Cystatin C Attenuates Perihematomal Secondary Brain Injury by Inhibiting the Cathepsin B/NLRP3 Signaling Pathway in a Rat Model of Intracerebral Hemorrhage.

Abstract

Secondary brain injury (SBI) is a noticeable contributor to the high mortality and morbidity rates associated with intracerebral hemorrhage (ICH), and effective treatment options remain limited. Cystatin C (CysC) emerges as a novel candidate for SBI intervention. The therapeutic effects and underlying mechanisms of CysC in mitigating SBI following ICH were explored in the current research. An in vivo ICH rat model was established by injecting autologous blood into the right caudate nucleus. Western blotting (WB) was utilized to assess the levels of CysC, cathepsin B (CTSB), and the NLRP3 inflammasome. Subsequently, the ICH rat model was treated with exogenous CysC supplementation or CysC knockdown plasmids. Various parameters, including Evans blue (EB) extravasation, brain water content, and neurological function in rats, were examined. RT-qPCR and WB were employed to determine the expression levels of CTSB and the NLRP3 inflammasome. The co-expression of CTSB, CysC, and NLRP3 inflammasome with GFAP, NeuN, and Iba1 was assessed through double-labeled immunofluorescence. The interaction between CysC and CTSB was investigated using double-labeled immunofluorescence and co-immunoprecipitation. The findings revealed an elevation of CysC expression level, particularly at 24h after ICH. Exogenous CysC supplementation alleviated severe brain edema, neurological deficit scores, and EB extravasation induced by ICH. Conversely, CysC knockdown produced opposite effects. The expression levels of CTSB and the NLRP3 inflammasome were significantly risen following ICH, and exogenous CysC supplement attenuated their expression levels. Double-labeled immunofluorescence illustrated that CysC, CTSB, and the NLRP3 inflammasome were predominantly expressed in microglial cells, and the interaction between CysC and CTSB was evidenced. CysC exhibited potential in ameliorating SBI following ICH via effectively suppressing the activation of the NLRP3 inflammasome mediated by CTSB specifically in microglial cells. These findings underscore the prospective therapeutic efficacy of CysC in the treatment of ICH-induced complications.