丹参二萜醌活血祛瘀抗肺癌作用及机制研究

Tumor inflammatory microenviroment is the key factor for development, invasion and metastasis of lung cancer. As reports shown, diterpenoid tanshinone (DT), including hydroxytanshinone IIA and crytotanshinone, has good effects on inhabiting tumor cell proliferation, inducing carcinoma cell apoptosis, inhibitatingtumor invasion and metastasis and tumor angiogenesis. It has discovered in ourprevious researches that diterpenoid tanshinone (DT) can inhibit the proliferation of lung cancer cell PC9, up-regulate expression of apoptosis associated protein, such as p-eIF2α and Caspase-12, which mediated by endocytoplasmic reticulum stress reaction, promote PC9 apoptosis and showing a good effect on anti lung carcinoma. According to the effects of DT on anti inflammation, down regulating chemotaxis wandering ability of monocyte and macrophage activiation,reducing immunoogic injury, we deduced that DT could cure lung cancer by activate apoptosis pathway mediated by ERS to promote lung cancer cells’ apoptosis, by regulating tumor inflammatory microenviroment and reducing cancer cells’ infiltration and metastasis. Techologies such as Wstern blot, RNA interference and pharmacology means and lung cancer xenograft animal model and cell models were carried out to confirmation the effect of DT on anti lung cancer and to illuminate the mechanisms of DT on promoting tumor cell apoptosis, infiltration and infiltration. The results of this work would greatly promote the development of DT in application of anti lung cancer treatment and endow new meaning of “Promoting blood circulation and removing blood stasis method” of traditional Chinese medicine on anti tumor therapy.

肿瘤炎症微环境是促进肺癌成瘤、侵袭转移和化疗等治疗后肺癌复发的重要原因。已知丹参酮IIA等丹参二萜醌（DT）成分具有抑制肿瘤细胞增殖、促进肿瘤细胞凋亡分化、抑制肿瘤细胞浸润转移和肿瘤血管新生等作用。前期研究表明，DT体外能显著抑制肺癌细胞增殖，上调ERS相关凋亡通路p-eIF2α、Caspase-12等蛋白表达促进肺癌细胞凋亡，显示了良好的抗肺癌潜能。结合丹参酮IIA等成分抗炎及抑制人白细胞游走趋化和巨噬细胞活化、降低血清免疫复合物、减轻免疫损伤等免疫调节作用，推测DT可通过激活肺癌细胞ERS介导的凋亡通路促进肺癌细胞凋亡以及调节肿瘤炎症微环境、减少肺癌细胞浸润侵袭而有效治疗肺癌。拟采用免疫印迹、RNA干扰和药理学手段等，通过肺癌移植瘤动物模型和体外细胞模型，明确DT抗肺癌作用，阐明DT促进肺癌细胞凋亡，抑制肺癌侵袭转移的作用机制，以促进DT的开发利用，发展中医药活血祛瘀抗肿瘤理论。

当前我国肺癌病死率已位居恶性肿瘤首位，是我国癌症防治的重点。选择有效的药物阻止肿瘤的复发和转移，已是当前肺癌治疗亟需解决的问题。丹参二萜醌（DT）为丹参脂溶性活性部位，前期研究发现，DT在体外对多种肺癌细胞均具有良好的抑制增殖和诱导凋亡作用，但体内抗肺癌作用及其机制未明。本项目拟在前期基础上，通过肺癌移植瘤动物模型及体外细胞模型，采用现代药理学方法及MoorFLPI激光多普勒、ELISA、免疫组化、流式细胞术、免疫印迹、RNA干扰技术等，进一步明确DT抗肺癌作用，并从内质网应激（ERS）介导的凋亡通路阐释其可能的作用机制。.整体动物实验结果显示，60 mg/kg DT能抑制体内肺癌移植瘤增长，能浓度依赖地改善荷瘤裸鼠外周微循环血流，降低荷瘤模型动物血清TNF-a、IL-6水平，抑制PC9肺癌移植瘤组织c-JUN和COX-2蛋白表达，改善肿瘤炎症微环境； 30 mg•kg-1、60 mg•kg-1 DT能显著增加肿瘤组织中肿瘤细胞凋亡率，增加肿瘤组织中Bip、IRE1a和TRAF2蛋白表达，提示DT可通过促进ERS凋亡途径介导肿瘤细胞凋亡抑制移植瘤生长。体外细胞实验结果显示，DT能浓度依赖地抑制PC9细胞增殖（作用24h的IC50为7.392±0.381μg/ml），破坏肿瘤细胞线粒体结构，诱导肿瘤细胞凋亡；可上调PC9细胞Bax及ERS凋亡途径相关Bag1、Bip、TRAR2、IRE1ɑ、Caspase-12、Claved-caspase9、P-PERK、P-EIF2a及CHOP蛋白的表达，下调Bcl-2、ATF4蛋白表达。提示DT诱导肺癌细胞凋亡的作用与其活化肺癌PC9细胞内质网应激凋亡通路IRE1α及PERK信号通路有关。此外，DT能破坏肺癌细胞A549细胞骨架，降低A549及人脐静脉内皮细胞的粘附率，降低HUVECs细胞成管能力及A549细胞粘附相关蛋白mRNA的表达，抑制细胞侵袭转移。.上述结果显示，DT具有明确的抗肺癌作用，该作用与调节ERS凋亡通路促进肺癌细胞凋亡，抑制炎症反应改善肿瘤炎症微环境，阻断肺癌侵袭转移有关。本项目的完成可进一步拓宽丹参及其成分的抗肿瘤应用范围，促进丹参二萜醌类成分的开发应用及肺癌特色治疗药物的研发；同时可创新中医药抗肿瘤理论，赋予中医药活血祛瘀法治疗恶性肿瘤的作用内涵，促进中医药在肿瘤治疗中发挥更大的作用。