基于单宁酸的ROS响应型水凝胶在巨噬细胞炎症中的抗炎抗氧化作用

doi:10.3969/j.issn.1674-8115.2026.07.007

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (7): 886-895.doi: 10.3969/j.issn.1674-8115.2026.07.007

• 论著 · 基础研究 • 上一篇

基于单宁酸的ROS响应型水凝胶在巨噬细胞炎症中的抗炎抗氧化作用

吴嘉龙¹, 陈旭卓¹, 黄恩奎², 尹修媛², 许谦和², 刘培念²(), 张善勇¹()

^1.上海交通大学医学院附属第九人民医院口腔外科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
^2.华东理工大学化学与分子工程学院，上海市功能性材料化学重点实验室，上海 200237

收稿日期:2025-11-21 接受日期:2026-01-21 出版日期:2026-07-28 发布日期:2026-07-28
通讯作者: 张善勇，主任医师，博士；电子信箱：zhangshanyong@126.com。
刘培念，教授，博士；电子信箱：liupn@ecust.edu.cn。
基金资助:
国家自然科学基金(82370979);上海交通大学医学院“双百人”项目(20152226)

Anti-inflammatory and antioxidant effects of tannic acid-based ROS-responsive hydrogel in macrophage inflammation

Wu Jialong¹, Chen Xuzhuo¹, Huang Enkui², Yin Xiuyuan², Xu Qianhe², Liu Peinian²(), Zhang Shanyong¹()

^1.Department of Oral Surgery, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology; Shanghai Research Institute of Stomatology, Shanghai 200011, China
^2.Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, Shanghai 200237, China

Received:2025-11-21 Accepted:2026-01-21 Online:2026-07-28 Published:2026-07-28
Contact: Zhang Shanyong, E-mail: zhangshanyong@126.com.
Liu Peinian, E-mail: liupn@ecust.edu.cn.
Supported by:
National Natural Science Foundation of China(82370979);“Two-hundred Talents” Program of Shanghai Jiao Tong University School of Medicine(20152226)

摘要/Abstract

摘要：

目的·开发一种基于单宁酸（tannic acid，TA）的活性氧（reactive oxygen species，ROS）响应型水凝胶，并评估其在脂多糖（lipopolysaccharides，LPS）诱导的RAW264.7巨噬细胞炎症模型中的抗炎和抗氧化作用。方法·使用苯硼酸改性透明质酸（phenylboronic acid-modified hyaluronic acid，HP）与TA络合制备HP-TA水凝胶，通过过氧化氢消耗实验检测HP-TA水凝胶的ROS响应特性。采用细胞计数试剂盒-8（cell counting kit-8，CCK-8）、细胞周期实验和活/死细胞染色，评估HP和TA对RAW264.7巨噬细胞的生物相容性。建立RAW264.7巨噬细胞炎症模型并给予HP-TA水凝胶干预，分别采用实时荧光定量PCR（quantitative real-time PCR，qPCR）和蛋白质印迹法（Western blotting）检测诱导型一氧化氮合酶（inducible nitric oxide synthase，Inos）、肿瘤坏死因子α（tumor necrosis factor-α，Tnf-α）等促炎基因以及NAD（P）H：醌氧化还原酶1［NAD（P）H dehydrogenase quinone 1，Nqo1］、血红素氧合酶1（heme oxygenase-1，Ho-1）等抗氧化基因在mRNA和蛋白水平的表达。采用2′，7′-二氯二氢荧光素二乙酸酯（2′，7′-dichlorodihydrofluorescein diacetate，DCFH-DA）探针结合流式细胞术以及一氧化氮含量检测分析HP-TA水凝胶的ROS消耗能力。结果·成功合成HP-TA水凝胶，且该水凝胶可对ROS产生响应。在安全浓度（HP 200 μg/mL，TA 25 μg/mL）下，二者对RAW264.7巨噬细胞均表现出良好的生物相容性。HP-TA水凝胶可显著抑制炎症模型细胞的促炎基因Inos、Tnf-α的mRNA和蛋白表达，同时促进抗氧化基因Nqo1、Ho-1的mRNA和蛋白表达（均P<0.05）。此外，HP-TA水凝胶能显著降低细胞内的总ROS水平（P<0.001）。结论·HP-TA水凝胶能有效抑制LPS诱导的巨噬细胞炎症反应，发挥抗ROS作用并上调抗氧化通路蛋白表达。该研究展示了HP-TA水凝胶用于ROS响应型智能药物递送系统的潜力。

关键词: 单宁酸, 活性氧响应, 脂多糖, RAW264.7, 抗炎

Abstract:

Objective ·To develop a reactive oxygen species (ROS)-responsive hydrogel based on tannic acid (TA), and to evaluate its anti-inflammatory and antioxidant effects in an lipopolysaccharide (LPS)-induced RAW264.7 macrophage inflammation model. Methods ·Phenylboronic acid-modified hyaluronic acid (HP) was complexed with TA to form the HP-TA hydrogel, and its ROS-responsive properties were assessed by using a hydrogen peroxide consumption assay. The biocompatibility of HP and TA in RAW264.7 macrophages was evaluated by cell counting kit-8 (CCK-8) assay, cell cycle assay, and live/dead staining. An LPS-induced inflammation model of RAW264.7 macrophages was established and treated with the HP-TA hydrogel. The mRNA and protein expression levels of pro-inflammatory genes, including inducible nitric oxide synthase (Inos) and tumor necrosis factor-α (Tnf-α), as well as antioxidant genes, including NAD(P)H: quinone oxidoreductase 1 (Nqo1) and heme oxygenase-1 (Ho-1), were analyzed by quantitative real-time PCR (qPCR) and Western blotting, respectively. A 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe combined with flow cytometry and nitric oxide content detection was performed to analyze the total ROS-scavenging capacity of the hydrogel. Results ·The HP-TA hydrogel was successfully synthesized and confirmed to be ROS-responsive. At safe concentrations (200 μg/mL for HP and 25 μg/mL for TA), both materials exhibited good biocompatibility with RAW264.7 macrophages. The HP-TA hydrogel significantly inhibited the mRNA and protein expression of the pro-inflammatory genes Inos and Tnf-α, and promoted the mRNA and protein expression of the antioxidant genes Nqo1 and Ho-1 in inflammatory macrophages (all P<0.05). In addition, the HP-TA hydrogel significantly reduced the total intracellular ROS level (P<0.001). Conclusion ·The HP-TA hydrogel can effectively suppress LPS-induced inflammatory responses in macrophages, exert antioxidant effects by scavenging ROS, and upregulate the expression of antioxidant pathway proteins. This study reveals the potential of the HP-TA hydrogel as a ROS-responsive intelligent drug delivery system.

Key words: tannic acid (TA), reactive oxygen species responsiveness, lipopolysaccharide (LPS), RAW264.7, anti-inflammatory effect

中图分类号:

R392.12

吴嘉龙, 陈旭卓, 黄恩奎, 尹修媛, 许谦和, 刘培念, 张善勇. 基于单宁酸的ROS响应型水凝胶在巨噬细胞炎症中的抗炎抗氧化作用[J]. 上海交通大学学报（医学版）, 2026, 46(7): 886-895.

Wu Jialong, Chen Xuzhuo, Huang Enkui, Yin Xiuyuan, Xu Qianhe, Liu Peinian, Zhang Shanyong. Anti-inflammatory and antioxidant effects of tannic acid-based ROS-responsive hydrogel in macrophage inflammation[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(7): 886-895.

图/表 9

表1 qPCR引物序列

Tab 1 Primer sequences for qPCR

Gene	Forward sequence (5′→3′)	Reverse sequence (5′→3′)
β-actin	GGCTGTATTCCCCTCCATCG	CCAGTTGGTAACAATGCCATGT
Inos	ACTCAGCCAAGCCCTCACCTAC	TCCAATCTCTGCCTATCCGTCTCG
Tnf-α	GCCTCTTCTCATTCCTGCTTGTGG	GTGGTTTGTGAGTGTGAGGGTCT
Ho-1	GAGCAGAACCAGCCTGAACT	AAATCCTGGGGCATGCTGTC
Nqo1	GGTAGCGGCTCCATGTACTC	CGCAGGATGCCACTCTGAAT

图1 HP聚合物的 1H-NMR表征结果

Fig 1 1H-NMR characterization of HP polymer

图2 SEM下HP-TA水凝胶的微观结构（×1 200）

Fig 2 Microstructure of HP-TA hydrogel observed by SEM (×1 200)

图3 HP-TA水凝胶的H2O2 消耗实验

Fig 3 H2O2 consumption assay of HP-TA hydrogel

图4 HP和TA对RAW264.7巨噬细胞增殖活性与细胞周期的影响Note： A. Detection of the proliferation activity of RAW264.7 macrophages treated with HP or TA at different concentrations and time points by CCK-8. ①P<0.001, compared with the 0 μg/mL group at 120 h; ②P=0.006, ③P=0.002, ④P<0.001, compared with the 0 μg/mL group at 24 h. B. Detection of cell cycle distribution in RAW264.7 macrophages treated with HP or TA at concentrations ranging from 0 to 200 μg/mL for 24 h.

Fig 4 Effects of HP and TA on the proliferation activity and cell cycle distribution of RAW264.7 macrophages

图5 在HP及TA的安全浓度下RAW264.7巨噬细胞的Calcein-AM/PI染色结果（×200）Note： BF—bright field.

Fig 5 Calcein-AM/PI staining of RAW264.7 macrophages treated with HP and TA at safe concentrations (×200)

图6 HP-TA水凝胶对LPS诱导的RAW264.7巨噬细胞中促炎及抗氧化基因的mRNA水平的影响Note： ①P<0.001, ②P=0.017, ③P=0.003, compared with the LPS group.

Fig 6 Effects of HP-TA hydrogel on the mRNA expression of pro-inflammatory and antioxidant genes in LPS-induced inflammatory RAW264.7 macrophages

图7 HP-TA水凝胶对LPS诱导的RAW264.7巨噬细胞中促炎及抗氧化基因的蛋白表达水平的影响Note： A. Western blotting analysis of protein expression in RAW264.7 macrophages. B. Statistical analysis of INOS, NQO1, and HO-1 protein expression in the three groups. C. Detection of TNF-α expression in RAW264.7 macrophages by ELISA. ①P<0.001, ②P=0.004, ③P=0.005, compared with the LPS group.

Fig 7 Effects of HP-TA hydrogel on the protein expression of pro-inflammatory and antioxidant genes in LPS-induced inflammatory RAW264.7 macrophages

图8 HP-TA水凝胶对LPS诱导的RAW264.7巨噬细胞炎症模型内ROS表达及NO分泌水平的影响Note： A. Microfluorescence images of RAW264.7 macrophages stained with ROS (×200). Green fluorescence represents ROS expression in RAW264.7 macrophages. B. Flow cytometric fluorescence distribution of RAW264.7 macrophages stained with ROS. C. Detection of the MFI of ROS. D. Detection of NO secretion in the culture supernatant of RAW264.7 macrophages. ①P<0.001, compared with the LPS group. MFI—mean fluorescence intensity.

Fig 8 Effects of HP-TA hydrogel on the ROS expression and NO secretion levels in the LPS-induced inflammatory model of RAW264.7 macrophages

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基于单宁酸的ROS响应型水凝胶在巨噬细胞炎症中的抗炎抗氧化作用

Anti-inflammatory and antioxidant effects of tannic acid-based ROS-responsive hydrogel in macrophage inflammation

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