吴茱萸主要成分在人结肠腺癌细胞和RAW264.7巨噬细胞共培养模型中的肠道抗炎作用

doi:10.5246/jcps.2020.12.077

中国药学(英文版) ›› 2020, Vol. 29 ›› Issue (12): 868-879.DOI: 10.5246/jcps.2020.12.077

吴茱萸主要成分在人结肠腺癌细胞和RAW264.7巨噬细胞共培养模型中的肠道抗炎作用

王石, 刘璐, 张友波^*(), 徐嵬, 杨秀伟^*()

北京大学医学部药学院天然药物及仿生药物国家重点实验室; 天然药物学系, 北京 100191

收稿日期:2020-08-26 修回日期:2020-10-10 接受日期:2020-11-06 出版日期:2020-12-30 发布日期:2020-12-30
通讯作者: 张友波, 杨秀伟
作者简介:
+ Tel.: +86-10-82801569, E-mail: xwyang@bjmu.edu.cn
+ E-mail: ybzhang@bjmu.edu.cn
基金资助:
National Key R&D Program of China (Grant No. 2018YFC1704500, 2018YFC1704506) and the National Natural Science Foundation of China (Grant No. 81773865).

Intestinal anti-inflammatory effects of main components of the fruits of Euodia rutaecarpa in a co-culture model of the human colorectal adenocarcinoma cells and RAW264.7 macrophages

Shi Wang, Lu Liu, Youbo Zhang^*(), Wei Xu, Xiuwei Yang^*()

State Key Laboratory of Natural and Biomimetic Drugs, Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing 100191, China

Received:2020-08-26 Revised:2020-10-10 Accepted:2020-11-06 Online:2020-12-30 Published:2020-12-30
Contact: Youbo Zhang, Xiuwei Yang

About author:

Dr. Youbo Zhang received his Ph.D. and was a staff fellow of Peking University at the year 2010. He went Natural Institutes of Health to complete his postdoctoral training in Dr. Frank J. Gonzalez’s laboratory during the year 2015-2016. Dr. Zhang’s research interests include chemistry of natural products, drug metabolism, drug-induced liver injury, and toxicometabolomics. He has published more than 40 papers on Free Radical and Biology and Medicine, Drug Metabolism and Disposition, and some other journals by now.

Dr. Xiuwei Yang got his doctorate in Toyama Medical and Pharmaceutical University of Japan in 1992. He worked as a postdoctoral research fellow at School of Pharmaceutical Sciences from Beijing Medical University in 1992–1994. In 1994.9, he joined Peking University School of Pharmaceutical Sciences and worked as a professor. His major research interests are the absorption, distribution, metabolism, excretion, toxicity and activity of drugs (ADMET/Act.), complex system exploration of traditional Chinese Medicine, biological transformation of natural drug and/or Chinese medicinal chemical compositions, innovative drug discovery and natural medicinal chemistry. His group has established human intestinal bacteria biological transformation system, human Caco-2 cell monolayer absorption model, modified rat everted intestinal sac model, blood brain barrier absorption cell model, liver microsomal transformation and metabolism, and tissue distribution of drugs. He has published more than 450 papers in journals, and edited more than 20 works. He serves as the editorial board member of Journal of Asian Natural Products Research, Journal of Chinese Pharmaceutical Sciences, China Journal of Chinese Medica and Chinese Journal of Pharmaceutical Analysis , etc.

摘要/Abstract

摘要：

生物碱是吴茱萸中一类主要活性成分, 具有抗肿瘤、抗炎、镇痛和抗菌作用。本文报道吴茱萸中六个主要成分吴茱萸碱、吴茱萸次碱、去氢吴茱萸碱、吴茱萸卡品碱、二氢吴茱萸卡品碱和1-甲基-2-十一烷基-4-(1 H)-喹诺酮潜在的抗炎作用。在人结肠腺癌细胞和RAW264.7巨噬细胞共培养体系中, 当以2 μg/mL脂多糖诱导RAW264.7巨噬细胞产生炎症反应时, 吴茱萸碱、吴茱萸次碱和去氢吴茱萸碱明显下调促炎性细胞因子(Il-6, Il-1β和Tnf-α)和炎性介质, 诸如环氧化酶-2(Cox-2)和诱导型一氧化氮合酶(iNos)的mRNA表达; 与模型组比较, 给药组跨上皮电阻增高, 紧密连接渗透率降低。总之, 受试化合物具有抗炎活性, 此结果为进一步研究吴茱萸的抗炎机制奠定了基础。

关键词: 吴茱萸, 抗炎作用, 紧密连接, Caco-2, RAW264.7

Abstract:

Alkaloids are the main active constituents of the nearly ripe fruits of Euodia rutaecarpa (Juss.) Benth., which possesses antitumor, anti-inflammatory, analgesic and antimicrobial activities. In this study, we aimed to assess the potential anti-inflammatory effects of six main components, evodiamine, rutaecarpine, dehydroevodiamine, evocarpine, dihydroevocarpine and 1-methyl-2-undecyl-4-(1H)-quinolone, in the nearly ripe fruits of E. rutaecarpa. In the co-culture system consisting of the human colorectal adenocarcinoma (Caco-2) cells and RAW264.7 macrophages, inflammation was induced in RAW264.7 cells with 2 μg/mL lipopolysaccharide. The results indicated that evodiamine, rutaecarpine, and dehydroevodiamine significantly down-regulated the mRNA expressions of pro-inflammatory cytokines (Il-6, Il-1β and Tnf-α) and inflammatory mediators, such as cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNos). Besides, the drug administration group showed a higher transepithelial electrical resistance and a lower tight junction permeability compared with the model group. Taken together, the tested compounds possessed anti-inflammatory effects. Our findings laid the foundation for further research on the anti-inflammatory mechanism of the nearly ripe fruits of E. rutaecarpa.

Key words: Euodia rutaecarpa, Anti-inflammation, Tight junction, Caco-2, RAW264.7

Supporting:

王石, 刘璐, 张友波, 徐嵬, 杨秀伟. 吴茱萸主要成分在人结肠腺癌细胞和RAW264.7巨噬细胞共培养模型中的肠道抗炎作用[J]. 中国药学(英文版), 2020, 29(12): 868-879.

Shi Wang, Lu Liu, Youbo Zhang, Wei Xu, Xiuwei Yang. Intestinal anti-inflammatory effects of main components of the fruits of Euodia rutaecarpa in a co-culture model of the human colorectal adenocarcinoma cells and RAW264.7 macrophages[J]. Journal of Chinese Pharmaceutical Sciences, 2020, 29(12): 868-879.

图/表 9

Figure 1. Chemical structures of evodiamine (1), rutaecarpine (2), dehydroevodiamine (3), evocarpine (4), dihydroevocarpine (5), and 1-methyl-2-undecyl-4-(1H)-quinolone (6).

Figure 2. A co-culture system was constructed with Caco-2 cells and RAW264.7 cells. LPS was added to the basolateral compartment of this co-culture system to induce inflammation.

Table 1. Inflammatory factors and associated sense and antisense sequences.

Figure 3. Dose-dependent cytotoxicity of the compounds to RAW264.7 cells.

Table 2. Inhibition of NO production in RAW264.7 cells induced by LPS by six alkaloid components of the nearly ripe fruits of E. rutaecarpa.

Figure 4. Time course of the compounds to TEER value. (A) Effects of EDM, RCP and DEDM to the TEER value; (B) Effects of ECP, DECP and MUQ to the TEER value.

Figure 5. The effects of EDM, RCP, DEDM, ECP, DECP, MUQ and CCM on the expressions of iNos and Cox-2 at the mRNA level. (A) Effects of EDM, RCP, DEDM, ECP, DECP, MUQ and CCM on the expressions of Cox-2 at the mRNA level; (B) Effects of EDM, RCP, DEDM, ECP, DECP, MUQ and CCM on the expressions of iNos at the mRNA level. *P < 0.05 vs. LPS, **P < 0.01 vs. LPS, ***P < 0.001 vs. LPS, #P < 0.1 vs. LPS, ##P < 0.01 vs. LPS, ###P < 0.001 vs. LPS.

Figure 6. The effects of EDM, RCP, DEDM and CCM on the expressions of Il-6, Tnf-α and Il-1β at the mRNA and protein levels. (A) (C) (E) Protein levels of Il-6, Tnf-α and Il-1β after treatment with LPS and EDM, RCP, DEDM and CCM; (B) (D) (F) mRNA expression levels of Il-6, Tnf-α and Il-1β after treatment with LPS and EDM, RCP, DEDM and CCM; *P < 0.05 vs. LPS, **P < 0.01 vs. LPS, ***P < 0.001 vs. LPS, #P < 0.1 vs. LPS, ##P < 0.01 vs. LPS, ###P < 0.001 vs. LPS.

Figure 7. The effects of ECP, DECP, MUQ and CCM on the mRNA expression and protein levels of cytokines. (A) (C) (E) Protein levels of Il-6, Tnf-α and Il-1β after treatment with LPS and ECP, DECP, MUQ and CCM; (B) (D) (F) mRNA expression levels of Il-6, Tnf-α and Il-1β after treatment with LPS and ECP, DECP, MUQ and CCM; *P < 0.05 vs. LPS, **P < 0.01 vs. LPS, ***P < 0.001 vs. LPS, #P < 0.1 vs. LPS, ##P < 0.01 vs. LPS, ###P < 0.001 vs. LPS.

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吴茱萸主要成分在人结肠腺癌细胞和RAW264.7巨噬细胞共培养模型中的肠道抗炎作用

Intestinal anti-inflammatory effects of main components of the fruits of Euodia rutaecarpa in a co-culture model of the human colorectal adenocarcinoma cells and RAW264.7 macrophages

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