1. Introduction
Hyperlipidemia is a complex and persistent metabolic disease caused by abnormalities in lipid homeostasis and is one of the most significant causative factors of cardiovascular disease, hypertension, and diabetes [
1]. The prevalence of hyperlipidemia is increasing worldwide each year, regardless of sex, age group, ethnicity, or race, with increasing mortality in aging populations, placing an additional burden on families, and posing a significant challenge to current healthcare systems [
2]. Hyperlipidemia is commonly caused by unhealthy diets and lifestyles, and owing to the adverse effects of hypolipidemic drugs, alternative treatments are currently attracting scholarly attention [
3]. Phytochemicals can be developed as natural, safe, and efficient hypolipidemic drugs because they are widely accessible. Phytochemicals with hypolipidemic effects include phytosterols, phenols, flavonoids, saponins, and alkaloids [
4].
With increasingly extensive and in-depth research on the hypolipidemic activity of flavonoids, it has been found that the consumption of flavonoid-rich foods can significantly reduce cholesterol levels and free radical scavenging capacity, thereby alleviating complications of hyperlipidemia [
5,
6]. The hypolipidemic activity of flavonoids is achieved by affecting multiple lipid metabolic pathways in the intestines and the liver and regulating imbalances in lipid metabolism, inhibiting lipid peroxidation and endogenous lipid biosynthesis, and promoting lipid redistribution and exogenous lipid metabolism. Consequently, a significant reduction of triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) levels occur [
7].
Flavonoids and polyphenols are widespread in the daily diet and are the primary phytochemicals found in vegetables, fruits, and tea [
8]. The hydroalcoholic extract of
Rosa roxburghii was found to have significant hypolipidemic effects [
9]. In recent years,
R. sterilis has received increasing attention as a close genetic relative of
R. roxburghii [
10]. He et al. have found that
R. sterilis contained rich polyphenols (79.39–108.4 mg GAE/g DW) and flavonoids (46.63–56.41 mg RE/g DW) in addition to essential elements, essential amino acids, and Vc. Moreover, the excellent antioxidant capacity was highly significantly correlated with the content of total flavonoids in
R. sterilis [
11]. Nevertheless, no research has been reported on the hypolipidemic effects of
R. sterilis. In the present study, to elucidate the key hypolipidemic components of RS and the mechanism of action for hypolipidemic activity, ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS), gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and molecular docking analysis were used to evaluate the hypolipidemic activity of
R. sterilis total flavonoid extract (RS) supplementation in the diet of an egg yolk powder-induced hyperlipidemic zebrafish model and investigate the mechanism of action. This study provides new ideas for RS intervention in hyperlipidemia and a theoretical basis for the additive value of
R. sterilis.
2. Materials and Methods
2.1. Preparation for RS
Fresh fruits of R. sterilis were obtained from Guizhou Lvyinhe Agricultural Development Co., Ltd. (Guizhou, China). The fresh fruits were washed and dried in an oven at 50 °C until constant weight. The dried whole fruits were crushed and sieved through a 40-mesh sieve. Subsequently, RS was extracted by ultrasound-assisted extraction at room temperature, with an extraction time of 70 min, an ethanol (AR, Ghtech, Guangzhou, China) volume fraction of 52%, and a material-to-liquid ratio of 1:23. The extraction was performed four times to obtain a high concentration of RS.
2.2. Zebrafish Experimental Design
The study protocol was approved by the Hunter Biotechnology Aquaculture Breeding Center (SYXK (Zhe) 2012-0171) and was accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All experiments were performed using melanin allele-mutant albino zebrafish bred in a naturally paired mating manner. The zebrafish were bred in water at 28 °C (200 mg of instant sea salt per 1 L of reverse osmosis water, 480–510 μS/cm conductivity, pH range of 6.9–7.2, and water hardness of 53.7–71.6 mg/L CaCO
3). Zebrafish were randomly selected and placed in six wells with 30 fish in each well and fed egg yolk powder (Bei**g Tianyuan, Bei**g, China) to establish a hyperlipidemia model. These zebrafish were grouped into five groups: model control, lovastatin (HPLC, Meilunbio, Dalian, China) treatment (0.081 μg/mL; 3 mL), and RS treatment (3, 10, and 30 μg/mL; 3 mL). After 2 days of drug administration, 10 zebrafish were randomly selected from each experimental group, stained with Oil Red O (ORO, Sigma-Aldrich, Shanghai, China), and photographed under a dissecting microscope (SZX7, Olympus Corporation, Tokyo, Japan). The images were analyzed with Image-Pro Plus version 6.0 image processing software, the vascular lipid optical density (S) of zebrafish was calculated, and the statistical results were expressed as mean ± SE. The rate of lipid reduction by RS was calculated using the following formula:
2.3. UPLC-Q-TOF-MS Analysis
Accurately weighed 0.050 g of RS was extracted with 10 mL of methanol (HPLC, Merck, Darmstadt, Germany) in an ultrasonic bath for 30 min. The mixture was filtered through a 0.22 μm nylon syringe filter (BKMAM, Changde, China) and the filtrate was transferred to an autosampler vial for analysis.
RS was analyzed in positive and negative ionization modes (UPLC, 1290; Q-TOF, 6550, Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was performed on an ACQUITY UPLC BEH C18 Column (2.1 mm × 100 mm × 1.7 μm; Waters, Milford, MA, USA). The column temperature was maintained at 28 °C, the flow rate of the mobile phase was 0.3 mL/min, and the injection volume was 5.0 μL. The mobile phase was composed of a 0.1% formic acid aqueous solution (A) and methanol (B). Elution was conducted using a linear gradient of 5–20% B within the first 10 min, 20–45% B within 10–30 min, 45–95% B within 30–35 min, 95–5% B within 35–36 min, and 5% B isocratic gradient elution for 36–37 min. The MS parameters were as follows: spray voltage, +4000 V/−3200 V; atomization temperature, 350 °C; sheath gas flow, 12 L/min; scan range, and m/z 50–1000.
High-accuracy precursor and product ions were obtained by UPLC-Q-TOF-MS, the elemental compositions were calculated, and the most reasonable molecular formula was obtained by comparing the previous literature and the ion breakage law of the compound.
2.4. Network Pharmacology Analysis of RS
The compounds identified by UPLC-Q-TOF-MS were used as the basis for web-based pharmacological analysis. The simplified molecular-input line-entry system strings of the above compounds were obtained from the PubChem database (
https://www.ncbi.nlm.nih.gov/, accessed on 27 September 2023) and uploaded to the SwissTargetPrediction database (
http://www.swisstargetprediction.ch/, accessed on 27 September 2023) to obtain their bioactivity targets [
12,
13]. The predicted targets were collated and imported into the UniProt database (
https://beta.uniprot.org/, accessed on 27 September 2023) to obtain standard gene names [
14]. Next, the keywords “hyperlipidemia” and “hypolipidemic” were used to obtain hyperlipidemia-related targets from the DisGeNET (
https://www.disgenet.org/, accessed on 27 September 2023), GeneCards (
https://www.genecards.org/, accessed on 27 September 2023), and Online Mendelian Inheritance in Man (OMIM) databases (
https://omim.org/, accessed on 27 September 2023) [
14,
15,
16]. The potential hypolipidemic targets of RS were screened by observing intersections with the standardized potential targets of compounds and “component-target-disease” interactions were established using Cytoscape.
The protein–protein interactions (PPI) network was generated by importing potential drug targets into the STRING database and the PPI network was constructed after filtering out datasets with minimum required interaction scores of less than 0.4 [
17]. The cytoHubba plugin in Cytoscape was utilized to calculate the degree value and filter the key target genes of the RS extract.
The GO biological processes and KEGG signaling pathways of key target genes of RS were annotated and visualized using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) (
http://david.abcc.ncifcrf.gov/, accessed on 28 September 2023) [
18]. With the background being set to Homo sapiens, data enrichment was performed using the hypergeometric test with
p < 0.01.
2.5. Molecular Docking Analysis
The reported 3D structures of key hyperlipidemia target proteins were retrieved from the PDB database (
https://www.rcsb.org/, accessed on 29 September 2023) [
19] and converted to a protein data bank (PDB) format using the OpenBabel program. The RS component structure files in the structure-data or MOL2 format were retrieved from the PubChem database (
https://pubchem.ncbi.nlm.nih.gov/, accessed on 29 September 2023) for hydrogenation and charging; the rotatable bond number was calculated [
12]. The co-crystal inhibitors of each protein target were re-docked to validate the reliability of the docking scheme before performing molecular docking on the new compounds. Semi-flexibly docked and processed RS compounds and key hyperlipidemia target proteins were analyzed using the AutoDock tool and the binding energy was calculated.
2.6. Statistical Analyses
Data obtained from the experiments were analyzed using GraphPad Prism 9 and the statistical significance of the results was analyzed by one-way ANOVA and Dunnett’s test. All experimental results are expressed as mean ± SE, and p < 0.05 was considered statistically significant.
4. Discussion
Flavonoids have demonstrated potential in the treatment of hyperlipidemia [
49,
50], but there are no studies on the hypolipidemic effects of RS. Therefore, we established a hyperlipidemic zebrafish model to evaluate the effects of RS in the treatment of hyperlipidemia. In this study, we demonstrated that RS exerts a pronounced hypolipidemic effect at concentrations between 10 and 30 μg/mL, showing superior hypolipidemic effects to lovastatin (0.081 μg/mL) in a dose-dependent manner. A total of 31 flavonoids were identified in this study, of which 22 compounds with biological activity were screened using active target screening. Topological analysis showed that RS16 and RS19 were enriched in most targets and highly correlated with core genes.
Hyperlipidemia is a typical chronic metabolic disease characterized by dyslipidemia and is caused by poor diet and lifestyle. High blood lipid levels in the body over a long period of time can directly or indirectly cause severe health complications, such as atherosclerosis, coronary heart disease, and pancreatitis [
51]. The current treatment for hyperlipidemia involves statins and fibrates, which are effective in treating hyperlipidemia; however, they can induce adverse effects such as respiratory infections and muscle pain [
52]. Natural products are useful adjuncts to conventional therapies for patients with metabolic disorders because of their low side effects. Therefore, based on the safety, health, and effectiveness of natural foods, the identification of novel hypolipidemic active ingredients has attracted attention [
53].
R. sterilis is a new type of healthy fruit that is rich in vitamin C, polyphenols, and flavonoids, and its juice can be consumed as a delicious drink. Evidence suggests that the
R. sterilis water extract confers a protective effect on biomolecules against free radical damage, which may be related to its high content of polyphenols and flavonoids [
54]. In addition, the survival rate of AHH-1 cells pretreated with the
R. sterilis flavonoid extract significantly increases 24 h after 5 Gy of 60Co irradiation [
55].
Natural products have great potential for the prevention and treatment of chronic diseases, owing to their multi-target modulatory capabilities [
56]. Recently, the widespread application of network pharmacology methods has provided new avenues for research on natural products [
57]. Dietary intake of flavonoids prevents obesity in healthy adults, and this positive effect is strongly associated with anthocyanins and proanthocyanidins; both RS16 and RS19 identified in this study belong to the anthocyanin family [
58]. Anthocyanins have been found to effectively reduce the levels of blood TG, TC, and LDL-C as well as non-esterified fatty acids. Additionally, anthocyanins have been shown to increase high-density lipoprotein cholesterol (HDL-C) levels, regulate the expression of proteins such as PPARγ, CCAAT/enhancer-binding protein (C/EBPs), and 2 homolog 1 (SIRT1), thereby alleviating atherosclerotic dyslipidemia [
59,
60]. Several active compounds in RS have been confirmed to exert strong hypolipidemic effects. RS17 has been widely used to treat chronic metabolic diseases in humans since ancient times. RS17 significantly reduces the serum levels of TC, TG, LDL-C, and very low-density lipoprotein cholesterol (VLDL-C) and markedly improves HDL-C levels [
61]. Zebrafish experiments in this study have also demonstrated the strong potential of active ingredients in RS to reduce blood lipid levels.
PPI network analysis showed that TNF, EGFR, VEGFA, PPARA, ESR1, HSP90AA1, MMP9, NR3C1, IGFBP3, and CYP19A1 were critical targets for RS-mediated hypolipidemic effects. TNF is an essential target for many chronic metabolic diseases and decreases lipoprotein lipase activity, thereby increasing serum TG levels [
62]. EGFR deficiency could limit lipid uptake, attenuate the inflammatory response, and impede the development of atherosclerosis. Conversely, the activation of EGFR will lead to the activation of the PI3K/AKT/mTOR signaling pathway, which plays an important role in pathophysiological processes such as hyperlipidemia and atherosclerosis [
63]. VEGFA, a member of the VEGF family, plays crucial roles in angiogenesis, regulation of vascular permeability, and maintenance of vascular physiological functions. Additionally, VEGFA is directly associated with the regulation of obesity [
64]. PPARA is a key transcription factor in lipid homeostasis, certain hepatic detoxification processes, and inflammation control, and the results of multiple studies suggest that many lipid-lowering drugs act by binding to and inducting PPARA [
65]. ESR1 mediates the physiological functions of estrogen and is associated with arterial hypertension, changes in blood lipid levels, coronary atherosclerosis, and changes in HDL-C levels in postmenopausal women [
66]. High lipid accumulation is typically accompanied by oxidative stress, and the downregulation of HSP90AA1 expression promotes nuclear factor erythroid 2-related factor 2 (Nrf2) activation and inhibits NF-κB expression in plaques, thereby exerting a hypolipidemic effect [
67]. MMP9 is secreted by vascular endothelial cells, smooth muscle cells, M lymphocytes, and T lymphocytes in hyperlipidemia-induced atherosclerotic plaques, and its overactivation leads to extracellular mesenchymal disruption, potential pathological remodeling, and restenosis [
68]. NR3C1 regulates genes involved in the control of development, metabolism, and immune responses and can regulate the flow of TGs to the liver through the angiopoietin-like 4 (ANGPTL4) pathway, thereby exerting hypolipidemic effects [
69]. IGFBP3 is the most abundant insulin-like growth factor in the serum. Its circulating levels closely correlate with daytime growth hormone secretion, reflecting spontaneous growth hormone secretion in healthy individuals. Moreover, reduced levels of IGFBP3 are associated with an increased risk of cardiovascular disease, including coronary artery disease and cardiovascular disease mortality [
70]. CYP19A1 regulates cholesterol-mediated organs of steroidogenesis. Its ability to regulate steroid hormone biosynthesis, thyroid hormone signaling pathways, and bile secretion is inextricably intertwined with lipid metabolism in the human body [
71].
The docking results of the active ingredients identified by UPLC-Q-TOF-MS with key proteins showed that RS1 with HSP90AA1 had the highest docking activity among all the ingredients of RS; docking activities of RS1, RS3, RS11, and RS22 with PPARA and of RS1, RS19, RS21, and RS22 with MMP9 were high. These results suggest that the active ingredients of RS directly bind to HSP90AA1, PPARA, and MMP9 to activate NF-κB, thus maintaining lipid homeostasis in the body [
72,
73]. These targets are strongly associated with human lipid levels and were the core targets enriched in this study, suggesting that they are potential targets for RS to exert hypolipidemic effects.
In the GO enrichment analysis, 41 intersecting genes were significantly associated with hypolipidemia-related metabolic biological processes, including the intracellular steroid hormone receptor signaling pathway, positive regulation of cholesterol efflux, and carbohydrate metabolic process. In addition, KEGG analysis revealed that metabolic pathways associated with cancer might play an important role in hypolipidemic effects, such as chemical carcinogenesis, receptor activation, pathways in cancer, prostate cancer, and proteoglycans in cancer. Alterations in lipid metabolism, which have been commonly disregarded in the past, are now accepted as hallmarks of cancer. Based on previous experimental observations, hyperlipidemia is strongly correlated with cancer, with common or partial hormonal metabolic mechanisms indicating that they have common drug targets [
74]. Cholesterol plays a vital role in key cellular processes and functions, particularly in cell membrane production. Cancer cells require high levels of cholesterol to increase cell differentiation for uncontrolled reproduction, thereby causing hyperlipidemia; therefore, current treatments for cancer generally prioritize cholesterol-limiting bioinhibitors [
75]. Furthermore, signaling pathways directly related to lipids were enriched in adipocyte lipolysis regulation as well as in lipids and atherosclerosis. These signaling pathways modulate the activity of lipolytic enzymes and auxiliary proteins in the body and adjust the rate of lipolysis via hormonal and biochemical signals, allowing for a maximal response of the adipose tissue to energy demand and availability [
76]. In this study, the above pathways were significantly enriched and demonstrated that RS can treat hyperlipidemia by modulating the above signaling pathways.