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Targeting connexin 43 provides anti-inflammatory effects after intracerebral hemorrhage injury by regulating YAP signaling
Journal of Neuroinflammation ( IF 9.3 ) Pub Date : 2020-10-28 , DOI: 10.1186/s12974-020-01978-z
Hailong Yu 1, 2, 3 , Xiang Cao 1 , Wei Li 3, 4 , Pinyi Liu 1 , Yuanyuan Zhao 3, 4 , Lilong Song 3, 4 , Jian Chen 1 , Beilei Chen 2, 3, 4 , Wenkui Yu 1 , Yun Xu 1
Affiliation  

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.

中文翻译:

靶向连接蛋白 43 通过调节 YAP 信号传导在脑出血损伤后提供抗炎作用

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稳态。与 Cx43 过表达类似,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,Cx43Hc 过度开放在脑出血 (ICH) 损伤中的作用尚不清楚。原代细胞中的血红素刺激和 C57BL/6J (B6) 小鼠中的胶原酶 IV 注射被用作体外和体内的 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。蛋白质印迹和免疫荧光用于测量蛋白质表达。qRT-PCR 和 ELISA 用于确定细胞因子的水平。应用共免疫沉淀 (Co-IP) 和 Duolink 原位邻近连接测定 (PLA) 来测量蛋白质之间的关联。在这项研究中,在 ICH 损伤后的小鼠中观察到 Cx43 表达上调和过度的 Cx43Hc 开放。Gap19 延迟治疗显着减轻了 ICH 损伤后的血肿体积和神经功能缺损。此外,Gap19 降低了血肿周围组织中的炎性细胞因子水平,并降低了体外和体内 ICH 损伤后的反应性星形胶质细胞增生。有趣的是,培养中氯化血红素刺激后,星形胶质细胞中的 Cx43 转录活性和表达显着增加。然而,Gap19 治疗通过泛素-蛋白酶体途径下调星形胶质细胞 Cx43 表达,而不影响 Cx43 转录。此外,我们的数据显示 Gap19 增加了 Yes 相关蛋白 (YAP) 核易位。这随后上调了 SOCS1 和 SOCS3 的表达,然后抑制了氯化血红素刺激的星形胶质细胞中的 TLR4-NFκB 和 JAK2-STAT3 通路。最后,YAP 抑制剂维替泊芬 (VP) 在体外逆转了 Gap19 的抗炎作用,并在 ICH 损伤后几乎完全阻断了其在体内的保护作用。这项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。Gap19 抑制异常星形胶质细胞 Cx43 表达和 Cx43Hc 开放在 ICH 损伤后具有抗炎和神经保护作用。这随后上调了 SOCS1 和 SOCS3 的表达,然后抑制了氯化血红素刺激的星形胶质细胞中的 TLR4-NFκB 和 JAK2-STAT3 通路。最后,YAP 抑制剂维替泊芬 (VP) 在体外逆转了 Gap19 的抗炎作用,并在 ICH 损伤后几乎完全阻断了其在体内的保护作用。这项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。Gap19 抑制异常星形胶质细胞 Cx43 表达和 Cx43Hc 开放在 ICH 损伤后具有抗炎和神经保护作用。这随后上调了 SOCS1 和 SOCS3 的表达,然后抑制了氯化血红素刺激的星形胶质细胞中的 TLR4-NFκB 和 JAK2-STAT3 通路。最后,YAP 抑制剂维替泊芬 (VP) 在体外逆转了 Gap19 的抗炎作用,并在 ICH 损伤后几乎完全阻断了其在体内的保护作用。这项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。Gap19 抑制异常星形胶质细胞 Cx43 表达和 Cx43Hc 开放在 ICH 损伤后具有抗炎和神经保护作用。这项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。Gap19 抑制异常星形胶质细胞 Cx43 表达和 Cx43Hc 开放在 ICH 损伤后具有抗炎和神经保护作用。这项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。Gap19 抑制异常星形胶质细胞 Cx43 表达和 Cx43Hc 开放在 ICH 损伤后具有抗炎和神经保护作用。
更新日期:2020-10-30
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