Hypoxia Mesenchymal Stem Cell Secretome Enhances IL-10 via STAT3 Pathway in a Rat PCOS Model

doi:10.21203/rs.3.rs-5348344/v1

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Hypoxia Mesenchymal Stem Cell Secretome Enhances IL-10 via STAT3 Pathway in a Rat PCOS Model

https://doi.org/10.21203/rs.3.rs-5348344/v1

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Background

Polycystic ovary syndrome(PCOS) is a condition of chronic anovulation and hyperandrogenism which commonly causes infertility. PCOS is closely associated with chronic inflammation triggered by glucose and saturated fat, causing hyperandrogenism. PCOS has been proven to affect patient’s quality of life and cause infertility, so a better therapeutic approach is needed. The secretome of MSCs is able to suppress the secretion of pro-inflammatory cytokines and growth factors. Administration of secretome MSCs can inhibit the inflammatory response by increasing IL-10 expression and inhibiting androgen secretion in PCOS model mice. Objective: To prove the effect of administration of Hypoxic Mesenchymal Stem Cell Secretome on IL-10 and STAT3 gene expression in PCOS model mice.

Method

In vivo experimental research with a post-test only control group design. The total sample was 24 wistar female rats, divided into four groups: healthy, negative control (PCOS rats were injected with 0.9% NaCL), T1 (PCOS rats were given Secretome at a dose of 200 µl) and T2 (PCOS rats were given Secretome at 400 µl) and were given treatment for 33 days. IL-10 and STAT3 gene expression was tested using the One Way Anova test followed by the Post Hoc LSD test.

Results

This study showed that the expression of the IL-10 and STAT3 genes was significant different in the T2 group compared to the negative control and there was a significant difference in IL-10 gene expression in groups T2 and T1 compared to negative control. And also there were differences in the expression of the STAT3 gene in the T2 And T1 groups.

Conclusion

Administration of Hypoxic Mesenchymal Stem Cell Secretomes had an effect on increasing IL-10 and STAT3 gene expression in PCOS rat models.

Stem Cell & Developmental Cell Biology

Mesenchymal Stem Cells

Hypoxia

IL-10 gene expression

STAT3 gene expression

Polycystic Ovary Syndrome (PCOS) is a prevalent endocrine disorder affecting reproductive-aged women, characterized by hyperandrogenism, anovulation, and polycystic ovaries [1]. The syndrome encompasses a range of metabolic and hormonal disturbances, often leading to irregular menstrual cycles, hirsutism, insulin resistance, and an increased risk of metabolic comorbidities such as obesity and type 2 diabetes mellitus [2].

Recent advances in regenerative medicine have highlighted the potential of stem cell-based therapies in addressing the complex pathophysiology of PCOS. Mesenchymal stem cells (MSCs), with their self-renewal and differentiation capabilities, have shown promise in restoring ovarian function and ameliorating metabolic imbalances associated with PCOS [3].

In particular, the hypoxic preconditioning of MSCs has emerged as a strategic approach to enhance their therapeutic potential [4]. Hypoxic conditions mimic the microenvironment of injured tissues, promoting the secretion of a distinct set of paracrine factors known as the secretome. This bioactive secretome, rich in cytokines, growth factors, and anti-inflammatory molecules, exhibits heightened regenerative and immunomodulatory properties compared to conventionally cultured MSCs [5].

The interplay between pro-inflammatory and anti-inflammatory factors plays a pivotal role in the progression and resolution of PCOS-related symptoms. Interleukin-10 (IL-10), an anti-inflammatory cytokine, exerts regulatory effects on the immune response and has been implicated in attenuating hyperandrogenism and insulin resistance in PCOS [6]. Similarly, Signal Transducer and Activator of Transcription 3 (STAT3) is a critical transcription factor involved in the regulation of various cellular processes, including inflammation and cell survival, which are perturbed in PCOS [7, 8].

Despite these promising insights, the specific impact of Mesenchymal Hypoxia Secretome Stem Cells (MHSSCs) on the expression of IL-10 and STAT3 in the context of PCOS remains underexplored. Elucidating the molecular mechanisms underlying the interaction between MHSSCs and these key regulatory elements offers a unique opportunity to comprehensively understand and potentially mitigate the multifaceted pathogenesis of PCOS.

This study seeks to bridge this knowledge gap by investigating the influence of MHSSCs on the expression profiles of IL-10 and STAT3 in a validated PCOS animal model. The findings hold significant potential for advancing our therapeutic approaches towards more targeted and effective interventions for individuals affected by this complex and challenging syndrome.

Mesenchymal Stem Cells (MSCs) Isolation from Umbilical Cord Blood

Umbilical cord blood for 21 day pregnant mice was collected from consenting donors after full-term deliveries. The mononuclear cell fraction was isolated by density gradient centrifugation using Ficoll-Paque™ Plus (GE Healthcare, USA). The obtained cells were cultured in T-75 flasks with low-glucose Dulbecco's Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). Cultures were maintained in a humidified atmosphere at 37°C with 5% CO2. Cells were passaged at 80–90% confluence using TrypLE™ Express (Gibco, USA) [9].

Validation of MSCs by Flow Cytometry Surface Marker Analysis

Passage 3 MSCs were harvested, counted, and resuspended in phosphate-buffered saline (PBS) supplemented with 2% FBS. The cells were incubated with anti-human CD73, CD90, CD105, CD34, CD45, and HLA-DR antibodies (BD Biosciences, USA) for 30 minutes at 4°C. After washing, cells were analyzed using a flow cytometer (BD Accuri™ C6, BD Biosciences, USA).

Validation of MSCs by Adipogenic and Osteogenic Staining

For adipogenic differentiation, passage 4 MSCs were cultured in adipogenic induction medium (Gibco, USA) for 21 days, followed by Oil Red O staining to visualize lipid droplets. For osteogenic differentiation, cells were cultured in osteogenic induction medium (Gibco, USA) for 21 days, followed by Alizarin Red staining to detect calcium deposits .

Induction of Hypoxia in MSCs

Passage 5 MSCs were subjected to hypoxia (5% O2) using a hypoxia chamber (STEMCELL Technologies, Canada) for 24 hours. Control cells were maintained under normoxic conditions (21% O2) [10].

PCOS Animal Model Induction

Adult female C57BL/6 mice were used. PCOS was induced by subcutaneous injection of dehydroepiandrosterone (DHEA, Sigma-Aldrich, USA) dissolved in sesame oil (6mg/100g body weight) daily for 30 days [11].

Testosterone Quantification by ELISA

Serum testosterone levels were measured using a commercially available mouse testosterone ELISA kit (Enzo Life Sciences, USA) following the manufacturer's instructions. Briefly, standards, controls, and serum samples were added to a microplate pre-coated with a testosterone-specific antibody. After incubation and washing steps, a testosterone-horseradish peroxidase (HRP) conjugate was added, followed by substrate solution. The reaction was stopped, and absorbance was read at 450 nm using a microplate reader (BioTek, USA). Testosterone concentrations were determined based on a standard curve.

Validation of PCOS by Estrus Cycle Analysis in HE Staining

After blood collection on day 23, mice were sacrificed, and ovaries were carefully excised, fixed in 10% formalin, and embedded in paraffin for histological analysis. Paraffin-embedded ovarian sections (5 µm thick) were deparaffinized, rehydrated, and stained with hematoxylin for 5 minutes. After rinsing, sections were counterstained with eosin for 1 minute. Slides were dehydrated and coverslipped. HE-stained sections were examined under a light microscope (Olympus BX53, Japan) by a blinded observer. Estrus cycle phases (proestrus, estrus, metestrus, and diestrus) were determined based on morphological characteristics of ovarian structures, vaginal epithelium, and leukocyte infiltration.

MHSSCs administration on PCOS animal model

Dehydroepiandrosterone (DHEA) injections were continued subcutaneously until day 30 to prevent ovarian self-recovery. Simultaneously, MHSSCs were administered from day 23 to day 30, with doses of 200 µL (T1) and 400 µL (T2) given intraperitoneally on days 23, 25, 28, and 30. The negative control group received NaCl treatment. From day 27 to 32, cytological examinations were performed to determine the estrus cycle. On day 33, testosterone levels were assessed, PCR analysis was conducted, and ovarian samples from all groups were collected for histological preparations using paraffin embedding and Hematoxylin-Eosin (HE) staining.

Analysis of IL-10 and STAT3 Gene Expression by qRT-PCR

Ovarian tissues were harvested, homogenized, and total RNA was extracted using TRIzol™ Reagent (Invitrogen, USA). cDNA synthesis was performed using SuperScript™ IV Reverse Transcriptase (Invitrogen, USA). qRT-PCR was conducted using PowerUp™ SYBR™ Green Master Mix (Applied Biosystems, USA) on a QuantStudio™ 3 Real-Time PCR System (Applied Biosystems, USA). Primers for IL-10, STAT3, and housekeeping gene (β-actin) were designed and validated (sequences available upon request). Relative gene expression was calculated using the ΔΔCt method [12, 13].

Statistical Analysis

The normality of the data distributions will be assessed using the Shapiro-Wilk test and The homogeneity of variances will be examined using the Levene's test. A p-value greater than 0.05 will indicate that the data is normally distributed and homogeneity of variances. Analysis of Variance (ANOVA) will be employed for comparing means across multiple groups, p < 0.05 indicated the significant differences among the treatment groups. The statistical analysis was analysed under SPPS 26 version.

MSCs isolation and validation

The MSCs was isolated form umbilical cords from 21-day-old pregnant rats. The isolated cells were then cultured in plastic flasks with specialized medium. Observation under a microscope after passage 5 revealed adherent cells with spindle-like morphology attached to the flask's surface (Fig. 1A-B). Validation of mesenchymal stem cell isolation was performed using flow cytometry to demonstrate the ability of MHSSCs to express various specific surface markers. This study showed that MHSSCs were capable of expressing CD90 (97.60%) and CD29 (97.70%), with low expression of CD45 (1.50%) and CD31 (3.20%) (Fig. 1C). Furthermore, the study assessed the differentiation potential of MHSSCs into various mature cell types. By providing specific medium, MHSSCs were induced to differentiate into osteocytes and adipocytes. The results indicated successful differentiation, as evidenced by calcium deposits and red-stained lipid droplets using Alizarin Red and Oil Red dye staining in respective osteogenic and adipogenic cultures (Fig. 1D-E).

Mesenchymal stem cells (MSCs) were then incubated under hypoxic conditions with 5% O2 concentration for 24 hours using a hypoxia chamber. Hypoxia-exposed MSCs exhibited denser growth compared to their appearance before culturing under hypoxic conditions (Fig. 1B).

Validation of PCOS Animal Model through Macroscopic and Microscopic Observation

The induction of DHEA in rats, based on macroscopic observations, revealed an increased number of palpable nodules compared to healthy rats, which are suspected to be follicular cysts, a characteristic sign of PCOS (Fig. 2A-B). Microscopic examination following DHEA induction displayed the presence of cystic follicles, further confirming a characteristic sign of PCOS (Fig. 3A-B). Furthermore, administration of DHEA induction in rats led to an elevation in testosterone levels, a key characteristic associated with the development of PCOS (Fig. 2).

MHSSCs induced IL-10 and STAT3 gene expression in PCOS animal model

As shown in Fig. 4A, the group treated with a higher dose of MHSSCs (400 µL) exhibited a significant upregulation in IL-10 gene expression compared to the control group (p < 0.05). Similarly, in Fig. 4B, the same group displayed a notable increase in STAT3 gene expression compared to the control group (p < 0.05).

Furthermore, a dose-response relationship was observed in the administration of MHSSCs. Specifically, the group receiving a higher dose demonstrated a more pronounced upregulation of both IL-10 and STAT3 gene expression levels compared to the group receiving a lower dose (200 µL) of MHSSCs. These findings highlight the potential of MHSSCs in modulating the expression of key genes involved in immune regulation and inflammation, particularly IL-10 and STAT3, in the PCOS animal model. The dose-dependent response further suggests the importance of optimizing the dosage for potential therapeutic applications. Overall, the results demonstrate the promising immunomodulatory effects of MHSSCs and underscore their potential as a targeted intervention in the management of PCOS-related inflammatory processes. Further studies are warranted to elucidate the underlying mechanisms and validate the clinical relevance of these findings.

Polycystic Ovary Syndrome (PCOS) is a condition characterized by prolonged anovulation and often accompanied by hyperandrogenism, which frequently leads to fertility issues [14]. PCOS is closely associated with chronic inflammation triggered by glucose and saturated fats, ultimately resulting in hyperandrogenism. This inflammatory state induces an increase in reactive oxygen species (ROS) levels, activating the nuclear transcription factor kappa beta (NF-κB) and enhancing the expression of pro-inflammatory cytokines such as IL-6 and TNF-alpha [15, 16].

Chronic inflammation occurs in the ovaries and surrounding tissues, marked by an abundance of Type 1 macrophages (M1), which produce pro-inflammatory cytokines like IL-6, TNF-α, and IL-1β, thus perpetuating chronic inflammation [17, 18]. Previous studies have shown that in some cases of PCOS, there is an imbalance between M1 activity and IL-10 levels [19]. Administration of MHSSCs in group T2, containing higher levels of IL-10 compared to group T1, can transform M1 into anti-inflammatory M2 macrophages. Differentiation of M1 into M2 balances the macrophage population within the ovaries, thereby halting the inflammatory process and reducing folliculogenesis, ultimately preventing PCOS [5].

IL-10 can halt inflammation by inhibiting NF-ΚB activation through several mechanisms, one of which is inhibiting IkappaB kinase (IKK) activation, an initial step in NF-κB activation [20]. Additionally, IL-10 can inhibit the TLR pathway by suppressing phosphorylation of mitogen-activated protein kinase (MAPK) and TGF-beta activated kinase 1 (TAK1), leading to reduced NF-ΚB activation induced by TLR receptor signaling. IL-10 also inhibits the activity of Tumor Necrosis Factor Receptor-Associated Factor 6 (TRAF6), a critical mediator in NF-ΚB activation [21–23]. IL-10's presence in MHSSCs, functioning as an anti-inflammatory agent, activates STAT3, leading to a significant increase in group T2. The transcription factor STAT3 binds to the promoter region of genes regulating pro-inflammatory cytokine expression, inhibiting their transcription and thereby reducing pro-inflammatory cytokine production in chronic PCOS inflammation [24–26]. STAT3 plays a crucial role in regulating regulatory T cell (Treg) function, a key player in alleviating inflammation and maintaining immunological tolerance [15, 27]. Activation of STAT3 in Treg enhances their ability to inhibit excessive immune responses and alleviate inflammation [28]. Moreover, STAT3 can direct macrophage differentiation towards the M2 phenotype, resulting in fewer pro-inflammatory cytokines and more anti-inflammatory cytokines, aiding in inflammation relief [15].

Other studies have shown a decrease in inflammation through the activation of the IL-10 STAT3 pathways. IL-10 binds to IL-10R, resulting in JAK 1 activation, inducing STAT3 phosphorylation [15]. The STAT3 protein translocates to the nucleus, activating mRNA SOSC3 sequences, which are then intracellularly expressed and can suppress pro-inflammatory signaling pathways, namely NF-κB [29, 30]. Suppression of the NF-κB pathway leads to a decrease in pro-inflammatory cytokine secretion, including IL-6 [31]. Additional research utilizing Fibrin Facilitates MSCs has demonstrated that MSCs regulate estradiol and progesterone, decrease gonadotropin (LH/FSH), testosterone (T), and TGF-β1, maintain regular estrus cycles, increase granulosa cell counts, and decrease immature cystic follicles [32, 33].

IL-10 from MHSSCs activates the STAT3 pathway through JAK-1 translocation from the intracellular membrane to the cytoplasm. This results in STAT3 phosphorylation, culminating in its translocation to the nucleus. Nuclear translocation of STAT3 activates the SOCS3 gene, which is subsequently synthesized intracellularly and can inhibit intracellular NF-kB signaling, preventing NF-κB translocation to the nucleus and the consequent expression of pro-inflammatory genes.

The significant increase in IL-10 and STAT3 expression in group K4 (MHSSCs 400µl) compared to group K2 rats indicates that MHSSCs have the potential to control inflammation. However, this study did not analyze the expression of pro-inflammatory cytokines, representing a limitation of this research.

The findings demonstrate that MHSSCs, particularly at a dose of 400µl, effectively increases IL-10 and STAT3 expression in PCOS, suggesting its potential as a therapeutic agent through multiple mechanisms: transformation of pro-inflammatory M1 to anti-inflammatory M2 macrophages, inhibition of NF-κB activation through IL-10-mediated pathways, and activation of the STAT3 signaling cascade.

Ethics approval

This study was approved by Komisi Bioetik Faculty of Medicine Universitas Islam Sultan Agung Semarang under No. 14/II/2024/Komisi Bioetik.

Acknowledgments

None to declare.

Competing interests

Authors have no known conflict of interest in relation to the publication of this work.

Funding

This study did not receive any external funding.

Underlying data

Data underlying the findings in this study could be requested to the corresponding author

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The authors declare no competing interests.

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Hypoxia Mesenchymal Stem Cell Secretome Enhances IL-10 via STAT3 Pathway in a Rat PCOS Model

Status:

Version 1

Abstract

Background

Method

Results

Conclusion

Figures

BACKGROUND

METHODS

Mesenchymal Stem Cells (MSCs) Isolation from Umbilical Cord Blood

Validation of MSCs by Flow Cytometry Surface Marker Analysis

Validation of MSCs by Adipogenic and Osteogenic Staining

Induction of Hypoxia in MSCs

PCOS Animal Model Induction

Testosterone Quantification by ELISA

Validation of PCOS by Estrus Cycle Analysis in HE Staining

MHSSCs administration on PCOS animal model

Analysis of IL-10 and STAT3 Gene Expression by qRT-PCR

Statistical Analysis

RESULTS

MSCs isolation and validation

Validation of PCOS Animal Model through Macroscopic and Microscopic Observation

MHSSCs induced IL-10 and STAT3 gene expression in PCOS animal model

DISCUSSION

CONCLUSION

Declarations

References

Additional Declarations

Status:

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