痤疮丙酸杆菌诱导髓核细胞焦亡在椎间盘退变中的作用及其机制研究

In recent years, multiple studies have reported low-virulent bacteria which is mainly Propionibacterium acnes (P. acnes) in disc material suggesting that such infection may contribute to disc degeneration. However, until now, there is still no pathophysiologic mechanism to explain why and how bacteria induces disc degeneration. In our previous study, we also found the presence of low-grade infection in patients undergoing surgery for disc degeneration by microbiological culturing and found that infection may play a role in the pathophysiology of Modic Changes (MCs). More recently, we found that bacteria could induce the nucleus pulposus cells death. Moreover, low-grade bacteria could significantly induce expression of caspase-1, GSDMD, IL-1β and IL-18 by nucleus pulposus cells via NLRP3. We hypothesized that low toxic bacteria may induce intervertebral disc degeneration by inducing nucleus pulposus cell pyroptosis. In this study, we will further explore the roles of low-virulence bacteria in nucleus pulposus cells pyroptosis during disc degeneration and the underlying mechanisms. We will test and verify the pyroptosis of nucleus pulposus cells caused by bacteria via morphological detection, tunel staining and western blot analysis in vivo and vitro for the first time. Then we will explore the underlying mechanism of pyroptosis that whether it is correlated with NLRP3/Caspase-1/GSDMD pathway or not by siRNA. At last, we will explore the correlation between the low toxicity infection and the prognosis of patients. Overall, it is the first time to elucidate that low toxic bacteria may induce pyroptosis of nucleus pulposus cells in degenerative discs, which will provide a new way for clinical treatment of intervertebral disc degeneration.

近来研究表明椎间盘退变与低毒性细菌感染有关，其中最常见的为痤疮丙酸杆菌（P.acnes）。我们新近在退变的椎间盘组织中培养出P.acnes且发现感染标本中髓核细胞坏死率增高。P.acnes感染髓核细胞后焦亡相关蛋白Caspase-1和GSDMD表达增强，炎症因子分泌增多，抑制NLRP3表达后焦亡蛋白表达减弱且炎症因子分泌减少。综上，我们推测P.acnes可能通过NLRP3/Caspase-1/GSDMD通路诱导髓核细胞焦亡导致椎间盘退变。本项目拟首先明确隐性感染在国人椎间盘退变性疾病中的发生率以及与患者预后相关性；通过形态学检测、分子标记等方法明确P.acnes可以导致髓核细胞焦亡；最后围绕NLRP3/Caspase-1通路研究P.acnes诱导髓核细胞焦亡的机制。本项目首次研究P.acnes通过诱导髓核细胞焦亡参与椎间盘退变，揭示感染导致椎间盘退变的新机制，为临床治疗提供新思路。

越来越多的文献研究证实退变的椎间盘内存在痤疮丙酸杆菌（P. acnes）的低毒性感染，但其发病率以及对椎间盘退变的作用机制如何，目前尚未阐明。在本研究中，我们首先检测了P. acnes在椎间盘退变患者中的感染率并统计分析P. acnes感染和椎间盘退变的相关性；然后通过细胞和动物实验，深入研究了P. acnes是否能够诱导髓核细胞发生经典通路细胞焦亡，髓核细胞的焦亡能否进一步引起椎间盘退变；最后通过ROS抑制剂和拮抗剂进一步探讨了P. acnes是否通过ROS/NLRP3通路激活了经典通路的髓核细胞焦亡。研究结果显示在80例退变的人椎间盘组织中，排除可能污染的标本后，有23例标本细菌培养阳性（28.7%），其中P. acnes感染20例（25%）。椎体终板退变Modic changes (MCs)和P. acnes的隐性感染密切相关（P<0.001）。在体外实验中髓核细胞和P. acnes共培养后，焦亡关键蛋白表达明显增加；焦亡产物的表达也明显增高，且呈现时间依赖性和浓度依赖性。此外，Trans well小室实验进一步验证了P. acnes感染的髓核细胞可以导致正常的髓核细胞炎症因子和分解代谢酶基因的表达增高。大鼠尾椎间盘注射P. acnes后，鼠椎间盘组织中焦亡相关蛋白表达明显增加，细胞外基质II型胶原和蛋白多糖的表达明显降低；小动物磁共振和组织染色进一步验证了感染的椎间盘发现明显退变；抑制细胞焦亡或清除焦亡产物可以缓解鼠椎间盘退变。最后，本项目通过Western blot和流式细胞检测进一步发现，感染的髓核细胞ROS和NLRP3的表达显著增高且具有相关性, 抑制ROS后NLRP3的表达降低，髓核细胞的焦亡率降低；抑制NLRP3的活性后髓核细胞焦亡率也明显降低。总而言之，本项目再次明确退变的椎间盘组织中存在低毒菌的隐性感染，首次研究P. acnes通过ROS/NLRP3信号通路诱导髓核细胞焦亡参与椎间盘退变，揭示P. acnes导致椎间盘退变的新机制，为临床治疗椎间盘退变提供新的思路。