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; Tong LIU; Sutong LIU; Lihui ZHANG; Yijia SONG; Wenjing WU; Beilei CUI; Yajie GUAN; Minghao LIU

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

VernacularTitle: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
Author: Junjiao XU ¹ ; Tong LIU ¹ ; Sutong LIU ¹ ; Lihui ZHANG ¹ ; Yijia SONG ¹ ; Wenjing WU ¹ ; Beilei CUI ¹ ; Yajie GUAN ¹ ; Minghao LIU ¹
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1. Liver Disease （Spleen & Stomach） Diagnosis and Treatment Center， The First Affiliated Hospital of Henan University of Chinese Medicine， Zhengzhou 450000， China
Publication Type:Journal Article
Keywords: Metabolic Dysfunction-Associated Fatty Liver Disease; 2，5-Dihydroxybenzoic Acid; Phosphatidylinositol 3-Kinases
From: Journal of Clinical Hepatology 2026;42(7):1586-1596
CountryChina
Language:Chinese
Abstract: ObjectiveTo 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. MethodsA 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. ResultsA 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）. ConclusionNetwork 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.