Affiliated of Drum Tower Hospital, Medical school of Nanjing University, Nanjing, Jiangsu, People's Republic of China.
Clinical Medical College of Yangzhou University, Yangzhou, Jiangsu, People's Republic of China.
J Neuroinflammation. 2020 Oct 28;17(1):322. doi: 10.1186/s12974-020-01978-z.
In the central nervous system (CNS), connexin 43 (Cx43) is mainly expressed in astrocytes and regulates astrocytic network homeostasis. Similar to Cx43 overexpression, abnormal excessive opening of Cx43 hemichannels (Cx43Hcs) on reactive astrocytes aggravates the inflammatory response and cell death in CNS pathologies. However, the role of excessive Cx43Hc opening in intracerebral hemorrhage (ICH) injury is not clear.
Hemin stimulation in primary cells and collagenase IV injection in C57BL/6J (B6) mice were used as ICH models in vitro and in vivo. After ICH injury, the Cx43 mimetic peptide Gap19 was used for treatment. Ethidium bromide (EtBr) uptake assays were used to measure the opening of Cx43Hcs. Western blotting and immunofluorescence were used to measure protein expression. qRT-PCR and ELISA were used to determine the levels of cytokines. Coimmunoprecipitation (Co-IP) and the Duolink in situ proximity ligation assay (PLA) were applied to measure the association between proteins.
In this study, Cx43 expression upregulation and excessive Cx43Hc opening was observed in mice after ICH injury. Delayed treatment with Gap19 significantly alleviated hematoma volume and neurological deficits after ICH injury. In addition, Gap19 decreased inflammatory cytokine levels in the tissue surrounding the hematoma and decreased reactive astrogliosis after ICH injury in vitro and in vivo. Intriguingly, Cx43 transcriptional activity and expression in astrocytes were significantly increased after hemin stimulation in culture. However, Gap19 treatment downregulated astrocytic Cx43 expression through the ubiquitin-proteasome pathway without affecting Cx43 transcription. Additionally, our data showed that Gap19 increased Yes-associated protein (YAP) nuclear translocation. This subsequently upregulated SOCS1 and SOCS3 expression and then inhibited the TLR4-NFκB and JAK2-STAT3 pathways in hemin-stimulated astrocytes. Finally, the YAP inhibitor, verteporfin (VP), reversed the anti-inflammatory effect of Gap19 in vitro and almost completely blocked its protective effects in vivo after ICH injury.
This study provides new insight into potential treatment strategies for ICH injury involving astroglial Cx43 and Cx43Hcs. Suppression of abnormal astroglial Cx43 expression and Cx43Hc opening by Gap19 has anti-inflammatory and neuroprotective effects after ICH injury.
在中枢神经系统（CNS）中，连接蛋白 43（Cx43）主要在星形胶质细胞中表达，并调节星形胶质细胞网络的稳态。类似于 Cx43 过表达，反应性星形胶质细胞上 Cx43 半通道（Cx43Hcs）的异常过度开放会加剧 CNS 病理中的炎症反应和细胞死亡。然而，异常 Cx43Hc 开放在脑出血（ICH）损伤中的作用尚不清楚。
原代细胞中血红素刺激和 C57BL/6J（B6）小鼠胶原酶 IV 注射用于体外和体内 ICH 模型。ICH 损伤后，使用 Cx43 模拟肽 Gap19 进行治疗。溴化乙锭（EtBr）摄取测定用于测量 Cx43Hcs 的开放。Western blot 和免疫荧光用于测量蛋白表达。qRT-PCR 和 ELISA 用于测定细胞因子水平。共免疫沉淀（Co-IP）和 Duolink 原位邻近连接分析（PLA）用于测量蛋白之间的关联。
本研究观察到 ICH 损伤后小鼠 Cx43 表达上调和 Cx43Hc 过度开放。延迟给予 Gap19 可显著减轻 ICH 损伤后的血肿体积和神经功能缺损。此外，Gap19 降低了组织中炎症细胞因子水平ICH 损伤后体外和体内的反应性星形胶质细胞增生。有趣的是，血红素刺激培养物中的星形胶质细胞中 Cx43 转录活性和表达明显增加。然而，Gap19 通过泛素-蛋白酶体途径下调星形胶质细胞 Cx43 的表达，而不影响 Cx43 转录。此外，我们的数据表明 Gap19 增加了 YAP 核易位。这随后上调了 SOCS1 和 SOCS3 的表达，然后抑制了血红素刺激的星形胶质细胞中的 TLR4-NFκB 和 JAK2-STAT3 途径。最后，YAP 抑制剂 verteporfin（VP）逆转了 Gap19 在体外的抗炎作用，并几乎完全阻断了其在 ICH 损伤后体内的保护作用。
本研究为涉及星形胶质细胞 Cx43 和 Cx43Hcs 的 ICH 损伤的潜在治疗策略提供了新的见解。通过 Gap19 抑制异常星形胶质细胞 Cx43 表达和 Cx43Hc 开放对 ICH 损伤后具有抗炎和神经保护作用。
