2，5-二羟基苯甲酸对代谢相关脂肪性肝病体外细胞模型的影响及其作用机制

徐俊姣; 刘彤; 刘素彤; 张丽慧; 宋艺佳; 吴文静; 崔蓓蕾; 管雅捷; 刘鸣昊

doi:10.12449/JCH260715

2，5-二羟基苯甲酸对代谢相关脂肪性肝病体外细胞模型的影响及其作用机制

DOI: 10.12449/JCH260715

徐俊姣^{1, 2},
刘彤^{1, 2},
刘素彤¹,
张丽慧¹,
宋艺佳^{1, 2},
吴文静^{1, 2},
崔蓓蕾^{1, 2},
管雅捷^{1, 2},
刘鸣昊^{1, 3, 4, 5, , ,}

1.
河南中医药大学第一附属医院肝病（脾胃）诊疗中心，郑州 450000
2.
河南中医药大学第一临床医学院，郑州 450003
3.
河南省中医药科学院，郑州 450046
4.
中西医防治重大疾病河南省协同创新中心，郑州 450046
5.
豫药全产业链研发河南省协同创新中心，郑州 450046

基金项目:

国家自然科学基金 (82575010);

国家自然科学基金 (81904154);

国家自然科学基金 (82205086);

河南省科技研发计划联合基金（优势学科培育类） (242301420096);

河南省科技研发计划联合基金（优势学科培育类） (242301420021);

河南省科技攻关计划 (242102310500);

河南省科技攻关计划 (232102310438);

河南省“双一流”创建学科中医学科学研究专项 (HSRP-DFCTCM-2023-1-10);

河南省卫生健康委员会中医药传承创新专项 (2023ZXZX1162);

河南省卫生健康委员会国家中医临床研究基地科研专项 (2022JDZX114);

第九届中国科协青年人才托举工程 (2023QNRC001)

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：徐俊姣负责提出研究思路，设计方案，获取数据及撰写原稿；刘彤、刘素彤和张丽慧负责校对和修改原稿；宋艺佳、吴文静、崔蓓蕾和管雅捷负责结果核查；刘鸣昊负责指导文章撰写并最后定稿。

详细信息

通信作者:
刘鸣昊， Liumh015@163.com （ORCID： 0009-0001-7712-4605）

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出版历程
- 收稿日期: 2026-01-03
- 录用日期: 2026-04-02
- 出版日期: 2026-07-25

Effect and mechanism of action of 2,5-dihydroxybenzoic acid on an in vitro cell model of metabolic dysfunction-associated fatty liver disease

Junjiao Xu^{1, 2},
Tong Liu^{1, 2},
Sutong Liu¹,
Lihui Zhang¹,
Yijia Song^{1, 2},
Wenjing Wu^{1, 2},
Beilei Cui^{1, 2},
Yajie Guan^{1, 2},
Minghao Liu^{1, 3, 4, 5
, ,
,}

1.
Liver Disease （Spleen & Stomach） Diagnosis and Treatment Center，The First Affiliated Hospital of Henan University of Chinese Medicine，Zhengzhou 450000，China
2.
The First Clinical Medical College，Henan University of Chinese Medicine，Zhengzhou 450003，China
3.
Henan Academy of Traditional Chinese Medicine，Zhengzhou 450046，China
4.
Henan Provincial Collaborative Innovation Center of Prevention and Treatment of Major Diseases by Chinese and Western Medicine，Zhengzhou 450046，China
5.
Henan Provincial Collaborative Innovation Center of Research and Development on the Whole Industry Chain of Yu-Yao，Zhengzhou 450046，China

Research funding:

National Natural Science Foundation of China (82575010);

National Natural Science Foundation of China (81904154);

National Natural Science Foundation of China (82205086);

Henan Provincial Science and Technology Research and Development Plan Joint Fund（Superior Discipline Cultivation Category） (242301420096);

Henan Provincial Science and Technology Research and Development Plan Joint Fund（Superior Discipline Cultivation Category） (242301420021);

Henan Provincial Science and Technology Tackling Program (242102310500);

Henan Provincial Science and Technology Tackling Program (232102310438);

“Double First-Class” Creation Discipline（Chinese Medicine） Scientific Research Special Project of Henan Province (HSRP-DFCTCM-2023-1-10);

Henan Provincial Health Commission’s Traditional Chinese Medicine Inheritance and Innovation Special Project (2023ZXZX1162);

Henan Provincial Health Commission National Clinical Research Base of Tradition Chinese Medicine Scientific Research Project (2022JDZX114);

The 9th Young Elite Scientist Sponsorship Program of the China Association for Science and Technology (2023QNRC001)

More Information

Corresponding author: Liu Minghao， Liumh015@163.com （ORCID： 0009-0001-7712-4605）

摘要

摘要: 目的探究2，5-二羟基苯甲酸（2，5-DHBA）治疗代谢相关脂肪性肝病（MAFLD）潜在靶点和调控磷脂酰肌醇3-激酶（PI3K）/蛋白激酶B（Akt）信号通路的分子机制。方法首先进行网络药理学研究，通过数据库筛选2，5-DHBA与MAFLD的共同作用靶点，进行分子对接与通路富集分析预测其潜在调控的生物学过程与信号通路。其次，进一步通过细胞（小鼠正常肝细胞AML-12）实验验证，通过细胞计数试剂盒8筛选2，5-DHBA 3个最佳干预浓度。将细胞分为5组：对照组、模型组（2 mmol/L的游离脂肪酸构建的MAFLD细胞模型）以及2，5-DHBA低、中、高3个干预剂量组（2 mmol/L的游离脂肪酸+3个不同浓度的2，5-DHBA）。采用油红O染色观察细胞内脂质沉积情况，荧光探针DCFH-DA检测细胞内活性氧的水平，生化法检测甘油三酯（TG）和总胆固醇（TC）含量；采用实时聚合酶链式反应与Western Blot检测基因及蛋白表达水平。多组间比较采用单因素方差分析，进一步两两比较采用Tukey’s检验。结果共筛选出32个2，5-DHBA与MAFLD的共同作用靶点。在1.25～20 μmol/L浓度范围内，2，5-DHBA处理组自2.5 μmol/L浓度起，呈剂量依赖性提高AML-12细胞活力。选取最低起效浓度 1.25 μmol/L、中间梯度浓度5 μmol/L及有效区间内最高安全浓度20 μmol/L作为低、中、高3个干预剂量。与模型组相比，1.25、5和20 μmol/L浓度的2，5-DHBA干预均能降低细胞内脂滴积累（P值均<0.05）；与模型组比较，3个剂量组TG和TC水平差异均有统计学意义（P值均＜0.05）。与对照组比较，模型组内ROS荧光强度显著升高（P<0.05）；与模型组相比，经5 μmol/L和20 μmol/L浓度的2，5-DHBA处理后，细胞内ROS水平呈剂量依赖性下降（P值均<0.05）；与模型组相比，5 μmol/L和20 μmol/L浓度的2，5-DHBA治疗显著下调了促脂生成基因固醇调节元件转录因子1、脂肪酸合成酶的mRNA表达水平，并上调了抗氧化核心基因核转录因子红系2相关因子2及其下游靶基因血红素加氧酶1的mRNA表达（P值均<0.05）。与对照组相比，模型组p-PI3K/PI3K、p-Akt/Akt表达均显著升高（P值均<0.05）；与模型组相比，经5 μmol/L的2，5-DHBA治疗后，p-PI3K/PI3K、p-Akt/Akt的蛋白表达水平均显著降低（P<0.05）。与对照组相比，模型组Nrf2、HO-1蛋白表达水平显著降低（P值均<0.05）；经1.25 μmol/L的2，5-DHBA治疗后Nrf2和HO-1蛋白水平均显著升高（P<0.05）。结论网络药理学联合细胞实验证实，2，5-DHBA能通过调控PI3K/Akt信号通路，减轻脂质沉积与氧化应激，进而发挥治疗MAFLD的作用。
- 代谢相关脂肪性肝病 /
- 2，5-二羟基苯甲酸 /
- 磷酸肌醇3-激酶类
Abstract: Objective To investigate the potential targets of 2，5-dihydroxybenzoic acid （2，5-DHBA） in the treatment of metabolic dysfunction-associated fatty liver disease （MAFLD） and the molecular mechanism by which it regulates the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway. Methods A network pharmacology analysis was performed at first， and related databases were used to obtain the common action targets of 2，5-DHBA and MAFLD， followed by molecular docking and pathway enrichment analysis to predict the potential biological processes and signaling pathways regulated by these targets. Then mouse normal hepatocytes AML-12 were used for cell experiments， and CCK-8 assay was used to determine the three optimal intervention concentrations of 2，5-DHBA. AML-12 cells were divided into control group， model group （cells cultured in a medium containing 2 mmol/L free fatty acid ［FFA］ to establish a cell model of MAFLD）， and three 2，5-DHBA intervention groups （cells cultured with FFA and 2，5-DHBA at the three different concentrations of 1.25， 5， and 20 μmol/L， respectively）. Oil red O staining was used to observe intracellular lipid accumulation； the fluorescent probe DCFH-DA was used to measure the level of intracellular reactive oxygen species （ROS）； biochemical assays were used to measure the content of triglyceride （TG） and total cholesterol （TC）； quantitative real-time PCR and Western Blot were used to measure the expression levels of related genes and proteins. A one-way analysis of variance was used for comparison between multiple groups， and the Tukey’s test was used for further comparison between two groups. Results A total of 32 common targets were obtained for 2，5-DHBA and MAFLD. Within a concentration range of 1.25 — 20 μmol/L， 2，5-DHBA treatment， starting from the concentration of 2.5 μmol/L， increased the viability of AML-12 cells in a dose-dependent manner. The minimum effective concentration of 1.25 μmol/L， the intermediate gradient concentration of 5 μmol/L， and the maximum safe concentration within the effective range of 20 μmol/L were selected for low-， middle-， and high-dose intervention， respectively. Compared with the model group， 2，5-DHBA intervention at concentrations of 1.25， 5， and 20 μmol/L could significantly reduce lipid droplet accumulation in cells （all P<0.05）， and there were significant differences in TG and TC between the model group and the three intervention groups （all P<0.05）. Compared with the control group， the model group had a significant increase in ROS fluorescence intensity （P<0.05）， and compared with the model group， there was a dose-dependent reduction in intracellular ROS level after treatment with 5 μmol/L or 20 μmol/L 2，5-DHBA （P<0.05）. Compared with the model group， 2，5-DHBA treatment at concentrations of 5 and 20 μmol/L significantly downregulated the mRNA expression levels of the lipogenic genes SREBf1 and FASN and upregulated the mRNA expression levels of the key antioxidant gene Nrf2 and its downstream target gene HO-1 （all P<0.05）. Compared with the control group， the expression of p-PI3K/PI3K and p-Akt/Akt in the model group was significantly increased （all P<0.05）. Compared with the model group， the protein expression levels of p-PI3K/PI3K and p-Akt/Akt were significantly decreased after 5 μmol/L 2，5-DHBA treatment （P<0.05）. Compared with the control group， the expression levels of Nrf2 and HO-1 protein in the model group were significantly decreased （all P<0.05）. The protein levels of Nrf2 and HO-1 were significantly increased after treatment with 1.25 μmol/L 2，5-DHBA （P<0.05）. Conclusion Network pharmacology and cellular experiments confirm that 2，5-DHBA can alleviate lipid deposition and oxidative stress by regulating the PI3K/Akt signaling pathway， thereby exerting a therapeutic effect on MAFLD.
- Metabolic Dysfunction-Associated Fatty Liver Disease /
- 2，5-Dihydroxybenzoic Acid /
- Phosphatidylinositol 3-Kinases

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注： a，GO功能富集分析结果，展示了BP、CC和MF中富集显著性前10的条目；b，KEGG通路富集分析前20的信号通路。MAFLD，代谢相关脂肪性肝病；GO，基因本体；KEGG，京都基因与基因组百科全书；HDAC8，组蛋白去乙酰化酶8；ERN1，内质网到细胞核信号转导1；CNR1，大麻素受体1；MPO，髓过氧化物酶；ELANE，中性粒细胞弹性蛋白酶；SERPINE1，丝氨酸蛋白酶抑制剂家族E成员1；MCL1，髓细胞白血病1；AXL，AXL受体酪氨酸激酶；PTGS2，前列腺素内过氧化物合酶2；MMP1，基质金属蛋白酶1；HSP90AA1，热休克蛋白90α家族A类成员1；EP300，蛋白乙酰转移酶P300；TLR4，Toll样受体4；MMP9；基质金属蛋白酶9；MMP2，基质金属蛋白酶2；MET，间质-上皮转化因子；KDR，激酶插入域受体；IGF1R，胰岛素样生长因子1受体；ESR1，雌激素受体1；EGFR，表皮生长因子受体；IL-17，白细胞介素17；Rap1，Ras相关蛋白1；HIF-1，低氧诱导因子1；PI3K-Akt，磷脂酰肌酶3-激酶-蛋白激酶B。

图 1 2，5-DHBA治疗MAFLD靶点的GO与KEGG富集分析

Figure 1. GO and KEGG enrichment analysis of 2，5-DHBA for MAFLD targets

下载: 全尺寸图片幻灯片

注： a，油红O染色结果（×400）；b，油红O染色定量分析；c，TC水平测定；d，TG水平测定。与模型组比较，*P<0.05。FFA，游离脂肪酸；TC，总胆固醇；TG，甘油三酯；2，5-DHBA，2，5-二羟基苯甲酸。

图 2 2，5-DHBA减轻FFA诱导的AML-12细胞脂质堆积

Figure 2. 2，5-DHBA alleviates FFA-induced lipid accumulation in AML-12 cells

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注： a，ROS荧光探针代表性图像（×400）；b，ROS定量分析；c～f，FASN、SREBF1、Nrf2和HO-1的mRNA表达水平。与模型组比较，*P<0.05。ROS，活性氧；FFA，游离脂肪酸；FASN，脂肪酸合成酶；SREBF1，固醇调控元件结合转录因子1；Nrf2，核转录因子红系2相关因子2；HO-1，血红素加氧酶1。

图 3 2，5-DHBA减轻FFA诱导的AML-12细胞脂质沉积和氧化应激

Figure 3. 2，5-DHBA alleviates FFA-induced lipid deposition and oxidative stress in AML-12 cells

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注： a、b，PI3K、p-PI3K的蛋白表达及相对表达量统计图；c，p-PI3K/PI3K的蛋白表达及相对表达量统计图；d、e，Akt、p-Akt的蛋白表达及相对表达量统计图；f，p-AKT/AKT的蛋白表达及相对表达量统计图。与模型组比较，*P<0.05。MAFLD，代谢相关脂肪性肝病；FFA，游离脂肪酸；PI3K，磷脂酰肌醇3-激酶；GAPDH，甘油醛-3-磷酸脱氢酶；p-PI3K，磷酸化磷脂酰肌醇3-激酶；Akt，蛋白激酶B；p-Akt，磷酸化蛋白激酶B。

图 4 2，5-DHBA调节PI3K/AKT信号通路改善MAFLD

Figure 4. 2，5-DHBA ameliorates MAFLD by modulating the PI3K/AKT signaling pathway

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注： a，Nrf2的蛋白表达及相对表达量统计图；b，HO-1的蛋白表达及相对表达量统计图。与模型组比较，*P<0.05。Nrf2，核转录因子红系2相关因子2；HO-1，血红素加氧酶1；GAPDH，甘油醛-3-磷酸脱氢酶；2，5-DHBA，2，5-二羟基苯甲酸；MAFLD，代谢相关脂肪性肝病。

图 5 2，5-DHBA抑制氧化应激改善MAFLD

Figure 5. 2，5-DHBA inhibits oxidative stress to ameliorate MAFLD

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表 1 引物序列

Table 1. Primer sequences

引物	方向	序列（5'-3'）
Nrf2	F	AAGCAGACGGAGGAGGAG
Nrf2	R	GGAGGTGTTCAGGCAAGG
SREBF1	F	CATCCACATGGCTCTGAGTG
SREBF1	R	CTCATCCACAAAGAAGCGGT
FASN	F	GAGAAAGCTTGCCAAACAGG
FASN	R	GAGGGTCTTGCAGGAGACAG
HO-1	F	GCTCAACATCCAGCTCTTTGAGG
HO-1	R	GACAAAGTTCATGGCCCTGGGA
GAPDH	F	GTTCCCACCCTAGAAAGTCCA
GAPDH	R	ACTTGGAGGAGGTTTGCTGG
注：Nrf2，核转录因子红系2相关因子2；SREBF1，固醇调控元件结合转录因子1；FASN，脂肪酸合成酶；HO-1，血红素加氧酶1；GAPDH，甘油醛-3-磷酸脱氢酶。

下载: 导出CSV

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