Zhang Yi-Yue, Tian Jing, Peng Zi-Mei, Liu Bin, Peng Ya-Wei, Zhang Xiao-Jie, Hu Zhong-Yang, Luo Xiu-Ju, Peng Jun
Department of Pharmacology, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 410078, China.
Hunan Provincial Key Laboratory of Cardiovascular Research, Xiangya School of Pharmaceutical Sciences, Central South University, Changsha, 410078, China.
Cardiovasc Drugs Ther. 2023 Feb;37(1):9-23. doi: 10.1007/s10557-021-07231-w. Epub 2021 Sep 8.
Pellino3, an ubiquitin E3 ligase, prevents the formation of the death-induced signaling complex in response to TNF-α by targeting receptor-interacting protein kinase 1 (RIPK1), and bioinformatics analysis predicted an interaction between Pellino3 and caspofungin, a common antifungal drug used in clinics. This study aimed to explore the effect of caspofungin on brain injury in ischemic stroke and the underlying mechanisms.
Ischemic stroke injury was induced in Sprague Dawley rats by occlusion of the middle cerebral artery (MCA) for 2 h, followed by 24 h reperfusion. PC12 cells were deprived of both oxygen and glucose for 8 h and then were cultured for 24 h with oxygen and glucose to mimic an ischemic stroke in vitro.
Animal experiments showed brain injury (increase in neurological deficit score and infarct volume) concomitant with a downregulation of Pellino3, a decreased ubiquitination of RIPK1, and an up-regulation of necroptosis-associated proteins [RIPK1, RIPK3, mixed lineage kinase domain-like protein (MLKL), p-RIPK1, p-RIPK3, and p-MLKL]. Administration of caspofungin (6 mg/kg, i.m.) at 1 h and 6 h after ischemia significantly improved neurological function, reduced infarct volume, up-regulated Pellino3 levels, increased RIPK1 ubiquitination, and down-regulated protein levels of RIPK1, p-RIPK1, p-RIPK3, and p-MLKL. PC12 cells deprived of oxygen/glucose developed signs of cellular injury (LDH release and necroptosis) concomitant with downregulation of Pellino3, decreased ubiquitination of RIPK1, and elevated necroptosis-associated proteins. These changes were reversed by overexpression of Pellino3.
We conclude that Pellino3 has an important role in counteracting necroptosis via ubiquitination of RIPK1 and caspofungin can suppress the brain cell necroptosis in ischemic stroke through upregulation of Pellino3.
泛素E3连接酶Pellino3通过靶向受体相互作用蛋白激酶1（RIPK1）来阻止肿瘤坏死因子-α（TNF-α）诱导的死亡信号复合物的形成，生物信息学分析预测Pellino3与卡泊芬净（一种临床常用的抗真菌药物）之间存在相互作用。本研究旨在探讨卡泊芬净对缺血性脑卒中脑损伤的影响及其潜在机制。
通过阻断大脑中动脉（MCA）2小时，随后再灌注24小时，在Sprague Dawley大鼠中诱导缺血性脑卒中损伤。将PC12细胞缺氧缺糖8小时，然后再用氧气和葡萄糖培养24小时，以在体外模拟缺血性脑卒中。
动物实验显示脑损伤（神经功能缺损评分和梗死体积增加）伴随着Pellino3的下调、RIPK1泛素化的减少以及坏死性凋亡相关蛋白[RIPK1、RIPK3、混合谱系激酶结构域样蛋白（MLKL）、p-RIPK1、p-RIPK3和p-MLKL]的上调。在缺血后1小时和6小时给予卡泊芬净（6mg/kg，肌肉注射）可显著改善神经功能、减少梗死体积、上调Pellino3水平、增加RIPK1泛素化，并下调RIPK1、p-RIPK1、p-RIPK3和p-MLKL的蛋白水平。缺氧缺糖的PC12细胞出现细胞损伤迹象（乳酸脱氢酶释放和坏死性凋亡），伴随着Pellino3的下调、RIPK1泛素化的减少以及坏死性凋亡相关蛋白的升高。这些变化通过Pellino3的过表达得以逆转。
我们得出结论，Pellino3通过RIPK1的泛素化在抵抗坏死性凋亡中起重要作用，卡泊芬净可通过上调Pellino3抑制缺血性脑卒中的脑细胞坏死性凋亡。
