基于Axin介导的TGFβ和 Wnt信号通路串话探讨OSF癌变及中药干预机制

OSF is a potential malignant disease of oral mucosa. The number of OSF cancer patients is increasing, but the mechanism of OSF carcinogenesis is still unclear. OSF carcinogenesis is closely related to abnormal hyperplasia of oral mucosal epithelium, and Wnt/ beta-catenin is the classical signaling pathway for regulation of epithelial cell proliferation and differentiation. Previous studies have found that the expression of TGFβ1 in the OSF tissue induced by betel nut increased significantly, and abnormal hyperplasia was observed in the epithelium, which suggested that OSF epithelial hyperplasia is related to crosstalk between Wnt and TGFβ signaling pathway. In the early stage of clinical discovery, Danxuankoukang had a good therapeutic effect on OSF. According to the overall pathogenesis of OSF cancer, namely "deficiency, blood stasis, phlegm and toxin”, crosstalk between TGFβ and Wnt signaling pathway could be interfered by modified danxuankoukang. Using cell culture, DNA quantitative analysis, qRT-PCR, gene transfection and silencing, etc, and selecting Axin, which is the key factor of Wnt and TGFβ signaling pathway crosstalking, as the main target of the study, for the first time, this project investigates the mechanism of anti-OSF carcinogenesis in traditional Chinese medicine based on crosstalk between TGFβ and Wnt signaling pathway mediated by Axin, and will provide a new experimental basis for the clinical prevention and treatment of OSF carcinogenesis.

OSF是一种口腔黏膜潜在恶性疾病。OSF癌变患者日渐增多，但目前OSF癌变的机理仍不甚明了。OSF癌变与口腔黏膜上皮异常增生密切相关，Wnt/β-catenin是上皮细胞增殖和分化调控的经典信号通路。前期研究发现，槟榔诱导的大鼠OSF组织TGFβ1表达异常增高，同时上皮可见异常增生现象，推测OSF上皮异常增生与Wnt和TGFβ信号通路串话有关。前期临床发现丹玄口康具有良好的治疗OSF的作用。根据OSF癌变“虚、瘀、痰、毒”总体病机，采用加味丹玄口康干预治疗，将可能干扰TGFβ、Wnt信号通路串话效应。本项目拟选择Wnt和TGFβ信号通路串话的关键因子Axin为研究的主要靶点，采用细胞培养、DNA定量分析、qRT-PCR、基因转染和沉默等技术方法，首次从Axin介导Wnt和TGFβ信号通路串话的角度研究中医药抗OSF癌变的作用机制，将为临床OSF癌变的防治提供新的实验依据。

OSF是一种口腔黏膜潜在恶性疾病。OSF癌变患者日渐增多，但目前OSF癌变的机理仍不甚明了。OSF癌变与口腔黏膜上皮异常增生密切相关，Wnt/β-catenin是上皮细胞增殖和分化调控的经典信号通路。前期研究发现，槟榔诱导的大鼠OSF组织TGFβ1表达异常增高，同时上皮可见异常增生现象，推测OSF上皮异常增生与Wnt和TGFβ信号通路串话有关。前期临床发现丹玄口康具有良好的治疗OSF的作用。根据OSF癌变“虚、瘀、痰、毒”总体病机，采用加味丹玄口康干预治疗，将可能干扰TGFβ、Wn t信号通路串话效应。本项目拟选择Wnt和TGFβ信号通路串话的关键因子Axin为研究的主要靶点，采用ELISA、免疫荧光、Westernblot、qRT-PCR、细胞平板克隆、流式细胞分析、基因转染和沉默等技术方法，结果发现：加味丹玄口康可通过作用于Wnt/β-catenin信号通路，调控TGF-β1、IL-6、GSK-3β、β-连环蛋白的表达，改善口腔黏膜下纤维化患者临床症状，缓解机体炎症，减轻疼痛，疗效显著；加味丹玄口康可有效改善OSF模型大鼠临床症状，通过调控Wnt1、β-catenin、CylinD1、Axin基因及蛋白表达，抑制大鼠口腔黏膜纤维化，逆转OSF大鼠癌前病变的形成；Axin1干扰口腔上皮细胞增殖活性和β-catenin、PCNA、Bcl-2蛋白表达均显著升高，Axin1、Bax、cleavedCaspase-3蛋白和Axin1mRNA的表达均显著降低，Axin1过表达口腔上皮细胞处于G1期的细胞比例、细胞增殖活性和β-catenin、PCNA、Bcl-2蛋白表达均显著降低，处于S期的细胞比例和Axin1、Bax、cleavedCaspase-3蛋白及Axin1mRNA的表达均显著升高，加味丹玄口康通过Axin1调节口腔上皮损伤细胞增殖活性。该研究初步阐明了加味丹玄口康通过调控Axin介导TGFβ、Wnt信号通路串话、拮抗口腔黏膜上皮异常增生而发挥抗OSF癌变的作用机制，为开发和应用中医药防治OSF癌变奠定理论和实践基础。