Hugan Decoction alleviates non-alcoholic fatty liver disease in rats by activating the AMPK/m-TOR signaling pathway and reducing lipid synthesis

doi:10.12122/j.issn.1673-4254.2025.12.14

Abstract

Abstract:

Objective To explore therapeutic mechanism of Hugan Tang (Hugan Decoction, HGT) for alleviating non-alcoholic fatty liver disease (NAFLD) in rats. Methods Network pharmacology analysis was used to predict the active components of HGT against NAFLD and their potential targets, and the core targets were identified using the protein-protein interaction network, followed by GO and KEGG pathway enrichment analyses. A rat model of high-fat diet (HFD)-induced NAFLD was used to test the effects of saline, silymarin, and low-, moderate-, and high-dose HGT on serum levels of ALT, AST, LDL, LDH, TG and TC, liver histopathology, and protein and mRNA expressions of ACC1, FASN, AMPK and m-TOR. In free fatty acid (FFA)-induced HepG2 cells, the effects of blank and HGT-medicated sera, compound C (an AMPK inhibitor), and MHY1485 (a mTOR agonist) were tested on cell viability, intracellular lipid deposition, TC and TG levels, and expressions of ACC1, FASN, AMPK and m-TOR. Results We identified 130 active components in HGT, 267 common targets with NAFLD, and 53 core gene nodes, nearly half of which were involved in lipid metabolism. HGT treatment of NAFLD was closely associated with lipid and atherosclerosis signaling, insulin resistance signaling, and AMPK signaling. In rat models of NAFLD, HGT significantly alleviated liver injury and lipid accumulation, and suppressed mRNA and protein expressions of ACC1 and FASN. In FFA-induced HepG2 cells, HGT-medicated serum obviously reduced TG and TC levels and inhibited ACC1 and FASN mRNA and protein expressions. The results of in vitro and in vivo experiments both demonstrated that HGT activated the AMPK/mTOR signaling pathway by promoting p-AMPK expression and suppressing p-mTOR expression, and its regulatory effects on p-AMPK, p-mTOR, ACC1, and FASN were differentially modulated by compound C and MHY1485. Conclusion HGT alleviates NAFLD in rats by activating the AMPK/m-TOR signaling pathway and reducing lipid synthesis.

Key words: Hugan Dcoction, non-alcoholic fatty liver disease, network pharmacology, lipid metabolism, AMPK/m-TOR

Du SHANG, Wen LI, Lihua CUI, Ming CHEN. Hugan Decoction alleviates non-alcoholic fatty liver disease in rats by activating the AMPK/m-TOR signaling pathway and reducing lipid synthesis[J]. Journal of Southern Medical University, 2025, 45(12): 2667-2678.

Figures/Tables 7

Tab.1 Primer sequences for RT-qPCR

Gene	Forward sequence (5'-3')	Reverse sequence (5'-3')
r-GAPDH	AGATGGTGAAGGTCGGTGTG	CTGGAAGATGGTGATGGGTT
r-ACC1	GGGAACATCCCCACGCTAAA	CATGCGTTGACAAGGTGGTG
r-FASN	TGTACCCTCTAGCTGGACCC	CCAGGCTAAGGGCAATGGAA
r-AMPK	TCAAAGCCGACCCAATGACA	CTTCCTTCGCACACGCAAAT
r-mTOR	ACCCATCCAACCTGATGCTG	TCGAGACCGGTAACCTCCAT
h-GAPDH	CAAATTCCATGGCACCGTCA	GACTCCACGACGTACTCAGC
h-ACC1	AGGAGCTGTCTATTCGGGGT	GGTCGCTCAGCCTGTACTTT
h-FASN	CCTGGCTGCCTACTACATCG	CACATTTCAAAGGCCACGCA
h-AMPK	TGCTTTTTGTCCAGGGCTTG	ATCCCCTAAAGAGAGGGCCA
h-mTOR	GCCACTGTGCGGATCATTTC	AGCGAATGTCAGGGTCAGGA

Fig.1 Network pharmacology analysis of Hugan Decoction (HGT) for treatment of NAFLD. A: Venn diagram of HGT-NAFLD common targets. B: PPI network and core target screening of HGT for NAFLD treatment. C: GO enrichment analysis of potential targets of HGT for the treatment of NAFLD. D: Bubble chart of KEGG enrichment analysis of potential targets of HGT for treatment of NAFLD.

Fig.2 HGT attenuates liver injury in NAFLD rats. Effects of HGT on body weight (A), liver index (B) and liver morphology (C) in NAFLD rats (Mean±SD, n=8). D: Effects of HGT on hepatic histopathological changes in NAFLD rats (HE staining,original magnification: ×200). E, F: Effects of HGT on liver function in NAFLD rats (Mean±SD, n=8). #P<0.05, ###P<0.001 vs ND group; *P<0.05, **P<0.01, ***P<0.001 vs HFD group.

Fig3 Effects of HGT on lipid deposition in NAFLD rats. A: Oil red O staining of liver tissues in NAFLD rats in different groups (×200). B-E: Effects of HGT on blood lipids in NAFLD rats. F, G: Effects of HGT on mRNA expressions of ACC1 and FASN in NAFLD rats (Mean±SD, n=8). H-J: Effects of HGT on protein expressions of ACC1and FASN (Mean±SD, n=8). #P<0.05, ##P<0.01, ###P<0.001 vs ND group; *P<0.05, **P<0.01, ***P<0.001 vs HFD group.

Fig.4 Effects of HGT-medicated serum on lipid deposition in FFA-induced HepG2 cells. A, B: CCK-8 assay for assessing the effect of different concentrations of FFA and HGT-medicated serum on HepG2 cell viability. C: Oil red O staining of HepG2 cells (×200). D, E: Effects of HGT-medicated serum on TG and TC in FFA-induced HepG2 cells. F, G: Effects of HGT-medicated serum on mRNA expressions of ACC1 and FASN in FFA-induced HepG2 cells. H, J: Effects of HGT-medicated serum on protein expressions of ACC1 and FASN in FFA-induced HepG2 cells. #P<0.05, ###P<0.001 vs CON group; *P<0.05, **P<0.01, ***P<0.001 vs different concentrations of CONs groups.

Fig.5 HGT regulates lipid synthesis in the liver of NAFLD rats and in FFA-induced HepG2 cells through the AMPK/m-TOR signaling pathway. A, B: Effects of HGT on mRNA expressions of AMPK and m-TOR in NAFLD rats (Mean±SD, n=8). C-E: Effects of HGT on AMPK/m-TOR pathway in NAFLD rats (Mean±SD, n=8). F, G: Effects of HGT-medicated serum on mRNA expressions of AMPK and m-TOR in FFA-induced HepG2 cells.H-J: Effects of HGT-medicated serum on AMPK/m-TOR signaling pathway in FFA-induced HepG2 cells. #P<0.05, ##P<0.01, ###P<0.001 vs ND/CON group; *P<0.05, **P<0.01, ***P<0.001 vs HFD/ different concentrations of CONs group.

Fig.6 HGT-medicated serum regulates lipid synthesis in FFA-induced HepG2 cells via the AMPK/mTOR signaling pathway. A, B: Effect of compound C on AMPK/m-TOR signaling pathway proteins and lipid synthesis proteins ACC1 and FASN in FFA-induced HepG2 cells. C, D: Effects of the m-TOR activator (MHY) on m-TOR proteins and ACC1 and FASN proteins in FFA-induced HepG2 cells. #P<0.05, ##P<0.01, ###P<0.001 vs CONs group; *P<0.05, **P<0.01 vs HGTs group.

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