Abstract
The immune system is closely associated with the pathogenesis of
polycystic ovary syndrome (PCOS). Macrophages are one of the important
immune cell types in the ovarian proinflammatory microenvironment, and
ameliorate the inflammatory status mainly through M2 phenotype
polarization during PCOS. Current therapeutic approaches lack efficacy
and immunomodulatory capacity, and a new therapeutic method is needed
to prevent inflammation and alleviate PCOS. Here, octahedral nanoceria
nanoparticles with powerful antioxidative ability were bonded to the
anti-inflammatory drug resveratrol (CeO[2]@RSV), which demonstrates a
crucial strategy that involves anti-inflammatory and antioxidative
efficacy, thereby facilitating the proliferation of granulosa cells
during PCOS. Notably, our nanoparticles were demonstrated to possess
potent therapeutic efficacy via anti-inflammatory activities and
effectively alleviated endocrine dysfunction, inflammation and ovarian
injury in a dehydroepiandrosterone (DHEA)-induced PCOS mouse model.
Collectively, this study revealed the tremendous potential of the newly
developed nanoparticles in ameliorating the proinflammatory
microenvironment and promoting the function of granulosa cells,
representing the first attempt to treat PCOS by using CeO[2]@RSV
nanoparticles and providing new insights in combating clinical PCOS.
Supplementary Information
The online version contains supplementary material available at
10.1186/s12951-023-02182-w.
Keywords: CeO[2]@RSV, PCOS, Macrophage polarization, Granulosa cells,
Inflammation
Introduction
Polycystic ovary syndrome (PCOS) is one of the most common endocrine
disorders and is characterized by polycystic ovaries, anovulation, and
hyperandrogenism [[51]1, [52]2]; PCOS affects approximately 20% of
women of reproductive age [[53]3, [54]4] and has lifelong consequences
for health and wellbeing [[55]5–[56]7]. Although polygenes and multiple
factors are involved in the pathophysiology of PCOS [[57]8], effective
biological targets and therapeutic drugs remain unsatisfactory. Thus,
innovative treatment strategies to improve therapeutic efficacy are
highly required.
Accumulating evidence strongly suggests that the proinflammatory
microenvironment in the ovary plays significant roles in the
pathogenesis of PCOS [[58]9]. Macrophages, the most abundant immune
cell type in the ovaries, are reported to exert considerable effects on
ovarian homeostasis and function [[59]10–[60]12]. Traditionally,
macrophages are categorized into classically activated (M1) and
alternatively activated (M2) subtypes, and the balance of M1 and M2
macrophages is essential for the immunological milieu within the ovary
[[61]13]. The abundance of macrophages with the proinflammatory M1
phenotype accelerates the inflammatory response in the ovaries
[[62]14], and potentially contributes to the progression of PCOS
[[63]15, [64]16]. Researches have demonstrated that an imbalance in the
ovarian immune status further impairs granulosa cell (GC)
proliferation, ovarian follicular development and ovulation [[65]14],
resulting in poor oocyte quality and IVF outcomes [[66]17]. Considering
that macrophages act as drivers and regulators of ovarian functions
[[67]18], achievements that either reduce the amount and function of M1
or increase M2 activity are essential for the treatment of PCOS.
Intriguingly, macrophages have been reported to be highly heterogeneous
and can shift from one phenotype to another in response to the
surrounding microenvironment [[68]19], and studies have also confirmed
that regulation of macrophage infiltration lowers the expression of
proinflammatory cytokines and remarkably ameliorates the function of
GCs and PCOS-associated clinical outcomes [[69]20, [70]21]. Therefore,
discouraging the immune activation of macrophages would be considered a
promising and significant therapeutic strategy to relieve the symptoms
of PCOS.
Recently, nanoceria (CeO[2]) has been widely applied in disease
diagnosis and therapy due to its favorable antioxidant capacity
[[71]22]. Studies have demonstrated that CeO[2] not only reduces
reactive oxygen species (ROS) production but also suppresses the
release of inflammatory cytokines by inhibiting M1 macrophage
polarization [[72]23, [73]24]. These results indicated the outstanding
potential of CeO[2] in scavenging excessive ROS and reducing macrophage
infiltration. Resveratrol (RSV), a natural polyphenol, acts as an
anti-inflammatory and antioxidant protective agent during PCOS [[74]25,
[75]26]. As a notable metabolic modulator, RSV can reduce the secretion
of inflammatory cytokines and promote the transformation of macrophages
into the M2 type [[76]27, [77]28]. However, the therapeutic application
of RSV remains limited because of its instability and poor systemic
bioavailability [[78]29, [79]30].
To address these issues, in the present study, we encapsulated the
powerful immunomodulatory drug RSV into CeO[2] through chemical bonding
(CeO[2]@RSV), which was delivered to the ovaries to regulate the immune
microenvironment and relieve the inflammatory response in PCOS (Scheme
[80]1). The synthesized CeO[2]@RSV nanoparticles (NPs) mainly possess
the following advantages: (1) The chemical bonding of RSV to CeO[2]
expands the antioxidant activity of the CeO[2] nanozymes. (2)
Surface-bound RSV may extend the anti-inflammatory capacity and play a
congenerous role in dual-directional immunoregulation via macrophage
polarization. (3) The CeO[2]@RSV nanocomposites also exhibited high
biocompatibility and drug delivery capabilities to the ovaries and
ameliorated ovarian injury in vivo. As a result, our NPs can
effectively suppress inflammation response-mediated injury and
alleviate endocrine dysfunction in PCOS mice, thereby achieving
prominent treatment in PCOS therapy. Our findings provide new insights
into the immunomodulation of PCOS and broaden the application of
nanomaterials as delivery systems.
Scheme 1.
[81]Scheme 1
[82]Open in a new tab
Schematic illustration of the synthesis process and anti-inflammatory
functions of CeO[2]@RSV NPs
Materials and methods
Synthesis of the nanocomposite
Preparation of CeO[2]@RSV
CeO[2] was prepared by a hydrothermal process, which was performed as
previously described [[83]31]. In brief, 0.1 mol Ce(NO[3])[3]·6H[2]O
(Siga, USA) and 0.01 mol Na[3]PO[4]·12H[2]O were dissolved in 10 mL and
30 mL of deionized water, respectively. Then, the two solutions were
mixed in a Teflon container under a magnetic stirrer for 1 h and
subsequently reacted for 12 h at 170 °C in a temperature-controlled
electric oven (DGG-9070BD, Shenxin, Shanghai, China). The above
products were washed and then dried at 50 °C in an oven.
Afterward, 3-aminopropyltriethoxysilane (APTES) was applied to prepare
amino-functionalized CeO[2] [[84]32]. One milliliter of APTES was added
to the aforementioned prepared 40 mg nanoparticles dispersed with 20 mL
of absolute isopropyl ethanol and stirred vigorously for 6 h at 85 °C.
A schematic image of the chemical bonding between RSV and APTES is
depicted in Additional file [85]1: Fig. S1. Subsequently, different
concentrations of RSV (5, 10, and 15 mg) were added to the
amino-functionalized CeO[2]@APTES (40 mg) and dispersed into 20 mL
ethanol, followed by mixing for 24 h. The final CeO[2]@RSV product was
centrifuged at 9500 rpm for 15 min and dried at 50 °C in an oven
overnight.
Characterization of synthesized nanocomposites
The morphology and structure of the NPs were characterized using
transmission electron microscopy (TEM, H-8100IV, Hitachi, Tokyo, Japan)
and scanning electron microscopy (SEM, JSM-IT300, JEOL, Tokyo, Japan).
XRD (Empyrean, PANalytical B.V. Netherlands) was used to examine the
crystalline structure of the nanoparticles by comparison with the
standard XRD profile of CeO[2], and the contents of Ce^4+ and Ce^3+
were determined by XPS (Escalab 250Xi, Thermo Fisher Scientific). The
zeta potential was determined using a zeta potential instrument
(Zetasizer, Nano-Z, Malvern Instruments Limited, UK). AIR-FTIR (VERTEX
70, Bruker, Germany) was used to measure functionalized CeO[2] with a
Bruker Vertex 80 V spectrometer. The size distribution of the
nanoparticles was determined by DLS (Zetasizer Nano-ZS ZEN3700, Malvern
Instruments Limited, UK). An Avance III HD 400 MHz NMR spectrometer
(Bruker-BioSpin, Rheinstetten, Germany) was used to confirm the
chemical bonding between RSV and APTES.
Drug loading efficiency (LE) and encapsulation efficiency (EE)
To determine the drug loading and encapsulation efficiency, the
prepared drug-loaded NPs were analyzed by spectrophotometry at a
wavelength of 372 nm. The drug loading and encapsulation efficiency
were calculated using the following equations.
[MATH: Drugloadingefficiency%=totaldrug-freedrug/weightofnanocomposites×
100%, :MATH]
[MATH: Encapsulationefficiency%=totaldrug-freedrug/totaldrug×100%. :MATH]
Patients and tissue samples
The study was approved by the Ethics Committee of the Renmin Hospital
of Wuhan University (Ethical Approval Number WDRY 2019-K077), and
written informed consent was obtained from each participant. Human
primary ovarian granulosa cells (GCs) were collected from patients at
the Reproductive Center of Renmin Hospital of Wuhan University. PCOS
patients (n = 10) were diagnosed according to the 2003 Rotterdam
Criteria [[86]33]. The baseline characteristics of the patients are
described in Additional file [87]1: Table S1.
GCs were obtained from PCOS patients who had undergone oocyte retrieval
with a long stimulation protocol and isolated by density gradient
centrifugation. Briefly, the follicular fluid was centrifuged at 300×g
for 10 min, and the supernatant was discarded. The pellet was suspended
in PBS (Servicebio, China), 50% Percoll (Biosharp, Wuhan) was added,
and the mixture was centrifuged at 1800 r/min for 20 min. The middle
white granulosa cell layer was aspirated and washed with PBS, and then
the cells were seeded in a cell culture plate for further experiments.
Animal models and treatment
Animal models
Female C57BL/6 mice (3 weeks) received adaptive feeding for 1 week. The
PCOS mouse model was constructed as previously described [[88]34], and
the mice were injected (s.c.) with dehydroepiandrosterone (DHEA)
(6 mg/100 g/d in olive oil) daily for 21 consecutive days. This study
was divided into five subgroups (n = 5/group): (1) sham group [olive
oil, subcutaneous (s.c.)], (2) PCOS group, (3) CeO[2] group [mice
injected with DHEA and treated with CeO[2] with the same amount of
CeO[2] as CeO[2]@RSV], and (4) RSV group [mice injected with DHEA and
treated with RSV with the identical amount of RSV as CeO[2]@RSV]. (5)
CeO[2] + RSV group [mice injected with DHEA and treated with a mixture
of RSV and CeO[2]], and (6) CeO[2]@RSV group [mice injected with DHEA
and treated with CeO[2]@RSV (1 mg/kg)]. For the JAK/STAT inhibitor
(WP1066) study, mice were injected with DHEA and treated with
CeO[2]@RSV+WP1066. All experimental groups were injected with the
corresponding solution by the tail vein every other week. Then the mice
were sacrificed by isoflurane anesthesia 2 weeks after the treatment.
Blood samples were collected from the eyes of mice and then centrifuged
and stored at − 80 °C for biochemical analyses and ELISA. All in vivo
studies were performed under the approval of Animal Care and Use
Committee of the Wuhan University (Ethical Approval Number 20190710).
In vivo fluorescence imaging
ICG-labeled NPs were synthesized as previously described [[89]35]. In
short, 40 mg CeO[2] NPs were added into 1 mL APTES (resuspended in
absolute isopropyl ethanol solution), and an indocyanine green (ICG)
solution (10 mg/mL, 1 mL) was added and stirred for 6 h. Twenty
milliliters of 10 mg RSV ethanol solution was then added to the mixed
solution and stirred for 24 h. ICG-labeled CeO[2]@RSV NPs (1 mg/kg)
were intravenously injected into mice, and the mice were sacrificed
2 weeks after the injection. Fluorescence imaging was recorded by an
IVIS Spectrum (Perkin Elmer, USA). Finally, the mice were killed, and
the ovarian tissues were detected by IVIS Spectrum.
Cytocompatibility biocompatibility assay for CeO[2]@RSV
A Cell Counting Kit-8 (CCK-8) assay was conducted to investigate the
cytotoxicity of our NPs in vitro. Cells were seeded at a density of
3 × 10^3 in a 96-well plate overnight and administered different
concentrations of NPs for 24 h, 48 h and 72 h. Then, 10 µl CCK-8
reagent was added to each well and incubated at 37 °C for 1 h. Cell
viability was determined by using a microplate reader (Ensight, Perkin
Elmer, Waltham, MA, United States) at 450 nm. All CCK-8 tests were
performed in triplicate and were repeated three times.
In vivo, healthy C57BL/6 mice (n = 5) were intravenously administered
NPs (1 mg/kg) as a bolus (100 μL) through the tail vein. After 2 weeks,
the primary organs (including the heart, liver, spleen, kidney, and
ovary) were collected and fixed in formalin, followed by H&E staining
and pathological analysis. The stained tissue samples were observed
under a microscope (Olympus BX53) at a magnification of 200×.
Cell culture and treatment
Cell culture
The human ovarian granulosa cell line (KGN cells) was acquired from the
Institute of Biochemistry and Cell Biology, the primary ovarian granule
cells (GCs) obtained from PCOS patients were grown in DMEM/F-12 medium
(Gibco, China), and the human monocyte cell line THP-1 was maintained
in RPMI-1640 medium (Gibco) with 10% FBS (Gibco) at 37 °C in 5% CO[2].
Effect of nanocomposites on immunoregulation via macrophage polarization in
vitro
Based on the cytotoxicity tests of the different concentrations of
CeO[2]@RSV (10, 50, and 100 µg/mL), the concentration of 50 µg/mL NPs
was suitable and used in the subsequent experiments.
THP-1 cells were incubated with phorbol 12-myristate 13-acetate (PMA,
Sigma, 16561-29-8, USA) for 24 h to differentiate into M0 macrophages.
To investigate the impact of NPs on the repolarization of M1
macrophages, M0 macrophages were cultured with 100 ng/mL LPS (Sigma,
L4391, USA) plus 20 ng/mL IFN-γ (PeproTech, 300-02, USA) to induce the
M1 phenotype. Similarly, a concentration of 50 µg/mL of prepared NPs
was added to M1 macrophages according to the CCK8 results of the
macrophages. After incubation for 48 h, the supernatant was centrifuged
at 2000 rpm for 10 min, and the cells and supernatant were collected
for further analysis. For inhibition of signaling pathways, macrophages
were pretreated with 5 µM WP1066 (MCE, China), 10 µM BAY11-7082, 100 nM
VX-11e or 10 µM SB 239063 for 12 h before further experiments.
Coculture model system
To investigate how macrophage polarization affects the proliferation
and apoptosis of KGN cells in vitro. A cell coculture model system was
used, wherein NP-treated M1 macrophages were added to the upper chamber
of a Transwell chamber system, and KGN cells were placed in the lower
chamber. Then, the cells were evaluated after coculturing for 48 h.
Flow cytometry
Fresh mouse spleens were acquired and ground and then filtered with a
strainer, and a red blood cell lysis solution (Servicebio, China) was
added to lyse the red blood cells. Ground spleen-derived cells or GC
cells in different treatment groups were separated in 200 μL of
transcription factor buffer (BD Pharmingen), and the corresponding
antibodies were incubated at 4 °C for 30 min. For apoptosis analysis,
an Annexin V-FITC/PI Apoptosis Detection Kit (ABclonal, China) was used
to stain cells at room temperature for 15 min in the dark. Next, the
cells were washed with PBS, and flow cytometry analysis was performed
using a Beckman CytoFLEX flow cytometer. The data were analyzed with
Flow Jo software (Tree Star, Inc., Ashland, OR USA).
Histological assessment
Tissues were fixated in 4% formaldehyde for 24 h, dehydrated and
embedded in paraffin. Then, the sections were cut at a thickness of 5
μm for hematoxylin and eosin (H & E) stain. The sections were stained
with hematoxylin and dehydrated by graduated ethanol, and the sections
were sealed with neutral resin. Representative tissue sections were
imaged under a light microscope (Nikon, Tokyo, Japan).
Immunofluorescence
After fixation with 4% formaldehyde, the cells were incubated with
primary antibodies against iNOS or CD206 (Affinity, Cat. No. DF4149 and
AF0199, dilution 1:500) at 4 °C overnight. The corresponding secondary
antibodies (1:200) were applied for 2 h, and the nuclei were stained
with 4′,6-diamidino-2-phenylindole (DAPI, Servicebio, China). The
results were observed with a fluorescence microscope (Olympus, Tokyo,
Japan).
Real-time polymerase chain reaction (RT-PCR)
Total RNA was extracted using TRIzol reagent (Accurate Biology, China)
according to the manufacturer’s instructions. Reverse transcription was
conducted with the PrimeScript RT reagent kit (Accurate). PCR was
performed by using a 7500 Real-Time PCR system (Applied Biosystems,
Foster City, CA, USA). The relative gene expression levels reported in
this study were analyzed with the 2^−ΔΔ Ct method. The primers used to
measure mRNA expression levels are shown in Additional file [90]1:
Table S2.
Western blot analysis
Proteins were extracted from tissues and cells with RIPA lysis buffer
containing protease inhibitors. Proteins were separated by SDS-PAGE and
transferred onto a PVDF membrane (EMD Millipore, Bedford, MA, USA). The
membranes were blocked in 5% BSA, and subsequently primary antibodies
against actin (ABclonal, Cat. No. A17910), Bcl2, Bax (Proteintech, Cat.
No. 68103-1-Ig, 60267-1-Ig), STAT3 (Proteintech, China, No.
10253-2-AP), p-STAT3 (Affinity, China, Cat. No. AF3293), NF-κB, p-NF-κB
(Cell Signaling, USA, Cat No. 8242, 3303), Erk1/2, p-Erk1/2 (Cell
Signaling, Cat No. 4696, 4376), p38 MAPK, and p-MAPK (Cell Signaling,
Cat No. 8690, 4511) were incubated overnight at 4 °C. HRP-conjugated
secondary antibodies (Abmart, Cat. No. T55756F) were utilized, and the
membranes were visualized using a chemiluminescence western detection
system (Bio-Rad, Hercules, CA, USA) and analyzed by ImageJ.
Measurement of serum biochemical markers
After collecting the supernatants of the mice, the levels of different
biochemical markers [testosterone (T), estradiol (E[2]), luteinizing
hormone (LH), IL-1β and IL-10] were measured by sandwich ELISA (R&D
Systems) according to the manufacturer’s instructions. The oral glucose
tolerance test (OGTT) was measured by tail vein blood sampling using a
blood glucose meter (ONETOUCH Ultra Vue, China). Blood glucose levels
were measured after fasting and then administered glucose (2 g/kg body
weight). The glucose uptake ability and the level of lactic acid were
evaluated by using a lactic acid determination kit (A019-2-1, Nanjing
Jiancheng) and glucose determination kit (A154-1-1, Nanjing Jiancheng)
according to the manufacturer’s instructions.
Malondialdehyde (MDA), catalase (CAT) and superoxide dismutase (SOD) tests
MDA (A003-1, Jiancheng, Nanjing), CAT (S0051, Beyotime) and SOD
(A001-3, Jiancheng, Nanjing) assay kits were used to assess the levels
of MDA, CAT and SOD. Then, the corresponding solutions were added to
the working solution following the manufacturer’s instructions.
ABTS free radical scavenging assay
An ABTS scavenging assay was used to evaluate the antioxidant activity
of NPs according to the corresponding instructions (SO121, Beyotime).
The different NP samples (10 μL) were mixed with peroxidase working
solution (20 μL) + ABTS working fluid (170 μL) and then examined at
734 nm absorbance.
Measurement of intracellular ROS levels
The cellular ROS levels were evaluated with 2′,7′-dichlorofluorescein
diacetate (DCFH-DA) as previously described [[91]36]. Briefly, the
treated KGN cells were suspended in 1 mL of serum-free medium
supplemented with 1 μL of the oxidative fluorescent dye DCFH-DA (S0033,
Beyotime) for 30 min at 37 °C. Finally, the intensity of the DCFDA
signal in the cells was detected by flow cytometry.
EdU and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling
(TUNEL) assay
The proliferation of KGN cells was assessed using an EdU cell
proliferation kit (C0078S, Beyotime, China). After treatment,
serum-free DMEM/F12 medium containing 1:1000 EdU was incubated for 2 h.
The apoptosis of ovarian tissues was assessed using a TUNEL apoptosis
assay kit (C1086, Beyotime, China). The cells and ovarian paraffin
sections were washed with PBS, fixed in 4% paraformaldehyde for 1 h and
permeabilized with 0.1% Triton X-100 for 5 min. Then, the cells and
paraffin sections were incubated with the TUNEL working solution in the
dark for 30 min at 37 °C. Three randomly selected fields in each sample
were observed, and the percentage of positive cells was calculated.
RNA sequencing
RNA sequencing was performed to investigate the underlying mechanisms
of the anti-inflammatory effect of CeO[2]@RSV. Briefly, PMA-induced M0
macrophages were seeded in 6 cm plates at a density of 5 × 10^6
cells/well, and then 100 ng/mL LPS plus 20 ng/mL IFN-γ and 50 μg/mL
CeO[2]@RSV were cultured for 48 h. TRIzol reagent was used to collect
the total RNA, and the gene expression level was analyzed by Wekemo
Technologies (Shenzhen, China). Gene Ontology (GO) enrichment analysis
and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment
analysis were performed using Wekemo BioinCloud
([92]https://bioincloud.tech/).
Statistics
All statistical analyses were conducted using GraphPad Prism 6
software. The results are presented as the mean ± SD. Paired t tests
were used when only two groups were compared. Evaluation of
significance was performed using one-way ANOVA when more than two
groups were compared. P < 0.05 was used to indicate statistical
significance.
Results
Characteristics of prepared NPs
Octahedral CeO[2] was functionalized with 3-aminopropyltriethoxysilane
(APTES) to form Si–O bonds, followed by chemical bonding of RSV to
CeO[2] through acidic hydroxyl groups of resveratrol replacing the
ethoxy groups provided by APTES, finally obtaining CeO[2]@RSV NPs. A
schematic of the chemical bonding between RSV and APTES is shown in
Fig. [93]1A. Different concentrations of RSV (5, 10, and 15 mg) were
attached to CeO[2]@APTES (40 mg) by mixing for 24 h. According to the
LE and EE results of the synthesized CeO[2]@RSV NPs, we found that
10 mg RSV obtained appropriate LE and EE levels and was selected for
subsequent research (Additional file [94]1: Fig. S2, Table S3). All NPs
were octahedral in shape with a uniform particle size of approximately
100 nm, as determined by TEM and SEM (Fig. [95]1B, C), and the average
sizes of CeO[2], CeO[2]@APTES and CeO[2]@RSV were 93, 105 and 120 nm,
respectively (Additional file [96]1: Fig. S3). The zeta potentials of
the three kinds of NPs changed from + 24.1 to − 5.6 mV, which was due
to the modification of RSV, as RSV has a negative potential.
(Fig. [97]1D). The formation of the synthesized particles was further
assessed by FTIR spectrometry. As shown in Fig. [98]1E, a peak at
1573 cm^−1 was associated with the NH[2] group, which was attributed to
the presence of APTES. The characteristic peak of stretching vibration
at approximately 1660 cm^−1 was found in CeO[2]@RSV, indicating the
chemical bond between CeO[2] NPs and RSV. The corresponding EDX mapping
images displayed the distribution of Ce, Si and C in the nanocomposites
(Additional file [99]1: Fig. S4). As shown in the X-ray diffraction
(XRD) patterns, the characteristic peaks of the three NPs could be
indexed to the ceria-based crystallographic structure (Additional file
[100]1: Fig. S5). These results demonstrated the successful
functionalization of CeO[2] with APTES and RSV.
Fig. 1.
[101]Fig. 1
[102]Open in a new tab
Characteristics of prepared NPs. A Schematic representation of the
synthesis of CeO[2]@RSV. B SEM images of CeO[2], CeO[2]@APTES and
CeO[2]@RSV. C TEM images, D zeta potential, E FTIR, F SOD activity and
G CAT activity of the indicated NPs. Data are shown as the mean ± SD.
n = 3, *P < 0.05. Scale bar: 100 nm
Given that the electron transfer between Ce^3+/Ce^4+ determines the
nanozyme activity of CeO[2] [[103]37, [104]38], we then examined the
proportion of Ce^3+ and Ce^4+ in the three NPs by X-ray photoelectron
spectroscopy (XPS) (Additional file [105]1: Fig. S6). The ratios of
Ce^4+/Ce^3+ in the three samples were 16.6%, 17.8% and 18.4%,
respectively, and slight changes in the Ce^3+ concentration indicated
the stable antioxidant activity of the nanoceria when combined with
RSV. The SOD and CAT activity assays demonstrated that all NPs
exhibited favorable antioxidant capacities, among which CeO[2]@RSV had
the greatest antioxidant ability (Fig. [106]1F, G). Furthermore, other
probes (2,2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate (ABTS)),
which are also commonly used in investigating antioxidant properties,
were used [[107]39], and Additional file [108]1: Fig. S7 shows similar
antioxidant efficiency of CeO[2]@RSV. Spectrophotometer analysis
indicated that the drug loading and encapsulation efficiencies were
6.13% and 67.92%, respectively. Furthermore, the diameter and zeta
potential exhibited minor fluctuations within 7 days (Additional file
[109]1: Fig. S8A, B), which confirmed the favorable stability of
CeO[2]@RSV under physiological conditions.
CeO[2]@RSV manipulates the infiltration of macrophages and reduces the
inflammatory response
Macrophage infiltration and macrophage-derived products are the main
pathological factors in PCOS [[110]14, [111]40]. Before applying our
constructed CeO[2]@RSV NPs, we first optimized the concentrations in
vitro. The cytocompatibility of CeO[2]@RSV was detected by a CCK-8
assay to assess the viability of the macrophages (Additional file
[112]1: Fig. S9). We found that a dose of 50 µg/mL resulted in
relatively high cell viability and served as the optimal concentration
for subsequent experiments. In addition, TEM revealed that CeO[2]@RSV
was present in the cytoplasm of macrophages, suggesting the favorable
uptake and biological activity of our synthesized NPs (Fig. [113]2A).
Fig. 2.
[114]Fig. 2
[115]Open in a new tab
CeO[2]@RSV manipulates the infiltration of macrophages and reduces the
inflammatory response. A Representative TEM images of macrophages
incubated with CeO2@RSV (50 μg/mL) for 48 h. Scale bar: 10 µm. The
arrowhead indicates intracellular NPs. B The diagram describes the
effect of CeO[2]@RSV on the repolarization of macrophages from M1 to
M2. mRNA expression levels of iNOS (C), IL-1β (D) and TNF-α (E) were
determined by RT-PCR in M1 macrophages treated with NPs. mRNA
expression levels of TGF-β (F) and IL-10 (G) in different experimental
treatment groups. H Immunofluorescence results of iNOS and CD206 in
different experimental treatment groups. Scale bar: 50 μm. I ELISA
results of IL-1β and IL-10 levels in the supernatants of the different
treatment groups. J Flow cytometry analysis of specific markers of M1
(CD86) and M2 (CD206) macrophages in different experimental treatment
groups. K Representative histograms and MFIs of DCF fluorescence in M1
macrophages treated with NPs. n = 3, *P < 0.05, **P < 0.01,
***P < 0.001
We next investigated the immunoregulatory effect of CeO[2]@RSV on
macrophage polarization. PMA-induced M0 macrophages were incubated with
CeO[2]@RSV, and the results showed that CeO[2]@RSV markedly promoted
the differentiation of M0 macrophages into M2 cells while decreasing
their differentiation into M1 cells (Additional file [116]1: Fig. S10).
In addition, LPS + IFN-γ was added to M0 macrophages prior to NPs
treatment (Fig. [117]2B). The mRNA expression levels of M1 polarization
markers (IL1-β, iNOS and TNF-α) were decreased compared with those in
the control group, with the CeO[2]@RSV group exhibiting the most
prominent effects (Fig. [118]2C–E), which indicated superior abilities
owing to the synergistic effect of CeO[2] and RSV. TGF-β and IL-10,
indicating M2 markers, were upregulated in the CeO[2]@RSV group
(Fig. [119]2F, G). Immunofluorescence also indicated increasingly high
levels of CD206 but diminished levels of iNOS in the CeO[2]@RSV group
(Fig. [120]2H). Furthermore, the level of proinflammatory IL1-β was
significantly suppressed in NPs-treated cells, and the level of
anti-inflammatory IL-10 increased most efficiently in the supernatant
of CeO[2]@RSV-treated cells (Fig. [121]2I). The flow cytometry results
indicated the same trend (Fig. [122]2J). The above results demonstrated
that the CeO[2]@RSV we prepared could manipulate the M2 phenotype
polarization of undifferentiated (M0) and differentiated (M1)
macrophages and promote the anti-inflammatory effect of M2 macrophages;
these changes in macrophage profiles are critical for remodeling the
ovarian immune microenvironment.
It is known that macrophages produce excessive ROS when activated by
diverse inflammatory stimuli [[123]41], and oxidative stress may
further exacerbate inflammatory reactions and arouse a vicious cycle
[[124]42]. In addition, CeO[2]@RSV NPs presented predominant SOD and
CAT activity; therefore, we evaluated the effect of NPs on ROS
production in inflammatory macrophages. As indicated in Fig. [125]2K,
macrophages pretreated with NPs exhibited distinctly reduced
intracellular ROS levels, with minimum DHE fluorescence. Moreover, MDA
and SOD, well-established markers of oxidative stress, were measured to
assess antioxidative capacity. As expected, compared to all other
groups, the CeO[2]@RSV group exhibited markedly increased SOD levels
and decreased MDA levels (Additional file [126]1: Fig. S11A, B).
Collectively, these data suggest that CeO[2]@RSV NPs inhibit oxidative
stress and increase the antioxidative ability of M1 macrophages.
CeO[2]@RSV-induced macrophage polarization promotes proliferation and
inhibits apoptosis in granulosa cells
The proliferation and apoptosis of GCs are thought to play essential
roles in the pathogenesis of PCOS [[127]43]. Recently, much attention
has been given to the effect of macrophage polarization on the
abilities of GCs, and the increased M1/M2 ratio suppresses GC
proliferation in antral follicles [[128]16]. Herein, granulosa cells
and NPs-treated M1 macrophages were cocultured in a noncontact
Transwell system to explore the effect of NPs-treated macrophage
polarization on the proliferation and apoptosis of KGN cells
(Fig. [129]3A). The results of CCK-8 and EdU assays illustrated that
the proliferation of KGN cells in the NPs-treated coculture group was
augmented compared with that in the group in which KGN cells were
cultured with M1 macrophages alone (Fig. [130]3B, C). In the apoptotic
pathway, the proapoptotic molecule Bax and the antiapoptotic molecule
Bcl2 are the most important participants that effectively regulate the
cell [[131]44]. Hence, to corroborate the effects of NPs-treated M1
macrophages on KGN cells, we tested the changes in the levels of these
genes. As expected, compared to all other groups, the CeO[2]@RSV
coculture group exhibited a markedly increased Bcl2/Bax ratio
(Fig. [132]3D, E). Furthermore, the apoptosis of KGN cells in different
groups was assessed using flow cytometry, and our results suggested
that the apoptosis rate of KGN cells was dramatically decreased in the
NPs-treated cocultured group compared with the M1 macrophage cocultured
group (Fig. [133]3F). Comparatively, CeO[2]@RSV yielded a more
efficient effect on the proliferation and apoptosis of KGN cells than
CeO[2] + RSV and CeO[2] alone. Furthermore, we observed analogous
phenomena in human primary ovarian GCs pretreated with CeO[2]@RSV, such
as a higher Bcl2/Bax ratio and a lower apoptosis rate (Additional file
[134]1: Fig. S12).
Fig. 3.
[135]Fig. 3
[136]Open in a new tab
CeO[2]@RSV-induced macrophage polarization promotes proliferation and
inhibits apoptosis in KGN cells. A Schema of the NPs-treated
macrophage-KGN cell coculture model. B Representative images and
quantification of EdU (+) in KGN cells. Scale bar = 50 μm. *P < 0.05,
**P < 0.01. C CCK8 results of KGN cells in different groups. *P < 0.05,
**P < 0.01, ***P < 0.001 vs. the CeO[2] group; ^#P < 0.05, ^##P < 0.01
vs. the CeO[2] + RSV group. D RT-PCR analysis of Bcl2/Bax in different
groups. E Western blot analysis of Bcl2/Bax in different groups. F
Apoptosis of KGN cells in each group was detected by flow cytometry.
n = 3, *P < 0.05, **P < 0.01, ***P < 0.001
CeO[2]@RSV directly changes the inflammation status and affects the apoptosis
of ovarian tissues in PCOS mice
Based on the excellent anti-inflammatory and antioxidant effects of
CeO[2]@RSV in vitro, we further established a DHEA-induced PCOS mouse
model and then administered CeO[2], RSV, CeO[2] + RSV and CeO[2]@RSV
for the next 2 weeks to investigate the effects in vivo (Fig. [137]4A).
First, an animal long-term toxicity test was conducted to examine the
biosafety of CeO[2]@RSV (1 mg/kg). Then, HE staining showed no
histological variation between the treatment groups and the control
group in the heart, liver, spleen, lung, kidney and ovary (Additional
file [138]1: Fig. S13). As shown in Additional file [139]1: Fig. S14A,
B, the PCOS model mice were intravenously injected with indocyanine
green (ICG)-labelled CeO[2]@RSV NPs, and the abdomen was enriched with
abundant nanoparticles. Furthermore, we witnessed an obvious
accumulation of NPs in ovarian tissue, among which the CeO[2]@RSV group
displayed prominent bioavailability.
Fig. 4.
[140]Fig. 4
[141]Open in a new tab
CeO[2]@RSV directly changes the inflammation status and affects the
apoptosis of ovarian tissues in PCOS mice. A Experimental protocol for
the therapeutic treatment of PCOS in mice. B ELISA results of IL1-β and
IL-10 levels in the serum of each group (n = 5). C RT-PCR analysis of
the mRNA expression levels of IL1-β, iNOS and IL-10 in the ovarian
tissues of each group. D The ratio of M1 (F4/80^+CD11c^+)/M2
(F4/80^+CD163^+) macrophages in the spleens of mice from the indicated
groups was analyzed by flow cytometry. E Representative images of TUNEL
assay results in mouse ovaries from the indicated groups. Scale bar:
50 µm. F Western blot analysis of the Bcl2/Bax ratio in the ovarian
tissues of each group. *P < 0.05, **P < 0.01, ***P < 0.001
PCOS is known for its chronic inflammation status, and macrophage
infiltration is the main pathological event that occurs in the ovaries
of PCOS patients [[142]45]. The ELISA results showed that the level of
the proinflammatory cytokine IL1-β was decreased in all NPs-treated
mice, and the level of the anti-inflammatory cytokine IL-10 was
significantly increased in the CeO[2]@RSV group compared with the PCOS
model mice (Fig. [143]4B), suggesting that inflammation was suppressed
by NPs treatment and that CeO[2]@RSV exhibited the greatest effect.
RT‒PCR analysis of the ovarian tissues also showed the same results
(Fig. [144]4C). Similarly, the M1/M2 ratio in the spleen of
CeO[2]@RSV-treated mice was greatly decreased (Fig. [145]4D),
indicating that CeO[2]@RSV successfully altered the macrophage
polarization state. Moreover, the apoptosis of ovarian GCs in PCOS mice
was further investigated. As shown in Fig. [146]4E, the TUNEL assay
showed that NPs treatment was able to suppress the apoptosis of ovarian
GCs in PCOS mice, among which the CeO[2]@RSV group contributed the most
efficiently. Similarly, western blot analysis confirmed a consistent
trend (Fig. [147]4F). Overall, these results suggested that CeO[2]@RSV
could effectively repolarize M1 macrophages into the M2 phenotype,
alleviate the inflammatory response, and ameliorate GC apoptosis in the
ovarian tissues of PCOS mice.
Effects of CeO[2]@RSV on ameliorating ovarian function and endocrine
disorders in PCOS mice
Reportedly, ovarian dysfunction including endocrine, follicular growth
and ovulation disorders, is an important characteristic of PCOS
[[148]46]. Given the excellent anti-inflammatory activity of
CeO[2]@RSV, we then assessed its effects on ovarian function and
endocrine disorders. As indicated in Fig. [149]5A, PCOS mice exhibited
abnormal estrous cycles, and this phenomenon was attenuated by NPs
treatment. Examination of hormones showed that the serum levels of
estradiol (E[2]), testosterone (T) and luteinizing hormone (LH) were
significantly higher in the PCOS group and subsequently decreased in
all experimental groups, especially those treated with CeO[2]@RSV
(Fig. [150]5B–D). In addition, the PCOS group showed the typical change
in ovarian morphology, with a considerable number of cystic follicles
(CF) and a relatively diminished number of corpora lutea (CL), and
administration of NPs reversed these effects (Fig. [151]5E–G).
According to the GTT results, PCOS mice exhibited impaired glucose
tolerance, while treatment with NPs significantly suppressed glucose
levels and the GTT area under the curve (AUC), and the CeO[2]@RSV group
alleviated the levels most efficiently (Fig. [152]5H, I). These data
suggested that CeO[2]@RSV treatment ameliorated endocrine disorders and
abnormal ovarian morphology and restored impaired glucose metabolism in
DHEA-induced PCOS mice.
Fig. 5.
[153]Fig. 5
[154]Open in a new tab
Effects of CeO[2]@RSV on ameliorating ovarian function and endocrine
disorders in PCOS mice. A Cytological assessment of vaginal smears
after inducing PCOS and treatment with NPs. B–D Serum levels of T, E[2]
and LH in the indicated groups. E The number of cystic follicles in the
ovaries of the mice in the indicated groups. F The number of corpora
lutea in the ovaries of the mice in the indicated groups. n = 5,
*P < 0.05, **P < 0.01, ***P < 0.001. G Representative HE staining of
ovarian sections from the experimental mice. Scale bar: 200 µm. H, I
Glucose tolerance tests and AUCs of the experimental mice. n = 5,
*P < 0.05, **P < 0.01, ***P < 0.001 vs. the CeO[2] group; ^#P < 0.05,
^##P < 0.01 vs. the RSV group. ^&P < 0.05 vs. the CeO[2] + RSV group
Anti-inflammatory mechanism of CeO[2]@RSV evaluated by transcriptome
To gain further insight into the anti-inflammatory mechanism of
CeO[2]@RSV, RNA sequencing was performed in M1 macrophages treated with
CeO[2]@RSV. Volcano plots demonstrated that the genes were
significantly different after treatment with CeO[2]@RSV (Fig. [155]6A).
As illustrated in Fig. [156]6B, the clustered heatmap shows that
treatment with CeO[2]@RSV had a suppressive effect on various
proinflammatory genes, such as IL13A, TNF, IL-6, CCL2 and IL32.
Notably, the expression of anti-inflammatory and antioxidative genes,
including GPX8 and TGFBR1, was distinctively upregulated after
CeO[2]@RSV treatment compared to that in the M1 macrophage group. GO
enrichment analysis of the differentially expressed genes demonstrated
significant changes in immune response, cytokine activity, and
chemokine activity (Fig. [157]6C). KEGG analysis identified
significantly enriched pathways, and the TNF signaling pathway,
cytokine‒cytokine receptor interaction, NF-κB signaling pathway,
NOD-like receptor signaling pathway, JAK-STAT signaling pathway, and
MAPK signaling pathway were strongly associated with the
anti-inflammatory effects of CeO[2]@RSV (Fig. [158]6D). To further
investigate the specific pathway after CeO[2]@RSV treatment, western
blot analysis was conducted and the results revealed that the
phosphorylation of STAT, NF-κB, Erk1/2 and MAPK was activated to
varying degrees. Among them, p-STAT3/STAT3 was the most significant
which suggested that the JAK-STAT signaling pathway played an important
role.
Fig. 6.
[159]Fig. 6
[160]Open in a new tab
Anti-inflammatory mechanism of CeO[2]@RSV evaluated by transcriptome. A
Volcano plots showing the genes regulated by treatment with CeO[2]@RSV.
B Clustered heatmap of representative inflammation-related genes (fold
change ≥ 2.0 and p < 0.05). C Bar plot of biological processes of
differentially expressed genes between the M1 and CeO[2]@RSV groups. D
KEGG pathway enrichment analysis of the differentially expressed genes
between the M1 and CeO[2]@RSV groups. E The protein expression of
STAT3/p-STAT3, NF-κB/p-NF-κB, Erk1/2/p-Erk1/2, and MAPK/p-MAPK was
detected by western blotting. n = 3, *P < 0.05, **P < 0.01,
***P < 0.001 vs. the M1 group
The JAK-STAT signaling pathway is responsive to the treatment of CeO[2]@RSV
during PCOS
Given the potent efficacy of CeO[2]@RSV and the transcriptome analysis,
relevant mechanisms of CeO[2]@RSV-mediated macrophage polarization and
the treatment of PCOS were explored, and relevant in vivo and in vitro
experiments were conducted for further verification. In cell
experiments, we found that a specific inhibitor of JAK (WP1066)
reversed the effect of CeO[2]@RSV on macrophage polarization, with
elevated levels of M1 phenotype markers and depressed levels of M2
phenotype markers (Fig. [161]7A, B). Additionally, western blot
analysis showed that after inhibiting JAK, STAT3 phosphorylation
decreased (Fig. [162]7C). At the same time, WP1066 inhibited the
increased Bcl2/Bax expression and promoted the apoptosis of KGN cells
after coculture with M1 macrophages (Fig. [163]7D–F). In animal
experiments, PCOS mice were then administered CeO[2]@RSV + WP1066, and
ELISA, western blotting, TUNEL and HE staining were performed. The
results indicated that after inhibiting JAK, the serum level of IL-1β
was enhanced, but IL-10 was reduced (Fig. [164]7G). The TUNEL assay
showed that the apoptosis of ovarian tissues was increased when WP1066
was administered (Fig. [165]7H). Furthermore, WP1066 treatment
deteriorated ovarian function, which was improved by CeO[2]@RSV, with
an increased number of CFs, a decreased number of CLs and deteriorated
glucose tolerance (Fig. [166]7I–L). All of these results suggested that
the JAK-STAT signaling pathway is a key signaling pathway in CeO[2]@RSV
therapy for PCOS, which involves changes in the immune
microenvironment, ovarian function, and glucose metabolism.
Fig. 7.
[167]Fig. 7
[168]Open in a new tab
The JAK-STAT signaling pathway is responsive to the treatment of
CeO[2]@RSV during PCOS. A ELISA results of IL-1β and IL-10 levels in
the supernatants of CeO[2]@RSV-treated M1 macrophages incubated with or
without WP1066. B Flow cytometry analysis of specific markers of M1
(CD86) and M2 (CD206) macrophages in different experimental treatment
groups. C The protein expression of STAT3 and p-STAT3 in the three
groups. D Representative images and quantification of EdU (+) in KGN
cells. E Western blot analysis of Bcl2/Bax in KGN cells. F Apoptosis of
KGN cells in each group was detected by flow cytometry. G ELISA results
of IL1-β and IL-10 levels in the serum of each group. H A TUNEL assay
was used to detect apoptosis in ovarian tissues in vivo. I
Representative HE staining of ovarian sections from the experimental
mice. J The number of CFs and CLs in the ovaries of the mice in each
group. *P < 0.05, **P < 0.01, ***P < 0.001. K, L Glucose tolerance
tests and AUCs of the indicated groups. n = 5, *P < 0.05, **P < 0.01,
***P < 0.001 vs. the PCOS group; ^#P < 0.05, ^##P < 0.01 vs. the
CeO[2]@RSV group
Discussion
PCOS is a disorder with reproductive, endocrine and metabolic
irregularities that poses enormous fertility challenges for women of
reproductive age [[169]47]. It is known that anti-inflammation is an
important strategy in the treatment of PCOS syndromes [[170]48,
[171]49]. Traditional agents for PCOS therapy (such as metformin and
oral contraceptives) mainly alleviate a series of complications, have
poor stability and cause severe side effects [[172]50, [173]51], and
there is a need to develop novel treatment options. Macrophages are key
mediators of the immune response in the ovarian immune microenvironment
due to their considerable plasticity and versatile roles in response to
external stimuli [[174]52]. It has been well documented that aberrant
macrophage polarization is critical for the maintenance of
proinflammatory phenotypes during PCOS and accelerates the injury of
ovarian tissues [[175]53]. Therefore, rebalancing proinflammatory
microenvironment agents would be beneficial for precise PCOS therapy.
Recently, CeO[2,] with potent anti-inflammatory and antioxidant
effects, has attracted considerable attention and protects against
numerous inflammation-related diseases, such as diabetic wounds, acute
kidney injury and ischemic stroke [[176]54, [177]55]. However, no study
has focused on the therapeutic effect of nanomaterials on PCOS, and
current CeO[2] nanoparticles have been limited by their
anti-inflammatory effects on the M2 phenotype. In the present study, we
characterized a novel nanoparticle, CeO[2]@RSV, which was synthesized
via chemical bonding of CeO[2] with traditional anti-inflammatory RSV,
thus exhibiting satisfactory effects in the treatment of PCOS. Compared
with traditional anti-inflammatory or natural enzymes, the chemically
bonded nanozyme CeO[2]@RSV shows superior capability in eradicating the
inflammatory response, scavenging excessive ROS and simultaneously
exhibiting excellent biocompatibility. Our results demonstrated that
CeO[2]@RSV possessed prominent SOD- and CAT-mimetic activities and has
tremendous potential to protect cells from a variety of types of
damage. Macrophages are the key regulators of GC proliferation,
follicular development and ovulation [[178]14], and the balance of the
M1/M2 ratio determines the pathophysiology of PCOS [[179]16]. After
being taken up by macrophages, CeO[2]@RSV can efficiently manipulate
the repolarization of macrophages, thereby reducing the proinflammatory
M1 phenotype and enhancing the anti-inflammatory M2 phenotype. In
addition, increasing evidence emphasizes the great potential of
crosstalk between macrophages and granulosa cells in the physiological
functions of ovaries [[180]14]. Upon inflammatory stimulation,
macrophage recruitment and subsequent proinflammatory cytokine release
induce GC apoptosis and follicular atresia [[181]56]. As expected, our
results demonstrated that CeO[2]@RSV-mediated macrophage transformation
promoted the proliferation and inhibited the apoptosis of ovarian
granulosa cells. Moreover, our results showed that CeO[2]@RSV treatment
ameliorated endocrine disorders and abnormal ovarian morphology, and
restored impaired glucose metabolism in DHEA-induced PCOS mice.
Therefore, all the results suggest that alterations in the local
microenvironment mediated by activated M1/M2 subtypes regulate the
function of the ovary during PCOS.
Within the ovary, macrophage activities are involved in various aspects
of ovarian function in PCOS pathogenesis [[182]57]. ELISA and flow
cytometry analysis revealed that treatment with CeO[2]@RSV dramatically
decreased the number of proinflammatory macrophages in PCOS mice, which
may contribute to the dampened proinflammatory microenvironment.
RNA-seq data and further experiments indicated that several signaling
pathways were related to the role of CeO[2]@RSV in macrophages,
including the JAK-STAT signaling pathway, NF-κB signaling pathway and
MAPK signaling pathway, of which the JAK-STAT signaling pathway
exhibited the most marked effects. The enrichment analysis revealed
that the immune response, cytokine activity, and chemokine activity are
vital changes after CeO[2]@RSV treatment, which are significantly
correlated with the improvement of PCOS. At the same time, the JAK/STAT
pathway emerges as a major regulator of macrophage polarization that
characteristically influences the production of proinflammatory
cytokines, which are vital for the development of inflammatory diseases
[[183]58, [184]59]. Further explorations confirmed that the therapeutic
abilities of CeO[2]@RSV could be inhibited by inhibitors of the
JAK/STAT pathway in vitro and in vivo.
Conclusion
Nanoparticles have emerged as a promising approach that possesses high
biocompatibility and drug delivery capabilities. The integration of
traditional anti-inflammatory drugs and various functions of synthetic
materials endow nanoparticles with widespread implications [[185]60].
In our study, RSV was loaded with CeO[2] via chemical bonding to obtain
the anti-inflammatory nanoparticle CeO[2]@RSV. CeO[2]@RSV nanoparticles
preferentially suppress inflammation response-mediated injury and
alleviate endocrine dysfunction, thereby reversing the pathological
changes that occur during the development of PCOS. Overall, our study
may elucidate the treatment and management of PCOS by using
nanoparticles as potential therapeutics.
Supplementary Information
[186]12951_2023_2182_MOESM1_ESM.docx^ (1.9MB, docx)
Additional file 1: Table S1. Baseline characteristics of the study
population (n = 10). Table S2. Primer sequences for RT‐PCR. Table S3.
EE and LE of different concentration of RSV onto CeO[2]@APTES. Figure
S1. The schematic graph of the chemical bonding between resveratrol and
APTES. Figure S2. Encapsulation efficiency and loading efficiency of
CeO[2]@APTES NPs with different RSV concentration. Figure S3. The
diameters of the NPs were determined by TEM. Figure S4. Representative
EDX mapping images of Ce, N, and C elements in CeO[2]@RSV
nanoparticles. Scale bar: 100 nm. Figure S5. XRD measurement of CeO[2],
CeO[2]@APTES and CeO[2]@RSV. Figure S6. XPS analysis of CeO[2] and
CeO[2]@RSV nanoparticles. Figure S7. ABTS^+ scavenging effects of
different samples. Figure S8. The stability of different samples. Zeta
potentials (A) and average size (B) of prepared NPs. Figure S9. Cell
viability of THP1 cells in CCK-8 assay incubated with CeO[2]@RSV NPs at
a concentration of 10, 50 and 100 µg/mL for 24, 48 and 72 h. Data are
shown as the mean ± SD. n = 3, *P < 0.05, **P < 0.01 vs. the control
group. Figure S10. CeO[2]@RSV could drive macrophage polarization and
reduce inflammatory response. (A). The diagram described the effect of
CeO[2]@RSA on macrophage polarization. (B). The expression levels of
polarization markers in these groups were determined by RT-PCR (C)
western blot (D) and flow cytometry analyses (E) with different
experimental treatment. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.
Figure S11. CeO[2]@RSV treatment attenuated oxidative stress. Levels of
SOD (A) and MDA (B) in M1 macrophages from each group 48 h after NP
treatment (n = 3). *P < 0.05, **P < 0.01, **P < 0.001. Figure S12.
CeO[2]@RSV-induced macrophage polarization promotes proliferation and
inhibits apoptosis in granulosa cells. (A). RT-PCR analysis of Bcl2/Bax
in different groups. (B). Western blot analysis of Bcl2/Bax in
different groups. (C). Apoptosis of granulosa cells in each group was
detected by flow cytometry. n = 3, *P < 0.05, **P < 0.01. Figure S13.
Evaluation on the biocompatibility of the CeO[2]@RSV from C57BL/6 mice.
Mice treated with PBS were defined as a control group. n = 5, scale
bar: 50 µm. Figure S14. (A). In vivo fluorescence imaging of mice and
Ex vivo fluorescence images of organs received intravenous injection of
CeO[2]@RSV NPs. (B). The fluorescence images were detected in the ovary
tissue injected with CeO[2], CeO[2] + RSV and CeO[2]@RSV NPs.
Acknowledgements