Following this peak, NLRP3, cleaved caspase-1, and IL-1levels declined, but still remained at high levels at 72 hours when compared with sham animals

Following this peak, NLRP3, cleaved caspase-1, and IL-1levels declined, but still remained at high levels at 72 hours when compared with sham animals. NLRP3 inflammasome may effectively reduce the inflammatory response following ICH. Spontaneous intracerebral hemorrhage (ICH) is a devastating disease, accounting for 15 to 20% of 4SC-202 all stroke types.1 There is currently no effective treatment for ICH.2,3 Increasing evidence indicates that inflammation mechanisms play a critical role in the pathophysiology of ICH.4 After ICH, blood components, including blood-borne leukocytes, enter into the brain parenchyma and activate resident immune cells, such as microglia. Leukocyte infiltration and activation of microglia enhance the production of proinflammatory cytokines. Among all the cytokines, interleukin (IL)C1is regarded as a pivotal therapeutic target in ICH,5 as the observations demonstrated that the overexpression of the IL-1antagonist via an adenovirus vector reduced brain edema and thrombin-induced inflammation in the ICH rat model.6 Our previous work has shown that the inhibition of caspase-1, the converting enzyme of active IL-1is processed following ICH remains unclear. Recently, several lines of evidence have suggested that inflammasome, one of the components of the innate immune system, is involved in the pathogenesis of sterile inflammatory response by processing caspase-1 and IL-1to an active stage following human central nervous system (CNS) disorders (such as spinal cord injury,8 traumatic brain injury,9 and ischemic stroke10). Among 20 members of the human NLR protein family that have been reported, the NLRP3 (NALP3, cryopyrin) inflammasome gains notable attention, as it can sense multiple stimulus, including tissue injury and microbial invasion.11 The NLRP3 inflammasome contains NLR family, pyrin domain containing 3 (NLRP3), 4SC-202 which is associated with the apoptosis-associated specklike protein containing a caspase recruitment domain (ASC), which recruits and activates caspase-1, therefore releasing cleaved IL-1processing. NLRP3 activation after ICH may be triggered by mitochondrial reactive oxygen species ROS (mROS), which are regulated by mitochondrial permeability transition pores (mPTPs; Supplementary Fig 2). To test this hypothesis, we first investigated the expression profiles of the NLRP3 inflammasome components, including NLRP3, caspase-1, and IL-1 0.05) after ICH and peaked at 12 hours. Following this peak, NLRP3, cleaved caspase-1, and IL-1levels declined, but still remained at high levels at 72 hours when compared with sham animals. Double immunostaining showed that NLRP3 is mainly expressed in microglia cells and not in other cell types, such as astrocytes (data not shown). Open in a separate window Figure 1 Expression profile of the NLRP3 inflammasome components after autologous arterial bloodCinduced intracerebral hemorrhage (ICH). (ACC) Western blot assay for the expression profiles of NLRP3 (A) and caspase-1 p20 subunit (B), and enzyme-linked immunosorbent assay assay for the expression profiles of interleukin (IL)C1(C) in the ipsilateral hemisphere in sham and ICH mice at 3, 6, 12, 24, and 72 hours following operation; n=6 mice per Rabbit Polyclonal to GRIN2B (phospho-Ser1303) group and per time point. Error bars represent meanstandard error of the mean. #levels detected by ELISA were also markedly reduced compared to ICH without treatment and siCtrl animals (in the ipsilateral hemisphere in sham, ICH, scrambled siRNA (ICH+siCtrl), and NLRP3 siRNA mixture (ICH+siNlrp3s) groups at 12 hours following operation; n=6 mice per group. Error bars represent meanstandard error of the mean. # 0.05; Fig 3). Following NLRP3 siRNA mixture administration, there was a significant improvement in the modified Garcia test at both 24 and 72 hours compared to ICH and siCtrl mice ( 0.05). Consistent with neurological improvement, the brain edema in the ipsilateral basal ganglia (ipsi-BG) after NLRP3 siRNA injection was also significantly reduced at both 24 (ipsi-BG: 80.880.29% vs ICH, 82.380.33%, 0.05; vs siCtrl, 82.680.35%, 0.05) and 72 hours (ipsi-BG: 4SC-202 81.640.30% vs ICH, 83.170.24%, 0.05; vs siCtrl, 82.900.43%, 0.05) compared to ICH and siCtrl. With regard.