ROS通过ATM/RelA信号通路诱导HMGB1外泌在椎间盘软骨终板退变中的作用机制研究

Chronic low back pain and neck and shoulder pain caused by degenerative intervertebral disc disease have seriously affected people's life. Endplate degeneration caused by apoptosis and calcification of cartilage endplate cells is an important reason of intervertebral disc degeneration. In our previous in vitro studies, we found that the expression of phosphorylated ATM protein increased during the apoptosis and calcification of cartilage endplate cells induced by reactive oxygen species (ROS). In addition, in vivo studies showed that the expression of phosphorylated ATM protein increased in degenerative intervertebral disc, while HMGB1 protein was secreted into the extracellular matrix. However, the specific mechanism between these two protein is unclear. Based on previous studies, we hypothesize that phosphorylation of ATM protein may caused in apoptosis and calcification of cartilage endplate cells through the regulation of HMGB1 exocytosis. In this study, the regulatory role of phosphorylated ATM protein on HMGB1 exocytosis was verified both in vivo and in vitro. Moreover, the relationship between phosphorylated ATM protein with RelA signaling pathway was explored, so as to the detect the explicit mechanism of "ATM-RelA-HMGB1" signal loop in regulating the degeneration of intervertebral disc cartilage endplate. This study will contribute to clarify the molecular mechanism of degenerative intervertebral disc disease and provide new ideas for further exploration of clinical prevention and treatment of intervertebral disc degenerative diseases.

椎间盘退行性变引起的慢性腰腿痛、颈肩痛严重影响了人们的生活，软骨终板细胞凋亡钙化引起的终板退变是椎间盘退变的一个重要原因。我们前期体外研究发现活性氧自由基（ROS）诱导椎间盘软骨终板细胞凋亡钙化的过程中，磷酸化ATM蛋白表达增多；进一步体内研究发现退变的椎间盘中，磷酸化ATM蛋白表达增多的同时伴随着HMGB1蛋白外泌至细胞外基质，但二者之间的具体作用机制尚不清楚。基于前期研究基础，我们推测磷酸化ATM蛋白可能通过调控HMGB1外泌，引发软骨终板细胞凋亡钙化。本研究将从体内、体外两方面验证磷酸化ATM蛋白对HMGB1外泌的调控作用，并探讨与RelA信号通路的关系，最终明确“ATM-RelA-HMGB1”正反馈信号环路调控椎间盘软骨终板退变的具体机制。本研究将有助于阐明椎间盘退行性疾病的分子机制，为进一步探索临床防治椎间盘退行性疾病提供新思路。

随着社会老龄化的加重,越来越多的老年人患有与衰老有关的退变性疾病,如心血管、关节炎、骨质疏松、糖尿病等。椎间盘退变是引起骨骼肌肉系统慢性疾病的一个重要原因,很多原因可以导致椎间盘的退变,衰老是椎间盘退行性病变的一个主要原因。人体的椎间盘是一个无血管的类软骨组织,包含水分、胶原、基质蛋白、以及大量的细胞外基质,其中衰老细胞本身基质合成代谢与分解代谢发生紊乱,进而细胞基质蛋白崩解增多,导致椎间盘老化退变。研究证明,许多内源性以及外源性因素和椎间盘的衰老退变有关,如氧气应激、渗透压应激等,其中,DNA损伤是引起椎间盘衰老退变的一个非常重要的因素。DNA损伤后的自我修复是维持细胞正常生理功能的第一步,如果DNA损伤后的修复功能不足,导致DNA损伤不断积累,最终导致椎间盘细胞的衰老与死亡。研究发现,一些导致DNA损伤的因素如电离刺激、吸烟等均可加速椎间盘的退变过程。. 共济失调突变基因(Ataxia telangiectasia mutated,ATM),是DNA依赖的一种PI-3K蛋白激酶,当DNA双链断裂时可导致ATM的磷酸化激活。在DNA损伤的细胞中,ATM磷酸化增强,磷酸化的ATM基因会对细胞增殖周期产生消极影响,进一步加强DNA的损伤,最后导致细胞衰老凋亡。活性氧自由基(Reactive oxygen species,ROS)可以直接导致细胞 DNA双链断裂,引起ATM的磷酸化,导致细胞的衰老。因此,本研究我们证实髓核细胞内DNA损伤可调控ATM基因磷酸化,减弱细胞基质代谢合成,加速分解,促进髓核细胞的衰老。并证实ATM参与的ROS/ATM细胞通路可调控髓核细胞的衰老情况以及ATM激活后可导致脊柱椎体骨质丢失及椎间盘老化退变。