a novel inhibitor of caspase-1,significantly improved the neurological function secondary to cerebral ischemia.However,the mechanisms underlying CZL80 have not been fully identified,and it remains unclear whether CZL80 is a promising drug for PSD therapy.The present study aims to explore the mechanism of CZL80,a novel caspase-1 inhibitor,against motor function injury after stroke,and to clarify the anti-PSD effect of CZL80.In this study,male C57/B6 and Caspase-1^-/-mice were employed to establish the photothrombosis（PT）-induced ischemic stroke model.Combined with western blotting and immunohistofluorescence staining,the expression and cell localization of Caspase-1 in the ischemic brains were determined,and the morphology and the number of microglia and neurons in the peripheral area of cerebral ischemia and infarction were observed.The mice showed neurological neurological impairment within 14 days after PT.Particularly,the acute and progressive neurological dysfunction can be observed on1 and 4-7 days after PT onset,respectively.CZL80 improves neurological function dose-dependently after cerebral ischemia when administered daily during day 1-7 after ischemia.Caspase-1 was up-regulated in a time-dependent manner on day 4-7 after cerebral ischemia.It showed that caspase-1 predominantly located in activated microglia rather than neurons or vascular endothelial cells.These results suggested that microglia caspase-1 activation may be involved in the progressive neurological impairment after ischemic stroke.Furthermore,CZL80 significantly decreased microglia activation after PT onset,but CZL80 did not reduce neuronal death after ischemia or promote cerebral angiogenesis after ischemia.Together,we concluded that the efficacy of CZL80 on reducing neurological damage can be attributed to its inhibition of microglia activation.In this study,a mouse PSD model was established by combining PT with chronic restrain stress（CRS）.After PT onset,mice were subjected to restraint stress for 2 h daily for 14 consecutive days.CZL80（30 mg/kg/d）was administered by intraperitoneal injection on day 1-7 or 1-14 after cerebral ischemia.The depression-like behavior of mice was determined by forced swimming test,tail suspension test and sucrose preference test.Western blotting was used to detect the expression of brain-derived neurotrophic factor（BDNF）in the prefrontal cortex,amygdala and hippocampus of depression-related brain regions.Golgi staining was carried out to detect the density of dendritic spines in the abovementioned brain regions.The results showed that administration of CZL80 at 1-14 days after ischemia significantly reduced the depression-like behavior of mice in a dose-dependent manner.As a comparison,CZL80administration at day 1-7 after PSD modeling was not effective.Furthermore,both the level of BDNF in the hippocampus and the density of neuronal dendritic spines of PSD model mice was significantly reduced.CZL80 significantly increased the expression of BDNF and the density of dendritic spines in the hippocampus.The PSD model was established in Caspase-1^-/-mice.Compared with wild-type littermates,the depression-like behavior of Caspase-1^-/-mice was significantly reduced,and CZL80 failed to ameliorate depression-like behavior after ischemia in Caspase-1^-/-mice,suggesting that CZL80 may alleviate depression-like behavior by inhibiting Caspase-1.In addition,CZL80 could also significantly improve CRS-induced depression-like behavior in mice,suggesting CZL80 as a potential drug for depression therapy.In conclusion,the present study found that inhibition of microglia activation underlies the effects of CZL80 in alleviating progressive neurological injury after cerebral ischemia.CZL80 ameliorates poststroke depressive in mice,possibly by upregulating BDNF and the density of dendritic spines in the hippocampus.