氧化纳米铈清除活性氧改善DSS诱导的小鼠结肠炎疾病活动度的研究

doi:10.3969/j.issn.1674-8115.2024.01.004

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (1): 35-42.doi: 10.3969/j.issn.1674-8115.2024.01.004

氧化纳米铈清除活性氧改善DSS诱导的小鼠结肠炎疾病活动度的研究

卢雨涵¹(), 石亚红¹, 龙满美², 王子³, 吴颖为¹()

^1.上海交通大学医学院附属第九人民医院放射科，上海 200011
^2.上海交通大学医学院附属第九人民医院病理科，上海 200011
^3.上海核工程研究设计院股份有限公司，上海 200233

收稿日期:2023-06-01 接受日期:2023-11-24 出版日期:2024-01-28 发布日期:2024-02-28
通讯作者: 吴颖为 E-mail:378390170@qq.com;wuyw0103@hotmail.com
作者简介:卢雨涵（1998—），女，硕士生；电子信箱：378390170@qq.com。
基金资助:
国家自然科学基金(82373114);上海交通大学医学院附属第九人民医院“交叉”研究基金(JYJC202107);上海交通大学医学院“双百人”项目(20191815)

Effect of ceria nanoparticles on activity of DSS-induced colitis in mice by eliminating active oxygen species

LU Yuhan¹(), SHI Yahong¹, LONG Manmei², WANG Zi³, WU Yingwei¹()

^1.Department of Radiology, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Department of Pathology, Shanghai Ninth People′s Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^3.Shanghai Nuclear Engineering Research & Design Institute Co. , LTD. , Shanghai 200033, China

Received:2023-06-01 Accepted:2023-11-24 Online:2024-01-28 Published:2024-02-28
Contact: WU Yingwei E-mail:378390170@qq.com;wuyw0103@hotmail.com
Supported by:
National Natural Science Foundation of China(82373114);"Cross" Research Fund Project of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202107);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20191815)

摘要/Abstract

摘要：

目的·探究氧化纳米铈-聚乙二醇（ceria nanoparticles-polyethylene glycol，CeNP-PEG）清除活性氧（reactive oxygen species，ROS）、改善葡聚糖硫酸钠（dextran sulphate sodium，DSS）诱导的小鼠结肠炎疾病活动度的效果。方法·首先应用醋酸铈的水合物、油胺与二甲苯等合成氧化纳米铈，经聚乙二醇-二硬脂酰磷脂酰乙醇胺（mPEG-DPSE）修饰并提纯后得到纯化的CeNP-PEG。应用透射电子显微镜（transmission electron microscopy，TEM）和动态光散射（dynamic light scattering，DLS）观察测定CeNP-PEG粒径和zeta电位等。体外培养小鼠巨噬细胞（Raw264.7）并诱导成为促炎表型（M1表型）。采用0.5 μg/mL和1.0 μg/mL CeNP-PEG分别处理M1表型巨噬细胞后应用Western blotting检测核因子-κB（nuclear factors-κB，NF-κB）信号通路相关蛋白表达的变化。构建DSS诱导的小鼠结肠炎模型并在造模期间予以尾静脉注射CeNP-PEG（1.0 mg/mL）3次，同时监测正常对照组（Normal组）、模型组（DSS组）和CeNP-PEG治疗组小鼠体质量、粪便性状与便血次数等，计算肠道炎症的疾病活动指数（disease activity index，DAI）。应用二氢乙啶（dihydroethidine，DHE）染色法检测小鼠肠道组织的ROS水平并通过实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）检测肠道组织炎症细胞因子γ干扰素（interferon-γ，Ifn-γ）、白细胞介素-6（interleukin-6，Il-6）、Il-1β和肿瘤坏死因子-α（tumor necrosis factor-α，Tnf-α）等基因的表达水平。结果·TEM和DLS结果显示合成的CeNP-PEG水合粒径为（6.96±0.27）nm，平均zeta电位为（-6.02±1.31）mV。Western blotting结果显示促炎表型巨噬细胞较对照组细胞p-P65表达增多，核因子-κB抑制蛋白α（NF-κB inhibitor-α，IκB-α）表达减少，经不同浓度CeNP-PEG干预后p-P65和IκB-α表达又趋于恢复。在小鼠结肠炎模型中，CeNP-PEG治疗组小鼠较DSS组体质量减轻更少（P=0.000），DAI评分更低（P=0.000）。肠道组织RT-qPCR结果显示，DSS组小鼠的Ifn-γ、Il-1β、Il-6和Tnf-α的mRNA水平较Normal组显著上调（均P=0.000），CeNP-PEG治疗后均显著回落。DHE染色结果显示，DSS组肠道组织的荧光强度较Normal组显著增强，CeNP-PEG治疗后荧光强度减弱。结论·CeNP-PEG可抑制肠道炎症因子的表达及促炎巨噬细胞NF-κB炎症通路的活化，消除肠道ROS，改善肠道炎症微环境并缓解DSS诱导的小鼠结肠炎的疾病活动度。

关键词: 炎症性肠病, 氧化纳米铈, 活性氧, 克罗恩病, 溃疡性结肠炎

Abstract:

Objective ·To investigate the effect of ceria nanoparticles-polyethylene glycol (CeNP-PEG) on scavenging reactive oxygen species (ROS) and alleviating disease activity in dextran sulphate sodium (DSS)-induced colitis mice. Methods ·CeNP was synthesized with the hydrates of cerium acetate, oleamine, and xylene, which was modified with polyethylene glycol-stearyl phosphatidylethanolamine (mPEG-DPSE) to obtain CeNP-PEG. Then CeNP-PEG was purified. The particle size and zeta potential of CeNP-PEG were measured by using transmission electron microscopy (TEM) and dynamic light scattering (DLS). Mouse macrophages (Raw264.7) were cultured in vitro and induced to a pro-inflammatory phenotype (M1 phenotype). M1 macrophages were treated with 0.5 μg/mL and 1.0 μg/mL CeNP-PEG, respectively, and then Western blotting was used to detect the expression changes of the proteins related with nuclear factor-κB (NF-κB) signaling pathway. DSS-induced colitis mice models were constructed, and CeNP-PEG (1.0 mg/mL) was intravenously administrated for 3 times via tail vein during the modeling period. Meanwhile, the body weight, fecal characteristics, and frequency of rectal bleeding in mice were monitored in the normal control group (Normal group), the model group (DSS group), and the CeNP-PEG treatment group. The disease activity index (DAI) was calculated to evaluate the intestinal inflammation. The level of ROS in mouse intestinal tissues was detected by dihydroethidine (DHE) staining and the mRNA expression levels of inflammatory cytokines interferon-γ (Ifn-γ), interleukin-6 (Il-6), Il-1β and tumor necrosis factor-α (Tnf-α) were detected by real-time quantitative PCR (RT-qPCR). Results ·The hydrated particle size of synthesized CeNP-PEG was (6.96±0.27) nm, and the average zeta potential was (-6.02±1.31) mV. Western blotting results showed that the expression of p-P65 increased in the pro-inflammatory macrophages compared with the control group. The expression of NF-κB inhibitor-α (IκB-α) decreased, and their expressions tended to recover after the intervention of different concentrations of CeNP-PEG. In the DSS-induced colitis models, mice in the CeNP-PEG treatment group lost less weight than those in the DSS group (P=0.000) and had lower DAI scores (P=0.000). The RT-qPCR results of intestinal tissues showed that the mRNA levels of Ifn-γ, Il-1β, Il-6 and Tnf-α in the DSS group were significantly up-regulated compared with those in the Normal group (P=0.000), and all of them significantly decreased in the CeNP-PEG treatment group. The results of DHE staining showed that the fluorescence intensity of intestinal tissues in the DSS group was significantly enhanced than that in the Normal group, and the fluorescence intensity decreased in the CeNP-PEG treatment group. Conclusion ·CeNP-PEG can inhibit the expression of intestinal inflammatory factors and the activation of NF-κB-related inflammatory pathway of pro-inflammatory macrophages, eliminate intestinal ROS, improve the intestinal inflammatory microenvironment, and alleviate the disease activity of DSS-induced colitis in mice.

Key words: inflammatory bowel disease, ceria nanoparticles (CeNP), reactive oxygen species (ROS), Crohn disease, ulcerative colitis

中图分类号:

R574.62

卢雨涵, 石亚红, 龙满美, 王子, 吴颖为. 氧化纳米铈清除活性氧改善DSS诱导的小鼠结肠炎疾病活动度的研究[J]. 上海交通大学学报（医学版）, 2024, 44(1): 35-42.

LU Yuhan, SHI Yahong, LONG Manmei, WANG Zi, WU Yingwei. Effect of ceria nanoparticles on activity of DSS-induced colitis in mice by eliminating active oxygen species[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(1): 35-42.

图/表 6

表1 RT-qPCR引物序列

Tab 1 Primer sequences for RT-qPCR

Gene	Sequence (5′→3′)
Tnf-α	Forward: CCTGTAGCCCACGTCGTAGC Reverse: AGCAATGACTCCAAAGTAGACC
Il-6	Forward: ATCCAGTTGCCTTCTTGGGACTGA Reverse: TGGCTAAGGACCAAGACCATCCAA
Il-1β	Forward: CTTCAGGCAGGCAGTATCACTC Reverse: TGCAGTTGTCTAATGGGAACGT
Ifn-γ	Forward: AGCAACAGCAAGGCGAAA Reverse: CTGGACCTGTGGGTTGTTGA
actin	Forward: CAGCCTTCCTTCTTGGGTATG Reverse: GGCATAGAGGTCTTTACGGATG

图1 CeNP和CeNP-PEG的表征Note： A/B. Representative images of CeNP (A) and CeNP-PEG (B) (×10 000). C/D. Hydrated particle size (C) and zeta potential (D) of CeNP-PEG.

Fig 1 Representation of CeNP and CeNP-PEG

图2 CeNP-PEG对小鼠巨噬细胞NF-κB促炎信号通路相关蛋白表达水平的影响

Fig 2 Effect of CeNP-PEG on the expression levels of NF-κB pro-inflammatory signaling pathway-associated proteins in murine macrophages

图3 CeNP-PEG缓解结肠炎小鼠疾病活动度的疗效评价Note： A. Relative body weight of the mice compared with the body weight on D1. ①P=0.001, compared with the DSS+CeNP-PEG group; ②P=0.001, compared with the DSS group; ③P=0.000, compared with the Normal group. B. DAI of the mice. ①P=0.000, compared with the Normal group; ②P=0.000, compared with the DSS group; ③P=0.000, compared with the DSS+CeNP-PEG group.

Fig 3 Evaluation of the effect of CeNP-PEG on reducing disease activity in the mice with colitis

图4 CeNP-PEG对结肠炎小鼠肠道促炎细胞因子表达水平的影响Note： A. Ifn-γ mRNA level. ①P=0.000, compared with the DSS group; ②P=0.000, compared with the DSS+CeNP-PEG group; ③P=0.000, compared with the Normal group. B. Il-1β mRNA level. ①P=0.000, compared with the DSS group; ②P=0.000, compared with the DSS+CeNP-PEG group; ③P=0.000, compared with the Normal group. C. Il-6 mRNA level. ①P=0.000, compared with the DSS group; ②P=0.000, compared with the DSS+CeNP-PEG group; ③P=0.000, compared with the Normal group. D. Tnf-α mRNA level. ①P=0.000, compared with the DSS group; ②P=0.000, compared with the DSS+CeNP-PEG group; ③P=0.000, compared with the Normal group.

Fig 4 Effect of CeNP-PEG on the expression of proinflammatory cytokines in the colonic tissues of colitis mice

图5 CeNP-PEG对结肠炎小鼠结肠组织ROS水平的影响

Fig 5 Effect of CeNP-PEG on ROS levels in the colonic tissues of colitis mice

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氧化纳米铈清除活性氧改善DSS诱导的小鼠结肠炎疾病活动度的研究

Effect of ceria nanoparticles on activity of DSS-induced colitis in mice by eliminating active oxygen species

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