SERPINA3K抑制TNF-α诱导的角膜内皮间隙连接功能障碍及其机制研究

Gap junctions mediated by Cx43 play an essential role in the maintenance of corneal endothelial homeostasis. Recently we found that inflammatory mediator TNF-α induced the gap junction dysfunction by disrupting the interaction between ZO-1 and Cx43 in corneal endothelium. Serine proteinase inhibitor A3K (SERPINA3K) was reported to inhibit inflammation and neovascularization, however, the inhibitory effect of SERPINA3k on gap junction dysfunction induced by TNF-α still remains unclear. Our previous researches had already confirmed that SERPINA3K can inhibit the destruction of ZO-1 structure induced by TNF-α, and accordingly we put forward our hypothesis that SERPINA3K can inhibit gap junction disruption by maintaining the interaction between ZO-1 and Cx43. To confirm this hypothesis, we will adopt tissue culture and primary cell culture model to observe the changed distribution and expression of Cx43 and ZO-1 induced by TNF-α, analyze the role of Src/Cx43 phosphorylation signaling pathway in regulating the gap junction via inhibition of interaction between ZO-1 and Cx43, preliminary assessment of effect of SERPINA3K on gap junction dysfunction and related mechanisms. This study helps to further understand the pathogenesis of the corneal endothelial keratopathy; moreover, the results will contribute greatly to provide a new method for treatment of corneal endothelial diseases.

Cx43介导的间隙连接在维持角膜内皮细胞的内环境稳态中发挥重要作用。近来我们发现，炎症介质TNF-α通过破坏ZO-1与Cx43的相互作用从而破坏角膜内皮细胞间隙连接。丝氨酸蛋白酶抑制剂SERPINA3K具有抗炎、抗新生血管等功能，然而其能否抑制TNF-α诱导的间隙连接功能障碍尚不明确。前期研究证实，SERPINA3K可以抑制TNF-α诱导ZO-1结构的破坏，因而推测SERPINA3K可通过维持ZO-1与Cx43的相互作用从而抑制间隙连接功能障碍。为证实该假说，本课题拟通过兔角膜内皮组织和原代细胞培养模型，观察TNF-α诱导Cx43分布和表达的变化，分析Src/Cx43磷酸化信号通路调控ZO-1与Cx43相互作用对间隙连接的影响，初步评估SERPINA3K抑制角膜内皮间隙连接功能障碍的作用及相关机制。本研究有助于进一步了解角膜内皮病变的发病机理，为临床角膜内皮疾病的治疗提供新的方法。

Cx43是角膜内皮细胞中分布最广泛和研究最深入的一种间隙连接蛋白，在维持角膜内皮细胞的内环境稳态和新陈代谢中发挥重要作用。肿瘤坏死因子α（TNF-α）被认为是引起角膜内皮功能障碍的最重要的炎症因子之一，然而TNF-α对Cx43介导的角膜内皮细胞间隙连接通道的影响尚不明确。本研究通过兔角膜内皮组织培养模型和原代兔角膜内皮细胞培养模型，采用免疫荧光、Western Blot、CO-IP等技术分别分析不同浓度的TNF-α对角膜内皮细胞间隙连接功能的影响。近来的研究结果证实，高浓度的TNF-α通过降低Cx43与ZO-1的相互作用，破坏Cx43在角膜内皮细胞中的结构分布，下调Cx43的蛋白表达水平，从而破坏兔角膜内皮间隙连接细胞通讯。该项研究有望为临床角膜内皮细胞失代偿的防治提供新的理论依据。丝氨酸蛋白酶抑制剂SERPINA3K具有抗炎抗氧化应激等功能，前期研究已证实SERPINA3K可抑制紧密连接蛋白ZO-1结构的破坏，因而推测其可通过维持Cx43与ZO-1的相互作用从而抑制间隙连接功能障碍，基于此部分的药效和分子机制，目前还在继续深入研究中。