基于微环境CAFs活化研究温下方调控Hh-Gli1通路抑制肺癌生长侵袭机制

CAFs activation in microenvironment is one of the main mechanisms of tumor cell growth and invasion. Previous studies have suggested that Wenxia Formula could inhibit the proliferation and migration of A549 cells, decrease CAFs activation. This study proposes the scientific hypothesis of “Wenxia Formula inhibits the growth and invasion of lung cancer by regulating the activation of Hh-Gli1 signaling pathway and decreasing the protein expression mediated by CAFs”. In vivo, the subcutaneous co injection transplantation tumor model is established, after the intervention of Wenxia Formula, tumor growth,metastasis and survival period are observed dynamically, the expression of α-SMA and E-cadherin is observed by immunohistochemistry and immunofluorescence, Hh signal pathway, growth and invasion related factor mRNA and protein expression are detected by qRT-PCR and WB to illuminate the mechanism of Wenxia Formula in inhibiting the lung cancer growth and invasion by CAFs activation. In vitro, the direct culture and conditional culture models of CAFs and A549 are established and intervened by Wenxia Formula medicated serum and antagonist, cell migration and invasion are detected, the cell factor content is detected by Liquichip, the expression of Gli1 and α-SMA is detected by immunofluorescence, Hh signal pathway, growth and invasion related factor mRNA and protein expression are detected by qRT-PCR and WB to study the effect of Wenxia Formula on the CAFs activation and to give counterevidence of Wenxia Formula in inhibiting the growth and invasion of lung cancer by regulating Hh pathway.

微环境CAFs活化是介导肿瘤生长侵袭主要机制之一。前期研究表明温下方抑制肺癌细胞增殖迁移，减少CAFs活化。由此提出“温下方干预CAFs活化及其介导的肿瘤细胞Hh-Gli1通路激活，调控相关蛋白表达，抑制肺癌生长侵袭”的科学假说。建立共注射移植瘤模型，动态观察温下方对肿瘤生长侵袭和生存时间影响，免疫组化、免疫荧光观察α-SMA、E-cadherin等表达，qRT-PCR和WB检测Hh通路、生长侵袭相关因子mRNA和蛋白表达，研究温下方调控CAFs活化介导的肿瘤生长侵袭机制；体外建立CAFs和A549直接培养和条件培养模型，温下方含药血清及拮抗剂干预，检测细胞迁移侵袭能力，液相芯片检测细胞因子含量，免疫荧光观察Gli1、α-SMA等表达，qRT-PCR和WB检测Hh通路、生长侵袭相关因子mRNA和蛋白表达。研究温下方对CAFs活化影响，反证其调控Hh通路抑制肺癌生长侵袭机制，阐释科学内涵。

目的：探讨温下方抗肿瘤有效提取部位的物质基础及主要作用机制。方法：系统溶剂萃取法制备石油醚、二氯甲烷、乙酸乙酯、正丁醇四个部位温下方；采用肺癌A549细胞、H1299细胞、H460细胞和H520细胞对四个提取部位和水煎液进行体外抗肿瘤活性筛选。建立CAFs-CM、NFs-CM和温下方有效提取部位干预的条件培养细胞模型，小室实验观察A549细胞侵袭的影响。HPLC法测定温下方不同提取部位主要化学成分的含量。UHPLC-Q-Orbitrap HRMS定性分析温下方有效提取部位化学成分。采用网络药理学和分子对接技术，预测温下方有效提取部位抗肿瘤的作用机制。建立肺癌A549裸鼠皮下移植瘤模型，随机分为A549组、模型组、顺铂组、温下方有效提取部位高中低剂量组和环巴胺组，观察温下方有效提取部位的抗肿瘤作用。采用免疫组化、免疫荧光、western blot和qRT-PCR技术检测裸鼠移植瘤中相关侵袭因子以及Hh-Gli1信号通路相关表达水平，探讨温下方有效提取部位抗肿瘤的作用机制。结果：体外抗肿瘤活性筛选实验发现，四个提取部位和水煎液对4种肺癌细胞的增殖均有抑制作用。其中温下方乙酸乙酯提取部位对4种肺癌细胞的体外抑制作用相比最强。侵袭实验证明温下方乙酸乙酯提取部位对A549细胞体外侵袭有抑制作用。利用UHPLC-Q-Orbitrap HRMS技术，温下方乙酸乙酯提取部位鉴别得到193个化合物。利用网络药理学，预测出温下方乙酸乙酯提取部位活性成分的潜在抗肿瘤靶点和可能的信号通路。分子对接发现，前五位核心靶点均可与多种配体化合物发生分子对接。温下方乙酸乙酯提取部位可以改善移植瘤裸鼠的生理状态；对裸鼠移植瘤的生长和侵袭均有抑制作用（P＜0.05）。温下方乙酸乙酯提取部位可以下调裸鼠移植瘤中α-SMA、FAP、FN、ColⅣ、N-cadherin、Vimentin、MMP2、MMP9蛋白和mRNA表达水平（P＜0.05）；提高E-cadherin、TIMP1蛋白和mRNA表达水平（P＜0.05）；下调Hh-Gli1信号通路中PTCH1、SMO和Gli1的蛋白和mRNA表达水平（P＜0.05）。结论：温下方乙酸乙酯提取部位在一定程度上可以抑制肺癌A549裸鼠移植瘤的生长和侵袭，其作用机制可能与其调控CAFs介导的Hh-Gli1信号通路从而抑制上皮细胞间充质转化、抑制细胞外基质重塑有关。