丹参酮协同三氧化二砷瘀毒同治调控巨噬细胞极化改善肝癌炎症微环境的机制研究

Despite the recent advance in diagnosis and treatment of Hepatocellular carcinoma,it remains a highly lethal disease due to recurrence of metastasis.Tumor-associated macrophages (TAMs) are a major component of leukocyte infiltrate of tumors and play a pivotal role in Hepatocellular carcinoma progression.The polarization of macrophages into tumor-suppressive M1 or tumor-promoting M2 types is a fundamental event in the establishment of the tumor microenvironment.TAMs acquire an M2-polarized phenotype and promote angiogenesis,metastasis,and suppression of adaptive immunity through the expression of cytokines,chemokines,growth factors and matrix metalloproteases.TAMs are key regulators of the link between inflammatory microenvironment and HCC cells.Redirecting tumor-associated macrophages to become tumoricidal effectors and improving the inflammatory microenvironment exhibit novel strategy for HCC therapy and prevention.Qi-stagnation and blood stasis,stasis toxin interties is a key pathogenesis for HCC development and progression,thus dispersing blood stasis and detoxifying would be the main treatment for HCC according to the basic theory of traditional Chinese medicine. Danshen is a representative TCM for promoting blood circulation to remove blood stasis,and arsenic trioxide is a representative TCM use of poisons as antidotes.Our previous studies demonstrated that the association of Tanshinone and Arsenic trioxide showed obvious synergistic effect toward Hepatocellular carcinoma.In the present project,we would systematically investigate the effect of Tanshinone and Arsenic trioxide on the hypoxic tumor microenvironment,polarization of tumor-associated macrophages,synthesis and secretion of inflammatory factors,and the activities of NF-κB,STAT-3,and HIF-1α signaling pathways.This study would also confirm the regulatory targets of the two drugs combination and illustrate its specific mechanisms.This work would enrich the scientific connotation of TCM on malignant tumor therapy and provide directions for TCM combination development target inflammatory tumor microenvironment.

肿瘤相关巨噬细胞(TAMs)介导的炎症微环境与肝癌的发生发展密切相关，调控TAMs的极化，阻断M2型TAMs对肿瘤组织的趋化浸润，减少其在微环境中数量和比例，可有效改善肝癌炎症微环境，提高患者预后生存质量。“瘀毒互结”是肝癌发生发展的关键病机，活血化瘀、以毒攻毒，瘀毒同治是中医治疗肝癌的基本大法。前期研究表明，丹参酮联合三氧化二砷存在显著的协同抗肝癌作用，初步证实了瘀毒同治治法的科学有效性。本项目以活血化瘀药丹参酮和以毒攻毒药三氧化二砷为研究对象，采用多种实验方法，体内外评价对肝癌缺氧环境、TAMs极化、炎症因子合成分泌和HIF-1α/STAT3/NF-κB通路活性的影响，明确丹参酮和三氧化二砷对TAMs、炎症微环境和肝癌细胞侵袭转移的影响和调控的靶点，阐明其调控的分子机制。项目的完成将丰富中医药防治恶性肿瘤的科学内涵，为联合用药瘀毒同治靶向炎症微环境抗肿瘤提供新的理论依据和数据支撑。

肝癌是严重危害人类健康的疾病之一，寻求积极有效的中西医治疗方案，是现阶段临床及科研工作者亟待解决的关键问题。三氧化二砷是中药砒霜的主要成分，鉴于其单独治疗肝癌疗效有限，三氧化二砷联合其他药物以实现增效减毒已是当前抗肝癌研究的热点。丹参酮为丹参的有效组分，前期研究发现丹参酮和三氧化二砷联合给药，能显著抑制肝癌细胞增值，促进凋亡。本项目拟通过体内外实验，进一步明确丹参酮协同三氧化二砷抗肝癌的起效基础，同时从巨噬细胞极化角度就其抗肝癌的机制开展研究。.通过体外细胞实验，明确了隐丹参酮是联合三氧化二砷治疗肝癌的有效成分，能抑制4株人源肝癌细胞株（7402、7721、Huh-7、HepG2）和2株鼠源肝癌细胞株（Hepa1-6、H22）的增殖，同时对小鼠正常巨噬细胞影响较小，二者最佳配比为1：10（三氧化二砷：隐丹参酮）。进一步的体外研究结果表明，二者联合使用可以显著抑制肝癌细胞的迁移，促进其凋亡，调控Bcl-2、Bax、p53、survivin等相关基因的表达。体内研究结果显示，二者联用可以抑制H22和HepG2肝癌移植瘤的生长，改善三氧化二砷造成的血象变化，促进肿瘤细胞的凋亡，抑制增殖和EMT。.使用隐丹参酮和三氧化二砷对Raw264.7和原代巨噬细胞进行干预发现，隐丹参酮能有效的促进M1型巨噬细胞极化，抑制M2型巨噬细胞极化。三氧化二砷可以抑制M2型巨噬细胞的极化，并特异性靶向M2型巨噬细胞，造成其死亡。二者联用能显著的上调M1型巨噬细胞的比例，下调M2型巨噬细胞的比例，提高巨噬细胞的吞噬能力，并影响炎症因子的水平调控肿瘤微环境。.机制研究结果表明，隐丹参酮和三氧化二砷联用显著提高了血清乳酸水平，降低肿瘤葡萄糖的摄取，抑制糖酵解。二者通过抑制HIF-1α/NF-κB通路，激活AMPK信号通路，抑制IL-6/STAT3通路，调控巨噬细胞极化，阻断EMT，发挥抗肝癌作用。.本项目的完成可助力抗肿瘤中药新药的研发，揭示瘀毒理论的科学内涵，为中医药抗肿瘤提供理论和数据支撑。