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. 2026 Feb 25;24:303. doi: 10.1186/s12951-026-04153-3

Ergostane steroid, as one of the major contributor to cranberry derived extracellular vesicle nanoparticles, restores ovarian function of murine premature ovarian failure

Zeyu Cui 1,#, Yanchen Lai 1,#, Te Liu 1,✉,#, Lian Yang 1, Yongyi Huang 2
PMCID: PMC13041353  PMID: 41742181

Abstract

Background

A high-fat and high-sugar (HFHS) diet can induce ovarian insufficiency and premature ovarian failure (POF) by inducing the abnormal expression of multiple metabolic pathways that makes treatment difficult. We previously evaluated cranberry (Vaccinium, Va)-derived extracellular vesicle nanoparticles (Va-PENs) for their therapeutic potential in HFHS diet-induced POF in mice and herein examined, in greater detail, the molecular mechanism(s) underlying Va-PENs action in the treatment of the condition.

Methods

Va-PENs were isolated, purified, and subsequently fed to our HFHS-POF model mice. Conventional H&E and immunohistochemical staining methods were used to observe the pathologic changes and protein expression in the murine ovarian tissues. Liquid chromatography/tandem mass spectrometry high-throughput metabolomic analysis was used to investigate the distribution and content of various metabolites in Va-PENs, cranberry, and murine serum. Quantitative polymerase chain reaction and western-blot analyses were used to determine the mRNA and protein expression levels in cell proliferation and aging and mitochondrial function in ovarian granulosa cells of the model mice.

Results

Oral administration of Va-PENs significantly improved the pathologic aging status of ovarian granulosa cells in the HFHS-POF mice, reduced the proportion of atretic follicles, and corrected sex hormone (anti-Müllerian hormone, estradiol, and follicle-stimulating hormone) concentrations and blood lipid disorders. High-throughput metabolomic analysis revealed ergostane steroids (particularly campestanol) as key bioactive components shared between Va-PENs and cranberry. Oral delivery of campestanol replicated the therapeutic effects of Va-PENs, restoring granulosa cell morphology, inhibiting follicular atresia, normalizing sex hormone levels, and reducing cellular senescence markers by modulating the expression of the cell cycle/senescence pathway.

Conclusion

Our findings indicated that Va-PENs exerted potent therapeutic effects in mice with HFHS diet-induced POF. One of the potential mechanisms was ergostane steroid, as one of the major contributor to Va-PENs, significant reduction in HFHS-POF symptoms via modulating the expression of the cell cycle/senescence pathway. This study presents a safe, plant-derived, nanovesicle-based strategy for restoring ovarian function after the induction of POF.

Graphical abstract

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Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04153-3.

Keywords: Premature ovarian failure, High-fat and high-sugar diet, Cranberry-derived extracellular vesicle nanoparticles, Ergostane steroids, Plant metabolites

Introduction

Premature ovarian failure (POF) in women is defined by amenorrhea, hypoestrogenism, elevated gonadotropins, and an absence of mature follicles prior to the age of 40; and it constitutes a major contributor to female infertility [1–4]. Increasing evidence links POF to endocrine and metabolic disturbances. For example, a high-fat, high-sugar (HFHS) diet—in addition to elevating the risk of obesity, cancer, and cardiovascular disease—also impairs ovarian function and oocyte quality [3, 5, 6]. Despite this, there are no effective therapies for ameliorating HFHS-induced ovarian dysfunction.

With continuous progress in experimental research, an increasing number of investigators have reported that extracellular vesicles/exosomes derived from stem cells exert potential therapeutic effects on POF [3, 4, 6–9]. Exosomes are nanoscale vesicles (30–150 nm) enclosed by lipid bilayers and are key mediators of intercellular communication—capable of delivering proteins, lipids, and nucleic acids to target cells [3, 8–12]. Although exosomes derived from mammalian stem cells are excellent therapeutic materials, they reflect disadvantages such as long preparation time and high cost [8, 9, 12, 13]. In recent years, studies on plant-derived extracellular vesicle nanoparticles (PENs) have reflected an intense area of interest as nanomedicine delivery materials and disease treatments due to their high safety, low cost, preparation in large quantities within a short timeframe, and inherent therapeutic activity [3, 10, 11, 14–19]. Our recent work revealed that oral administration of cranberry (Vaccinium, Va)-derived extracellular vesicle nanoparticles (Va-PENs) attenuated POF in mice by modulating the gut microbiota and reducing granulosa cell PANoptosis (a convergence of pyroptosis, apoptosis, and necroptosis) [3]. However, the molecular biological mechanism underlying the therapeutic action of Va exos in POF remains unclear.

Vaccinium macrocarpon Ait (cranberry) is a herbaceous plant of the Rhododendron family and the Vaccinium genus, with spherical berries that are purplish-red in color [20–24]. Cranberries contain numerous active substances, with high concentrations of hyperoside, pentahydroxyflavonoid, proanthocyanidins, ellagic acid, phenolic acid, resveratrol, and myricetin [20–24]. Numerous studies have indicated that cranberry not only manifests significant antioxidant, anti-tumor, antiviral, and anti-infective functions but also protects the liver and gastrointestinal systems and relieves oral and gastric ulcers [20–24]. While Va-PENs have shown therapeutic efficacy in POF, the bioactive components responsible remain unknown. Given that PENs can deliver plant metabolites to recipient tissues, we hypothesized that Va-PENs exert their therapeutic effects in HFHS-induced POF by transferring specific metabolites. We herein therefore applied metabolomics to identify these metabolites and to evaluate the contribution of ergostane steroids, with the aim of clarifying a mechanistic basis for their reproductive benefits.

Materials and methods

Isolation and enrichment of Va-PENs

As previously reported [3], fresh cranberries were washed to remove debris, weighed, and incubated with cellulase (3 g/100 mL) and pectinase (0.2 g/100 mL) for 12 h. The mixture was centrifuged at 2800 × g for 20 min, and the supernatant was centrifuged again at 850 × g for 40 min at 4 °C to remove plant fibers. The clarified juice was concentrated to approximately 100 mL using a hollow-fiber module, layered over 7 mL of 0.971 M sucrose in an ultracentrifuge tube, and centrifuged at 150 000 × g for 90 min at 4 °C. The sucrose layer was collected, diluted in sterile PBS, and passed through an ultrafiltration tube to remove sucrose. Va-PENs were then resuspended in PBS and quantified by protein content using a BCA protein assay kit (Beyotime, Shanghai, China).

HFHS-POF model construction and treatment

Following established methods [3, 5, 6], female C57BL/6 mice (10 weeks old, n = 24) were purchased from the Experimental Animal Centre of Shanghai University of Traditional Chinese Medicine. Mice were randomly allocated to four groups (n = 8 per group). The control group (wild type [WT]) was fed standard chow without intervention. The HFHS + Va-PENs group was fed a high-fat diet (8 g/kg) and gavaged daily with 200 µL of 30% high-fructose solution plus 100 µL of Va-PENs at a concentration of 1 × 106 particles each. The HFHS + PBS group was fed a high-fat diet and gavaged daily with 200 µL of 30% high-fructose plus an equal volume of physiological saline. The HFHS + Untreated group (POF control group) was only fed a high-fat diet and gavaged daily with 200 µL of 30% high-fructose. All treatments lasted for 2 months. This study was approved by the Ethics Committee of Shanghai Institute of Geriatrics and Traditional Chinese Medicine (SHAGESYDW202009), and it complied with Chinese national regulations on animal experimentation.

Testing of blood lipids

Peripheral blood was collected, and serum levels of total cholesterol (TC), triglycerides (TGs), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) were measured by chemiluminescence according to the manufacturer’s instructions (Beyotime Biotechnology Co., Ltd., Zhejiang, China).

ELISA

Peripheral blood was centrifuged at 3000 rpm for 10 min to obtain serum. Concentrations of estradiol-17 (E2), follicle-stimulating hormone (FSH), and anti-Müllerian hormone (AMH) were determined using ELISA kits according to the manufacturer’s protocols (Beyotime Biotechnology Co., Ltd., Zhejiang, China).

Hematoxylin and Eosin (H&E) staining

Paraffin-embedded tissue sections were dewaxed, rehydrated through a graded ethanol series, stained with hematoxylin and eosin (H&E) for nuclei and eosin for cytoplasm, sealed with neutral resin, and examined under a light microscope.

Immunofluorescence staining

Paraffin sections were dewaxed, rehydrated, and blocked with immunohistochemical blocking buffer (Beyotime) for 30 min at 37 °C. The sections were subsequently incubated with primary antibodies for 45 min at 37 °C, washed three times, and then incubated with secondary antibodies under the same conditions. After washing, slides were sealed with an antifade mounting medium (Sigma-Aldrich, St. Louis, MO, USA) and imaged with a fluorescence microscope.

RNA extraction, reverse transcription, and quantitative polymerase chain reaction (qPCR)

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNase I (Sigma-Aldrich). RNA was quantified and reverse-transcribed into cDNA with a ReverTra Ace-α First Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan). qPCR was then performed on a RealPlex4 real-time PCR system (Eppendorf, Hamburg, Germany) using SYBR Green RealTime PCR Master Mix (Toyobo). Amplification consisted of 40 cycles at 95 °C for 15 s, 58 °C for 30 s, and 72 °C for 42 s. We calculated relative gene expression using the 2−ΔΔCt method, normalized to 18 S rRNA.

Western-blot analysis

Cells were lysed in 300 µL of lysis buffer on ice for 20 min and centrifuged at 10,000 × g for 10 min, and the supernatant was mixed with 6× SDS loading buffer and boiled at 100 °C for 10 min. Proteins were separated by SDS-PAGE, transferred to PVDF membranes, blocked with 5% BSA at room temperature, incubated with primary antibodies for 45 min at 37 °C, washed with TBST, and incubated with secondary antibodies for 45 min. Signals were detected using an ECL substrate (Beyotime Biotechnology Co., Ltd., Zhejiang, China).

LC-MS/MS analysis

We performed metabolomic analysis on a Waters ACQUITY™ I-Class PLUS UPLC system coupled to a Xevo G2-XS QTOF mass spectrometer (Waters, Milford, MA, USA). Samples were analyzed in positive and negative ion modes using an ACQUITY Ultraperformance (UPLC) HSS T3 column (1.8 μm, 2.1 mm ×100 mm). Mobile phases were 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) for both modes, and injection volume was 1 µL. Data were acquired in MSe mode, with low-collision energy at 2 V and high-collision energy ranging from 10 to 40 V.

Statistical analysis

All experiments were performed at least in triplicate. Data are expressed as mean±SD. Statistical comparisons were performed with Student’s t-test or one-way ANOVA using GraphPad Prism v7. One-way ANOVA was used to determine the homogeneity of the study groups. Also, a t-test was used to determine the probable differences between the participants who took the pre-test at baseline. In the Turkey’s Post-hoc test, One-way ANOVA was utilized to assess the differences among the three study groups regarding the mean scores of the study variables. A P value less than 0.05 was considered to be statistically significant.

Results

Va-PENs exhibited typical physicochemical properties of extracellular vesicle nanoparticles and contains abundant plant metabolites

We obtained a sufficient quantity of Va-PENs via mechanical disruption and gradient density ultracentrifugation (Fig. 1A). TEM and nanoparticle tracking analysis (NTA) confirmed that Va-PENs exhibited typical exosomal morphology, with particle diameters ranging from 80 to 240 nm (Fig. 1B, C) and possessing a zeta potential of −2.989 ± 0.757 mV (Fig. 1D). A majority of previous studies have primarily characterized plant exosomes by TEM morphology and particle size, with limited analyses of protein markers; and no consensus has been reached regarding their defining markers [10, 11, 17]. Therefore, following these reports [10, 11, 17], we assessed the RNA content and protein profile of Va-PENs. Triton X-100 treatment markedly elevated total RNA content in solution, confirming the presence of abundant encapsulated RNAs, a recognized characteristic of exosomes (Fig. 1E); and SDS-PAGE analysis showed that Va-PENs proteins were predominantly distributed in the 40–130 kDa range (Fig. 1F). To determine the composition of substances contained within the Va-PENs, we adopted LC-MS/MS high-throughput metabolomic analysis; and LC-MS/MS profiling revealed 1225 metabolites (Fig. 1G, Table Supplementary Data S1), with the most abundant categories (log10 peak value ≥ 4.0) being antibiotics (23%), acids (13%), esters (8%), and glycosides (6%) (Fig. 1H). Further subclassification identified high levels of 2’-norberbamunine (an antibiotic), N-acetylneuraminate (an acid), 22-hydroxydocosanoate (an ester), and secologanin (a glycoside) (Fig. 1I–K). HMDB-based prediction indicated that Va-PENs metabolites participated in metabolic pathways that included isoquinoline alkaloid biosynthesis, ABC transporters, porphyrin metabolism, and brassinosteroid biosynthesis (Fig. 1L). These findings confirmed that our Va-PENs exhibited typical extracellular-vesicle characteristics and encapsulated a diverse array of plant-derived metabolites.

Fig. 1.

Fig. 1

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Va-PENs possess typical exosomal characteristics and contain abundant plant metabolites. (A) Separation and enrichment protocol for Va-PENs. (B) Transmission electron microscopic analysis confirmed that Va-PENs exhibited typical exosomal morphology (scale bar = 500 nm). (C) Nanoparticle tracking analysis (NTA) showed that the particle size of Va-PENs ranged from 80 to 240 nm. (D) Zeta potential of Va-PENs was approximately −2.989 ± 0.757 mV. (E) Triton X-100 treatment markedly elevated the total RNA content in solution from Va-PENs. **p < 0.01 vs. Triton X-100 (untreated group, −) (t test; n = 3). (F) SDS-PAGE showed that the protein distribution of Va-PENs was principally between 40 and 130 kDa. (G) Heatmap of LC-MS/MS profiling revealed 1225 metabolites in Va-PENs. (H) Classification and content statistics of metabolites in Va-PENs (log10 peak value ≥ 4.0). (I) Positive mass spectrometric chromatogram of LC-MS/MS-detected metabolites in Va-PENs. (J) Negative mass spectrometric chromatogram of LC-MS/MS-detected metabolites in Va-PENs. (K) Primary and secondary classification of high-abundance metabolites in Va-PENs identified high levels of 2’-norberbamunine (an antibiotic), N-acetylneuraminate (an acid), 22-hydroxydocosanoate (an ester), and secologanin (a glycoside). (L) HMDB-based prediction indicated that Va-PENs metabolites participated in metabolic pathways that included isoquinoline alkaloid biosynthesis, ABC transporters, porphyrin metabolism, and brassinosteroid biosynthesis

Va-PENs improved signs of ovarian insufficiency in HFHS-POF mice

To evaluate whether Va-PENs ameliorated HFHS-induced POF, they were administered orally to the HFHS-POF model mice (Fig. 2A). H&E staining subsequently revealed that the ovarian interstitial spaces were enlarged and that granulosa cell staining was intensified; and we observed numerous atretic follicles in the HFHS-PBS group. In contrast, ovaries from Va-PENs-treated mice exhibited compact tissue architecture, normal granulosa cell morphology, and the frequent presence of healthy follicles (Fig. 2B, Figure Supplementary Data S1). Ovarian weight was significantly reduced in the HFHS-PBS and HFHS-untreated groups compared with WT controls, whereas Va-PENs treatment significantly increased ovarian weight relative to HFHS-PBS and HFHS-untreated groups (Fig. 2C). Follicle counts showed a higher proportion of atretic follicles in HFHS-PBS and HFHS-untreated group ovaries than in the WT group, while Va-exo administration markedly reduced this proportion (Fig. 2D).

Fig. 2.

Fig. 2

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Va-PENs significantly improve POF and dyslipidemia symptoms in HFHS-POF mice. (A) Treatment strategy for HFHS-POF mice with Va-PENs. (B) H&E staining revealed that ovarian interstitium was dense, that granulosa cell staining was complete, and that the number of atretic follicles was significantly reduced in the ovarian tissues of the Va-PENs group (magnification, 400×). (C) Ovarian weight was significantly elevated in the Va-PENs group compared with that in the HFHS-PBS and HFHS-untreated groups. **p < 0.01 vs. HFHS-untreated group (Un); ##p < 0.01 vs. WT group (one-way ANOVA). (D) Follicle counts showed a lower proportion of atretic follicles in the Va-PENs group relative to that in the HFHS-PBS, HFHS-PBS, and HFHS-untreated groups. **p < 0.01 vs. HFHS-untreated group (Un); ##p < 0.01 vs. WT group (t-test; n = 6, ΔΔp < 0.01; one-way ANOVA). (E) ELISA analysis demonstrated that AMH and E2 levels were significantly higher in the Va-PENs group than in the HFHS-PBS and HFSH-untreated groups. **p < 0.01 vs. HFHS-untreated group (Un); ##p < 0.01 vs. WT group; t-test; n = 6; ΔΔp < 0.01; One-way ANOVA. (F) Serum lipid analysis indicated that HDL was reduced and TGs and LDL were increased in the HFHS-PBS and HFHS-untreated groups and that these alterations were reversed in the Va-PENs group. **p < 0.01, *p < 0.05 vs. HFHS-untreated group (Un); ##p < 0.01, #p < 0.05 vs. WT group; t-test; n = 6; ΔΔp < 0.01; One-way ANOVA;. (G) Red fluorescence signal from DiI-labeled Va-PENs in ovarian cells of HFHS-POF mice (magnification, 400×). (H) Cell cycle/senescence pathway showing reduced blue fluorescence in ovarian tissue of the Va-exo group (magnification, 400×)

ELISA analysis demonstrated that AMH and E2 levels were significantly lower in HFHS-PBS and HFHS-untreated groups relative to the WT group but that the levels were restored in Va-PENs-treated mice. Conversely, FSH levels were elevated in HFHS-PBS and HFHS-untreated groups but significantly lowered in the Va-PENs treatment group (Fig. 2E). Serum lipid analysis also indicated that HDL was reduced and that TGs and LDL were increased in HFHS-PBS and HFHS-untreated groups compared with the WT group; importantly, these alterations were reversed in the Va-PENs group (Fig. 2F). According to the previous reports [25–27], in order to analyze the pharmacokinetic and biodistribution of Va-PENs in murine ovaries, Va-PENs have been labeled by fluorescent dye DiI. Subsequently, the mice were orally administered 100 µL of Va-PENs at a concentration of 1 × 106 particles of each once on one day. We found that red fluorescence distribution can only be detected in ovarian tissue on 72 h after the first oral administration of Va-PENs-Dil (Figure Supplementary Data S2A-S2C). When the oral administration was stopped, the red fluorescence in the ovaries can be maintained for about 6 days (Figure Supplementary Data S2A-S2C). Fluorescence microscopy of DiI-labeled Va-PENs confirmed their efficient uptake by ovarian tissues (Fig. 2G), and filipin staining revealed extensive blue fluorescence in HFHS-PBS ovarian tissues—indicating lipid accumulation; whereas Va-PENs treatment markedly diminished this signal (Fig. 2H). Collectively, these findings demonstrated that Va-PENs significantly improved lipid metabolism, ovarian reserve, and sex hormone balance in mice with HFHS diet-induced POF.

Va-PENs reduced ovarian granulosa cell senescence and improved mitochondrial function

SA-β-gal staining showed that the proportions of β-gal–positive cells (GPF+) in the ovarian tissues of HFHS-PBS and HFHS-untreated groups were significantly elevated compared to the WT group, whereas Va-PENs treatment markedly attenuated this proportion (Fig. 3A). Similarly, immunofluorescence analysis revealed that the proportion of γH2A.X–positive ovarian granulosa cells (γH2A.X+/AMH+) was significantly increased in the HFHS-PBS and HFHS-untreated groups compared with the WT group but that it was significantly diminished in the Va-PENs–treated group (Fig. 3B). In contradistinction, expression of the proliferation marker Ki67 exhibited an opposite pattern, as it was reduced in the HFHS-PBS and HFHS-untreated groups and restored in the Va-PENs group (Fig. 3B). These results indicated that Va-PENs mitigated ovarian granulosa cell senescence in HFHS-POF mice.

Fig. 3.

Fig. 3

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Va-PENs alleviate ovarian granulosa cells aging in HFHS-POF mice. (A) SA-β-gal staining showing a reduction in β-gal-positive cells in ovarian tissue (magnification, 400×). **p < 0.01 vs. HFHS-untreated group (Un); ##p < 0.01 vs. WT group; t-test; n = 6; ΔΔp < 0.01; One-way ANOVA; Magnification, 400×. (B) Immunofluorescence staining showing reduced γH2A.X-positive and increased Ki67-positive granulosa cells in the Va-PENs group. **p < 0.01 vs. HFHS-untreated group (Un); ##p < 0.01 vs. WT group; t-test; n = 6; ΔΔp < 0.01; One-way ANOVA; Magnification, 400×

In addition, mitochondrial membrane potential (MΨ) assays showed that JC-1 monomer fluorescence was elevated and aggregate fluorescence was reduced in HFHS-PBS and HFHS-untreated groups compared with the WT group, while Va-PENs treatment reversed these changes (Fig. 4A). Western-blot analysis demonstrated that the expression of mitochondrial complex subunits NDUFB (I), SDHB (II), UQCRC2 (III), COXIV (IV), and ATP5A1 (V) was significantly attenuated in the HFHS-PBS group compared to WT, but it was significantly higher in the Va-PENs group than in the HFHS-PBS group (Fig. 4B).

Fig. 4.

Fig. 4

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Va-PENs enhance mitochondrial function in ovarian cells of HFHS-POF mice. (A) JC-1 staining for the mitochondrial membrane potential (ΨM) assay showed that Va-PENs-treated ovaries had elevated JC-1 aggregate fluorescence but reduced monomer fluorescence (magnification, 400×). **p < 0.01 vs. HFHS-untreated group (Un); ##p < 0.01 vs. WT group; t-test; n = 6; ΔΔp < 0.01; One-way ANOVA. (B) Western blots showing increased expression of mitochondrial complex I–V markers in an ovary from the Va-PENs group. (C) Biochemical assays showing reduced LPO and MDA levels and increased SOD, GSH, and ATP levels in Va-PENs-group ovarian tissues. **p < 0.01, *p < 0.05 vs. HFHS-untreated group (Un); ##p < 0.01, #p < 0.05 vs. WT group; t-test; n = 6; ΔΔp < 0.01; One-way ANOVA

Biochemical assays revealed that the oxidative stress markers LPO and MDA were elevated in the HFHS-PBS and HFHS-untreated groups, whereas protective factors that included SOD, GSH, superoxide anion scavenging capacity, ATP, and pyruvic acid were reduced. However, Va-PENs treatment significantly diminished LPO and MDA levels while increasing all protective markers (Fig. 4C). Taken together, these findings demonstrated that Va-PENs improved mitochondrial membrane potential, preserved oxidative phosphorylation capacity, and enhanced overall mitochondrial function in ovarian tissues of mice with HFHS diet-induced POF.

Va-PENs encapsulated cranberry metabolites

To investigate the relationship between metabolites in Va-PENs and those in the parent cranberry (Va), we exploited LC-MS/MS to profile cranberry metabolites (Fig. 5A, B, Table Supplementary Data S1), and our analysis revealed that Va contained 1010 metabolites (Fig. 5C). Based on chemical classification, the most abundant categories (log10 [peak value] ≥ 4.0) were antibiotics (20%), acids (14%), glycosides (12%), esters (11%), and alcohols (9%) (Fig. 5D). Further subclassification identified high levels of 22-hydroxydocosanoate (an ester), decanoic acid (an acid), campest-4-en-3β-ol (an alcohol), mycobactin S (an antibiotic), and 5’-butyrylphosphoinosine (a glycoside) (Fig. 5E). Most metabolites were enriched in organooxygen compounds, fatty acyls, prenol lipids, and steroids or steroid derivatives (Fig. 5F). KEGG-pathway prediction indicated involvement in brassinosteroid biosynthesis, starch and sucrose metabolism, galactose metabolism, fatty acid biosynthesis, and related signaling pathways (Fig. 5G).

Fig. 5.

Fig. 5

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LC-MS/MS analysis of cranberry metabolites and their relationship with Va-PENs. (A) Positive mass spectrometric chromatogram of cranberry metabolites. (B) Negative mass spectrometric chromatogram of cranberry metabolites. (C) Heatmap of LC-MS/MS analysis revealed that cranberry contained 1010 metabolites. (D) Classification and content statistics of cranberry metabolites (log10 [peak value] ≥ 4.0). (E) Primary and secondary classification of high-abundance cranberry metabolites identified high levels of 22-hydroxydocosanoate, decanoic acid, campest-4-en-3β-ol, mycobactin S, and 5’-butyrylphosphoinosine. (F) Classification and statistics of high-abundance cranberry metabolites were enriched in organooxygen compounds, fatty acyls, prenol lipids, and steroids or steroid derivatives. (G) KEGG-pathway prediction indicated involvement in brassinosteroid biosynthesis, starch and sucrose metabolism, galactose metabolism, fatty acid biosynthesis, and related signaling pathways. (H) Overlap of metabolites between cranberry and Va-PENs showed 53 high-abundance common metabolites (log10 [peak value] ≥ 4.0). (I) Classification and content statistics of shared metabolites. (J) Primary and secondary classification of shared metabolites revealed that decanoic acid, campest-4-en-3β-ol, mycobactin S. tubulosine (biotins), and 22-hydroxydocosanoate were among the most abundantly shared compounds

Venn diagram analysis depicted 53 high-abundance metabolites common to both Va and Va-PENs (log10 [peak value] ≥ 4.0) (Fig. 5H). These shared metabolites were primarily antibiotics (21%), acids (15%), biotin compounds (9%), alcohols (9%), and esters (9%) (Fig. 5I). Detailed analysis revealed that decanoic acid, campest-4-en-3β-ol, mycobactin S, tubulosine (biotin), and 22-hydroxydocosanoate were among the most abundant shared compounds (Fig. 5I). Collectively, these results demonstrated that Va-PENs encapsulated a substantial proportion of cranberry-derived metabolites.

Va-PENs transfered metabolites to serum of recipient HFHS-POF mice

We performed a systematic LC-MS/MS analysis of plant-derived metabolites in the serum of HFHS-POF mice treated with Va-PENs (Fig. 6A, B, Table Supplementary Data S1), and our results revealed 932 metabolites in the mouse serum (Fig. 6C). Based on chemical classification, the most abundant categories (log10 [peak value] ≥ 4.0) were antibiotics (18%), biotin compounds (15%), acids (12%), esters (9%), and alcohols (8%) (Fig. 6D). Detailed categorization showed particularly high levels of taurocholate (an acid), gibberellin A12 (an antibiotic), 12-ethyl-8-isobutyl-3-vinylbacteriochlorophyllide d (biotin), 26-hydroxybrassinolide (an ester), and campest-4-en-3β-ol (an alcohol) (Fig. 6E). Most metabolites were concentrated in fatty acyls, prenol lipids, and steroids or steroid derivatives (Fig. 6F). HMDB-based functional prediction indicated that plant metabolites in the serum were significantly upregulated in pathways that involved fatty acyls, prenol lipids, and steroid derivatives; while pathways related to carboxylic acids, derivatives, and organooxygen compounds were downregulated (Fig. 6G).

Fig. 6.

Fig. 6

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LC-MS/MS analysis of plant metabolites in mouse serum. (A) Positive mass chromatogram of plant metabolites in serum. (B) Negative mass chromatogram of plant metabolites in serum. (C) Heatmap of LC-MS/MS analysis revealed 932 plant metabolites in the serum. (D) Classification and content statistics of plant metabolites in serum (log10 [peak value] ≥ 4.0). (E) Primary and secondary classification analysis showed particularly high levels of taurocholate, gibberellin A12, 12-ethyl-8-isobutyl-3-vinylbacteriochlorophyllide d, 26-hydroxybrassinolide, and campest-4-en-3β-ol in serum. (F) Classification and statistics of high-abundance plant metabolites (fatty acyls, prenol lipids, and steroids or steroid derivatives) in serum. (G) HMDB predictive analysis showed increased serum metabolites in pathways of fatty acyls, prenol lipids, and steroid derivatives. (H) HMDB predictive analysis of decreased serum metabolites in pathways of carboxylic acids, derivatives, and organooxygen compounds

To determine which cranberry metabolites were transferred to HFHS-POF mice via Va-PENs, we conducted intersection analysis of LC-MS/MS datasets from Va, Va-PENs, and serum. Spearman correlation analysis showed r values close to 1, indicating high reproducibility among replicates (Fig. 7A). Principal component analysis (PCA) revealed a clear separation between serum profiles and those of Va or Va-PENs (Fig. 7B). Venn diagram analysis primarily identified 43 metabolites common to all three datasets (Fig. 7C): antibiotics (21%), alcohols (12%), biotin compounds (12%), and acids (9%) (Fig. 7D). Further subclassification revealed that gibberellin A12 (an antibiotic), decanoic acid (an acid), campest-4-en-3β-ol (an alcohol), and tubulosine (a biotin) were present at particularly high levels (Fig. 7E). KEGG-pathway analysis indicated that these shared metabolites were enriched in signal-transduction pathways that included biosynthesis of secondary metabolites and other metabolic pathways (Fig. 7F). Prediction with LIPID MAPS further suggested involvement in lipid metabolism, particularly sterol [ST] and prenol lipids [PR] (Fig. 7G). HMDB predictions indicated key roles for ergostane steroid biosynthesis (steroids and derivatives), fatty acid and conjugate metabolism (fatty acyls), and carbohydrate metabolism (organooxygen compounds) (Fig. 7G). However, Through mass spectrometry identification, we found that both Va-PENs and Serum have their own unique metabolites. Among them, Va-PENs contained 29 metabolites, while serum contained 56 metabolites not present in the former. These metabolites were not transferred to the serum of HFHS-POF-treated mice via Va-PENs (Fig. 7C). This indicated that not all metabolites in Va-PENs were transferred to the mouse serum, suggesting the presence of a specific mechanism. Collectively, these findings indicated that Va-PENs delivered specific cranberry-derived metabolites—notably ergostane steroids and fatty acids—into HFHS-POF mice.

Fig. 7.

Fig. 7

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LC-MS/MS analysis of cranberry-derived metabolites in Va-PENs-treated HFHS-POF mice. (A) Spearman rank-correlation analysis of three LC-MS/MS datasets. (B) Principal component analysis (PCA) of three LC-MS/MS datasets. (C) Overlap of metabolites identified 43 metabolites common to all three datasets. (D) Classification and content statistics of shared metabolites. (E) Primary and secondary classification of shared particularly high-level metabolites were gibberellin A12, decanoic acid, campest-4-en-3β-ol, and tubulosine. (F) KEGG-pathway analysis indicated that these shared metabolites were enriched in signal transduction pathways that included biosynthesis of secondary metabolites. (G) LIPID MAPS prediction further suggested involvement of lipid metabolism, particularly sterol lipids and prenol lipids. (H) HMDB predictions indicated key roles for ergostane steroid biosynthesis (steroids and derivatives), fatty acid and conjugate metabolism (fatty acyls), and carbohydrate metabolism (organooxygen compounds)

Ergostane steroid (Campestanol), as one of the major contributor to Va-PENs, restored HFHS-POF mice ovarian function

Because the ergostane steroid (Campestanol) was as one of the major contributor to Va-PENs, to determine whether Va-PENs delivering ergostane steroids comprised one of the crucial mechanisms of therapeutic effects on POF, the HFHS-POF model mice were treated to campestanol by oral administration (Fig. 8A). H&E staining revealed that ovaries from the HFHS-ES group exhibited a more compact tissue architecture than those from the HFHS-PBS and HFHS-untreated groups, with granulosa cell morphology markedly restored; and we also noted fewer atretic follicles (Fig. 8B, Figure Supplementary Data S3). Ovarian weight was significantly greater in the HFHS-ES group compared with the HFHS-PBS and HFHS-untreated groups (Fig. 8C), and follicle counts showed a lower proportion of blocked follicles in the HFHS-ES group relative to the HFHS-PBS and HFHS-untreated groups (Fig. 8D).

Fig. 8.

Fig. 8

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Oral administration of ergostane steroids (campestanol) alleviated POF symptoms by modulating the cell cycle/senescence pathway. (A) Treatment strategy for HFHS-POF mice with ES (campestanol). (B) H&E staining revealed that ovarian granulosa cell morphology was markedly restored and that fewer atretic follicles were observed in ovarian tissue of the HFHS-ES group (magnification, 400×). (C) Ovarian weight was significantly greater in the HFHS-ES group compared to that in the HFHS-PBS and HFHS-untreated groups. **p < 0.01 vs. HFHS-untreated group (Un); t-test; n = 6; ΔΔp < 0.01; One-way ANOVA. (D) Follicle counts showed a lower proportion of blocked follicles in the HFHS-ES group relative to the HFHS-PBS and HFHS-untreated groups. **p < 0.01 vs. HFHS-untreated group (Un); t-test; n = 6; ΔΔp < 0.01; One-way ANOVA. (E) ELISA measurements indicated that serum AMH and E2 levels were significantly higher, while FSH levels were significantly lower in the HFHS-ES group compared with the control group. **p < 0.01 vs. HFHS-untreated group (Un); t-test; n = 6; ΔΔp < 0.01; One-way ANOVA. (F) SA-β-gal staining showed a reduced proportion of β-gal-positive cells in the HFHS-ES group ovary (magnification, 400×). **p < 0.01 vs. HFHS-untreated group (Un); t-test; n = 6; ΔΔp < 0.01; One-way ANOVA. (G) Immunofluorescence showing reduced γH2A.X-positive and increased Ki67-positive granulosa cells in the HFHS-ES group (magnification, 400×). **p < 0.01 vs. HFHS-untreated group (Un) (one-way ANOVA). (H) Western-blot analysis showing increased expression of mitochondrial complex I–V markers in HFHS-ES group ovarian tissue. (I) qPCR assay showed that the expression levels of p53, p21, and p16 in the HFHS-ES group were significantly lower than those in the HFHS-PBS group and HFHS-untreated group. **p < 0.01 vs. HFHS-untreated group; *p < 0.05 vs. HFHS-untreated group; t-test; n = 6; ΔΔp < 0.01; one-way ANOVA. (J) Western-blot assay showed that the expression levels of p53, p21, and p16 in the HFHS-ES group were significantly lower than those in the HFHS-PBS group and HFHS-untreated group

ELISA measurements indicated that serum AMH and E2 levels were significantly higher, while FSH levels were significantly lower in the HFHS-ES group compared with the HFHS-PBS and HFHS-untreated groups (Fig. 8E). SA-β-gal staining also showed a reduced proportion of β-gal-positive cells in the HFHS-ES group (Fig. 8F). Similarly, IF staining showed a decreased proportion of γH2A.X-positive granulosa cells, which was accompanied by an increased proportion of Ki67-positive granulosa cells in the HFHS-ES group (Fig. 8G). In addition, western-blot analysis revealed that the expression of mitochondrial complex subunits NDUFB (I), SDHB (II), UQCRC2 (III), COXIV (IV), and ATP5A1 (V) was significantly elevated in the HFHS-ES group compared to the HFHS-PBS group (Fig. 8H).

The results of our qPCR assay showed that the expression levels of cell senescence factors (p53, p21, and p16) in the HFHS-ES group were significantly lower than those in the HFHS-PBS group and HFHS-untreated group; while the expression levels of cell-cycle regulatory factors (Cdk1, Cdk2, Ccna, Ccne, and E2f1) and Bcl-2 were in the were the opposite of the above-mentioned genes (Fig. 8I). The tendencies manifested in our western blot assay and qPCR results were also consistent (Fig. 8J). Collectively, these findings suggested that oral administration of ergostane steroids (campestanol) markedly improved ovarian reserve and sex hormone profiles, while it delayed granulosa cell senescence in HFHS-POF mice by modulating the cell cycle/senescence pathway (Fig. 9).

Fig. 9.

Fig. 9

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The ergostane steroid, as one of the major contributor to Va-PENs, significant reduction in HFHS-POF symptoms via modulating the expression of the cell cycle/senescence pathway

Discussion

Growing evidence has revealed that abnormal glucose and lipid metabolism (such as that induced by an HFHS diet) profoundly alters ovarian structure and function and ultimately induces premature ovarian insufficiency and failure (POF) [3, 4, 28–32]. Our recent investigation revealed that oral administration of Va-PENs attenuated POF in mice by modulating their gut microbiota and reducing granulosa cell PANoptosis [3]. Compared with exosomes produced by animal cells, PENs exhibit lower immunogenicity and are inherently safe [3, 11, 16, 18, 19, 33–35]. The lipid and protein composition of the PENs membrane not only promotes cellular uptake and transport but also ensures complete degradation, thereby minimizing side-effects and toxicity to the greatest extent possible [3, 11, 16, 18, 19, 33–35]. Investigators have recently demonstrated the broad potential of plant-derived extracellular vesicles in the biomedical arena, highlighting their multifunctionality in drug delivery, intercellular communication, and disease-treatment interventions [3, 11, 16, 18, 19, 33–35]. Several PENs have been identified and reported to promote interspecific communication and to possess anti-cancer and anti-inflammatory properties [3, 11, 16, 18, 19, 33–35]. The aforementioned advantages reflect the great potential of PENs in the field of disease cell therapy. Our preliminary study revealed that the Va-PENs not only mitigated granulosa cell aging and injury but also reduced ovarian inflammation and corrected dyslipidemia. These results confirm that plant-derived extracellular vesicle nanoparticles—particularly Va-PENs—retain the therapeutic properties of their source plants—including lipid-lowering, anti-inflammatory, antioxidant, and anti-aging actions [3].

In this study, in order to analyze the pharmacokinetic and biodistribution of Va-PENs in murine ovaries, Va-PENs have been labeled by fluorescent dye DiI and were orally administered. We found that red fluorescence distribution can only be detected in ovarian tissue on 72 h after the first oral administration of Va-PENs-Dil. When the oral administration was stopped, the red fluorescence in the ovaries can be maintained for about 6 days. Then, since Va-PENs manifest significant efficacy in treating HFHS-POF, further elucidation of its underlying molecular mechanism (s) of action is crucial for its future development and application. Since PENs function as carriers that transport intracellular substances to extracellular destinations, it is highly plausible that Va-PENs exert their therapeutic effects by delivering specific bioactive compounds from cranberry into mice with HFHS diet-induced POF. To investigate this, we analyzed metabolites from Va-PENs, cranberry (Va), and serum from HFHS-POF mice treated with Va-PENs using LC–MS/MS; and our results showed that Va-PENs contained various plant-derived bioactive substances that included fatty acids, alcohols, carbohydrates, phytosterols, and other biologically active compounds essentially consistent with those detected in cranberry. A comparison with existing reports confirmed that the LC–MS/MS–identified substances corresponded closely to the known bioactive constituents of cranberry [20, 21, 23, 24]. Additional analysis revealed that Va-PENs, cranberry, and serum from Va-PENs-treated mice with HFHS diet-induced POF all contained plant-derived fatty acids, steroids, biotin, and carbohydrates—confirming that Va-PENs successfully transferred cranberry’s bioactive components into the HFHS-POF mice. The key question, however, is which of these compounds primarily mediate (s) the beneficial effects of Va-PENs on POF. By ranking and analyzing the shared metabolites among Va-PENs, cranberry, and serum from HFHS-POF mice treated with Va-PENs, we ascertained that ergostane steroids comprised the most abundant class. In the current analysis, Va-PENs were particularly rich in campestanol, campesterol, campest-4-en-3β-ol, and obtusifoliol. Plant sterols such as sitosterol and campesterol are widely used as cholesterol-lowering dietary supplements [36]; and campestanol is a phytosterol found in plant-based foods that has been shown to lower serum cholesterol and low-density lipoprotein levels and to alleviate metabolic disorders in mice [18, 34, 37, 38]. Phytosterols such as stigmasterol and campesterol have been proposed as potential inhibitors of steroidal 5α-reductase type 2 (an enzyme critical for the conversion of testosterone to dihydrotestosterone), suggesting their possible use in treating benign prostatic hyperplasia [14]. Campesterol ester derivatives have also elicited potent anti-inflammatory effects, reducing paw edema in experimental arthritis models while protecting tissues and improving organ function [39]. Moreover, meta-analyses have found no association between plant sterol intake and cardiovascular disease risk, thus supporting their safe application [15].

Our findings indicate that plant-derived ergostane steroids possess diverse therapeutic properties. In view of the ergostane steroid (Campestanol) was as one of the major contributor to Va-PENs, to test the hypothesis that Va-PENs ameliorate HFHS-POF by delivering ergostane steroids, we administered campestanol orally to mice with HFHS-POF, and our pathologic and molecular analyses supported this hypothesis. The ovaries of campestanol-treated mice with HFHS diet-induced POF showed that granulosa cell morphology was largely restored and that atretic follicle numbers were significantly reduced; while serum AMH and E2 levels were augmented and FSH levels lowered. The improvement of premature ovarian failure symptoms was only observed in the ergostane steroid (Campestanol) treatment group and the Va-PENs treatment group. However, there was no significant improvement in ovarian pathological symptoms and hormone levels in HFHS-POF mice (Untreated group) without any treatment. These results strongly suggest that Va-PENs improve ovarian insufficiency—in part by transporting ergostane steroids from cranberry to the HFHS-POF model mice.

Limitations and future perspectives

There are still some limitations to this study: firstly, in currently, the PENs have not yet been fully standardised, we can only speculate that they are PENs by detecting the total protein, total RNA encapsulation and release, as well as some physicochemical parameters. Therefore, the aforementioned issues also limit the further development and application of PENs. Secnodly, in view of the Va-PENs contained a complex mixture of metabolites (including fatty acids, sterols, biotin-related compounds, and sugars, etc.). Campestanol only partially reproduced the biological effects of Va-PENs. And, Campestanol was not an equivalent substitute for the full biological activity of Va-PENs, synergistic effects or contributions from other metabolites cannot be excluded. In this study, we planned to determine the effect of ergostane steroid (Campestanol) on restoration for ovarian function of HFHS-POF model mice because the ergostane steroid (Campestanol) was one of the major contributor to Va-PENs. Although independent administration of campestanol partially replicated the observed therapeutic outcomes, this did not confirm campestanol as the sole or principal active compound responsible for the effects we observed. We will therefore investigate the synergistic effects of other active compounds within Va-PENs in future studies. In addition, although we have revealed some molecular biology mechanisms, our research was still relatively superficial. For in-depth mechanism research, we will conduct detailed studies in the exploration of subsequent topics.

Conclusions

Our findings indicated that Va-PENs exerted potent therapeutic effects in mice with HFHS diet-induced POF. One of the potential mechanisms is ergostane steroid, as one of the major contributor to Va-PENs, significant reduction in HFHS-POF symptoms via modulating the expression of the cell cycle/senescence pathway. This study offers a safe, plant-derived, nanovesicle-based strategy for restoring ovarian function after POF induction.

Supplementary Information

Supplementary Material 1. (428.3KB, pdf)
Supplementary Material 2. (583.6KB, pdf)
Supplementary Material 3. (638.2KB, xlsx)
Supplementary Material 4. (1MB, pdf)

Acknowledgements

We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript. We declared no potential conflicts of interest.

Author contributions

Zeyu Cui, Yanchen Lai, Te Liu performed the majority of the experiments in the study. Lian Yang, Yongyi Huang, Te Liu contributed to the analysis of experimental data. Te Liu contributed to the study design, manuscript writing and provided experimental funding support. All authors read and approved the final manuscript.

Funding

This work was supported by grant from Traditional Chinese Medicine - traditional Chinese patent medicines and clinical evaluation platform (A1-U21-205-01010202).

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All experimental procedures were conducted in conformity with institutional guidelines for the care and use of laboratory animals and protocols, which were approved by the Animal Care and Use Committee of Shanghai Institute of Geriatrics and Traditional Chinese Medicine (SHAGESYDW202009).

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Zeyu Cui, Yanchen Lai and Te Liu contributed equally to this work and shared the first authorship.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1. (428.3KB, pdf)
Supplementary Material 2. (583.6KB, pdf)
Supplementary Material 3. (638.2KB, xlsx)
Supplementary Material 4. (1MB, pdf)

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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