目的 合成羽扇豆醇-3-吡啶季銨鹽衍生物，并進(jìn)行其體內(nèi)外抗乳腺癌MCF-7活性及作用機(jī)制研究。方法 通過酯化、?；统甥}反應(yīng)得到目標(biāo)化合物。采用紫外分光光度法在305 nm下測定羽扇豆醇和羽扇豆醇-3-吡啶季銨鹽的水溶性；以羽扇豆醇-3-吡啶季銨鹽0、1.25、2.5、5、10、20、40 μmol/L作用乳腺癌MCF-7細(xì)胞24、48、72 h，使用MTT法對其細(xì)胞增殖抑制能力進(jìn)行測定；采用羽扇豆醇-3-吡啶季銨鹽（7、14、28 μmol/L）作用MCF-7細(xì)胞48 h后，采用流式細(xì)胞術(shù)（AnnexinV-FITC/PI法）檢測細(xì)胞凋亡，JC-1染色法檢測細(xì)胞線粒體膜電位，DCFH-DA染色法檢測細(xì)胞活性氧（ROS）水平；同時在激光共聚焦顯微鏡下觀察細(xì)胞凋亡、線粒體膜電位變化以及ROS水平；Western blotting法檢測MCF-7細(xì)胞中線粒體凋亡途徑相關(guān)蛋白表達(dá)變化。通過構(gòu)建4T1小鼠荷瘤模型，設(shè)置對照組和羽扇豆醇-3-吡啶季銨鹽（20、40 mg/kg）組，與對照組進(jìn)行比較，觀察羽扇豆醇-3-吡啶季銨鹽在體內(nèi)抑制乳腺癌的活性。結(jié)果 合成羽扇豆醇-3-吡啶季銨鹽，其結(jié)構(gòu)經(jīng)¹H-NMR、¹³C-NMR和HR-ESI-MS確證。紫外分光光度法結(jié)果顯示羽扇豆醇-3-吡啶季銨鹽相對羽扇豆醇水溶性顯著提高；MTT提示羽扇豆醇-3-吡啶季銨鹽對乳腺癌MCF-7細(xì)胞有顯著的抑制能力（P＜0.01、0.001），且抑制率呈濃度、時間相關(guān)趨勢；流式細(xì)胞術(shù)以及激光共聚焦顯微鏡觀測結(jié)果表明羽扇豆醇-3-吡啶季銨鹽能夠有效誘導(dǎo)細(xì)胞凋亡，線粒體膜電位顯著下降，活性氧水平明顯升高；羽扇豆醇-3-吡啶季銨鹽能夠顯著上調(diào)B細(xì)胞淋巴瘤2（Bcl-2）相關(guān)X蛋白（Bax）、細(xì)胞色素C（Cyt C）、Bax、cleaved Caspase-9、cleaved Caspase-7蛋白表達(dá)，下調(diào)Bcl-2蛋白表達(dá)（P＜0.01、0.001）；羽扇豆醇-3-吡啶季銨鹽在體內(nèi)抑制腫瘤生長效果顯著，并未引起明顯毒性。結(jié)論 成鹽后的羽扇豆醇-3-吡啶季銨鹽水溶性明顯增加，并顯著提高了體內(nèi)外抗乳腺癌活性，本研究拓展了羽扇豆醇抗腫瘤研究的新思路。

Objective To study the effect and mechanism of a quaternary ammonium salt derivative of lupeol pyridine on breast cancer in vitro and in vivo. Methods The target compound was obtained by esterification, acylation and salt forming reactions. The water solubility of lupeol and lupeol-3-pyridine quaternary ammonium salt were determined by ultraviolet spectrophotometry at 305 nm. Breast cancer MCF-7 cells were treated with lupeol and lupeol-3-pyridine quaternary ammonium salt (0, 1.25, 2.5, 5, 10, 20, 40 μmol/L) for 24, 48, 72 h, and the cell proliferation inhibition ability was measured by MTT assay. MCF-7 cells were treated with 7, 14, and 28 μmol/L lupeol and lupeol-3-pyridine quaternary ammonium salt for 48 h, and the apoptosis was detected by flow cytometry (AnnexinV-FITC/PI method). The mitochondrial membrane potential was detected by JC-1 staining, and the reactive oxygen species (ROS) level was detected by DCFH-DA staining. At the same time, apoptosis, mitochondrial membrane potential changes and ROS levels were observed under confocal laser microscopy. Western blotting was used to detect the expression of proteins related to mitochondrial apoptosis pathway in MCF-7 cells. The tumor bearing model of 4T1 mice was established, and the control group and lupinol-3-pyridine quaternary ammonium salt (20 and 40 mg/kg) groups were set up to compare with the control group, and the anti-breast cancer activity of lupinol-3-pyridine quaternary ammonium salt in vivo was observed. Results Lupeol-3-pyridine quaternary ammonium salt derivative was synthesized and the structure was confirmed by ¹H-NMR, ¹³C-NMR and HR-ESI-MS. UV spectrophotometry showed that the water solubility of lupeol-3-pyridine quaternary ammonium salt was significantly higher than that of lupeol. MTT assay showed that lupeol-3-pyridine quaternary ammonium salt had a significant inhibitory effect on breast cancer MCF-7 cells (P < 0.01, 0.001), and the inhibition rate was concentration-time dependent. Flow cytometry and laser confocal microscopy showed that lupeol-3-pyridine quaternary ammonium salt could effectively induce cell apoptosis, significantly decrease mitochondrial membrane potential, and significantly increase the level of reactive oxygen species. Lupeol-3-pyridine quaternary ammonium salt could significantly up-regulate the expression of Bax, Cyt C, Bax, cleaved Caspase-9, and cleaved Caspase-7 proteins, and down-regulate the expression of Bcl-2 protein (P < 0.01, 0.001). Lupeol-3-pyridine quaternary ammonium salt significantly inhibited tumor growth in vivo without causing obvious toxicity. Conclusion The water solubility of Lupeol-3-pyridine quaternary ammonium salt significantly increased, and improved the anti-breast cancer activity in vivo and in vitro. The study has expanded the new idea of anticancer research of lupeol.

R284.3;R285.5

黑龍江省教育廳基本科研業(yè)務(wù)費基礎(chǔ)研究項目(2020-KYYWF-0024)