Integration proteomics analysis to identify AMPK as key target pathways of TCM formula for high fat diet induced obesity in mice

Abstract

Background

Livsooth Authentic Herbal Formula (LAH) is a novel Chinese herbal medicine that has been previously shown to prevent non-alcoholic fatty liver disease (NAFLD). However, its efficacy in treating obesity and its underlying mechanisms remain unclear. This study uniquely investigates the therapeutic effects of LAH on high-fat diet (HFD)-induced obese mice, focusing on its multi-targeted regulation of metabolic pathways. This research highlights the potential of a multi-component herbal formula in simultaneously activating the AMPK pathway, regulating lipid metabolism, and enhancing antioxidant defenses. By integrating network pharmacology predictions with proteomics analysis, in vivo, and in vitro experiments, this study provides a comprehensive understanding of LAH's mode of action.

Materials and methods

Mice were fed a high-fat diet (HFD) for 8 weeks, followed by oral treatment with LAH at doses of 615 mg/kg and 2460 mg/kg for 10 weeks. Each treatment group consisted of 6 mice. Body weight, blood biochemistry, and antioxidant enzyme activities were measured. Network pharmacology and proteomics analyses were conducted to identify mechanisms, and in vitro studies validated molecular pathways.

Results

This study utilized network pharmacology to investigate the therapeutic effects and mechanisms of LAH on obesity. Through relevant databases, 19 major chemical components and 605 potential targets were identified. KEGG pathway analysis identified the AMPK signaling pathway as a key target of LAH. Animal experiments showed that LAH reduced body weight by 16.55 % compared to HFD-induced mice. In addition to weight reduction, LAH significantly improved serum metabolic parameters. Glucose, triglyceride, and cholesterol levels were significantly reduced, and liver function improved, with ALT decreasing from 142.00 ± 32.63 U/L (HFD) to 63.57 ± 33.16 U/L (H-LAH), and AST from 147.20 ± 12.92 U/L (HFD) to 81.71 ± 31.31 U/L (H-LAH). It also enhanced liver antioxidant enzyme activity and reversed oxidative stress. Proteomics analysis revealed that LAH treatment downregulated the expression of FASN, HMGCR, and SREBP1 while upregulating PRKAA1, PRKAA2, ACACA, SOD1, and GSTP1, which are linked to the AMPK pathway and antioxidant mechanisms. In vitro experiments confirmed that LAH ameliorates hepatic steatosis by activating the AMPK pathway, as evidenced by its regulation of p-ACC, p-AMPK, CPT1A, FAS, and SREBP1 protein expression, identifying it as a critical regulator of obesity and NAFLD.

Conclusion

LAH reduces the expression of FAS and SREBP1 proteins via the AMPK pathway, while enhancing the activity of antioxidant enzymes. These effects promote weight loss and improve hepatic lipid metabolism, supporting its potential as a therapeutic agent for obesity and related metabolic disorders. This provides a theoretical foundation for using LAH in weight management.

Graphical abstract

Abbreviations:

AMPK

AMP-activated protein kinase

ACC

Acetyl-CoA carboxylase

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BP

biological processes

CC

cellular components

CAT

Catalase

CHOL

Cholesterol

CPT1A

Carnitine palmitoyltransferase 1A

DAPI

4′,6-diamidino-2-phenylindole

DMEM

Dulbecco's Modified Eagle Medium

FAS

Fatty acid synthase

FBS

Fetal bovine serum

FFAs

Free fatty acids

GAPDH

Glyceraldehyde-3-phosphate dehydrogenase

GLU

Glucose

GO

Gene Ontology

GPx

Glutathione peroxidase

GR

Glutathione reductase

GSH

Glutathione

HFD

High fat diet

HPLC

High-performance liquid chromatography

HS

Hovenia acerba Lindl

IF

Immunofluorescence

KEGG

Kyoto Encyclopedia of Genes and Genomes

LAH

Livsooth authentic herbal formula

LJF

Lonicera Japonica Thunb

MF

molecular functional entries

NAFLD

Non-alcoholic fatty liver disease

ND

Chow diet

OA

Oleic acid

PBS

Phosphate-buffered saline

PR

Pueraria lobata (Willd.) Ohwi

PVDF

Polyvinylidene fluoride

Q-TOF-MS

Quadrupole time-of-flight mass spectrometry

ROS

Reactive oxygen species

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SF

Siraitia grosvenorii (Swingle) C

SOD

Superoxide dismutase

SREBP1

Sterol regulatory element-binding protein 1

TCM

Traditional Chinese medicine

TG

Triglyceride

1. Introduction

According to the World Health Organization (WHO), obesity is escalating globally at an alarming rate, particularly in developed countries. Poor dietary habits and genetic factors are cited as primary contributors to this phenomenon.¹ Obesity is closely linked to several diseases, including nonalcoholic fatty liver disease (NAFLD), cardiovascular disease, type 2 diabetes and stroke. These factors complicate the management of metabolic syndrome, highlighting the urgent need for the development of anti-obesity medications (AOMs).²

Traditional Chinese Medicine (TCM) has been used in China for thousands of years due to its valuable therapeutic effects and its reputation for causing fewer side effects compared to conventional medicines. TCM formulations are combinations of various Chinese herbal medicines with multi-target and multi-mechanism characteristics, exerting potential therapeutic effects on obesity caused by multiple factors.^3,4 Livsooth authentic herbal formula (LAH) is a TCM compound composed of Pueraria lobata (Willd.) Ohwi (PR), Lonicera Japonica Thunb. (LJF), Hovenia acerba Lindl. (HS) and Siraitia grosvenorii (Swingle) C. (SF). PR In “Treatise on febrile and miscellaneous diseases”, an ancient book of TCM, PR has been known for its effects in relaxing tendons, reducing fever, promoting body fluids, and relieving diarrhea. Modern pharmacological studies have shown its effects in treating fever, thirst, acute dysentery, diabetes, hypolipidemia, and other symptoms.5, 6, 7 On the other hand, LJF is widely used in the treatment of scabies and swelling, and has been found to regulate inflammatory responses and elicit antiviral and antioxidant effects.⁸ Also, LJF modulates gut permeability and alter intestinal microbiota to alleviate obesity.⁹ HS has been used to treat liver disease and alcohol poisoning.¹⁰ A previous study showed that HS extract decrease lipid accumulation in a mouse model of acute hyperlipidemia by activating AMPK and PPARα/CPT-1 pathways.^11,12 SF has been known as the “Fruit of the Immortals.” This herb was traditionally used for treating cough, sore throat, and pharyngitis in China. It is believed to have immunomodulatory, antioxidant, anti-cancer, anti-obesity, and anti-asthmatic effects.¹³ Prior research has demonstrated that LAH effectively reduces liver fat accumulation and enhances liver function in a mouse model with HFD-induced hepatic steatosis.¹⁴.

AMP-activated protein kinase (AMPK) is an important metabolic regulator that controls how energy is used and stored in the body. Activating AMPK has been shown to have positive effects in managing type 2 diabetes and metabolic disease.^15,16 The regulation of AMPK signaling is essential and is influenced by tissue-specific expression, with various factors, such as stress, impacting its activity. The genes PRKAA1/2, PRKAB1/2, and PRKAG1/2/3 encode the subunits that form AMPK. AMPK exerts its effects through multiple mechanisms, including enhancing insulin sensitivity in peripheral tissues, inhibiting de novo lipogenesis, and regulating key genes involved in lipid metabolism, such as sterol regulatory element-binding protein 1 (SREBP1) and acetyl-CoA carboxylase (ACC).¹⁷ Additionally, AMPK contributes to mitochondrial stability, increases antioxidant enzyme activity, and reduces oxidative stress by inhibiting reactive oxygen species (ROS) production.^18,19 These mechanisms emphasize the crucial function of AMPK in regulating energy balance, metabolic activities, and cellular protection systems, especially in addressing obesity and its related metabolic issues.

Oxidative stress is a critical factor in obesity-related metabolic dysfunctions and has been implicated in the progression of NAFLD and other metabolic diseases. Enhancing the activity of antioxidant enzymes, such as glutathione (GSH), glutathione reductase (GR), and glutathione peroxidase (GPx), can play a significant role in mitigating obesity and its complications.²⁰ GSH, a primary antioxidant enzyme, scavenges free radicals and protects cells from oxidative damage, while GR regenerates reduced GSH to maintain its balance within cells. Obesity-associated oxidative stress may impair GR activity, leading to decreased GSH levels. GPx, which converts GSH to GSSG, is essential for maintaining cellular antioxidant defenses and preventing oxidative damage Chen et al., 2024. By targeting multiple pathways, LAH offers a comprehensive therapeutic strategy for obesity and its complications. However, the precise mechanisms underlying these effects remain to be fully elucidated.

Despite the growing interest in the use of Traditional Chinese Medicine (TCM) for obesity treatment, significant gaps remain in understanding the precise molecular mechanisms underlying its effects. LAH has shown promising results in reducing liver fat accumulation and improving liver function, but its therapeutic potential for obesity, particularly in metabolic regulation and associated diseases, is not fully understood. Furthermore, the combination of herbal ingredients in LAH, each with distinct pharmacological properties, suggests that its anti-obesity effects may stem from a complex interplay of molecular targets and signaling pathways. However, the lack of comprehensive studies investigating how these herbal compounds interact at a cellular level leaves the therapeutic effects of LAH on obesity requiring further investigation. Addressing these unknowns will be pivotal for translating its therapeutic effects into clinical practice and improving patient outcomes in obesity treatment. While the anti-obesity effects of PR, LJF, HS and SF have been recognized, research is still insufficient to fully elucidate its underlying mechanisms, particularly from the perspectives of network pharmacology and proteomics.

In recent years, network pharmacology has been introduced to comprehensively evaluate the pharmacological effects of TCM and explore the molecular mechanisms involved. A “multi-component, multi-target” perspective is offered by network pharmacology as opposed to the traditional “one target, one drug” approach. The molecular and systemic mechanisms of the herbal prescriptions and how their active components exert their effects have been studied through network-based pharmacological analysis.²¹ Network pharmacology has emerged to modernize traditional medicine. Network pharmacology has emerged to modernize traditional medicine. This study used network pharmacology to build multi-level biological information networks such as “active components target of TCM”, protein-protein interaction (PPI) network, and target gene functional pathway. The potential components and molecular targets of LAH were analyzed, revealing the pharmacological mechanisms underlying the treatment of obesity by LAH. The potential components and molecular targets of LAH were analyzed, revealing the pharmacological mechanisms underlying the treatment of obesity by LAH.

Proteomics has the potential to uncover valuable insights into the proteins and key biological processes involved in drug therapy, and it has become an important tool in ethnopharmacology research. Protein expression, localization, and activity vary under various conditions; therefore, studying protein expression in different cell types under different conditions helps in easily identifying and understanding the biological information.²² In this study, we measured lipid levels, biochemical indicators, and oxidative stress levels, and performed proteomics analysis of differentially expressed proteins in liver tissue. This revealed key proteins and protein network characteristics involved in LAH's improvement of obesity. These findings provide further insights into the mechanisms and targets of LAH for treating obesity.

This study aims to employ proteomics to explore and identify the anti-obesity mechanisms of LAH. By integrating proteomics with network pharmacology, this research will contribute to a deeper understanding of how the herbal compounds in LAH synergistically work to combat obesity and its associated metabolic dysfunctions.

2. Materials and Methods

2.1. Network pharmacology analysis of LAH on obesity

The context of TCM often consist of various components and bioactive compounds. A high oral bioavailability (OB ≥ 30 %) is desirable for active substances because it indicates efficient absorption from the gastrointestinal tract and reaching therapeutic concentrations in the body. In Drug Bank, a DL index ≥0.18 indicates high drug ability.²³ Therefore, the current study selected LAH compounds with DL index ≥0.18 and OB ≥ 30 % as active substances. A total of 30 different bioactive compounds were identified for further analysis after validation in the literature or other databases. The TCMSP database (http://lsp.nwu.edu.cn/tcmsp.php) was used to predict potential targets for LAH. After removing duplicated genes, we obtained 762 targets for further analysis. Using the keywords “obesity” and “metabolic syndrome,” we collected disease-related targets from the Genecards database (http://www.genecards.organd) the Online Mendelian Inheritance in Man (OMIM) database (http://www.omim.org). Removing duplicates, and then 18,708 potential targets related to the obesity and metabolic syndrome were obtained. Construction and visualization of an interaction network containing LAH potential targets, active components, and obesity-related targets using Cytoscape 3.7.1 software. (https://cytoscape.org).

We conducted gene function and pathway enrichment analyses to investigate the functional and signaling pathways associated target proteins of LAH. A Gene Ontology (GO) function enrichment analysis was conducted using the Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/), which investigated the involvement of cellular components (CC), molecular functions (MF), and biological processes (BP). Additionally, pathway enrichment analysis was carried out using the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway database (https://david.ncifcrf.gov/) to elucidate the pathways associated with LAH effects. Adjusted p-value ≤0.0001 and count ≥10 was chosen in functional annotation clustering.

2.2. Preparation of LAH

LAH from PR, LJF, HS and SF was purchased from Infinitus Pharmaceutical. Brion Research Institute of Taiwan authenticated all medicinal materials. The drug extraction ratio, extraction solvent type and concentration were consistent with previous studies.¹⁴

2.3. Animal procedures and treatments

Male C57BL/6 mice (5 weeks) were provided by the National Laboratory Animal Center (Taiwan). During the first week, the mice were acclimated to laboratory conditions and provided with food and water at a temperature of 23 °C under a 12-h light/12-h dark cycle. A schematic representation of the animal experimental method is shown in Fig. 1A. C57BL/6 mice were randomly divided into four groups. Blank group was fed with a normal diet and orally administered ddH₂O. HFD group was fed with a commercial diet containing 60 % fat (No. 58Y1; TestDiet, Inc.) and orally administered ddH₂O. After eight weeks of HFD feeding, obesity model was established, and two groups were orally administered LAH (615 and 2460 mg/kg) for 10 weeks. Mice (n = 6 per group) were periodically measured for obesity-related biomarkers and body weight for 18 weeks. A representation of the animal experimental methods is shown in Fig. 1A. After a 12-h fasting period, the mice were euthanized using CO₂, and blood and tissue samples were subsequently collected. No animal deaths were observed throughout the 18-week experimental period. All animal research procedures were reviewed and approved by the Taipei Medical University Institutional Animal Care and Use Committee (IACUC) (License No. LAC-2018-0114). The dose selection of LAH (615 and 2460 mg/kg) in this study was based on the principles outlined in the Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers issued by the U.S. Food and Drug Administration (FDA) in 2005. According to this guidance, for a 60-kg adult, the dose conversion between humans and experimental animals, such as mice, follows a factor of 12.3. This means that the recommended daily intake per kilogram of body weight (mg/kg/day) for humans is multiplied by 12.3 to determine the equivalent dose for mice. The human dosage is based on a 60 kg body weight, and the recommended daily dosage for adults is 3g/day. Experimental results from preliminary studies indicate that these doses have shown promising effects in animal models and are non-hepatotoxic.¹⁴.

Fig. 1.

Analysis of LAH's bioactive compounds and potential targets for obesity prevention. (A) Venn diagrams of potential targets of LAH and obesity. (B) LAH-component-target-network by Cytoscape 3.7.1. The 19 points in yellow circles represent compounds of four herbs in LAH, and the points in blue circles represent potential targets related to obesity. (C) The KEGG pathway enrichment analyses of 605 target proteins. The size of the circles represents the count of genes, and the color represents the p-value. p < 0.05. (D) The GO enrichment analyses of 605 putative targets.

2.4. Histological and ORO staining

The liver tissue was fixed in 4 % formalin, followed by a dehydration process using increasing concentrations of ethanol, and subsequently rinsed with xylene. Paraffin embedding was performed, and sections of 5 μm thickness were obtained using a microtome. The sections were subjected to staining using hematoxylin and eosin (H&E) for the purpose of examining the gross morphology. Another portion of liver tissue was dehydrated using sucrose solution with concentrations ranging from 10 % to 30 % and were then embedded in paraffin. Samples were embedded in OCT compound, cut into 10 μm thickness, and stained with Oil Red O (ORO) to visualize lipid content.

2.5. Serum biochemical analysis

Serum was separated from blood samples by centrifuging at 3000 rpm for 20 min at 4 °C. In accordance with the manufacturer's protocol, GLU, TG, CHOL, ALT, and AST levels were determined using VetTest 8008 (IDEXX Lab Inc., Westbrook, ME, USA).

2.6. Analysis of oxidative stress biochemical parameters

Liver tissue homogenates were prepared by agitating liver tissues at a high speed in an ice-cold buffer solution (1 mM EDTA, and 10 mM Tris-HC, 0.25 M sucrose). Following centrifugation for 10 min (12,000 rpm 4 °C), the mixture supernatant was obtained and stored at −80 °C for further determinations. Various biochemical parameters were measured in the liver homogenates including SOD (Rondox SD125; Antrim, UK), LDL and HDL (Randox CH201; Antrim), TG (TR213; Antrim), GR (Randox GR 2368; Goldberg, DM), GSH (Cat. No. 703002), CAT (Cayman No. 707002), and GPx (Randox RS 504; Paglia).

2.7. Proteomic analysis

Liver tissue samples from each group were subjected to proteomic analysis using the method described by Duong et al.²⁴. UPLC-Q-TOF/MS (Synapt G2; Waters Corporation, Milford, MA) was used for proteomic analysis. An Acquity UPLC HSS C18 column (1.7 μm, 2.1 × 100 mm²) set at 40 °C was used. The mobile phase was composed of a solution containing 0.1 % formic acid in water (A) and 0.1 % formic acid in acetonitrile (B). UPLC elution conditions were 0–25 min, 1–40 % B; 25–27 min, 40.0–85.0 % B; 27–31 min, 85.0–85.0 % B; 31–32 min, 85.0–1.0 % B; and 32–50 min, 1.0 % B. The flow rate was established at 0.5 mL/min with an injection volume of 5 μL. The autosampler was kept at a temperature of 10 °C.

Label-free relative quantification was carried out using Progenesis QI version 3.1 (Nonlinear Dynamics, USA). Three technical replicates of MS spectral data from each sample group (Blank, HFD, and H-LAH) were aligned and compared to obtain quantitative measurements of matched peptides across all MS runs. The filtering criteria for peptide ions included MS peaks within the 50–3000 m/z range, a retention time of 5–60 min, and charge states of 2+, 3+, and 4+. The identified peaks were exported for MS/MS analysis, which was conducted against the UniProt human proteome database (release 2019_03). The search parameters used were an 8-ppm precursor mass tolerance, a 0.5 Da fragment mass tolerance, and trypsin as the enzyme, allowing for up to two missed cleavages. The search results were imported back into Progenesis QI. The peptides identified were then further filtered and considered “confident” based on the following criteria: a confidence threshold of |Log Prob| > 1.3, an ANOVA p-value <0.05, selection of only unique sequence peptides with non-conflicting MS/MS (defined as MS/MS compounds assigned to a single peptide), and removal of incorrect protein sequences, including reverse sequences and keratin contaminants. Only proteins containing at least two peptides were included for relative quantification. The relative quantification of proteins in the Blank, HFD, and H-LAH groups was based on the MS peak intensities of the selected peptides.

2.8. Cell culture and cell viability assay

The HepG2 hepatic cell line was maintained in DMEM containing 10 % fetal bovine serum (FBS), 1 % L-glutamine, and 1 % penicillin/streptomycin. AML12 cells were cultured in DMEM/F12 medium with 0.005 mg/mL insulin, 0.005 mg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone and 10 % of FBS. The cells were incubated at 37 °C with 5 % CO₂ for 24 h. HepG2 cells and AML12 cells were seeded into 96-well plates overnight, cultured with LAH at concentrations of 25, 50, 100, and 200 μg/ml 24 h. Cell viability was determined using cell counting kit-8 (CCK-8) assay (ab228554, Abcam, Burlingame, CA, USA). CCK-8 solution was added to each well for 3 h, following which cell viability was determined by measuring the absorbance at 450 nm. To establish a cellular model of lipid accumulation, cells (2 × 10⁵/ml) were seeded into 6-well culture plate. Pre-treatment with 0.5 mM oleic acid (OA) for 6 h was performed, followed by treatment with various concentrations of LAH (25–200 μg/mL) for 24 h. Subsequently, cells were harvested to assess the molecular mechanisms of lipid metabolism.

2.9. Oil red O staining

HepG2 cells and AML12 cells were seeded in 24-well cell culture plates and incubated for 24 h. To induce lipogenesis, pre-treatment with 0.5 mM oleic acid (OA) for 6 h was performed, followed by treatment with various concentrations of LAH (25–200 μg/mL) for 24 h. After incubation, the cells were washed with PBS and fixed with formalin for 1 h at 24 °C, then washed with 1xPBS three times to remove residual formalin. The cells were stained with ORO in 60 % isopropanol for 30 min at 24 °C and then washed three times with distilled water. Following washing, the stained ORO dye was eluted with 100 % isopropanol, transferred to a 96-well plate and quantified spectrophotometrically at 520 nm using an ELISA plate reader (BioTek, Winooski, VT, USA).

2.10. Western blotting assay

Samples were extracted using RIPA buffer and then centrifuged at 12,000 rpm for 30 min to obtain protein extracts. The protein concentration was assessed utilizing the Bradford protein assay method. Western blot was performed following the previously described protocol.¹⁴ The protein targets related to fat metabolism were CPT1, SREBP1, p-AMPK, AMPK, ACC, and p-ACC (Cell Signaling Technology, Denvers, MA, USA). Protein bands were quantified using ImageJ software, and the ratios of each target protein were normalized relative to the GAPDH.

2.11. Immunofluorescence

The AML12 cell line was subjected to fixation in 4 % paraformaldehyde for a duration of 30 min, followed by permeabilization using 0.2 % Triton X-100 for 5 min at ambient temperature. Subsequently, the cells underwent blocking with 5 % bovine serum albumin (BSA) for a period of 1 h, followed by incubation with 1:200 dilution of primary antibody at 4 °C overnight in a moist chamber. Incubate the corresponding secondary antibody (1:200 dilution each; Alexa Fluor® 647, ab150079; Abcam and Alexa Fluor® 488, ab150077; Abcam) were then applied for 1 h at room temperature in the absence of light. Finally, nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI). The slides were visualized using a Stellaris 8 Confocal Microscope (Leica, Germany). Statistical analysis was conducted utilizing the Image J software.

2.12. Statistical analyses

The data were expressed as the mean ± standard deviation. All data in this study were continuous and were assessed for normality using the Kolmogorov-Smirnov test. Variations among multiple groups were assessed through one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test to determine intergroup differences. Statistical significance was considered at a p-value of less than 0.05. GraphPad Prism software (version 8.0; GraphPad Software Inc., San Diego, CA, USA) was used to perform all statistical analyses.

3. Results

3.1. Network pharmacological analysis of potential targets of LAH in relation to obesity

To evaluate the potential mechanisms of LAH in treating obesity, this study employed network pharmacology analysis to investigate the potential components and molecular targets of LAH. This approach facilitated the identification of the pharmacological mechanisms underlying the treatment of obesity by LAH from a systems biology perspective. From the TCMSP and PubChem databases, 19 potential active components in LAH were identified, including 6 compounds from PR, 14 compounds from LJF, 5 compounds from HS, and 3 Compounds from SF. After removing duplicate gene targets, a total of 671 potential targets were obtained. Additionally, 18,708 obesity-related gene targets were collected from the Genecards database. By performing a Venn intersection of the targets of SNS and obesity, 605 overlapping targets were identified as potential targets for LAH against obesity effects (Fig. 1A). To anticipate the pharmacological mechanisms of LAH on obesity among the active compounds of LAH, we utilized Cytoscape to construct the relevant relationships between the associated targets and obesity-related genes (Fig. 1B). To clarify the biological functions of LAH on obesity, we conducted KEGG pathway and GO enrichment analyses on the involved targets. The significantly enriched KEGG pathways among the 605 putative targets include “Lipid and atherosclerosis,” “AGE-RAGE signaling pathway in diabetic complications,” “PI3K-Akt signaling pathway,” “Non-alcoholic fatty liver disease,” “Insulin signaling pathway,” and “AMPK signaling pathway.” (Fig. 1C) These signaling pathways are closely associated with lipid metabolism. The current study showed that stimulating the phosphorylation of AMPK can significantly reduce lipid accumulation and promote fat metabolism,²⁵ which is consistent with our previous findings that LAH can improve NAFLD and regulate fatty acid metabolism. This suggests that the anti-obesity effects of LAH may be related to the AMPK signaling pathway. The results of GO enrichment analysis also demonstrated that LAH may contribute to the improvement of obesity by regulating cholesterol metabolic process, response to insulin, response to oxidative stress via enzyme binding and identical protein binding (Fig. 1D).

3.2. Effects of LAH on serum biochemical parameters

Serum biochemical values are assessed to monitor and evaluate metabolic alterations. After eight weeks of HFD. Levels of ALT, AST, GLU, TC, and TG were significantly higher in the HFD, L-LAH, and H-LAH groups than in the blank group. However, after 10 weeks of LAH treatment, serum levels of ALT, AST, GLU, TC, and TG were significantly decreased (Table 1). The findings demonstrate that LAH effectively reverses increased levels of biochemical markers in mice with HFD-induced obesity.

Table 1.

Serum biochemical analysis. ALT, AST, GLU, TC, and TG levels were measured in the serum of mice in different experimental groups. Values are expressed as the mean ± standard deviation (SD; n = 6/group). ∗p < 0.05 compared to the blank group. +p < 0.05, ++p < 0.01, +++p < 0.001 and ++++p < 0.0001 compared to HFD group. compared to HFD group.

Groups	ALT (U/L)	AST (U/L)	GLU (mg/dL)	TC (mg/dL)	TG (mg/dL)
8 weeks after HFD fed
Blank	50.90 ± 8.09	76.10 ± 42.84	146.13 ± 11.49	46.39 ± 12.26	68.23 ± 11.35
HFD	90.60 ± 25.25∗	141.80 ± 67.02	178.30 ± 20.26∗	110.09 ± 20.82∗	108.92 ± 13.03∗
L-LAH	90.57 ± 21.72∗	140.71 ± 39.01∗	168.71 ± 28.24∗	109.43 ± 15.10∗	100.00 ± 21.32∗
H-LAH	92.29 ± 32.85∗	151.86 ± 52.29∗	178.29 ± 21.62∗	112.14 ± 11.29∗	99.00 ± 16.23∗
18 weeks after HFD fed
Blank	44.00 ± 3.33	67.40 ± 25.67	142.60 ± 20.75	56.00 ± 10.08	53.80 ± 14.14
HFD	142.00 ± 32.63∗	147.20 ± 12.92∗	263.20 ± 33.41∗	147.40 ± 31.89∗	138.60 ± 26.43∗
L-LAH	80.57 ± 27.09+++	93.43 ± 41.01++	123.00 ± 45.41++++	116.57 ± 13.50+	123.57 ± 25.62
H-LAH	63.57 ± 33.16++++	81.71 ± 31.31+++	98.57 ± 38.65++++	100.71 ± 27.42+++	103.57 ± 26.64+

3.3. LAH reverses HFD-induced obesity in mice

Obesity model was established using HFD-induced mouse and the experimental setup is depicted in Fig. 2A. Following 8 weeks of HFD administration, the body weights of mice exhibited significant increase in the HFD, L-LAH, and H-LAH groups compared to the blank group. After 10 weeks of LAH treatment, body weights were lower in the L-LAH and H-LAH groups than in the HFD group (Fig. 2B–D). Additionally, treatment with LAH reduced organ weights of the liver and epididymal white adipose tissue. (Fig. 2E and F). Meanwhile, H&E staining revealed that HFD-fed mice exhibited excessive lipid accumulation with massive lipid droplet vacuoles, ballooning, and lobular inflammation (Fig. 3A) which was reversed by LAH treatment. Furthermore, ORO staining revealed that lipid accumulation in hepatocytes was markedly decreased in LAH-treated mice than in mice with HFD-induced obesity (Fig. 3A). Hepatic cholesterol (CHOL), low-density lipoprotein (LDL), and triglyceride (TG) levels were significantly higher in the HFD group than in the blank group. The administration of LAH treatment effectively mitigated this effect in mice fed a high-fat diet. (Fig. 3B). The results indicate that LAH effectively alleviates hepatic steatosis and histopathological alterations in mice with HFD-induced metabolic syndrome.

Fig. 2.

LAH alleviates obesity in HFD-induced mice. (A) Schematic diagram of HFD-induced obesity mice and LAH treatment. (B) After 18 weeks, photographs of representative mice from each group are shown. (C) Changes in the body weight of mice fed either normal diet (Blank), HFD, HFD containing 615 mg/kg (L-LAH), and HFD containing 2460 mg/kg (H-LAH) LAH extract. (D) Final body weight, (E) Final liver weight. (F) Epididymal white adipose tissue weight. Data are represented as the mean ± SD (n = 6/group). ∗p < 0.05 compared to the blank group. +p < 0.05, ++p < 0.01, +++p < 0.001 compared to HFD group.

Fig. 3.

Effects of LAH on Hepatic Lipid Accumulation and Antioxidant Enzyme Activity in HFD-Induced Obesity Mice. (A) Representative photographs of liver sections stained with H&E and ORO in each treatment group of mice (100 × magnification). (B) Concentrations of total cholesterol, low-density lipoprotein (LDL), and triglycerides within the hepatic tissue. (C) Superoxide dismutase (SOD), (D) catalase (CAT), (E) glutathione (GSH), (F) glutathione reductase (GR), and (G) glutathione peroxidase (GPx) levels in the liver tissue. Data are represented as the mean ± SD (n = 6/group). ∗p < 0.05 compared to the blank group. +p < 0.05 compared to HFD group. ++p < 0.01 compared to HFD group. +++p < 0.001 compared to HFD group. ++++p < 0.0001 compared to HFD group.

3.4. LAH relieves oxidative damage in mice with obesity

Oxidative stress exacerbates the development of obesity, while fat accumulation further exacerbates oxidative and inflammatory conditions. Therefore, we evaluated whether hepatic antioxidant enzymes were changed after LAH treatment of obese mice. Results showed that HFD-induced obesity decrease liver SOD and CAT levels whereas both L-LAH and H-LAH significantly upregulated them (Fig. 3C and D). In contrast to the HFD group, the activities of GSH, GR and GPx in the liver of the L-LAH group and the H-LAH group were significantly increased (Fig. 3E, F, G). These findings suggest that LAH can ameliorate GSH metabolism in the liver, consequently bolstering hepatic antioxidant capabilities and augmenting the functionality of antioxidant enzymes.

3.5. Creation of a protein-protein interaction network and discovery of central genes

Proteomics analysis was conducted to determine the mechanism by which LAH improves lipid metabolism abnormalities. First, 1037 proteins were identified via Q-TOF-MS analyses. To clarify the mechanism of action of LAH in treating obesity, we conducted proteomic analysis and screened for significantly differentially expressed proteins between the HFD and H-LAH groups. In total, 137 differentially expressed proteins, including 62 upregulated and 75 downregulated proteins were identified. The heat map revealed significantly differential proteins between HFD group and H-LAH group (Fig. 4A). Principal Component Analysis (PCA) was employed to visually assess the similarities and disparities in the liver proteome across the three groups. PCA elucidated distinct group differences between the blank and HFD groups, as well as with HFD and H-LAH groups (Fig. 4B).

Fig. 4.

Proteomics Data Analysis and Pathway Enrichment in HFD-Induced Obesity Mouse Model. (A) Proteome heatmap with the clustering dendrogram of samples. The color red signifies elevated levels of expression, while blue signifies reduced levels of expression. (B) Score plots of principal component analysis (PCA) of the blank and HFD groups and the HFD and H-LAH groups (n = 6 per group). (C) KEGG pathway terms. Adjusted p-value ≤0.05 and count ≥5 were chosen for functional annotation clustering. (D) KEGG map of the AMPK signaling pathway. Green rectangles represent confirmed organism-specific gene products, red stars represent genes with significant differences between HFD and H-LAH. (E) A bubble plot was utilized for conducting GO enrichment analysis, where the color scale denoted the p-value, and the dot size indicated the gene count per term.

To comprehensively understand the main mechanisms of action of LAH, we used KEGG enrichment analysis to analyze the differential proteins. The results showed that these proteins are highly related to metabolic pathways, alcoholic liver disease, the AMPK signaling pathway, the insulin signaling pathway, fatty acid metabolism, and non-alcoholic fatty liver disease (Fig. 4C). These differential proteins involve multiple points and pathways, among which the AMPK pathway has piqued our interest. Studies have shown that activating the AMPK pathway helps reduce fat accumulation in tissues and promote cellular energy balance. As shown in Fig. 4D, LAH can regulate multiple AMPK pathway-related proteins, such as: AMPK, PP2A, SIRT1, GS, HMGR, SREBP1c, FAS, and ACC1. In order to comprehensively understand the function, localization and biological pathways of LAH in organisms, differential proteins were annotated through GO analysis. Fig. 4E shows an overview of GO analysis, presenting bubble diagrams of the top 10 significantly enriched terms in the Biological Process (BP), Molecular Function (MF), and Cellular Component (CC) categories, respectively. In biological processes, LAH play a key role in lipid metabolism, cholesterol biosynthesis, and lipid synthesis. In terms of molecular functions, LAH are notably associated with oxidoreductase activity and protein binding. Regarding cellular components, LAH are closely linked to the cytosol, cytoplasm, and macromolecular complexes.

3.6. Proteomics analysis reveals mechanisms of LAH in improving lipid metabolism abnormalities

The PPI network was created using the STRING database, which involved identifying 137 differentially expressed proteins (DEPs). The network was visualized using Cytoscape software. (Fig. 5A). The MCC algorithms of the cytoHubba plug-in of Cytoscape (v3.7.2) were used to calculate the PPI network of the 137 DEPs. Furthermore, top 14 hub genes were screened out with MCC algorithms, including Fasn, Hmgcr, Srebf1, Sirt1, Acaca, Prkaa1, Pxdn, Hmgcs2, Fabp1, Ctnnb1, Acaa1b, Cyp2e1, Prkaa2 and Sdha (Fig. 5B). The top three ranked protein were FAS, HMGCR and SREBF1. The AMPK signaling pathway negatively regulates FASN, HMGCR, and SREBF1 to reduce lipid synthesis and promote lipid oxidation, which is crucial for counteracting the effects of a high-fat diet and reducing obesity-related complications. Proteomic analysis showed that protein levels were significantly upregulated in the HFD group, whereas protein levels were downregulated in the LAH group (Fig. 5C–E). PRKAA1/2 aka AMPK and ACACA were significantly increase in the H-LAH group compared to HFD group (Fig. 5F–H). In addition, SOD is an antioxidant enzyme, while GSTP1 is glutathione S-transferase, both of which are antioxidant enzymes that can protect cells from oxidative damage induced by HFD. LAH increased the expression of SOD and GSTP1 compared to the HFD group (Fig. 5I and J). These results indicate that LAH reverses HFD-induced obesity through multi-target effects. In addition to the proteomic analysis presented, p-AMPK protein expression in mouse liver was verified using Western blotting. As shown in the results, LAH increased the expression of p-AMPK protein compared with the HFD group (Fig. S1).

Fig. 5.

Proteomic analysis of potential protein changes in LAH treatment of HFD-induced obesity. (A) PPI analysis describe the differential proteins are involved in different network. (B) Visualization for Top 14 protein by CytoHubba software. Top 3 ranked proteins (C) FASN, (D) HMGCR, and (E) SREBF1 identified by CytoHubba analysis. Proteins involved in. Proteins related to AMPK pathway (F) PRKAA1, (G) PRKAA2, and (H) ACACA. Proteins associated with antioxidant function, (I) SOD1 and (J) GSTP1. Data are presented as the mean ± SD (n = 6/group). ∗p < 0.05 compared to the blank group. +p < 0.05 compared to HFD group.

3.7. LAH reduces fat accumulation in HepG2 and AML12 cells induced by OA through the AMPK pathway

Obesity can lead to metabolic disorders, affect liver function, lead to excessive fat accumulation in liver cells, and lead to NAFLD.²⁶ In vitro experiments were conducted on OA-induced HepG2 and AML12 cells to determine the effect of LAH on hepatic steatosis. LAH at concentrations of 50, 100, 200, and 400 μg/ml did not induce hepatotoxicity in HepG2 and AML12 cells, as determined by the CCK-8 assay (Fig. 6A and B). Compared with the OA group, LAH treatment significantly decreased OA-induced lipid accumulation (Fig. 6C and D). To understand the activity of LAH in ameliorating obesity-induced hepatic steatosis, the AMPK signaling pathway was detected using Western blot, LAH significantly increased p-AMPK, p-ACC, and CPT1A protein expression levels compared to those in the OA group (Fig. 7A and B). SREBP-1 is a key transcription factor that controls the production of fatty acids by influencing the transcription of ACC1 and FAS.²⁷ A significant reduction in FAS and SREBP1 protein levels was observed in LAH treatment group compared to OA group. Similarly, the immunofluorescence findings (Fig. 7C and D) revealed that LAH reduces the expression of OA-induced SREBP1 and FAS in AML12 cells. As a result, it was found that LAH regulates lipid accumulation through the AMPK signaling pathway.

Fig. 6.

LAH regulates OA-induced lipid accumulation in HepG2 and AML12 cells. CCK-8 assay of HepG2 cells (A) and AML12 cells (B) viability after treatment with LAH. (C, D) Lipid droplets were stained by ORO staining. Quantitative lipid accumulation of ORO stain at 520 nm. Data are represented as the mean ± SD (n = 3/group). ∗p < 0.05 compared to the blank group. +p < 0.05 compared to OA group.

Fig. 7.

LAH regulates lipogenesis by AMPK signaling pathway. (A and B) The protein expressions of p-AMPK, AMPK, p-ACC, ACC, CPT1A, FAS, and SREBP1 were analyzed in the protein lysates from HepG2 and AML12 cells using Western blotting. Quantification of p-AMPK/AMPK, p-ACC/ACC, CPT1A/GAPDH, FAS/GAPDH and SREBP1/GAPDH ratios was performed using ImageJ. Data are represented as the mean ± SD (n = 3/group). ∗p < 0.05 compared to the blank group. +p < 0.05 compared to OA group. (C) Immunofluorescence staining of SREBP1 and FAS expression in OA-induced AML12 cells. (D) Quantitative analysis of the expression of SREBP1 and FAS in immunofluorescence staining using ImageJ. Data are represented as the mean ± SD (n = 2/group). ∗p < 0.05 compared to the blank group. +p < 0.05 compared to OA group.

4. Discussion

Obesity is a global chronic, recurrent, and progressive disease process, often accompanied by elevated blood glucose levels, dyslipidemia, NAFLD, and coronary heart disease.¹ However, efforts to find multi-targeted drugs to treat obesity have not yet yielded good results. In this research, we used proteomics, in vivo model, and in vitro model to study a new TCM prescription to provide a new regimen to be used in the treatment of obesity. C57BL/6 mice were chosen as the in vivo model due to their genetic similarity to humans, their well-characterized metabolism, and their ability to replicate key features of human obesity, including diet-induced weight gain and associated metabolic dysfunctions.²⁸

Previous studies have indicated that the principal component of LAH is puerarin, which regulates lipid accumulation in OA-induced HepG2 cells through the AMPK pathway.¹⁴ Puerarin modulates inflammation by suppressing the TNF-α/NF-κB pathway in obese mice, presenting a potential treatment for obesity and its related complications.²⁹ In internet pharmacology, it is also revealed that kaempferol and quercetin in LAH are highly related to LAH against obesity. Kaempferol inhibits the activities of Akt, SREBP-1 and reduces mTOR protein expression, resulting in the stimulation of hepatic autophagy and consequent decrease in hepatic lipid content in dyslipidemia-induced mice.³⁰ Quercetin enhances hepatic insulin sensitivity, reduces hepatic fat content, and improves hepatic steatosis by regulating autophagy.³¹. Interestingly, these compounds belong to the flavonoid compounds, which have shown potential therapeutic effects against metabolic diseases such as obesity, type 2 diabetes, or NAFLD. Flavonoids act on multiple targets and affect various pathological processes, thereby enhancing their therapeutic efficacy.³² However, further studies are needed to validate the therapeutic effects of these compounds in humans, establish optimal dosages, and explore their synergistic roles.

Proteomics provided key insights into the mechanisms underlying LAH's therapeutic effects. LAH treatment reversed the upregulation of FASN (Fatty Acid Synthase), HMGCR (3-Hydroxy-3-Methylglutaryl-CoA Reductase), and SREBF1 (Sterol Regulatory Element-Binding Protein 1) induced by HFD, highlighting its role in suppressing lipid and cholesterol synthesis. FASN, a key enzyme catalyzing fatty acid synthesis, and HMGCR, the rate-limiting enzyme in the cholesterol synthesis pathway that converts HMG-CoA into mevalonate,³³ were significantly downregulated, reducing lipogenesis and cholesterol production. SREBF1, a transcription factor that regulates lipid metabolism genes including FASN and HMGCR, was also suppressed, further limiting lipid synthesis. Additionally, LAH treatment upregulated PRKAA1 and PRKAA2, which encode the catalytic subunits of AMPK, confirming activation of this central metabolic pathway.²⁹ Enhanced antioxidant enzyme activities (e.g., SOD, GSH, GPx, GR) and increased expression of SOD and GSTP1 were also observed, indicating LAH's ability to mitigate oxidative stress, a key factor in obesity-related metabolic dysfunction. Biochemical analyses further supported these findings. LAH significantly reduced serum levels of ALT, AST, triglycerides, cholesterol, and glucose in HFD-induced obese mice, indicating improvements in liver function and systemic metabolic profiles. Enhanced antioxidant enzyme activities, including GSH, GPx, and GR, were also observed in the liver, demonstrating LAH's ability to mitigate oxidative stress, a major contributor to obesity-related metabolic dysfunction. Histological validation provided direct evidence of LAH's effects at the tissue level. H&E staining revealed reduced lipid accumulation and inflammatory cell infiltration in the liver of LAH-treated mice, indicating an amelioration of hepatic steatosis and inflammation. ORO staining further demonstrated a significant decrease in lipid droplet size and density in both liver and adipose tissues, reflecting reduced triglyceride accumulation. These integrated findings suggest that AMPK plays a central role in mediating the therapeutic effects of LAH. AMPK activation suppresses lipogenesis through downregulation of FASN and SREBF1 while enhancing fatty acid oxidation, contributing to reduced lipid accumulation and improved metabolic profiles. Additionally, the reduction of oxidative stress and inflammation further supports LAH's systemic benefits in obesity management. Furthermore, to corroborate the findings of the proteomics analysis, in vivo models were employed alongside in vitro model assessments to evaluate the efficacy of LAH. An in vitro steatosis model was established using HepG2 (hepatoma cell line) and AML12 cells (a normal mouse hepatocyte cell line) cells with oleic acid. Compared to the model group, LAH significantly increased the phosphorylation of AMPK and ACC, as well as the protein expression of CPT1A, while reducing the levels of SREBP1 and FAS proteins, suggesting the inhibition of lipid accumulation. These results are consistent with those observed in the in vivo model and proteomics analysis results which further support the potential of LAH as a therapeutic agent against obesity and NAFLD.

Several limitations must be addressed to fully understand the therapeutic potential of LAH. The absence of an LAH-only group in normal mice hampers the determination of whether LAH can activate the AMPK signaling pathway under non-obese conditions. Future studies should include this group to determine whether the effects of LAH on AMPK activation are specific to obesity-induced metabolic dysfunction or also occur in healthy states. Additionally, as these results rely on animal models and in vitro studies, they may not be fully translatable to humans due to interspecies differences in metabolism and drug responses. Therefore, further research on the pharmacokinetics and pharmacodynamics of LAH in clinical settings are needed, and well-designed randomized controlled trials should be conducted to validate these findings in human populations.

Despite these limitations, the findings have important implications for obesity treatment and patient care strategies. LAH's ability to target multiple pathways offers a comprehensive approach to treating obesity and its associated comorbidities, such as NAFLD and dyslipidemia. Currently available anti-obesity drugs are GLP-1 receptor agonists or orlistat.³⁴ As a potential adjuvant therapy, LAH can be used as a supplement in the future and combined with existing anti-obesity drugs. We look forward to the LAH can enhance the efficacy of current treatments, address treatment resistance, and potentially reduce the doses of traditional medications required, thereby minimizing side effects. However, rigorous clinical trials are essential to confirm these benefits and further establish its role in obesity management.

5. Conclusion

This study explored the potential of LAH as a novel therapeutic option for obesity and related metabolic disorders. By integrating network pharmacology, proteomic, biochemical, and histological findings, this study demonstrated that LAH alleviates HFD-induced obesity through multi-target regulation of AMPK activation and metabolic pathways. These insights underscore the potential of LAH as a therapeutic agent for obesity and related metabolic dysfunctions.

Informed consent statement

Not applicable.

Author contributions

Chia-Jung Lee and Yu-Ju Chen designed the experiments, analyzed the data, and wrote the manuscript. Ching-Chiung Wang, De-Shan Ning, Kun-Teng Wang, Hong-Wei Zhao and Ming-Chung Lee analyzed the data. Ching-Chiung Wang, Wan Chun Chiua and Chiu-Li Yeh contributed to the experimental design and discussion. John Louie Dela Vega edited the manuscript. All the authors have read and agreed to the published version of the manuscript.

Institutional review board statement

Not applicable.

Data availability statement

Not applicable.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article: