高渗微环境中TonEBP调控自噬在椎间盘退变的作用及机制研究

TonEBP, responds to hypertonicity as part of a cell-protective mechanism, but little is known how TonEBP serves as an important transcription factor for intervertebral disc to survive in hyperosmotic microenvironment. Previous studies found a high level of basal autophagy occur in NP cells, meanwhile, an accumulation of TonEBP with increasing osmolarity. Importantly however, autophagy in NP cells is irresponsive to changes in osmolarity. When treated with Bafilomycin A1 to block the flux, ULK1 is activated and the accumulation of autophagy related proteins between different osmotic mediums change, indicating absence of active autophagic flux. So we hypothesize that hyperosmotic stress not only induce the autophaogosomes formation, but also meanwhile increase autophagic flux to maintain the baseline of autophagy through TonEBP/ULK1 signaling pathway. To prove the above hypothesis, mCherry-EGFP-LC3B transduction were used to detect the autophagosomes and autophagolysosomes to dynamic detection of autophagy; molecular biology techniques were used to detect the activity of mTOR/ ULK1 and autophagy related proteins when stable knockdown of TonEBP to investigate the effect of TonEBP on autophagy. Our findings may demonstrate the mechanism of TonEBP involves in the regulation of autophagy under hyperosmotic condition, and may provide a basis for future research to develop effective and safe therapy for intervertebral disc degeneration.

TonEBP是椎间盘适应高渗微环境的重要转录因子，但其作用机制未明。预实验发现椎间盘中存在较高水平的自噬，且高渗能诱导髓核细胞内TonEBP的表达。有趣的是，高渗对椎间盘自噬水平的影响不大，而加入TonEBP抑制剂及Baf A1处理后，ULK1信号通路被激活、自噬相关蛋白发生变化，提示高渗可能通过TonEBP/ULK1调控自噬。鉴于此，我们提出以下科学假设：高渗诱导TonEBP，通过ULK1，促进自噬形成的同时加速其降解，从而维持髓核细胞内自噬水平的稳态，以利细胞适应高渗环境。为此，本研究拟利用mCherry-EGFP-LC3慢病毒转染髓核细胞，动态观察高渗对自噬潮的影响；检测TonEBP敲除后mTOR/ULK1及自噬相关蛋白的变化，探讨TonEBP在高渗调控自噬中的作用。本研究有望揭示TonEBP参与高渗环境下椎间盘存活的机制，为TonEBP作为椎间盘退变治疗的一种新的选择提供理论依据。

椎间盘退变是一系列脊柱退行性疾病前提和基础病理过程，其引起颈腰痛及神经症状，影响人们正常生活的同时给个人和社会造成了巨大的经济负担。因此对椎间盘退变进行各方面研究以能有效延缓其退变、对抗其损害是非常重要的。研究表明椎间盘髓核细胞高渗微环境中自噬的调控可能成为延缓椎间盘退变的核心策略，而TonEBP正是椎间盘适应高渗微环境的重要转录因子，但其作用机制未明。本研究发现椎间盘中存在较高水平的自噬，且高渗能诱导髓核细胞内TonEBP的表达。有趣的是，高渗对椎间盘自噬水平的影响不大，而加入TonEBP抑制剂及Baf A1处理后，ULK1信号通路被激活、自噬相关蛋白发生变化，提示高渗可能通过TonEBP/ULK1调控自噬。鉴于此，我们进一步利用Tandem mCherry-EGFP-LC3慢病毒转染髓核细胞，应用免疫荧光标记对自噬小体、自噬溶酶体进行追踪定位，动态检测自噬潮，发现Tandem mCherry-EGFP-LC3转染显示高渗微环境下髓核细胞LC3绿色/黄色、红色荧光无明显变化，Baf A1处理后高渗环境下绿色/黄色、红色荧光数量增加；通过构建Sh-TonEBP慢病毒载体敲除TonEBP后高渗作用下自噬相关蛋白SQSTM1水平上升，同时自噬上游信号通路mTOR/ULK-1ser757发生变化，表明高渗可能通过影响自噬潮，在促进自噬形成的同时加速其降解，从而维持髓核细胞内自噬水平的稳态；高渗对自噬稳态的调节可能通过TonEBP/mTOR/ULK-1ser757信号通路。本研究揭示了TonEBP参与高渗环境下椎间盘存活的可能机制，为TonEBP作为椎间盘退变治疗的一种新的选择提供理论依据。