成纤维细胞P2Y2受体激活的信号通路对失神经支配喉肌纤维化的作用

Long-term denervation-induced muscular atrophy and fibrosis contributed to unsatisfied function recovery of laryngeal muscles following reinnervation, in which excessive activation or dysregulation of fibroblasts is the key cellular event. Emerging evidences indicated that purinergic receptor P2Y2 may be involved in the fibrotic process of several organs following injury, and it possibly mediated various signaling pathways to differentially controlled cellular activities in different cell types, which implicating the complexity of P2Y2 signaling networks. In our previous study, upregulation of P2Y2 receptor was detected in denervated human laryngeal muscles and activated NIH-3T3 fibroblast cell line as well. Inhibition of P2Y2 receptor of 3T3 cell in vitro significantly downregulated the transcript level of fibrosis-related genes, such TGF, CTGF, alpha-SMA and collagen I. Thus, we hypothesize that P2Y2 signaling may mediate activation of fibroblasts or other cellular events which promote the fibrotic process in denervated laryngeal muscles. However, the mechanism of P2Y2 signaling networks regulating skeletal muscle fibroblasts remains unclear. For this purpose, P2Y2 knock-down and specific inhibitor in cultured 3T3 fibroblasts are used to classify the definite effect of P2Y2 on fibroblasts activities, including survival, proliferation, migration, differentiation and secretion. Signaling Pathway-Finder Microarray is then adopted to analyze the potential upstream signal factors of P2Y2 combined with published online database. Then high-throughput RNAi screening using lentivirus-bases vectors is performed to investigate P2Y2 signaling networks regulating the activities of fibroblasts. And the pivotal cascades of P2Y2 signaling are validated by constituted activation and inactivation of signaling components. Finally, the effect of P2Y2 on the denervation-induced fibrosis in laryngeal muscles is demonstrated in vivo using a recurrent laryngeal nerve injury animal model with P2Y2 knock-out and wild-type mice. The present study aims at exploring the role of P2Y2 signaling networks in the fibrotic process of denervated laryngeal muscles, which is expected to highlight the regulatory effect of P2Y2 receptor in the fibrotic process, and to provide a novel and potential therapeutic strategy for skeletal muscle diseases in clinic.

长期失神经支配喉肌的萎缩纤维化导致喉返神经自发性再生或神经修复后喉功能恢复不完全。申请人研究发现失神经支配人喉肌成纤维细胞的嘌呤能受体P2Y2受体表达上调，阻断成纤维细胞P2Y2受体能下调纤维化相关因子的表达。根据理论分析和前期工作基础，我们提出假设，失神经支配喉肌的成纤维细胞P2Y2受体表达上调，通过其下游信号转导网络调控成纤维细胞激活分泌过量细胞外基质，导致肌肉组织发生不可逆性纤维化。本课题拟采用信号通路芯片、功能性RNAi文库的高通量筛选和组成型激活/失活技术，明确P2Y2受体调控喉肌成纤维细胞激活的作用和信号转导网络及关键通路；利用基因敲除小鼠模型，探讨P2Y2受体对失神经支配喉肌纤维化的作用及可能机制。从分子、细胞和组织水平，阐明P2Y2受体调控成纤维细胞激活的信号转导机制及对失神经支配喉肌纤维化的作用。为更好地延缓失神经支配喉肌的纤维化，寻找有潜力的药物作用靶点提供理论依据。

成纤维细胞在失神经支配骨骼肌萎缩纤维化进程中发挥重要的调控作用，课题组前期工作发现失神经支配喉肌成纤维细胞的嘌呤能受体P2Y2受体表达上调，阻断成纤维细胞P2Y2受体能下调纤维化相关因子的表达。因此提出假设， P2Y2受体及其下游信号转导通路调控成纤维细胞激活参与失神经支配骨骼肌萎缩纤维化。本课题首先成功构建了P2Y2 受体基因敲除小鼠，并完成了基因敲除（KO组）和野生型（WT组）小鼠成纤维细胞的分离培养，构建了P2Y2 受体过表达慢病毒并转染KO组成纤维细胞。Transwell迁移实验证实P2Y2受体对成纤维细胞迁移有促进作用；免疫组化、定量PCR和WB证实P2Y2受体对成纤维细胞分化有促进作用，可上调α-SMA、I型胶原和CTGF的表达水平，同时可检测到可提高p-AKT、p-ERK1/2、p-PKC的表达水平。为明确这些信号分子的作用，采用AKT、ERK和PKC抑制剂同时处理，均可降低α-SMA、I型胶原和CTGF的表达水平，其中ERK1/2抑制剂的效应最为显著；AKT和ERK抑制剂减弱P2Y2促细胞迁移的效应最为显著，上述结果提示了P2Y2受体的激活可能主要通过AKT和ERK1/2信号通路参与调节成纤维细胞的迁移和分化。构建的坐骨神经切断结扎伤模型的结果显示同一时间点的KO组小鼠腓肠肌的萎缩纤维化程度较WT组要轻微，提示了下调P2Y2受体的表达可以减缓失神经支配诱导的骨骼肌纤维化进程。综上所述，本课题从分子、细胞和组织水平，阐明P2Y2受体主要是通过激活AKT和ERK1/2信号通路调控成纤维细胞分化和迁移能力，参与失神经支配的骨骼肌萎缩纤维化进程。其作为一个潜在的作用靶点，有望为将来促进骨骼肌修复和功能重建提供可靠的理论和实验基础。