BRD4调控自噬在糖尿病椎间盘退变中的作用及其机制

Diabetes mellitus is a risk factor for intervertebral disc degeneration (IVDD). Our group has reported that diabetes mellitus may cause IVDD in rats (the results were published on Journal of Orthopaedic Research in 2013), however the mechanism was unclear. In previous studies (published on Cell Death & Disease in 2016 and 2018), we found that autophagy is a protective mechanism against IVDD process. Pathological factors that contribute to diabetes such as oxidative stress, inflammatory cytokines and advanced glycation end products (AGEs) may cause nucleus pulposus cells impairment;They may also lead to increased expression of BRD4 as well as decreased expression of SIRT1，which is a synergistic factor of BRD4; while BRD4 has been reported to be correlated to autophagy. Therefore we hypothesize that increased expression of BRD4 induced by diabetes may inhibit autophagy in nucleus pulposus cells, which may in turn lead to failed protection mechanism of autophagy against cellular damage and further aggravate IVDD in diabetic conditions. We will carry out the following studies to prove our hypothesis. Firstly, we will apply in vitro and in vivo experiments to confirm the high expression of BRD4 in diabetic nucleus pulposus tissues; Secondly, using condition knockout mice and over-expression or knock-down viruses, we regulate the expression of BRD4 in the nucleus pulposus in vivo and in vitro, we aim to detect the role of BRD4 in diabetes IVDD; Thirdly, we will clarify the mechanism from the view of SIRT1/BRD4/autophagy pathway; Finally, we will use BRD4 inhibitor or SIRT1 agonist to treat diabetic IVDD in rats. This study will help to clarify the mechanism of diabetic IVDD, and also will provide new therapeutic targets for the treatment of diabetic IVDD.

糖尿病是椎间盘退变（IVDD）危险因素，课题组在大鼠中证明糖尿病可引发IVDD（J Orthop Res, 2013），但机制不清。自噬是IVDD保护机制（Cell Death Dis, 2016 & 2018）。课题组发现糖尿病病理因素（氧化应激，炎症因子，AGEs等）可导致髓核细胞损伤，预实验显示此类病理因素可促进BRD4并抑制其协同作用蛋白SIRT1表达，文献报道BRD4与自噬相关。由此假说：糖尿病通过BRD4高表达抑制细胞自噬，使髓核细胞损伤保护机制失效引发IVDD。课题拟：1.体内外实验确证糖尿病IVDD髓核中BRD4高表达；2.通过基因鼠及病毒等工具体内外调控髓核中BRD4的表达，检测其对糖尿病IVDD的作用；3.从SIRT1/BRD4调控自噬角度阐释作用机制；4.验证BRD4抑制剂或SIRT1激动剂对糖尿病IVDD治疗效果。本研究阐释糖尿病IVDD新机制，为其治疗提供新靶点。

椎间盘退变（IVDD）会导致腰痛，是世界范围内导致残疾的主要原因。而糖尿病是新发现的椎间盘退变危险因素之一。基质金属蛋白酶-13(MMP-13)是髓核细胞(NPCs)细胞外基质(ECM)的主要分解代谢因子之一，参与了糖尿病IVDD的发生。溴结构域蛋白4（BRD4）是溴结构域和超末端蛋白家族的成员，与慢性炎症有关。在此，我们报道了BRD4和MMP-13的表达在糖尿病髓核组织以及晚期糖基化终产物(AGEs)处理的NPCs中升高。此外，我们还研究了BRD4对MMP-13的调控作用。我们发现MMP-13在AGEs处理的NPCs中受到MAPK和NF-κB信号通路以及自噬的调控。接下来，我们探讨了BRD4在MAPK、NF-κB信号传导和自噬调节中的作用。结果表明，BRD4是这3个因子的上游调节因子，抑制BRD4可能抑制MAPK和NF-κB信号通路，激活AGEs诱导的NPCs自噬。最后，我们证明无论是在体外还是体内抑制BRD4都可以抑制糖尿病NPCs中MMP-13的表达，对糖尿病大鼠ECM起到保护作用。.在此基础上，我们还研究了BRD4协同作用蛋白SIRT1在糖尿病IVDD中的作用及其机制。在目前的研究中，我们发现高糖能够促进体外培养的NPCs凋亡和衰老的发生。同时，在体内糖尿病NP组织和体外高糖刺激下的NPCs中也发现凋亡和衰老的主要转录因子p53的乙酰化增加。SIRT1是一种NAD+依赖性脱乙酰酶，我们发现NP组织中SIRT1的表达降低，而高糖能够抑制NP细胞中SIRT1的表达和活性。此外，我们证明了butein可以抑制P53的乙酰化，并通过SIRT1激活保护NPCs免受高糖诱导的凋亡和衰老，因为SIRT1抑制剂Ex527可以抵消butein在高糖NP细胞中的保护作用。体内实验结果表明，butein能改善糖尿病大鼠的IVDD过程，同时能提高NP组织中SIRT1的表达，降低乙酰化p53的表达。.综上，我们的研究表明，抑制BRD4可能通过抑制MAPK和NF-κB信号传导、激活自噬，从而抑制糖尿病IVDD中MMP-13的表达，并且糖尿病IVDD可能受到BRD4抑制剂的抑制。SIRT1/P53轴也参与了糖尿病IVDD的发病机制，并可作为糖尿病IVDD的潜在治疗靶点。