Mesenchymal stem cells derived extracellular vesicles ameliorate ovarian aging through inhibiting LGALS3BP/NF-κB induced inflammation.

EVs characterization—including identity, size distribution, and purity was performed using Western blot and transmission electron microscopy (TEM). CD63 and Alix were enriched in UCMSC-EVs (Fig. S1A), and the particles displayed the characteristic cup-shaped morphology with diameters of 20–150 nm (Fig. S1B). After 4 times of UCMSC-EVs treatment, the ovarian tissues of 60-week-old aging-mice were isolated. The flowchart of the detailed experimental procedure is displayed in Fig.  1 A. Macromorphological assessment of the ovary under visible light showed that the ovarian volume and weight of mice received UCMSC-EVs treatment were significantly increased when compared with those in natural 60-week-old aging mice (Fig.  1 B, S2 A and B). Simultaneously, HE staining showed that it was clearly observed numerous blood cells in the ovarian follicles of 60-week-old aging mice, and Masson staining reveals that the ovarian stroma has undergone atrophy, exhibiting numerous vacuolar structures and a markedly increased level of fibrosis. However, in the 60-week-old mice subjected to UCMSC-EVs intervention, the ovarian morphology remained intact, with no obvious signs of stroma atrophy or fibrosis (Fig.  1 C, D). Furthermore, WB results indicated that natural 60-week-old aging mice showed markedly reduced Anti-müllerian hormone (AMH), follicle-stimulating hormone receptor (FSHR), estrogen receptor alpha (ER-α), and cytochrome p450 family 19 subfamily a member 1 (CYP19A1) levels, indicating diminished reserve, responsiveness, and steroidogenic capacity. In contrast, ovarian function in the 60-week-old mice subjected to UCMSC-EVs intervention shows significant improvement (Fig.  1 E, F). Additionally, the expressions of aging-related proteins p53, p16, p21, Bax and Bcl-2 in the ovary were detected by WB. The results indicated that, compared to naturally aged 60-week-old mice, UCMSC-EVs treatment significantly delayed ovarian aging and enhanced anti-apoptotic capacity in 60-week-old mice (Fig.  1 G, H and I). Moreover, aging cells secrete pro-inflammatory factors such as IL-1β, IL-6, and TNF-α, which activate more immunocytes, exacerbating tissue damage and further accelerating the aging process. As expected, after UCMSC-EVs intervention, the levels of pro-inflammatory factors in the ovaries were significantly reduced, while the expression of anti-inflammatory factors was enhanced (Fig.  1 J and K). These results suggested that UCMSC-EVs transplantation can effectively delay ovarian aging though reducing ovarian inflammation. Fig. 1 UCMSC-EVs improved ovarian aging by reducing ovarian inflammation. A Scheme of animal experiment. Mice ( n  = 5) were administered UCMSC-EVs at 44, 48, 52, and 56 weeks, and sacrificed on 60 weeks, Mice ( n  = 5) were received saline at the corresponding ages as control. Ovarian tissue was collected at the end of experiment. B The Macroscopic ovarian sizes were observed under visible light. C Representative images of HE stained ovarian tissue sections. Scale bars = 100 μm. D Representative images of Masson stained fibrosis level of ovarian stroma. Scale bars = 100 μm. E Expressions level of ER-α, CYP19A1 FSHR and AMH of the whole ovary from 60 W and 60 W-UCMSC-EVs mice were determined by the western blot. F Densitometric analysis of band intensities of western blots are shown in ( E ). G Expressions level of p53, p16, p21, Bax and Bcl-2 of the whole ovary from 60 W and 60 W-UCMSC-EVs mice were determined by the western blot. H , I Densitometric analysis of band intensities of western blots are shown in ( G ). J Expressions level of IL-10, IL-6, IL-1β and TNF-α of the whole ovary from 60 W and 60 W-UCMSC-EVs mice were determined by the western blot. K Densitometric analysis of band intensities of western blots are shown in ( J ). Data are presented as means ± SEM values. * p  < 0.05; ** p  < 0.01; *** p  < 0.001. Note: The HE and Masson images are derived from representative sections of the same experimental groups and are presented to optimally demonstrate the respective pathological features UCMSC-EVs improved ovarian aging by reducing ovarian inflammation. A Scheme of animal experiment. Mice ( n  = 5) were administered UCMSC-EVs at 44, 48, 52, and 56 weeks, and sacrificed on 60 weeks, Mice ( n  = 5) were received saline at the corresponding ages as control. Ovarian tissue was collected at the end of experiment. B The Macroscopic ovarian sizes were observed under visible light. C Representative images of HE stained ovarian tissue sections. Scale bars = 100 μm. D Representative images of Masson stained fibrosis level of ovarian stroma. Scale bars = 100 μm. E Expressions level of ER-α, CYP19A1 FSHR and AMH of the whole ovary from 60 W and 60 W-UCMSC-EVs mice were determined by the western blot. F Densitometric analysis of band intensities of western blots are shown in ( E ). G Expressions level of p53, p16, p21, Bax and Bcl-2 of the whole ovary from 60 W and 60 W-UCMSC-EVs mice were determined by the western blot. H , I Densitometric analysis of band intensities of western blots are shown in ( G ). J Expressions level of IL-10, IL-6, IL-1β and TNF-α of the whole ovary from 60 W and 60 W-UCMSC-EVs mice were determined by the western blot. K Densitometric analysis of band intensities of western blots are shown in ( J ). Data are presented as means ± SEM values. * p  < 0.05; ** p  < 0.01; *** p  < 0.001. Note: The HE and Masson images are derived from representative sections of the same experimental groups and are presented to optimally demonstrate the respective pathological features Additionally, the morphology of endometrium is regulated by ovarian hormone levels, indirectly reflecting ovarian function. Therefore, we collected uterine tissues of mice and performed HE and Masson staining. The results showed that in 60-week-old mice, the endometrial glands displayed significant dysplasia, cystic dilation, irregular shapes, and columnar glandular epithelium arranged in a pseudo-layered or layered pattern (Fig. S3 A). Moreover, the level of endometrial fibrosis was significantly increased (Fig. S3 B). In contrast, in 60-week-old mice treated with UCMSC-EVs intervention, the glandular structure of the endometrium was more regular, with a greater number of glands and a lower level of fibrosis (Fig. S3 A-D). To further investigate the potential mechanisms by which UCMSC-EVs improve ovarian function in aged mice, RNA-seq analysis was conducted on ovarian tissues between the 60-week-old with and without UCMSC-EVs treatment, identifying a total of 932 differentially expressed genes (DEGs). Enrichment analysis of these DEGs, including heatmaps and volcano plots of gene expression and gene ontology (GO) enrichment analysis (Fig.  2 A-E), indicated that UCMSC-EVs reversed age-associated inflammatory signatures, as evidenced by the marked down-regulation of pathways related to inflammatory response in treated ovaries compared with naturally 60-week-old mice (Fig.  2 F). Fig. 2 UCMSC-EVs improved ovarian aging in vivo by inhibiting inflammation. A Heatmap indicated differential genes expression (DEGs) in ovarian tissues between 60 W and 60 W-UCMSC-EVs mice. B Volcano plot showing the DEGs in ovarian tissues between 60 W and 60 W-UCMSC-EVs mice. Green dots represent downregulated DEGs, and red dots represent upregulated DEGs. C Cellular component related differential pathways based on above-screened DEGs by GO analysis. D Biological process related differential pathways enriched based on above-screened DEGs mice by GO analysis. E Molecular function related differential pathways enriched based on above-screened DEGs by GO analysis. F Heatmap indicated changes of inflammation-related DEGs in ovarian tissues between 60 W and 60 W-UCMSC-EVs mice UCMSC-EVs improved ovarian aging in vivo by inhibiting inflammation. A Heatmap indicated differential genes expression (DEGs) in ovarian tissues between 60 W and 60 W-UCMSC-EVs mice. B Volcano plot showing the DEGs in ovarian tissues between 60 W and 60 W-UCMSC-EVs mice. Green dots represent downregulated DEGs, and red dots represent upregulated DEGs. C Cellular component related differential pathways based on above-screened DEGs by GO analysis. D Biological process related differential pathways enriched based on above-screened DEGs mice by GO analysis. E Molecular function related differential pathways enriched based on above-screened DEGs by GO analysis. F Heatmap indicated changes of inflammation-related DEGs in ovarian tissues between 60 W and 60 W-UCMSC-EVs mice Based on in vivo experimental results and RNA-seq analysis, this study subsequently employed D-gal to induce senescence in ovarian GCs (KGN and SVOG), thereby constructing an in vitro aging model. Initially, the optimal concentration (150mM) and exposure duration (48 h) for D-gal-induced aging damage in KGN and SVOG cells were determined using the CCK-8 assay (Fig.  3 A and B). Following this, UCMSC-EVs were administered to assess their efficacy in ameliorating cellular damage of KGN and SVOG. Flow cytometry results indicated that D-gal significantly induced apoptosis in cells, particularly late-stage apoptosis; however, treatment with UCMSC-EVs markedly improved the cell apoptosis induced by D-gal (Fig.  3 C and D). Consistent with the flow cytometry findings, viability assays utilizing live/dead staining demonstrated that in cells treated with D-gal, red fluorescence-labled cells (indicating apoptotic cells) significantly increased, while green fluorescence-labled cells (indicating live cells) decreased compared to the cells in control group. Conversely, following UCMSC-EVs treatment, there was a reduction in red fluorescence-labled cells and an increase of green fluorescence-labled cells (Fig.  3 E and F). Additionally, WB analysis revealed that D-gal induction significantly upregulated the expression of senescence-associated proteins (p53, p16 and p21) in KGN and SVOG cells, while downregulating the expression of proliferation-associated proteins (Kiel 67 (Ki67), Lamin B1). While treatment with UCMSC-EVs significantly decreased the levels of senescence-associated proteins and upregulated the expression of proliferation-associated proteins ( P  < 0.05) (Fig.  3 G, H and I). These findings indicated that UCMSC-EVs treatment markedly improved the viability of ovarian GCs injured by D-gal. Fig. 3 UCMSC-EVs ameliorated D-gal-induced apoptosis and inflammation in GCs. A , B CCK-8 assay to measure KGN and SVOG cells viability under different D-gal concentrations. C , D KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs and then stained with annexin V-FITC/PI were determined using flow cytometry. E , F Live/Dead staining for KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs (green fluorescence for live cells, red fluorescence for dead cells). Scale bar = 100 μm. G Expressions level of Ki67, Lamin B1, p53, p16 and p21 from the KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs were determined by the western blot. H , I Densitometric analysis of band intensities of western blots are shown in ( G ). J , K ROS assay to evaluate the intracellular ROS levels of KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs. L Expressions level of IL-1β, IL-6, IL-10 and TNF-α from the KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs were determined by the western blot. M , N Densitometric analysis of band intensities of western blots are shown in ( L ). * p  < 0.05; ** p  < 0.01; *** p  < 0.001 UCMSC-EVs ameliorated D-gal-induced apoptosis and inflammation in GCs. A , B CCK-8 assay to measure KGN and SVOG cells viability under different D-gal concentrations. C , D KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs and then stained with annexin V-FITC/PI were determined using flow cytometry. E , F Live/Dead staining for KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs (green fluorescence for live cells, red fluorescence for dead cells). Scale bar = 100 μm. G Expressions level of Ki67, Lamin B1, p53, p16 and p21 from the KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs were determined by the western blot. H , I Densitometric analysis of band intensities of western blots are shown in ( G ). J , K ROS assay to evaluate the intracellular ROS levels of KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs. L Expressions level of IL-1β, IL-6, IL-10 and TNF-α from the KGN and SVOG cells treated with D-gal or D-gal + UCMSC-EVs were determined by the western blot. M , N Densitometric analysis of band intensities of western blots are shown in ( L ). * p  < 0.05; ** p  < 0.01; *** p  < 0.001 During cellular aging, the release of inflammatory factors activates cellular oxidative stress responses. Upon reaching a certain threshold of oxidative stress, this activation triggers apoptotic signaling pathways within cells, thereby inducing cell apoptosis. Flow cytometry analysis of ROS demonstrated a significant increase in intracellular ROS levels in KGN and SVOG cells following D-gal induction, whereas UCMSC-EVs treatment markedly ameliorated this increase in ROS levels (Fig.  3 J and K). Furthermore, WB analysis revealed that D-gal induction significantly upregulated the protein level of pro-inflammatory factors (IL-1β, IL-6, p16, and TNF-α) in KGN and SVOG cells while downregulated the protein level of the anti-inflammatory factor IL-10. UCMSC-EVs administration reversed these alterations, significantly suppressing pro-inflammatory mediators while upregulating anti-inflammatory factor expression. (Fig.  3 L, M and N). These findings indicated that UCMSC-EVs was likely to improve intracellular inflammation by reducing oxidative damage in ovarian granulosa cells, thereby inhibiting cell apoptosis. UCMSC-EVs were isolated from conditioned medium (CM) and subjected to LC–MS/MS proteomic profiling to evaluate their potential protein cargo in ovarian aging rescue. The results revealed a total of 739 proteins present in UCMSC-EVs. Based on protein abundance and relevant literature, we selected LGALS3BP as a potential target for further research (Fig.  4 A). Subsequently, we verified the expression levels of LGALS3BP protein in both UCMSCs and UCMSC-EVs using Western Blot analysis, with results indicating that UCMSC-EVs were enriched in LGALS3BP protein (Fig.  4 B). To evaluate LGALS3BP-mediated mechanisms in UCMSC-EV-driven ovarian rejuvenation, LGALS3BP was transiently knocked down in UCMSCs. Subsequent WB results identified si-LGALS3BP2 as the optimal construct, which was advanced for further studies (Fig. S 4 A, B). The results of CCK-8 assay indicated that UCMSC-EVs with low expression of LGALS3BP (UCMSC-EVs si−LGALS3BP ) were unable to rescue the viability of SVOG cells induced by D-gal (Fig.  4 C). Subsequently, flow cytometry analysis revealed that, compared to the cells with UCMSC-EVs treatment, UCMSC-EVs si−LGALS3BP treatment significantly reduced the therapeutic effect against apoptosis in D-gal-injured ovarian GCs (Fig.  4 D and E). Additionally, WB analysis demonstrated that knockdown of LGALS3BP in UCMSC-EVs impaired UCMSC-EV-mediated improvements in senescence-related proteins (p53, p16, p21 and Lamin B) and the pro-apoptotic protein (Bax) (Fig.  4 F, G, H and I). Furthermore, the result of ROS assay indicated that, UCMSC-EVs si−LGALS3BP treatment did not attenuate D-gal–induced ROS generation in KGN or SVOG cells. (Fig.  4 J and K). Therefore, the above evidence suggested that UCMSC-EVs treatment was likely to ameliorate D-gal-induced GCs aging through delivering LGALS3BP and subsequently mitigate the cell apoptosis and improve the cell viability. Fig. 4 UCMSC-EVs ameliorated D-gal-induced GCs apoptosis through transmitting LGALS3BP. A The LGALS3BP protein was identified via LC–MS/MS analysis from UCMSC-EVs. B Expressions level of LGALS3BP were determined by the western blot in UCMSCs and UCMSC-EVs. C CCK-8 assay to measure the D-gal-induced SVOG cells viability treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP . D , E D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP and then stained with annexin V-FITC/PI were determined using flow cytometry. F Expressions level of p53, p16, p21, Lamin B1, Bax and Bcl-2 in the D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. G , H and I Densitometric analysis of band intensities of western blots are shown in ( F ). J , K ROS assay to evaluate the intracellular ROS levels of D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP . * p  < 0.05; ** p  < 0.01; *** p  < 0.001 UCMSC-EVs ameliorated D-gal-induced GCs apoptosis through transmitting LGALS3BP. A The LGALS3BP protein was identified via LC–MS/MS analysis from UCMSC-EVs. B Expressions level of LGALS3BP were determined by the western blot in UCMSCs and UCMSC-EVs. C CCK-8 assay to measure the D-gal-induced SVOG cells viability treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP . D , E D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP and then stained with annexin V-FITC/PI were determined using flow cytometry. F Expressions level of p53, p16, p21, Lamin B1, Bax and Bcl-2 in the D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. G , H and I Densitometric analysis of band intensities of western blots are shown in ( F ). J , K ROS assay to evaluate the intracellular ROS levels of D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP . * p  < 0.05; ** p  < 0.01; *** p  < 0.001 To further investigate the role of LGALS3BP in UCMSC-EVs ameliorating D-gal-induced ovarian aging, the UCMSC-EVs or UCMSC-EVs si−LGALS3BP were transplanted into D-gal-induced aging mice. As expected, compared to the mice in UCMSC-EVs group, UCMSC-EVs si−LGALS3BP treatment cannot reverse the decline in ovarian volume, stromal atrophy, and the reduction in the number of antral follicles of mice induced by D-gal (Fig.  5 A, B and C). Consistently, the protein level of AMH, ER-α, CYP19A1 and FSHR showed that UCMSC-EVs si−LGALS3BP cannot alleviate the damage to ovarian reserve and hormonal stimulation response induced by D-gal (Fig.  5 D, E, F and S5 A). Next, pro-aging proteins (p53, p16, p21, Bax) were upregulated while Bcl-2 was downregulated in UCMSC-EVs si−LGALS3BP group compared to those in D-gal-induced aging mice treated with UCMSC-EVs (Fig.  5 G, H and I). Furthermore, to investigate whether UCMSC-EVs reduce the inflammatory response in the ovaries of aging mice via LGALS3BP, we conducted F4/80 staining on the ovarian tissues. The results demonstrated that UCMSC-EVs significantly decreased the expression of F4/80 in the ovaries, whereas UCMSC-EVs si−LGALS3BP treatment did not exhibit this anti-inflamatory effect (Fig.  5 J and S5 B). Simultaneously, we confirmed UCMSC-EVs si−LGALS3BP cannot reduce the expression of proteins (IL-6, IL-1β, TNF-α, p-NF-κB and p-nuclear factor kappa B inhibitor alpha (p-IκBα)) associated with the inflammatory signaling pathways in ovarian tissue induced by D-gal (Fig.  5 K, L and M). Taken together, these data supported the idea that UCMSC-EVs inhibited inflammation via delivering LGALS3BP, therefore ameliorated ovarian aging. Fig. 5 UCMSC-EVs ameliorated D-gal-induced ovarian aging in vivo through transmitting LGALS3BP. A The Macroscopic ovarian sizes were observed under visible light. B Representative images of HE stained ovarian tissue sections. Scale bars=200 μm. C The quantities of primordial, primary, secondary, and maturing follicles per slide in each group were analyzed. D Representative immunohistochemistry images for AMH were shown. Scale bar = 100 μm. E Expressions level of ER-α, CYP19A1, FSHR and AMH in the ovaries of the D-gal-induced mice treated with UCMSC-EVs or UCMSC-EVs si-LGALS3BP were determined by the western blot. F Densitometric analysis of band intensities of western blots are shown in ( E ). G Expressions level of p53, p16, p21, Bax and Bcl-2 in the ovaries of the D-gal-induced mice treated with UCMSC-EVs or UCMSC-EVssi-LGALS3BP were determined by the western blot. H, I Densitometric analysis of band intensities of western blots are shown in ( G ). J Representative images of immunofluorescence were shown for F4/80 (red). Scale bar = 50 μm. K Expressions level of IL-1β, IL-6, TNF-α, NF-κB, p-NF-κB, IκBα and p-IκBα in the ovaries of the D-gal-induced mice treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. L, M Densitometric analysis of band intensities of western blots are shown in ( K ). * p < 0.05, ** p < 0.01, *** p < 0.001 UCMSC-EVs ameliorated D-gal-induced ovarian aging in vivo through transmitting LGALS3BP. A The Macroscopic ovarian sizes were observed under visible light. B Representative images of HE stained ovarian tissue sections. Scale bars=200 μm. C The quantities of primordial, primary, secondary, and maturing follicles per slide in each group were analyzed. D Representative immunohistochemistry images for AMH were shown. Scale bar = 100 μm. E Expressions level of ER-α, CYP19A1, FSHR and AMH in the ovaries of the D-gal-induced mice treated with UCMSC-EVs or UCMSC-EVs si-LGALS3BP were determined by the western blot. F Densitometric analysis of band intensities of western blots are shown in ( E ). G Expressions level of p53, p16, p21, Bax and Bcl-2 in the ovaries of the D-gal-induced mice treated with UCMSC-EVs or UCMSC-EVssi-LGALS3BP were determined by the western blot. H, I Densitometric analysis of band intensities of western blots are shown in ( G ). J Representative images of immunofluorescence were shown for F4/80 (red). Scale bar = 50 μm. K Expressions level of IL-1β, IL-6, TNF-α, NF-κB, p-NF-κB, IκBα and p-IκBα in the ovaries of the D-gal-induced mice treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. L, M Densitometric analysis of band intensities of western blots are shown in ( K ). * p < 0.05, ** p < 0.01, *** p < 0.001 We next elucidated LGALS3BP-mediated inflammatory mechanisms in D-gal-induced senescent GCs. The NF-κB signaling axis, essential for transcriptional control of cytokines and cell survival programs during infection, requires IκBα phosphorylation as its primary activation step. In response to stimuli such as inflammation, immune reactions, cell proliferation, differentiation, and survival, IκBα undergoes phosphorylation. Therefore, we analyzed NF-κB signaling in D-gal induced GCs with UCMSC-EVs or UCMSC-EVs siLGALS3BP treatment by western blotting. Results demonstrated significantly elevated p-NF-κB expression in the UCMSC-EVs si−LGALS3BP group compared with the UCMSC-EVs group, whereas p-IκBα expression showed marked downregulation (Fig.  6 A and B). Consistently, in D-gal induced KGN and SVOG cells, the expression level of IL-10 was significantly upregulated, while the expression level of IL-6, IL-1β and TNF-α (NF κB-targets) was markedly downregulated after UCMSC-EVs treatment. However, UCMSC-EVssi-LGALS3BP treatment failed to suppress IL-6, TNF-α, and IL-1β expression in D-gal-induced cells (Fig.  6 C, D and E). This LGALS3BP knockdown effect was further demonstrated by reduced nuclear translocation of NF-κB p65 (Fig.  6 F). These results suggested that LGALS3BP can rejuvenate ovarian aging by negatively regulating the NF-κB inflammatory pathway. Fig. 6 LGALS3BP inhibited inflammation through the NF-κB pathway. A Expressions level of IκBα and NF-κB activation from the D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. B Densitometric analysis of band intensities of western blots are shown in ( A ). C Expressions level of IL-10, IL-6, IL-1β and TNF-α in the D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. D , E Densitometric analysis of band intensities of western blots are shown in ( C ). F The expression level of NF-κB was analyzed in the cytoplasmic and nuclear fractions. β-tubulin and lamin B were used as the loading controls for the cytosol and nucleus, respectively. * p  < 0.05; ** p  < 0.01; *** p  < 0.001 LGALS3BP inhibited inflammation through the NF-κB pathway. A Expressions level of IκBα and NF-κB activation from the D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. B Densitometric analysis of band intensities of western blots are shown in ( A ). C Expressions level of IL-10, IL-6, IL-1β and TNF-α in the D-gal-induced KGN and SVOG cells treated with UCMSC-EVs or UCMSC-EVs si−LGALS3BP were determined by the western blot. D , E Densitometric analysis of band intensities of western blots are shown in ( C ). F The expression level of NF-κB was analyzed in the cytoplasmic and nuclear fractions. β-tubulin and lamin B were used as the loading controls for the cytosol and nucleus, respectively. * p  < 0.05; ** p  < 0.01; *** p  < 0.001

SVOG and KGN, two human granulosa cell lines were obtained from OTWO Biotechnology (HTX2650 and HTX2045, China). UCMSCs were supplied by the Zhongyuan Stem Cell Research Institute, and HEK293T cells were procured from the Zhong Qiao Xin Zhou Biotechnology (ZQ0033; ZQXZBIO, China). High-glucose DMEM (ZQ-121, ZQXZBIO) containing 10% FBS and 1% Pen-Strep was used for cell culture at 37 °C with 5% CO₂ humidification. To investigate the role of UCMSC-EVs in ameliorating cellular senescence, SVOG and KGN cells were inoculated into plates and then incubated for 24 h in advance. Cells with an adhesion rate of 50% were randomly divided into a Vehicle group (Vehicle), D-galactosidase (D-gal) cell aging model group (D-gal) and UCMSC-EVs treatment group (D-gal + UCMSC-EVs). D-gal (100µM) was added to the D-gal and UCMSC-EVs groups and they were treated for 48 h. Then the UCMSC-EVs group was then subjected to EVs (40 µg/ml), while the other groups received an equal volume of serum-free DMEM. Samples were collected and/or tested after 24 h. To determine whether UCMSC-EVs ameliorate cellular senescence by delivering LGALS3BP, SVOG and KGN cells were inoculated into plates and incubated for 24 h in advance. Cells with an adhesion rate of 50% were randomly divided into a Vehicle group (Vehicle), D-gal cell aging model group (D-gal), UCMSC-EVs treatment group (D-gal + UCMSC-EVs) and UCMSC-EV si−LGALS3BP treatment group (D-gal + UCMSC-EV si−LGALS3BP ). D-gal (100µM) was added to the D-gal, UCMSC-EVs and UCMSC-EV si−LGALS3BP groups and they were treated for 48 h. Then the UCMSC-EVs group was then subjected to EVs (40 µg/ml), the UCMSC-EV si−LGALS3BP was then subjected to EVs (40 µg/ml) without LGALS3BP, while the other groups received an equal volume of serum-free DMEM. Samples were collected and/or tested after 24 h. Briefly, P3-P5 UCMSCs at 70% confluence were washed twice with PBS and cultured in serum-free DMEM for 36 h. The conditioned supernatant was collected, centrifuged at 2000×g for 10 min to remove debris, and filtered through a 0.45-µm filter. To concentrate EVs, the supernatant was then subjected to sequential ultracentrifugation at 10,000×g for 1 h and 100,000×g for 2 h at 4 °C (BECKMAN COULTER, optima XPN-100, USA). The pelleted EVs were washed with PBS and subsequently re-suspended in fresh PBS after a second ultracentrifugation step. The final EVs preparation was either utilized directly in subsequent experiments or stored at -80 °C for later use. The particle concentration and size distribution were quantified by nanoparticle tracking analysis (NTA) (ZetaView, Particle Metrix, Germany). EVs identity was confirmed by transmission electron microscopy (TEM) for morphology, and Western blot was performed for surface markers of EVs (CD63 (25682-1-AP, Proteintech, China) and Alix (2171 S, CST, USA)) expression. LGALS3BP siRNAs (Sangon Biotech) were transfected into 70% confluent UCMSCs using LipoFiter 3.0 (Hanbio, HB-LF3-1000) following the manufacturer’s protocol. siRNA (5 µL) and LipoFiter (12 µL) were each diluted in 200 µL Opti-MEM (Invitrogen, 11058021), incubated separately for 5 min, combined for 20 min, and then applied to UCMSCs for 8 h. Following transfection, cells were maintained in fresh medium, and the optimal interference efficiency was observed between 48 and 72 h. Target sequences: #1, 5′-GAGACUUCCUCAGGUACUU-3′ (sense) and 5′-AAGUACCUGAGGAAGUCUC-3′ (antisense); #2, 5′-TGTGTGACAACCTGTGGGAC-3′ (sense) and 5′-ATGATGGGGCCTGATCCTTG-3′ (antisense). In the first stage, Female C57BL/6J mice (40 weeks) were purchased from Vital River Laboratory Animal Technology (Beijing, China) and maintained at a specific, pathogen-free facility. All animal procedures conformed to the ARRIVE guidelines and were approved by the Xinxiang Medical University Ethics Committee. The naturally aged mice were administered UCMSC-EVs at 44, 48, 52, and 56 weeks, while the control group received saline at the corresponding ages. At 60 weeks, the mice were euthanized under anesthesia, and ovarian tissues were harvested for subsequent experiments. In the second stage, D-gal-induced mouse aging models were employed to validate whether UCMSC-EVs ameliorate ovarian aging through LGALS3BP. Female C57BL/6J mice (5 weeks) were randomly and blindly divided into four groups: Vehicle group (Vehicle), D-gal group (D-gal), UCMSC-EVs treatment group (D-gal + UCMSC-EVs) and UCMSC-EVsi-LGALS3BP treatment group (D-gal + UCMSC-EVsi-LGALS3BP). The D-gal, D-gal + UCMSC-EVs, D-gal + UCMSC-EV si−LGALS3BP group received intraperitoneal injections of 150 mg/kg/day of D-gal (G0750, Sigma-Aldrich, USA) for 6 weeks, whereas the mice in the Vehicle group received equivalent volumes of physiological saline. Followed by a one-week interval, the D-gal + UCMSC-EVs group received UCMSC-EVs (100 µg dissolved in 200 µL of PBs, i.v.) once a week for four weeks, D-gal + UCMSC-EV si−LGALS3BP received UCMSC-EVs (100 µg dissolved in 200 µL of PBs, i.v.) once a week for four weeks. After two weeks, the ovarian samples of the mice in each group were harvested for subsequent experiments. Tissues were fixed in 4% paraformaldehyde and paraffin-embedded. Ovarian and uterine sections were stained with hematoxylin and eosin (H&E) following the manufacturer’s protocol. To quantify follicles, an inverted microscope was used to count those at each developmental stage across consecutive sections of the entire ovary. Only follicles containing visible oocyte nuclei were tallied to prevent duplication. Routine sections underwent Masson’s trichrome staining. For quantification of fibrosis, five representative sections per ovarian and uterine tissue were analyzed to determine the proportion of fibrotic area. The photomicrographs shown for H&E and Masson’s trichrome staining were selected based on their clarity and their representation of the typical morphological alterations observed consistently across each experimental group. SVOG and KGN cells were plated in a 10cm 2 plate (4 × 10 6 cells). Cells were exposed to 100 µM D-gal for 48 h, and were then treated with UCMSC-EVs or UCMSC-EV si−LGALS3BP for 24 h. Following treatment, nuclear and cytoplasmic fractions were isolated with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, P0028, China), and total protein was solubilized in RIPA buffer. Equal loads were separated by SDS-PAGE, transferred to PVDF, blocked in 3% BSA for 1 h at room temperature, probed overnight at 4 °C with primary antibodies (Table S1), incubated for 1 h at room temperature with secondary antibodies (Table S1), and visualized using ECL. SVOG and KGN cells were plated in a 10cm 2 plate (4 × 10 6 cells). Cells were exposed to 100 µM D-gal for 48 h, and were then treated with UCMSC-EVs or UCMSC-EV si−LGALS3BP for 24 h. Following treatment, intracellular ROS were quantified with the DCFH-DA probe (5 µM, 37 °C, 30 min) followed by flow cytometric analysis (S0033S, Beyotime, China). SVOG and KGN cells were plated in a 96-well plate (3000 cells/well). To determine the optimal concentration of D-gal, cells were treated with a range of concentrations at 0, 5, 10, 25, 50, 100, 150, and 200 µM for 48 h. To assess the effects of UCMSC-EVs or UCMSC-EV si−LGALS3BP , cells were exposed to 100 µM D-gal for 48 h to induce senescence, and were then treated with UCMSC-EVs or UCMSC-EV si−LGALS3BP for 24 h. Following treatment, cell viability was conducted. 5 µL of Cell counting kit-8 reagent (C0037, Beyotime, China) was added to each well, and the plates were incubated at 37 °C for an additional 3 h. Absorbance at 450 nm was measured on a microplate reader. SVOG and KGN cells were plated in a 24 well plate (1.5 × 10 4 cells/well). Cells were exposed to 100 µM D-gal for 48 h, and were then treated with UCMSC-EVs or UCMSC-EV si−LGALS3BP for 24 h. Following treatment, cell apoptosis in SVOG and KGN cells were conducted. Cells were stained with Calcein-AM (2 µM) and Propidium Iodide (4 µM) for 15 min at 37 °C in the dark, following the manufacturer’s protocol (C1371S, Beyotime, China). Fluorescence images were captured using an inverted fluorescence microscope. Mice from 60-week and 60-week + UCMSC-EV groups were euthanized, ovaries harvested, and RNA-Seq libraries prepared. Differential expression (DESeq2 v1.4.5; Q ≤ 0.05) followed by KEGG and GO enrichment analyses identified the significantly altered genes and pathways. Protein content of UCMSC-EVs was quantified by BCA, and aliquots containing equal protein amounts were adjusted to identical volumes for downstream assays. Subsequently, Tris(2-carboxyethyl) phosphine (TCEP) and cysteine-amine (CAA) were added to facilitate a reduction and alkylation reaction at 60 °C for 30 min. Following this, the samples were treated with 100 mM Tris-HCl to dilute the concentration below 2 M. An enzyme-to-protein ratio of 1:50 was used, adding trypsin for an overnight incubation at 37 °C with shaking to allow for proteolytic digestion. The digestion was halted by the addition of trifluoroacetic acid (TFA). The supernatant was then processed for desalting using Styrenedivinylbenzene-Reversed Phase Sulfonate (SDB-RPS) spin columns. For mass spectrometry analysis, a Bruker timsTOFPro instrument with ion mobility-quadrupole-time of flight mass spectrometer was utilized. Sample injection and separation were conducted using an online coupled Ultimate 3000 RSLCnano liquid chromatography system. Statistical analyses were performed using GraphPad Prism 9.0. Data are presented as mean ± SEM. Differences between two groups were assessed by independent Student’s t -test, while comparisons among multiple groups used one-way ANOVA. p  ≤ 0.05 was considered statistically significant.

In this study, we have demonstrated that UCMSC-EVs alleviate ovarian aging through delivering protein LGALS3BP, which inhibits inflammation by suppressing NF-κB signaling. This suggests that UCMSC-EVs exert a therapeutic effect against ovarian aging by suppressing inflammatory responses. As the orchestrator of follicular development and oocyte competence, the ovarian microenvironment is vulnerable to maternal pathophysiologies—notably endometriosis, PCOS, and aging—which perturb its homeostasis, leading to aberrant folliculogenesis and compromised gamete quality [ 23 ]. Chronic low-grade inflammation has been confirmed to induce oxidative stress and tissue fibrosis within the ovary [ 24 , 25 ]. During ovarian aging, the levels of pro-inflammatory cytokine in follicular fluids are often elevated, leading to oxidative stress and impaired follicular development [ 26 ]. In the context of inflammatory aging (inflammaging), senescent cells release senescence-associated secretory phenotype factors, contributing to persistent inflammation and oxidative stress in the ovary [ 27 , 28 ]. Our study indicated that ovarian tissues from 60-week-old mice exhibit a reduction in primordial follicles and significant fibrosis, with elevated levels of inflammation, and apoptosis in injured ovarian GCs. Thus, chronic low-grade inflammation improvement in the ovary may offer a promising therapeutic approach to ameliorate the follicular microenvironment and mitigate ovarian dysfunction. Recently, EVs are emerging as key participants in diverse cellular communication systems, playing a pivotal role in modulating various signaling pathways and facilitating intercellular information transfer [ 29 ]. Their broad array of properties has enabled their successful application across multiple fields, including immunomodulation, and regenerative medicine [ 29 , 30 ]. MSCs play a fundamental role in the field of regenerative medicine [ 31 ]. Recent studies investigating the mechanisms underlying MSCs-based therapies have highlighted the growing significance of MSCs’ paracrine action in promoting favorable outcomes, even in the absence of substantial cell engraftment [ 32 ]. Hopefully, MSC-EVs are rich in various bioactive factors, including RNA, cytokines, immune modulators, and chemokines, all of which exhibit significant immunomodulatory properties [ 33 ]. These MSC-EVs carry multiple adhesion molecules, such as CD29, CD44, and CD73, enabling their homing to sites of injury and inflammation [ 34 ]. In a mouse model of acute kidney injury (AKI), MSC-EVs predominantly accumulate in the inflamed kidneys, while in a model of intracerebral hemorrhage, MSC-EVs are also detected in the damaged brain tissue [ 35 , 36 ]. However, most intravenously administered MSC-EVs preferentially accumulate in the liver, spleen, and lungs, likely due to the active mononuclear phagocyte system (MPS) in these organs [ 37 ]. In macrophage-depleted mice, the clearance rate of EVs from circulation is significantly slower, highlighting the critical role of the MPS in the biodistribution of EVs. Studies have demonstrated that MSCs alleviate colitis symptoms by inhibiting colonic macrophages via EVs. An increase in the number of IL-10-producing M2 macrophages was observed in MSC-EV-treated mice, alongside a reduction in the expression of pro-inflammatory cytokines and chemokines (TNF-α, IL-1β, IL-6) derived from macrophages, thus alleviating colonic inflammation [ 38 , 39 ]. Consistently, our research indicated that UCMSC-EVs significantly enhanced the ovarian function in 60-week naturally aged mice, reduced the extent of interstitial fibrosis, and markedly downregulated the ovarian expression levels of TNF-α, IL-1β, and IL-6. Furthermore, RNA-seq analysis revealed that the expression of genes associated with inflammatory pathways in the ovarian tissues of 60-0week-old mice received UCMSC-EV transplantation exhibits significant improvement compared to that in the mice without UCMSC-EV transplantation. GCs engage in intricate gap junction-mediated intercellular communication with oocytes, thereby modulating the growth and maturation of the oocytes [ 40 ]. In mammalian studies, it has been observed that coculture of cumulus cells with embryos can surmount developmental arrest and significantly bolster embryonic development [ 41 ]. Accordingly, ovarian GCs (KGN, SVOG) were selected to investigate the impact of UCMSC-EVs on aged ovaries by simulating cellular senescence and damage with D-galactosidase (D-gal), thereby exploring the potential mechanisms of UCMSC-EVs’ restorative actions on ovarian aging. Consistent with the findings from in vivo experiments, KGN and SVOG cells cultured with UCMSC-EVs demonstrated enhanced capacity of anti-apoptosis, and following culture with UCMSC-EVs, there was a significant reduction in cellular oxidative stress and inflammatory factor, leading to an improvement in D-gal -induced cellular senescence. However, the mechanisms by which UCMSC-EVs mitigate cellular senescence remain unclear. Therefore, to elucidate the specific core factors within UCMSC-EVs that play a curcial role in enhancing the viability of aged ovarian cells, this study conducted a proteomic analysis of UCMSC-EVs. The results revealed a high expression of LGALS3BP within the UCMSC-EVs. LGALS3BP was initially identified by two independent research groups as a 90 kDa tumor-associated antigen. It was detected using the SP2 monoclonal antibody in CG-5 human breast cancer cells and, separately, with the L3 monoclonal antibody in Calu-1 human lung cancer cells [ 42 – 44 ]. Concurrently, it was also characterized as a novel ligand for galectin-3 (formerly Mac-2), a beta-galactoside-binding lectin, has been implicated in the modulation of cell-cell and cell-matrix interactions, and is involved in the recruitment, activation, and clearance of neutrophils [ 45 , 46 ]. On the other hand, LGALS3BP is thought to regulate macrophage adhesion, chemotaxis, and apoptosis. Initial investigations into the function of LGALS3BP centered on its implications in the context of human immunodeficiency virus (HIV) infection, where it was shown as a serological indicator for the advancement to acquired immune deficiency syndrome (AIDS) [ 47 ]. As research progresses, the comprehensive understanding of its functions continues to expand. Hou et al., indicated LGALS3BP plays an active role on the process of angiogenesis by facilitating the upregulation of angiogenic factors via the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway [ 20 ]. Additionally, Studies have demonstrated that in a murine model of colitis induced by dextran sulfate sodium (DSS), LGALS3BP expression in the colonic tissue of model mice significantly increases, and LGALS3BP-deficient mice exhibit more pronounced weight loss, rectal bleeding, diarrhea, colon shortening, and spleen enlargement. Further staining reveals increased apoptosis, severe inflammation, and larger ulcer areas, indicating that the absence of LGALS3BP exacerbates inflammation in mice [ 48 ]. Consistent with previous reports, the current study found that UCMSC-EVs exerted their anti-inflammatory effects by transmitting LGALS3BP, which in turn ameliorating the functionality of aging ovaries. However, when silencing LGALS3BP in UCMSCs, the isolated UCMSC-EVs si−LGALS3BP exhibited significantly impaired anti-inflammatory, anti-apoptotic, and pro-reparative effects on D-gal-injured cells, suggesting that UCMSC-EVs si−LGALS3BP markedly reduces the efficacy in improving D-gal-induced cellular senescence and damage. These findings suggested that LGALS3BP may serve as a key factor in UCMSC-EVs’ capacity to improve the viability of aged ovarian granulosa cells, with its underlying mechanism requiring further investigation. Generally, NF-κB is activated in response to DNA damage, mitochondrial dysfunction (through the generation of reactive oxygen species), or proinflammatory cytokines in the surrounding environment [ 49 , 50 ]. Simultaneously, NF-κB regulates the expression of genes that encode proteins involved in controlling cell proliferation, apoptosis, morphogenesis, and differentiation [ 51 ]. Whether considering the proinflammatory cytokines associated with human aging (e.g., IL-1, IL-6, TNF-α) or the proinflammatory components of the senescence-associated secretory phenotype (SASP), which is widely implicated in inflammaging, a common theme emerges: many of the genes involved in these processes are regulated by NF-κB [ 52 , 53 ]. Here, we revealed that LGALS3BP modulates the inflammatory response in ovaries by repressing the activity of the NF-κB signaling pathway, consequently alleviating ovarian aging. In summary, our study confirmed that UCMSC-EVs significantly enhanced the function of aged ovaries. Subsequent RNA-sequencing results validated the alleviation of ovarian inflammation in mice with UCMSC-EVs treatment. Furthermore, we confirmed that UCMSC-EVs delivered LGALS3BP protein to modulate NF-kB-related inflammatory pathways, thereby ameliorating ovarian aging. To this end, when devising novel regenerative medicine strategies, effectively managing inflammation should be seriously considered. Consequently, MSC-EVs’ mediation of anti-inflammatory and pro-regenerative effects can serve therapeutic purposes, and further elucidating the underlying mechanisms of MSC-EVs’ anti-inflammatory properties lays a robust theoretical foundation for their clinical translation.

Contemporary societal trends toward deferred childbearing contrast sharply with the comparatively accelerated senescence of the female gonad, which outpaces chronological aging of most somatic organs. This temporal discordance precipitates a pronounced diminution in reproductive capacity, with a clinically evident inflection point occurring at approximately 37 years of age, followed by a precipitous decline rendering natural conception exceptional beyond 45 years. These age-dependent reproductive constraints engender substantial medical and sociodemographic challenges [ 1 , 2 ]. Ovarian aging, distinct from other organ senescence, leads to a decline in endocrine and reproductive capacity and contributes to the exacerbation of multi-organ dysfunctions [ 3 ]. Manifestations of ovarian aging include weight gain, dry skin, reduced skin elasticity, diminished immune response, and a higher prevalence of conditions such as cardiovascular diseases, digestive disorders, and urinary system issues [ 4 , 5 ]. Consequently, ovarian aging is considered the catalyst for overall female senescence and the primary trigger for the aging of multiple organ systems [ 6 ]. Research has established that a hallmark of ovarian aging involves the progressive loss of resting follicles and a concomitant reduction in oocytes possessing the competence for fertilization and subsequent embryonic development [ 7 ]. It has been extensively confirmed that ovarian aging is affected by various intraovarian and extraovarian factors, including oxidative stress, DNA damage, and telomere attrition (telomere theory), which have been thoroughly studied [ 8 ]. Especially, above-mentioned factors induced chronic inflammation is recognized to seriously impair reproductive lifespan. With advancing age, systemic inflammation escalates, elevating circulating concentrations of inflammatory mediators including cytokines and biomarkers such as C-reactive protein and plasma IL-6 [ 9 ]. Recent evidence reveals that ovarian tissue demonstrates premature activation of senescence-associated gene expression and inflammatory pathways, indicating heightened vulnerability to inflammaging compared to other somatic tissues [ 10 ]. Inflammation markers and immune cell populations were examined in the ovaries of C57BL/6 female mice at 2, 6, 12, and 18 months of age. Aging mice exhibited reduced follicular reserves concomitant with ovarian accumulation of CD4⁺ T cells, B cells, and macrophages. Concordantly, both circulating concentrations and intra-ovarian transcripts of pro-inflammatory mediators—including IL-1α/β, TNF-α, IL-6, and inflammasome-related genes—rose markedly over time [ 9 ]. In recent studies, extracellular vesicles (EVs) have been identified as significant contributors to the paracrine effects of mesenchymal stem cells (MSCs) [ 11 ]. Encapsulated within their membrane are proteins, nucleic acids, and diverse intercellular signaling molecules [ 12 ]. Functioning as key mediators in tissue microenvironments, these EVs facilitate paracrine communication and biomolecular transfer, thereby modulating physiological and pathological processes while offering novel therapeutic opportunities [ 13 ]. The advent of MSC-based therapies for premature ovarian insufficiency (POI) has been catalyzed by progress in MSCs’ research, with EVs derived from MSCs emerging as pivotal facilitators of these therapeutic actions [ 14 ]. Human umbilical cord derived MSCs (UCMSCs), harvested from Wharton’s jelly, exhibit robust proliferative capacity, low immunogenicity, and abundant availability, making them an optimal choice for EVs production. Recent study demonstrated that UCMSC-EV coculture enhances granulosa cell proliferation in primordial and primary follicles in vitro, concurrently upregulating early follicular marker expression while suppressing apoptosis [ 15 ]. Additionally, Li et al., demonstrated that hUCMSC-derived exosomes deliver miR-21-5p to suppress PTEN expression, thereby inhibiting apoptosis and ultimately preserving ovarian function [ 16 ]. Protein mass spectrometry of EVs in this study identified Galectin 3-binding protein (LGALS3BP)—a ubiquitous multifunctional glycoprotein belonging to the scavenger receptor cysteine-rich (SRCR) domain family—as highly concentrated in UCMSC-EVs. This protein was initially characterized in tumor transformation and cancer progression contexts [ 17 ]. LGALS3BP, an evolutionarily conserved extracellular matrix glycoprotein, orchestrates immunity, proliferation, migration, cell adhesion, and angiogenesis, and critically governs the anchoring and migratory behavior of human neural progenitors [ 18 – 20 ]. Recent studies have reported that upregulated LGALS3BP inhibit the secretion of IL-4, IL-5, and IL-13 and to activate antiviral response [ 21 , 22 ]. Furthermore, Cho et al. demonstrated that LGALS3BP is a physiological regulator of inflammation through regulating nuclear factor kappa-light-chain-enhancer of activated B Cells (NF-κB) signaling. However, it remains unclear whether UCMSC-EVs exerts its therapeutic effects on ovarian aging through LGALS3BP. Therefore, elucidating the underlying mechanisms is imperative for developing therapeutic strategies to delay ovarian aging, thereby extending reproductive longevity and mitigating menopause-associated complications affecting the musculoskeletal, cardiovascular, and neurological systems. To this end, we examined the amelioration of UCMSC-EVs in ovarian aging and further investigated the role of LGALS3BP in inflammation inhibition during ovarian aging.

Supplementary Material 1. Supplementary Material 1. Supplementary Material 2. Supplementary Material 2.