针刺通过SIRT1介导的LC3去乙酰化修饰调控自噬干预“炎-癌”转化及其表观遗传学机制研究

Inflammation and tumor have close link that called "inflammation-tumor" transformation. SIRT1 and autophagy play an important role in the pathogenesis of inflammation-tumor-related diseases and SIRT1 could also regulate autophagy, which becomes an intervention target of "inflammation-tumor" transformation. According to our previous research, we confirmed that acupuncture activates SIRT1 to regulate downstream inflammatory factors to improve the inflammatory state and established an objective "inflammation-tumor" transformation model successfully. The purpose of this study was to investigate the effect of acupuncture on AOM / DSS "inflammation-tumor" transformation model mice, combined with SIRT1 agonists and inhibitors as control, and observed the SIRT1-mediated LC3 deacetylation regulation of autpphagy in various pathological stages of "inflammation-tumor" transformation. Trying to evaluate the potential mechanism of acupuncture’s intervention in "inflammation-tumor" transformation. Chromatin immunoprecipitation(ChIP) assay and ChIP-seq technique will be applied to investigate the effect of acupuncture activating SIRT1 to the acetylation of NFkB, AP-1, P53 gene’s promoter and histone modification in global DNA profiling. The whole reserch looking for possible epigenetic intervention and treatment targets which will provide the experimental basis for acupuncture’s intervention in "inflammation-tumor" transformation and related diseases.

炎症与肿瘤存在密切联系即“炎-癌”转化。SIRT1与自噬在炎症-肿瘤相关疾病的病理机制中作用关键且SIRT1对自噬发挥重要调控作用，成为潜在干预靶点。前期工作证实针刺通过SIRT1调控下游炎症因子活性改善炎症状态，并建立客观的“炎-癌”转化模型。本项目拟采用针刺干预AOM/DSS“炎-癌”转化模型小鼠，结合SIRT1激动剂及抑制剂，在“炎-癌”转化各阶段动态观测SIRT1介导的LC3去乙酰化修饰对自噬的调控作用并评价针刺的干预效应，探讨针刺通过激活SIRT1调控自噬干预“炎-癌”转化的潜在机制。利用ChIP、ChIP-seq技术，分析检测SIRT1对“炎-癌”转化关键因子NF-kB、AP-1、P53基因启动子区域去乙酰化修饰位点，鉴定分析针刺对转录因子乙酰化修饰的调控机制；并对全基因组蛋白乙酰化分析，寻找可能的表观遗传学干预及治疗靶点。以上研究结果将为针刺干预“炎-癌”转化提供实验依据。

结合课题组前期研究结果：“标本配穴”针刺减少肥胖相关的炎性病理改变，促进SIRT1介导的抗炎通路途径改善胰岛素抵抗肥胖及SIRT1去乙酰化修饰NF-κB，减少炎症因子的转录。本课题在此基础上，进一步探索针刺通过SIRT1介导的LC3去乙酰化修饰调控自噬干预“炎-癌”转化及其表观遗传学机制研究，进行针刺及其诱导的细胞自噬在肠道“炎-癌”转化中的作用机制探究。本研究在应用AOM+DSS联合应用构建小鼠结直肠“炎-癌”转化模型基础上，针刺足（后）三里、丰隆作为治疗手段，标本配穴电针治疗后动态检测小鼠的DAI评分、组织病理改变、组织蛋白和分子检测，同时在体外实验中通过蛋白和分子水平检测证实机制水平的研究完整性。结果显示：1.针刺干预肠道“炎-癌”转化进展，抑制肠道和外周血中炎症因子（TNF-α、IL-6等）、肠道肿瘤分子表达，改善小鼠肠道DAI评分，动态延缓肠道炎症-癌前病变-癌症的组织病理病变；2.针刺促进肠道炎症和癌症组织细胞发生自噬改变，进而抑制肠道炎症改变，同时抑制癌变的进展，且证实上述变化与针刺促进SIRT1高表达直接相关；3. 针刺抑制炎性结肠癌变主要通过SIRT1作用于下游的转录因子、micRNA及直接作用于自噬相关分子LC3直接或间接干预细胞自噬过程，在结肠中自噬抑制“炎-癌”转化过程的发生和发展。4.针刺促进SIRT1表达增高，直接改变LC3的乙酰化和去乙酰化平衡，即促进其表观遗传发生改变，另外本研究证实SIRT1可以改变下游转录因子FOXQ1和micRNA的转录起始区域组蛋白的乙酰化状态间接促进LC3改变，促进自噬发生，抑制炎症，抑制肿瘤，从而实现抑制肠道“炎-癌”转化进展的目的。该项目“标本配穴”针刺可通过促进SIRT1高表达促进LC3及其表观遗传改变从而抑制肠道组织“炎-癌”转化进展，为针刺在改善肠道炎症和抑制肠道“炎-癌”转化提供了实验证据。研究结果成为临床应用针刺防治肠道组织“炎-癌”转化及其相关疾病提供新的科学依据。