Abstract

Background

   Purslane (Portulaca oleracea) is a medicinal and edible plant. Purslane
   extract (POEE) exhibits lipid-lowering, anti-inflammatory, and
   antioxidant properties. Traditionally, this extract has been used to
   treat various inflammatory conditions, including skin inflammation,
   enteritis, and dysentery. However, its therapeutic potential and
   molecular mechanisms in atherosclerosis (AS) remain unclear.

Methods

   Ultra-performance liquid chromatography-quadrupole/time-of-flight mass
   spectrometry (UPLC-Q/TOF-MS) and the Traditional Chinese Medicine
   Systems Pharmacology Database were employed to identify the active
   components of POEE. Network pharmacology was used to predict POEE’s
   mechanisms for alleviating AS. An in vitro foam cell model was
   established by treating RAW264.7 macrophages with oxidized low-density
   lipoprotein (ox-LDL), and the protective effects of POEE were assessed
   via the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide
   (MTT) assay, while intracellular lipid accumulation was identified
   using Oil Red O staining. Protein expression related to cholesterol
   metabolism was analyzed by Western blot (WB). For in vivo validation,
   AS was induced in rats through a high-fat diet and carotid artery
   injury. After 4 weeks of daily POEE administration, the therapeutic
   efficacy was tested by measuring serum lipid levels, cardiac function,
   histopathological changes, and the cholesterol transport-related
   protein expression.

Results

   The bioactive compounds identified in POEE were categorized into 10
   groups, including flavonoids (24), terpenoids (16), phenols (6), and
   alkaloids (4), and others. Network pharmacology predictions implicated
   POEE in modulating the “Lipid and Atherosclerosis” pathway. POEE
   significantly reduced total cholesterol (TC) and free cholesterol (FC)
   levels in ox-LDL-stimulated macrophages, attenuating foam cell
   formation. Furthermore, POEE enhanced reverse cholesterol transport
   (RCT) by upregulating the expressions of ATP-binding cassette
   transporters ABCA1 and ABCG1 to promote cholesterol efflux, while
   suppressing CD36 and MSR1 expressions to inhibit cholesterol uptake. In
   vivo, POEE administration lowered serum triglycerides (TG), TC, FC, and
   LDL-C levels; elevated HDL-C; and ameliorated carotid artery lesions in
   AS rats. Concordantly, ABCA1 expression was upregulated and that of
   MSR1 was downregulated in POEE-treated carotid tissues.

Conclusion

   POEE alleviates atherosclerosis by enhancing RCT through regulation of
   cholesterol efflux and uptake pathways. POEE may be a promising
   therapeutic candidate for AS.

   Keywords: Portulaca oleracea, bioactive ingredients, atherosclerosis,
   cholesterol metabolism, reverse cholesterol transport

1 Introduction

   Atherosclerosis (AS) is a chronic inflammatory disorder involving lipid
   accumulation, inflammation, cellular death, and fibrosis in the
   arterial tunica intima ([40]Williams et al., 2020). Currently, the
   primary mechanisms of prevention and management of AS focus on lipid
   regulation, anti-inflammation, and antihypertensive control ([41]Wang
   et al., 2019; [42]Devesa et al., 2023). Central to AS pathogenesis is
   foam cell formation—a consequence of dysregulated cholesterol
   homeostasis involving influx, esterification, and efflux imbalances
   ([43]Ren et al., 2021). The hallmark pathological feature of AS is
   lipid-rich plaques, characterized by abundant macrophage-derived foam
   cells within the arterial intima ([44]Zhao et al., 2019). Conversely,
   reverse cholesterol transport (RCT) exported excess cholesterol from
   foam cells to the liver for conversion into bile acids and subsequent
   fecal excretion ([45]Chen et al., 2023). Thus, RCT represents a
   critical and potentially pivotal defense mechanism in mitigating the
   progression of AS.

   RCT is a cholesterol metabolism pathway, and cholesterol efflux can
   remove free cholesterol from macrophages through active transfer or
   passive transmembrane diffusion mediated by cholesterol transporters
   ([46]Chen et al., 2023). Subsequently, HDL or apolipoprotein (Apo) A1
   captures and releases cholesterol. The cholesterol transporters,
   ATP-binding cassette (ABC) transporters (ABCA1 and ABCG1), play major
   roles in active free cholesterol efflux ([47]Bi et al., 2017).
   Macrophage scavenger receptor 1 (MSR1) and cluster of differentiation
   36 (CD36) are scavenger family A and B receptor proteins, respectively
   ([48]Bieghs et al., 2010). They are the primary receptors for
   phagocytosis and uptake of oxidized low-density lipoprotein (ox-LDL) by
   macrophages cells, accounting for 90% of the ox-LDL load of
   macrophages. This dynamic imbalance between ABCA1-/ABCG1-facilitated
   cholesterol export and CD36-/MSR1-dependent ox-LDL uptake constitutes a
   pivotal pathological mechanism driving foam cell transformation and
   atherosclerotic plaque progression ([49]Duan et al., 2017).

   Portulaca oleracea, widely distributed in temperate and tropical
   regions, is recognized by the World Health Organization as one of the
   most extensively used medicinal plants and a “global panacea”
   ([50]Zhang et al., 2020). Its phytochemical components include
   flavonoids, terpenoids, alkaloids, coumarin, organic acids, and
   polysaccharides ([51]Li et al., 2024). These bioactive constituents
   exhibited multi-target effects, including anti-inflammatory,
   anti-tumor, and antimicrobial activities and metabolic management
   ([52]Gao et al., 2020; [53]Park et al., 2019; [54]Noorbakhshnia and
   Karimi-Zandi, 2017). The traditional use of P. oleracea extract (POEE)
   was in treating inflammatory disorders including skin inflammation,
   enteritis, and dysentery ([55]Li et al., 2024). Emerging evidence
   supports that the P. oleracea plant has anti-atherosclerotic potential
   ([56]Wang et al., 2024; [57]Hao et al., 2024). Experimental studies
   demonstrated that the stem extract of P. oleracea has protective
   effects on hyperlipidemia ([58]El-Newary, 2016). In addition, Portulaca
   grandiflora, the plant belonging to the same family as P. oleracea,
   played an important role in attenuating atherosclerotic lesion
   progression in experimental models. However, the precise mechanisms
   underlying POEE action have not yet been fully elucidated. Therefore,
   the objective of the current study was to explore the potential role of
   POEE against AS, with particular emphasis on its modulation of RCT
   pathways. The flow chart of this study is shown in [59]Figure 1.

FIGURE 1.

   [60]FIGURE 1
   [61]Open in a new tab

   Flowchart of this study.

2 Materials and methods

2.1 Chemicals and reagents

   DMEM-high glucose culture medium was purchased from American Hyclone
   Inc., and fetal bovine serum was purchased from Gibco Co. (United
   States). Ox-LDL was obtained from Guangdong YiYuan Biotech Co. Ltd.
   (China) ([62]Liu et al., 2014). Shanghai Mclean Biochemical Technology
   Co. Ltd. (China) supplied the cell lysis buffer used for Western blot
   and immunoprecipitation (IP). Oil Red O dye was purchased from Sigma
   (United States). The TC and free cholesterol (FC) detection kits were
   purchased from Shanghai Yuan Ye Biotechnology Co. Ltd. The antibodies
   against ABCA1, ABCG1, CD36, and MSR1 were bought from Abcam Company
   (United States). Rutin (purity >98.0%), matrine (purity >98.0%), and
   glutamic acid (analytically pure) standards were purchased from Beijing
   Solarbio Technology Co. Ltd., Dalian Meilun Biotechnology Co. Ltd., and
   Sinopharm Chemical Reagent Co. Ltd., respectively.

2.2 Preparation of POEE

   Manual crushing and high-speed grinding created a fine powder. The
   powder was sifted with a 40-mesh sieve. Based on the extraction
   optimization process of POEE ([63]Kong et al., 2018), extraction was
   performed at the temperature of 50°C; 70% ethanol was used, with an
   extraction interval of 53 min and a solid-to-liquid ratio of 1:15
   (g/mL). POEE was obtained after filtrate drying and volume
   confirmation. The concentration of the purslane extract was prepared
   according to the mass concentration required for the test.

2.3 Content determination of active fractions in POEE

2.3.1 Determination of the total flavonoid content in POEE

   Two milliliters of POEE followed by 0.5 mL of 5% sodium nitrite was
   pipetted into a volumetric flask, shaken, and left to stand. After
   that, 0.5 mL of 10% aluminum nitrate was added and mixed. At the end of
   the reaction, 5 mL of 4% sodium hydroxide was added to the flask and
   was filled to volume. After mixing, the solution was left undisturbed
   for 20 min, which led to the formation of the POEE reaction mixture
   ([64]Yu et al., 2007). Using rutin as the standard, the total flavonoid
   content was assessed by measuring the mixture’s absorbance at 510 nm
   ([65]Wang et al., 2012).

2.3.2 Determination of the total alkaloid content in POEE

   The content of total alkaloid in POEE was determined according to
   [66]Gan et al. (2010). Matrine was conformed as the standard to measure
   the total alkaloid content.

2.3.3 Determination of the total amino acid content in POEE

   The content of total amino acid in POEE was determined according to
   [67]Wang (2006). In addition, the total amino acid content was measured
   using glutamic acid as the standard.

2.3.4 Chemical profiling of POEE based on UPLC-Q-TOF-MS/MS

   The parameter setting of UPLC-Q-TOF-MS/MS was referred to in [68]Zhang
   C. et al. (2023). The sample injection volume was 2 μL.

   A comprehensive score of 0.7 or more was used to identify the compounds
   in POEE by comparing the retention time, molecular weight (with an
   error of less than 10 ppm), secondary fragmentation spectra, collision
   energy, and other details. The methods employed for identification and
   statistical analysis of the compounds were comparable to those used in
   [69]Fei et al. (2022).

2.4 Network pharmacology analysis

2.4.1 Screening of active components

   All identified POEE components were input into the TCMSP as references