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Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve

  • Authors:
    • Yi Ying
    • Yue Wu
    • Ji Zhou
    • Yaming Wang
    • Hongtao Yin
    • Gang Lu
    • Ruolang Pan
    • Liangzhong Lv
  • View Affiliations / Copyright

    Affiliations: Department of Traditional Chinese Medicine, Xianju People's Hospital, Hangzhou Medical College, Taizhou, Zhejiang 317300, P.R. China, Center for Clinical Pharmacy, Cancer Center, Department of Pharmacy, Zhejiang Provincial People's Hospital (Affiliated People's Hospital), Hangzhou Medical College, Hangzhou, Zhejiang 310014, P.R. China, Department of Gynecology, Xianju People's Hospital, Hangzhou Medical College, Taizhou, Zhejiang 317300, P.R. China, Department of Psychology and Human Development, Institute of Education, University College London, London, WC1E 6BT, UK, Key Laboratory of Cell‑Based Drug and Applied Technology Development in Zhejiang Province, Institute for Cell‑Based Drug Development of Zhejiang Province, S‑Evans Biosciences, Hangzhou, Zhejiang 311122, P.R. China
    Copyright: © Ying et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 311
    |
    Published online on: September 15, 2026
       https://doi.org/10.3892/mmr.2026.14021
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Abstract

The development of more effective and affordable strategies for the treatment of diminished ovarian reserve (DOR) is of notable importance due to its increasing prevalence in young patients and the limited efficacy of current clinical interventions in restoring fertility. The present study investigated the efficacy of umbilical cord‑derived mesenchymal stem cell (UCMSC)‑derived exosome‑based hyperin delivery in ameliorating cyclophosphamide (CP)‑induced DOR in mice, and elucidated the potential underlying mechanisms of resulting therapeutic benefits. Exosomes were isolated from UCMSCs and encapsulated with hyperin using ultrasonication. The DOR mouse model itself was established via treatment with CP; these model mice were then treated with hyperin, UCMSC‑exosome controls or hyperin‑encapsulated UCMSC‑exosomes. After treatment, body weight, estrous cycles and general health were monitored. Ovarian tissues were evaluated histologically, and serum levels of anti‑Müllerian hormone, follicle‑stimulating hormone and estradiol were measured via enzyme‑linked immunosorbent assay. Ovarian expression of the autophagy markers LC3 II, Beclin‑1 and P62 was analyzed via western blotting. CP treatment led to a notable deterioration in general health, body weight and ovarian function, as evidenced by altered estrous cycles and reduced follicle counts. Treatment with hyperin‑loaded UCMSC‑exosomes resulted in improvements in body weight, the restoration of estrous cycles. Notably, compared with the model group, mice treated with hyperin‑loaded UCMSC‑exosomes demonstrated increased primordial follicle counts and decreased atretic follicle counts. Treatment with hyperin‑loaded UCMSC‑exosomes modulated autophagic activity within the ovaries, with reduced LC3‑Ⅱ and beclin‑1 levels and increased P62 levels following treatment with hyperin‑loaded UCMSC‑derived exosomes. UCMSC‑derived exosomes loaded with hyperin effectively mitigated the adverse effects of CP on ovarian function, potentially via the modulation of autophagic processes. These findings supported the therapeutic potential of engineered exosomes for treating DOR, highlighting a novel approach for fertility preservation and reproductive medicine. 
View Figures

Figure 1

UCMSC-derived exosomes achieve
effective hyperin drug delivery. (A) The morphology of UCMSCs in
passage 5 reached 90% confluence (scale bar, 50 µm). Then, the
culture medium was replaced with a specific lineage induction
medium. Representative inverted light microscopic images showing
cells 2–3 weeks post-lineage induction after staining with (B)
Alizarin Red S, (C) Oil Red O and (D) Alcian blue (scale bar, 50
µm). (E) Flow cytometry results indicated that UCMSCs were positive
for CD73, CD90 and CD105 and negative for CD19, HLA-DR and CD45.
(F) Representative transmission electron microscopy image of
hyperin-loaded exosomes (scale bar, 200 nm). (G) Western blot
assays revealed the positive expression of typical exosome markers
in UCMSC-derived exosomes. (H) Analysis of drug-loading capacity of
UCMSC-derived exosomes indicated that the exosomal loading rate of
hyperin was ~10% and remained stable within 24 h. (I) Analysis of
stability of hyperin-loaded exosomes in PBS, 10 and 50% mouse
serum. (J) Fluorescence microscopy was used to analyze the uptake
of PKH67-labeled exosomes (green signals) in KGN cells,
demonstrating that hyperin drug loading did not affect cellular
internalization (scale bar, 50 µm). EV-C, control extracellular
vesicle; EV-H, hyperin-loaded exosomes.

Figure 2

Evaluation of the therapeutic effect
of hyperin on a CP-induced DOR mouse model. (A) Experimental
schematic flow diagram, showing how mice were randomly assigned to
five treatment groups: Control, DOR, OA, EV-C and EV-H groups.
Therapeutic administration of hyperin was initiated on day 1 and
performed once every other day. On day 7, a weekly dose of CP was
administered to mice all groups except the control group via
intraperitoneal injection to induce DOR-like pathology (red arrow).
Mice were systematically monitored and a subset of mice were
sacrificed on day 29 (black arrow). (B) Body weights of mice across
the five treatment groups were recorded longitudinally at
designated time points throughout the 4-week experimental period.
*P<0.05 and **P<0.01, vs control. DOR, diminished ovarian
reserve; CP, cyclophosphamide; OA, oral administration; EV-C,
control extracellular vesicle; EV-H, hyperin-loaded exosomes.

Figure 3

Detection of estrous cycle changes
and sex hormone levels in mice of each group. (A) As the experiment
progressed, changes in the proportion of mice in each stage of the
estrous cycle were recorded across treatment groups. (B) By day 29,
a subset of mice in each group were sacrificed. Hormone levels in
the serum of each group of mice were detected and evaluated.
*P<0.05 and **P<0.01. DOR, diminished ovarian reserve; OA,
oral administration; EV-C, control extracellular vesicle; EV-H,
hyperin-loaded exosomes; AMH, anti-Müllerian hormone; E2,
estradiol; FSH, follicle-stimulating hormone.

Figure 4

Delivery of hyperin via exosomes
attenuates symptoms of cyclophosphamide-induced DOR in mice. (A)
Representative images of hematoxylin and eosin staining of ovaries
from mice in each treatment group (scale bar, 500 and 100 µm).
Black arrow, atretic follicle, blue arrow indicated antral
follicle, yellow arrow indicated secondary follicle, red arrow
indicated primary follicle, and green arrow indicated primordial
follicle. (B) Number of follicles of different grades in mice in
each treatment group. (C) The proportion of developing follicles in
each group. (D) The proportion of atretic follicles in each group.
(E) Ovarian index of mice in each group. n=5. (F) Comparison of
conception time of mice in each group. n=6. *P<0.05 and
**P<0.01. DOR, diminished ovarian reserve; OA, oral
administration; EV-C, control extracellular vesicle; EV-H,
hyperin-loaded exosomes.

Figure 5

Expression of LC3-II, beclin-1 and
P62 proteins in the ovaries of mice in each group. (A) Western blot
assays were conducted to evaluate LC3-II, beclin-1 and P62 protein
levels across treatment groups, and (B) grayscale values were
semi-quantitative analyzed using ImageJ. β-actin was used as an
internal reference. *P<0.05 and **P<0.01. DOR, diminished
ovarian reserve; OA, oral administration; EV-C, control
extracellular vesicle; EV-H, hyperin-loaded exosomes.

Figure 6

Exosome-based hyperin delivery
inhibited 4-HC-induced autophagy in KGN cells. KGN cells were
pre-treated with or without EV-C or EV-H for 48 h and then exposed
to 10 µM 4-HC for 24 h. (A) Protein levels of autophagy markers,
such as LC3-II, P62 and beclin-1, were detected by western blotting
and (B) semi-quantitatively analyzed using ImageJ. Mitochondrial
membrane potential was also assessed in different treatment groups
via JC-10 staining followed by (C) flow cytometry and (D)
microplate reader analysis. (E) Representative transmission
electron microscopy images for detecting mitophagic vacuoles in KGN
cells as signs of autophagy (scale bar, 1 µm). *P<0.05 and
**P<0.01. 4-HC, 4-hydroperoxycyclophosphamide; EV-C, control
extracellular vesicle; EV-H, hyperin-loaded exosome.
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Copy and paste a formatted citation
Spandidos Publications style
Ying Y, Wu Y, Zhou J, Wang Y, Yin H, Lu G, Pan R and Lv L: Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve. Mol Med Rep 34: 311, 2026.
APA
Ying, Y., Wu, Y., Zhou, J., Wang, Y., Yin, H., Lu, G. ... Lv, L. (2026). Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve. Molecular Medicine Reports, 34, 311. https://doi.org/10.3892/mmr.2026.14021
MLA
Ying, Y., Wu, Y., Zhou, J., Wang, Y., Yin, H., Lu, G., Pan, R., Lv, L."Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve". Molecular Medicine Reports 34.5 (2026): 311.
Chicago
Ying, Y., Wu, Y., Zhou, J., Wang, Y., Yin, H., Lu, G., Pan, R., Lv, L."Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve". Molecular Medicine Reports 34, no. 5 (2026): 311. https://doi.org/10.3892/mmr.2026.14021
Copy and paste a formatted citation
x
Spandidos Publications style
Ying Y, Wu Y, Zhou J, Wang Y, Yin H, Lu G, Pan R and Lv L: Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve. Mol Med Rep 34: 311, 2026.
APA
Ying, Y., Wu, Y., Zhou, J., Wang, Y., Yin, H., Lu, G. ... Lv, L. (2026). Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve. Molecular Medicine Reports, 34, 311. https://doi.org/10.3892/mmr.2026.14021
MLA
Ying, Y., Wu, Y., Zhou, J., Wang, Y., Yin, H., Lu, G., Pan, R., Lv, L."Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve". Molecular Medicine Reports 34.5 (2026): 311.
Chicago
Ying, Y., Wu, Y., Zhou, J., Wang, Y., Yin, H., Lu, G., Pan, R., Lv, L."Therapeutic effects of exosome‑based hyperin delivery in a cyclophosphamide‑induced mouse model of diminished ovarian reserve". Molecular Medicine Reports 34, no. 5 (2026): 311. https://doi.org/10.3892/mmr.2026.14021
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