Liu Lihan, Wang Yue, Wang Xiaolin, Zhang Guowen, Sha Sha, Zhou Rong, Du Yimei, Wu Chunfeng, Chen Lei
Department of Physiology, Nanjing Medical University, No. 101, Longmian Ave, Nanjing, Jiangsu Province, 211166, P.R. China.
Research Center of Ion Channelopathy, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei Province, 430022, P.R. China.
Acta Neuropathol Commun. 2025 Apr 10;13(1):73. doi: 10.1186/s40478-025-01990-5.
Pyroptosis contributes to the neuronal damage that occurs during epilepsy. Calcium-activated neutral protease (calpain) dissociates cysteinyl aspartate specific proteinase-1 (caspase-1, cas-1) from the cytoskeleton, and the activated cas-1 is responsible for the production of N-terminus of gasdermin D (N-GSDMD), the final executor of pyroptosis. Blocking transient receptor potential vanilloid 4 (TRPV4) can reduce neuronal injury in temporal lobe epilepsy (TLE) model mice. This study investigated the role of TRPV4 in pyroptosis during TLE. In the hippocampus of pilocarpine-induced status epilepticus (PISE) mice, the ratio of inactive calpain 1 protein level to its total protein level (inactive/total calpain 1) significantly decreased, while the ratio of inactive calpain 2 protein level to its total protein level remained unchanged. The protein levels of NLRP3, cleaved cas-1 (c-cas-1), interleukin (IL)-1β, and N-GSDMD increased, with more GSDMD-immunofluorescence-positive (GSDMD) cells and fewer surviving pyramidal neurons observed in the hippocampus of PISE mice. Calpain inhibition with MDL-28170 reversed these changes, except for the elevated NLRP3 levels. Inhibitors targeting NLRP3 (MCC950) and cas-1 (Ac-YVAD-cmk) blocked the increase in c-cas-1, IL-1β, and N-GSDMD levels in the hippocampus of PISE mice. TRPV4 inhibition via HC-067047 increased the inactive/total calpain 1 ratio, decreased NLRP3, c-cas-1, IL-1β, and N-GSDMD protein levels, reduced GSDMD cells number, and improved pyramidal neuron survival in the hippocampus of PISE mice. Conversely, TRPV4 activation with GSK1016790A decreased the inactive/total calpain 1 ratio, elevated NLRP3, c-cas-1, IL-1β, and N-GSDMD levels, and increased GSDMD cells number in the hippocampus. In the hippocampus of GSK1016790A-injected mice, the inactive/total calpain 1 ratio was increased by MDL-28170, and c-cas-1, IL-1β, and N-GSDMD protein levels were markedly attenuated by MDL-28170, MCC950, and Ac-YVAD-cmk, respectively. In conclusion, TRPV4 inhibition mitigates pyroptosis in PISE mice by downregulating the calpain 1-NLRP3/cas-1-GSDMD pathway, ultimately reducing neuronal damage.
细胞焦亡会导致癫痫发作时出现的神经元损伤。钙激活中性蛋白酶（钙蛋白酶）使半胱天冬酶-1（caspase-1，cas-1）从细胞骨架上解离，激活的cas-1负责产生gasdermin D的N端（N-GSDMD），这是细胞焦亡的最终执行者。阻断瞬时受体电位香草酸受体4（TRPV4）可减轻颞叶癫痫（TLE）模型小鼠的神经元损伤。本研究探讨了TRPV4在TLE期间细胞焦亡中的作用。在毛果芸香碱诱导的癫痫持续状态（PISE）小鼠的海马中，无活性钙蛋白酶1蛋白水平与其总蛋白水平的比值（无活性/总钙蛋白酶1）显著降低，而无活性钙蛋白酶2蛋白水平与其总蛋白水平的比值保持不变。NLRP3、裂解的cas-1（c-cas-1）、白细胞介素（IL）-1β和N-GSDMD的蛋白水平升高，在PISE小鼠的海马中观察到更多GSDMD免疫荧光阳性（GSDMD）细胞，而存活的锥体神经元减少。用MDL-28170抑制钙蛋白酶可逆转这些变化，但NLRP3水平升高除外。靶向NLRP3（MCC950）和cas-1（Ac-YVAD-cmk）的抑制剂可阻断PISE小鼠海马中c-cas-1、IL-1β和N-GSDMD水平的升高。通过HC-067047抑制TRPV4可增加无活性/总钙蛋白酶1比值，降低NLRP3、c-cas-1、IL-1β和N-GSDMD蛋白水平，减少GSDMD细胞数量，并改善PISE小鼠海马中锥体神经元的存活。相反，用GSK1016790A激活TRPV4可降低无活性/总钙蛋白酶1比值，升高NLRP3、c-cas-1、IL-1β和N-GSDMD水平，并增加海马中GSDMD细胞数量。在注射GSK1016790A的小鼠海马中，MDL-28170可增加无活性/总钙蛋白酶1比值，而MDL-28170、MCC950和Ac-YVAD-cmk分别可显著降低c-cas-1、IL-1β和N-GSDMD蛋白水平。总之，抑制TRPV4可通过下调钙蛋白酶1-NLRP3/cas-1-GSDMD途径减轻PISE小鼠的细胞焦亡，最终减少神经元损伤。
