Introduction
Obesity, as a chronic and sophisticated modern global epidemic [
1], has been found to reduce life expectancy and increase the risk of numerous illnesses [
2]. Obesity prevalence in the United States increased from 30.5% to 42.4% between 1999 and 2018. Meanwhile, according to the most recent WHO report on obesity in Europe, 59% of adults and nearly one-third of children in Europe are overweight or obese. This imposes a substantial burden on millions of households [
3‐
5]. Early intervention against obesity is an appropriate approach to prevent complex multimorbidity [
6]. However, with certain anti-obesity medications producing mediocre outcomes and some reports of specific side effects, treating obesity is still difficult [
7]. Particularly, many of these medications exhibit a high incidence of adverse effects related to cardiovascular diseases, leading to their withdrawal from the market [
8], which further underscores the challenge of effectively managing obesity. Against this backdrop, there is growing interest in exploring the potential of Chinese herbal medicines derived from natural resources for the management of obesity and obesity-related diseases [
9‐
11].
Oleanolic acid (OA), a pentacyclic triterpenoid, is an active ingredient in herbs such as Fructus Ligustri Lucidi and Folium Camellia Sinensis, as well as in numerous common foods including olive leaves, Apple, Grape, Ginger, and Mango [
12]. In China, OA has been utilized as a hepatoprotective medication [
13], and there is clinical proof of its effectiveness in treating hyperlipidemia [
14]. Furthermore, multiple clinical studies have demonstrated the potential of various OA derivatives to prevent or treat a range of diseases, including cancer, diabetes, and viral infections [
13,
15,
16]. Previous research has identified OA as possessing diverse pharmacological properties, such as antiviral, antibacterial, anticancer, anti-inflammatory, antioxidant, hepatoprotective, and gastroprotective effects [
17‐
19]. OA has been shown to improve aberrant alterations in lipid parameters, reduce hepatic microvesicular steatosis, increase leptin content, significantly reduce visceral fat, improve glucose tolerance, and elevate insulin levels [
20,
21]. It also reduced systemic inflammation, promoted hepatic lipogenesis, and enhanced the taste perception of dietary fat in mice fed the HFD [
21]. Meanwhile, OA derivatives, such as Nano-OA, exhibit similar lipid-lowering effects [
22]. Although the hypolipidemic effect of OA has been confirmed [
23,
24], further research is required to elucidate the mechanisms underlying its potential role in combating obesity.
Network pharmacology is an important approach for investigating the biological effects of small molecules derived from various natural resources by constructing biomedical interaction networks to assess drug molecular mechanisms [
25,
26]. Particularly, network pharmacology is an efficient tool for identifying active ingredients and potential targets of Chinese medicine [
27]. Molecular docking and molecular dynamics techniques can provide an in-depth account of intermolecular interactions can graphically explain the mechanism of interactions, and have equally important applications in drug development [
28]. Consequently, combining molecular simulation-assisted validation with network pharmacological analysis screening is a useful approach to examine the anti-obesity mechanism of OA.
To shed light on the underlying mechanism of OA’s anti-obesity effect, the present study employs a combination of network pharmacology, molecular docking, and molecular dynamics simulation techniques to screen and analyze key targets and pathways. Moreover, in vivo experiments are conducted to validate the principal targets and pathways. As far as we know, this represents the first systematic investigation into the anti-obesity effect and mechanism of OA. The study findings will not only serve as an experimental foundation for utilizing OA as an anti-obesity agent but also offer a theoretical basis for the application of OA-derived drugs and food, such as olive oil, in the management and prevention of obesity.
Discussion
OA has demonstrated efficacy in the contexts of anti-inflammatory, hypoglycemic, anti-atherosclerotic, and diabetic treatments [
43‐
45]. A systematic review examining OA’s effects on metabolic syndrome-related indicators, such as central adiposity, lipid profiles, blood pressure, hyperglycemia, and biomarkers of oxidative stress, demonstrated its potential in regulating the lipid spectrum and treating insulin resistance and metabolic syndrome [
46]. In a previous study, we found that OA can reduce insulin resistance by lowering inflammatory cytokine levels [
47]. Moreover, OA has been shown to reduce adipose tissue inflammation by regulating macrophage infiltration and polarization, enhance taste perception, reduce systemic inflammation, promote hepatic adipogenesis, and increase lipid preference [
21,
44]. Short-term OA administration to neonatal rats can counteract fructose-induced oxidative stress without impacting long-term health [
48]. OA has also been found to regulate redox and PPAR
\(\gamma\) signaling to reduce PCB-induced obesity and insulin resistance [
43]. However, systematic research on its anti-obesity properties is lacking. As a result, we comprehensively investigated the mechanism of OA’s anti-obesity action in this study using network pharmacology, molecular docking, Molecular dynamics simulation and animal trials. We first analyzed and screened the core targets of OA exerting anti-obesity effects using network pharmacology, verified the binding effect of OA to the core targets using molecular docking methods, and further verified the binding stability and binding capacity using molecular dynamics simulations. At the same time, animal experiments were used to verify the core targets and pathways.
The current study is primarily focused on elucidating the underlying mechanism by which OA facilitates weight loss, which is based on the shared target of OA and obesity. A comprehensive search and screening of multiple databases yielded a total of 42 OA-obesity-associated targets, which were subjected to GO enrichment analysis and KEGG pathway enrichment analysis to better comprehend the correlation outcomes. The PPAR signaling pathway was identified as the key pathway based on the results of GO function and KEGG enrichment analysis. The PPAR signaling pathway has been widely recognized as the underlying mechanism of herbs’ lipid-lowering effects [
49‐
51]. The peroxisome proliferator-activated receptors (PPARs) are ligand-activated nuclear hormone receptors [
52] that can be activated by fatty acids and their derivatives to regulate the transcription of lipid metabolizing enzymes [
53]. PPARs consist of three isoforms, namely PPAR
\(\alpha\),
\(\beta\)/
\(\delta\), and PPAR
\(\gamma\) [
54], each of which plays a distinct role in lipid metabolism. PPAR
\(\alpha\) is primarily present in tissues with high energy demands, such as the liver, kidney, and heart [
55]. It mainly regulates the expression of downstream genes and proteins that are associated with lipid metabolism and liver metabolism [
56], and is a therapeutic target of fibrates (selective PPAR-agonists) [
57]. PPAR
\(\beta\)/
\(\delta\) is a critical regulator of muscle lipid homeostasis and is involved in systemic lipid regulation by
\(\beta\)/
\(\delta\) agonists (bezafibrate, telmisartan, etc.) [
58]. PPAR
\(\gamma\) is primarily located in adipose tissue [
59] and is a crucial mediator of energy balance and cell differentiation. It can promote adipose differentiation and lipid synthesis, leading to morphological changes and adipocyte enlargement [
60,
61]. Furthermore, it is a major regulator of glucose metabolism and a therapeutic target of type 2 diabetes medication thiazolidinediones (TZDs) [
53,
61].
Based on the PPI network, we can speculate that PPARG, PPARA, and several other molecules are the primary targets for OA-regulated lipids. PPARG(peroxisome proliferator-activated receptor gamma) and PPARA(peroxisome proliferator-activated receptor Alpha), the aforementioned PPAR
\(\gamma\) and PPAR
\(\alpha\), have long been recognized as critical regulators of obesity and are commonly employed in clinical treatment protocols61.MAPK3 (mitogen-activated protein kinase 3), also known as ERK1 (extracellular regulated protein kinases), is a class of intracellular serine/threonine protein kinases that are involved in various cellular processes such as cell proliferation, cell survival, cell growth, cell metabolism, cell migration, and cell differentiation [
62]. It plays a role in various diseases such as hepatic lipid metabolism, liver lipid metabolism, cardiac metabolic disorders, etc [
63,
64]. Prior research has demonstrated that Leptin, Protocatechuic Acid, and other molecules can combat obesity and atherosclerosis through MAPK3/ERK1 [
65,
66]. NR3C1 (Nuclear Receptor Subfamily 3 Group C Member 1), which encodes the glucocorticoid receptor, is involved in the inflammatory response, cell proliferation, and differentiation in target tissues [
67]. It has been reported to exert beneficial effects on obesity, impaired glucose metabolism, and dyslipidemia [
68‐
70]. Similarly, PTGS2(prostaglandin-endoperoxide synthase 2) and CYP19A1(cytochrome P450 family 19 subfamily A member 1) are also predicted to be pivotal target for reducing or reversing hyperlipidemia and obesity [
68,
71,
72]. Additionally, the CNR1 (cannabinoid receptor 1) has been identified as a promising drug target for the treatment of obesity, with CNR1 knockout in mice resulting in improvements in insulin resistance, ER stress, and lipid accumulation [
73]. Conversely, overexpression of HSD11B1 (corticosteroid 11-beta-dehydrogenase isozyme 1), which catalyzes the conversion of the active form of cortisol [
74], has been found to result in visceral obesity, insulin-resistant diabetes, and dyslipidemia [
75]. AGTR1 (angiotensin II receptor type 1) is a key player in the renin-angiotensin system (RAS) that can increase blood pressure and insulin resistance while also inhibiting lipolysis, maintaining energy homeostasis, and reducing inflammation [
76]. Molecular docking and molecular dynamic simulation were used to validate the molecular mechanism of OA intervention in obesity. The molecular docking studies revealed that OA has a high affinity for the key targets identified by network pharmacology. The results of molecular dynamics simulation revealed that OA binding to the core targets was all more stable, indicating that the previously hypothesized core proteins were indeed the critical linkages in OA’s anti-obesity action.
To validate the predicted targets and pathways of OA in treating obesity, we established a high-fat diet-induced obesity mouse model and investigated the effects of OA on inflammatory responses and glucolipid metabolism in mice. We also focused on validating the primary core target, peroxisome proliferator-activated receptor gamma (PPAR
\(\gamma\)), which was identified in our prediction through network pharmacology and molecular docking approaches. Our results demonstrate that OA treatment significantly ameliorated high-fat diet-induced metabolic dysfunction in the treated mice, as evidenced by lowered FBG levels, blood cholesterol levels, tissue damage, and reduced inflammatory response. Our findings are consistent with earlier studies [
17,
21,
43]. We also observed that high-fat feeding increased the expression of PPAR
\(\gamma\) in adipose tissues while decreasing the expression of mitochondrial uncoupling protein 1 (UCP1). Conversely, after OA treatment, the expression of PPAR
\(\gamma\) decreased, while that of UCP1 increased. UCP1, a downstream target of PPAR
\(\gamma\), is highly expressed in the mitochondria of brown adipose and beige adipose tissues [
77], and is involved in regulating energy expenditure and metabolic dynamic homeostasis through multiple cellular pathways, affecting the production of reactive oxygen species in adipocyte mitochondria [
78‐
80]. The increase in PPAR
\(\gamma\) and the decrease in UCP1 expression represent an increase in fat synthesis and storage, as well as a decrease in energy consumption in obese mice, which is consistent with the developmental mechanism of obesity. However, the negative effects of these changes were counteracted by the participation of OA. Our findings corroborate previous predictions from network pharmacology and molecular docking on OA’s anti-obesity targets and pathways. Overall, our results suggest that OA can help treat obesity by regulating PPAR
\(\gamma\) and activating the PPAR signaling pathway.
To gain a better understanding of the effects of OA on lipid accumulation in obese mice, we investigated the regulatory impact of OA on the expression of the proteins SREBP1 and TGR5. SREBP1 (Sterol-regulatory element binding proteins1), a key regulator of fatty acid metabolism, participates in lipid absorption, lipid synthesis, and saturated fatty acid oxidation, and is increased in obese hosts [
81,
82]. TGR5, the G-protein-coupled bile acid receptor, controls the metabolism of cholesterol, bile acids, fats, and carbohydrates, as well as insulin and systemic energy expenditure, and has been shown to boost metabolic rate, uptake of oxygen, and decrease obesity and hepatic steatosis in a mouse model of obesity [
83‐
85]. In line with the findings of PPAR
\(\gamma\)/UCP1, obese animals exhibited higher levels of SREBP1 and lower levels of TGR5, whereas OA was able to down-regulate SREBP1 and up-regulate TGR5 expression, suggesting that one possible anti-obesity strategy for OA would involve up-regulating TGR5/UCP1 to encourage fat consumption and down-regulating PPAR
\(\gamma\)/SREBP1 to decrease fat synthesis and alter lipid accumulation. Furthermore, GLP-1 is glucagon-like peptide-1, an enteroglucagon generated from the intestine that increases insulin secretion from pancreatic
\(\beta\)-cells [
86] while also decreasing food intake and body weight [
87]. It and its receptor have a significant potential for the therapy of glycolipid metabolic disorders. Our study revealed that OA treatment can increase the secretion of TGR5/GLP-1, which supports our previous finding that modulation of TGR5/GLP-1 can improve both inflammatory and glucolipid metabolism disorders. This finding suggests that OA’s ability to combat obesity may involve this mechanism.
In conclusion, this study combines network pharmacology, molecular docking, molecular dynamic simulation, and animal experimental verification for the first time to study the effectiveness and mechanism of action of OA in the treatment of obesity. The findings reveal the potential of OA in the treatment of obesity and provide a novel direction for the development of OA-derived drugs and food, thereby contributing to the discovery and development of natural resources related to obesity. Furthermore, this study explored the effects of medications on obesity through lipid accumulation, providing researchers with new insights for developing obesity drugs. Undeniably, there are some limitations in our study. Network pharmacology, molecular docking, and molecular dynamic simulation are reliant on data and algorithms, and their outcomes may differ from actual results due to database and software limitations. Additionally, due to time and resource limitations, we were unable to experimentally confirm all of the expected targets, or to combine animal and cell research with clinical validation and other methods, preventing us from fully revealing the anti-obesity mechanism of OA. We will conduct additional tests in the future to examine the potential molecular pathways behind the anti-obesity effects of OA in greater detail.