Department of Neurosurgery, Guangxi Medical University Cancer Hospital, Nanning, China (J.Y.).
Department of Neurosurgery, Second Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, China (W.X.).
Stroke. 2021 Dec;52(12):4021-4032. doi: 10.1161/STROKEAHA.120.033285. Epub 2021 Nov 1.
Neuronal pyroptosis is a type of regulated cell death triggered by proinflammatory signals. CCR5 (C-C chemokine receptor 5)-mediated inflammation is involved in the pathology of various neurological diseases. This study investigated the impact of CCR5 activation on neuronal pyroptosis and the underlying mechanism involving cAMP-dependent PKA (protein kinase A)/CREB (cAMP response element binding)/NLRP1 (nucleotide-binding domain leucine-rich repeat pyrin domain containing 1) pathway after experimental intracerebral hemorrhage (ICH).
A total of 194 adult male CD1 mice were used. ICH was induced by autologous whole blood injection. Maraviroc (MVC)-a selective antagonist of CCR5-was administered intranasally 1 hour after ICH. To elucidate the underlying mechanism, a specific CREB inhibitor, 666-15, was administered intracerebroventricularly before MVC administration in ICH mice. In a set of naive mice, rCCL5 (recombinant chemokine ligand 5) and selective PKA activator, 8-Bromo-cAMP, were administered intracerebroventricularly. Short- and long-term neurobehavioral assessments, Western blot, Fluoro-Jade C, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and immunofluorescence staining were performed.
The brain expression of CCL5 (chemokine ligand 5), CCR5, PKA-Cα (protein kinase A-Cα), p-CREB (phospho-cAMP response element binding), and NLRP1 was increased, peaking at 24 hours after ICH. CCR5 was expressed on neurons, microglia, and astrocytes. MVC improved the short- and long-term neurobehavioral deficits and decreased neuronal pyroptosis in ipsilateral brain tissues at 24 hours after ICH, which were accompanied by increased PKA-Cα and p-CREB expression, and decreased expression of NLRP1, ASC (apoptosis-associated speck-like protein containing a CARD), C-caspase-1, GSDMD (gasdermin D), and IL (interleukin)-1β/IL-18. Such effects of MVC were abolished by 666-15. At 24 hours after injection in naive mice, rCCL5 induced neurological deficits, decreased PKA-Cα and p-CREB expression in the brain, and upregulated NLRP1, ASC, C-caspase-1, N-GSDMD, and IL-1β/IL-18 expression. Those effects of rCCL5 were reversed by 8-Bromo-cAMP.
CCR5 activation promoted neuronal pyroptosis and neurological deficits after ICH in mice, partially through the CCR5/PKA/CREB/NLRP1 signaling pathway. CCR5 inhibition with MVC may provide a promising therapeutic approach in managing patients with ICH.
神经元细胞焦亡是一种由促炎信号触发的受调控的细胞死亡方式。CCR5（C-C 趋化因子受体 5）介导的炎症参与了多种神经疾病的病理过程。本研究旨在探讨 CCR5 激活对实验性脑出血（ICH）后神经元细胞焦亡的影响及其涉及 cAMP 依赖性 PKA（蛋白激酶 A）/CREB（cAMP 反应元件结合）/NLRP1（核苷酸结合域富含亮氨酸重复吡咯烷域包含 1）通路的潜在机制。
共使用 194 只成年雄性 CD1 小鼠。采用自体全血注射法诱导 ICH。ICH 后 1 小时，通过鼻腔给予 CCR5 选择性拮抗剂 Maraviroc（MVC）。为了阐明潜在机制，在 ICH 小鼠中给予 MVC 前，通过侧脑室给予特异性 CREB 抑制剂 666-15。在一组新生小鼠中，通过侧脑室给予重组趋化因子配体 5（rCCL5）和选择性 PKA 激活剂 8-Bromo-cAMP。进行短期和长期神经行为评估、Western blot、Fluoro-Jade C、末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）和免疫荧光染色。
ICH 后 24 小时，脑组织中 CCL5（趋化因子配体 5）、CCR5、PKA-Cα（蛋白激酶 A-Cα）、p-CREB（磷酸化 cAMP 反应元件结合）和 NLRP1 的表达增加，于 24 小时达到峰值。CCR5 表达于神经元、小胶质细胞和星形胶质细胞上。MVC 改善了 ICH 后 24 小时同侧脑组织的短期和长期神经行为缺陷，并减少了神经元细胞焦亡，同时增加了 PKA-Cα 和 p-CREB 的表达，降低了 NLRP1、ASC（凋亡相关斑点样蛋白包含一个 CARD）、C-caspase-1、GSDMD（gasdermin D）和 IL（白细胞介素）-1β/IL-18 的表达。MVC 的这种作用被 666-15 所消除。在新生小鼠注射后 24 小时，rCCL5 诱导了神经功能缺损，降低了大脑中的 PKA-Cα 和 p-CREB 表达，并上调了 NLRP1、ASC、C-caspase-1、N-GSDMD 和 IL-1β/IL-18 的表达。rCCL5 的这些作用被 8-Bromo-cAMP 逆转。
CCR5 激活促进了 ICH 后小鼠的神经元细胞焦亡和神经功能缺损，部分通过 CCR5/PKA/CREB/NLRP1 信号通路。用 MVC 抑制 CCR5 可能为 ICH 患者的治疗提供一种有前景的方法。
