基于调控巨噬细胞极化的恰玛古多糖抗肿瘤分子机制研究

Brassica rapa L., an edible and medicinal plant, has been traditionally used as a Uyghur folk medicine over the long history. It had been reported that Brassica rapa L. in combination with chemotherapy can improve survival rate of cancer patients in Xinjiang Uygur Autonomous Region. However, its molecular mechanism is still unclear. We had isolated polysaccharide from Brassica rapa L. in our previous research, named as BRP (Brassica rapa polysaccharide). Compared with Brassica rapa L., BRP can more strongly stimulate macrophage phagocytosis and activate NF-κB (nuclear transcription factor κB) signal pathway. This indicated M1 like macrophages polarized with BRP. At present, it is very promising strategy for anti-tumor through promoting M1-like polarization or inhibiting M2-like polarization of tumor-associated macrophages. So in this project, we will systematically study the molecular mechanism of BRP regulate polarization of macrophages. This is realized by detecting transcription and secretion level of IL-12, TNFα, iNOS, ArgI, and TGF-β, through testing PI3Kγ-Akt-mTOR-NF-κB-C/EBPβ signal pathway and analyzing polarization types of macrophage treated with BRP. We will further determine anti-tumor effect of BRP in vivo 4T1 (murine breast cancer cell) and LLC (murine lung cancer cell) xenograft, and analyze the relationship between anti-tumor effect of BRP and polarization of macrophages in solid tumors. This research will promote clinical application of BRP and innovative medicines in minority regions, which has important and profound value.

恰玛古在维吾尔族医药中有着悠久的应用历史，是一种传统的药食同源植物。恰玛古作为药用食物结合化疗提高肿瘤患者生存率在新疆已有临床报道，但作用机制不明。前期研究从恰玛古中分离得到其多糖组分（BRP），与恰玛古相比，能够更强的提高巨噬细胞吞噬能力并活化NF-κB信号通路，使巨噬细胞呈现典型M1极化特征。促进肿瘤相关巨噬细胞M1极化或抑制其M2极化是当前极具有应用前景的抗肿瘤新策略，因此本项目拟通过极化标志IL-12、TNFα、iNOS、ArgI和TGF-β等转录和分泌水平检测、PI3Kγ-Akt-mTOR-NF-κB-C/EBPβ信号通路分析、结合巨噬细胞极化状态的流式确证，系统阐述BRP调控巨噬细胞极化的分子机制。在乳腺癌和肺癌小鼠移植瘤中深入研究BRP的抗肿瘤作用及其与调控巨噬细胞极化之间相关性，如能证实BRP抗肿瘤作用及分子机制，对推动其进一步应用及民族地区药物创新具有重要而深远的意义。

恰玛古在维吾尔族医药中有着悠久的应用历史，是一种传统的药食同源植物。恰玛古作为药用食物结合化疗提高肿瘤患者生存率在新疆已有临床报道，但作用机制不明。前期研究从恰玛古中分离得到其多糖组分（BRP），与恰玛古相比，能够更强的提高巨噬细胞吞噬能力并活化NF-κB信号通路，使巨噬细胞呈现典型M1极化特征。促进肿瘤相关巨噬细胞M1极化或抑制其M2极化是当前极具有应用前景的抗肿瘤新策略。基于该研究基础，本项目阐述BRP调控巨噬细胞极化的分子机制，深入研究BRP的抗肿瘤作用及其与调控巨噬细胞极化之间相关性。我们首先在体外实验中发现恰玛古多糖具有调控巨噬细胞极化表型转化的作用，通过调节RAW264.7小鼠巨噬细胞促进M1型标志物表达，降低M2标志物的表达，说明恰玛古多糖可通过调节巨噬细胞由M2型向M1型转化来增加体内免疫。将巨噬细胞和肿瘤细胞共培养，给予恰玛古多糖处理可以显著降低癌细胞的增殖和活力。随后我们构建了LLC荷瘤小鼠模型，以CSF-1R抑制剂PLX-3397为阳性对照药，给药2周，发现BRP抑制LLC移植小鼠模型的肿瘤生长，免疫组化检测BRP上调CD11b和HLA-DR的表达，降低CD163和CD206的表达，增加CD3淋巴细胞数量，并减少CD161+ 细胞和CD4+/CD8+ 细胞的比例，表明 BRP 增加了T细胞和NK细胞的数量。为了进一步研究 BRP 对巨噬细胞极化的作用机制，通过 RT-PCR分析M1和M2 TAM的标记基因，western blot检测STAT通路蛋白水平，发现BRP在体内调控巨噬细胞极化表型由M2转化为M1型，以达到抗肿瘤的作用。该项目从体内体外解析BRP调控巨噬细胞极化的分子机制，及与抗肿瘤作用的相关性，对推动其进一步应用及民族地区药物创新具有重要而深远的意义。