β‐Lactoglobulin affects the oxidative status and viability of equine endometrial progenitor cells via lncRNA‐mRNA‐miRNA regulatory associations

Abstract The β‐lactoglobulin (β‐LG) was previously characterized as a mild antioxidant modulating cell viability. However, its biological action regarding endometrial stromal cell cytophysiology and function has never been considered. In this study, we investigated the influence of β‐LG on the cellular status of equine endometrial progenitor cells under oxidative stress. The study showed that β‐LG decreased the intracellular accumulation of reactive oxygen species, simultaneously ameliorating cell viability and exerting an anti‐apoptotic effect. However, at the transcriptional level, the reduced mRNA expression of pro‐apoptotic factors (i.e. BAX and BAD) was accompanied by decreased expression of mRNA for anti‐apoptotic BCL‐2 and genes coding antioxidant enzymes (CAT, SOD‐1, GPx). Still, we have also noted the positive effect of β‐LG on the expression profile of transcripts involved in endometrial viability and receptivity, including ITGB1, ENPP3, TUNAR and miR‐19b‐3p. Finally, the expression of master factors of endometrial decidualization, namely prolactin and IGFBP1, was increased in response to β‐LG, while non‐coding RNAs (ncRNAs), that is lncRNA MALAT1 and miR‐200b‐3p, were upregulated. Our findings indicate a novel potential role of β‐LG as a molecule regulating endometrial tissue functionality, promoting viability and normalizing the oxidative status of endometrial progenitor cells. The possible mechanism of β‐LG action includes the activation of ncRNAs essential for tissue regeneration, such as lncRNA MALAT‐1/TUNAR and miR‐19b‐3p/miR‐200b‐3p.

or allergenicity. 3,4 However, since β-LG degrades into smaller peptides, which undergo self-assembly to form structures that mimic the extracellular microenvironment of many cell types, it can also find comprehensive biomedical application in regenerative medicine. For instance, fibrils formed from whey protein isolate containing β-LG supported the metabolic activity of human bone marrow stromal cells (hBMSC) and promoted their attachment and spreading, eventually facilitating functional differentiation into bone-forming cells. 5 Furthermore, β-LG fibrils functionalized with chitosan-formed biomimetic nanotopographies improved the adipogenic differentiation of hBMSCs. 6 When deliberating the bioactivity of β-LG, we also paid attention to the homology with human glycodelin (Gd), which participates in the metabolic activity and differentiation of the human endometrial cells. 1,7 Given that β-LG modulates the differentiation of progenitor cells 9,10 and may affect their oxidative status, we were interested in whether it will influence the metabolism of endometrial progenitor cells.
Indeed, the oxidative status of the endometrial progenitor cells significantly impacts their functional differentiation and cellular plasticity. Previously, we have shown that obesity-induced oxidative stress affects the metabolism of equine endometrial progenitor cells (Eca EPCs), decreasing the dynamics of the mitochondrial network and deteriorating the regenerative potential of cells related to proliferative activity and multipotency maintenance. 8 Moreover, oxidative stress impairs the decidualization of endometrial stromal cells (ECSs), inducing their apoptosis and declining mitochondrial membrane potential. 11 On the contrary, mounting evidence indicates that resistance to oxidative stress and intracellular reactive oxygen species (ROS) levels increase during the decidualization of ESCs. 9,10 Decidualization of EPCs is essential to assuring embryo implantation, thus a prerequisite for a successful pregnancy. This process is governed by ovarian hormones, that is oestrogen and progesterone, that cooperate in regulating pregnancy and menstruation. 9 Decidualization of endometrial stromal cells is associated with cytophysiological changes in endometrial stromal cells, related to their transformation into specialized secretory decidual cells characterized by expression of phenotypic markers, that is prolactin (PRL) and insulin-like growth factor-binding protein-1 (IGFBP-1). 9,12 In addition, the involvement of non-coding RNAs (ncRNAs) in the process of decidualization is now intensively studied to reveal novel molecular pathways and find their physiological and clinical relevance. 13 Besides elements of the PRL axis, other molecular markers gain importance as factors essential for proper embryo implantation and blastocyst attachment. For example, ectonucleotide pyrophosphatase-phosphodiesterase 3 (ENPP3) undergoes cyclic changes in the endometrium regulating its receptivity, 14 while lncRNA TUNAR (TCL1 upstream neural differentiation-associated RNA) modulates proliferation and decidualization of endometrial stromal cells. 15 In addition, Yu et al. showed that overexpression of miR-375 during decidualization of endometrial stromal cells in vitro significantly reduced the expression of PRL, IGFBP1 and another decidualization marker, namely forkhead box protein O1 (FOXO1). 10 Furthermore, metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA, was found to improve the decidualization of ESCs via binding with miR-498-3p, which resulted in the upregulation of histone deacetylase 4 (HDAC4) and maintained cells viability. 16 However, Liang et al.
showed that MALAT-1 increases during endometriosis and might promote the proliferation and migration of endometriotic stromal cells in a miR-200c-dependent manner. 17 Therefore, the current progress in studies on the interaction between long ncRNAs, small non-coding RNA and coding RNA (i.e. lncRNA-miRNA-mRNA axis) is essential to identify novel targets for combating diseases associated with endometrium.
In this study, for the first time, we have tested the influence of β-LG on equine EPCs. The main objective of the research was to analyse the potential antioxidant activity of β-LG and correlate it with the expression of PRL and insulin-like growth factor-binding protein-1 (IGFBP1). Moreover, we were interested in whether β-LG may affect the EPCs' viability. Based on the expression profile of essential biomarkers, we proposed a potential molecular axis of action. Our investigation may contribute to revealing the mechanism in which β-LG at the molecular level regulates regeneration and functional differentiation of endometrium.

| Experimental culture conditions
The biological activity of β-LG was tested using EPCs established at passage 4. Cells were isolated accordingly to the protocol published previously. 8  For the experiment, cells were plated into 6-well culture dishes at 100,000 cells/well. The cultures were divided into four groups: (i) control (CTRL)-maintaining the regular condition of growth, cultured in CGM; (ii) treated with β-LG; (iii) with induced oxidative stress (OS); (iv) with induced oxidative stress and treated with β-LG.
To induce oxidative stress, EPCs cultures were treated with 100 μM of H 2 O 2 in CGM for 2 h in a 5% CO 2 incubator at 37°C. Experimental cultures were treated with 5 mg/mL of β-LG for 48 h under oxidative stress and normal culture conditions. The β-LG was dissolved in CGM.

| Evaluation of β -LG influence on EPCs cytophysiology-Oxidative status, viability and morphology
The influence of β-LG on oxidative status and viability was de-

| Evaluation of β -LG influence on the transcriptome in EPCs-Reverse transcriptionquantitative polymerase chain reaction (RT-qPCR)
Total RNA was isolated by the phenol-chloroform method and extracted with Extrazol® (Blirt DNA, Gdansk, Poland). Obtained RNA was purified from genomic DNA using a PrecisionDNAse kit  Table S1. The obtained data were normalized using the RQ MAX algorithm described previously. 8,19

| The influence of β -LG on the expression of proteins associated with decidualization-Western blot
The protocol of protein detection was published previously. Here, cultures were lysed using an ice-cold RIPA buffer containing 1% protease and phosphatase inhibitor (Sigma-Aldrich/Merck, Poznan, Polska). As previously, protein concentration in the samples was as-  Table S2.

| Statistical analysis
The results obtained in the study are presented as the mean with standard deviation (±SD). The values included in the analysis re-

| β -LG has an antioxidant effect on equine endometrial progenitor cells
To test whether β-LG exerts an antioxidant effect on equine EPCs, we first analysed its impact on intracellular ROS accumulation under control and oxidative stress conditions. The β-LG was tested in vitro at a 5 mg/mL concentration, established previously based on preliminary screening assays. At this concentration, β-LG significantly affected the cellular metabolism of EPCs Furthermore, antioxidant properties of β-LG were assessed based on its modulatory effect on mRNA levels measured for genes coding catalase (CAT; Figure 1G), superoxide dismutase 1 (SOD -1; Figure 1H), glutathione peroxidase (GPx; Figure 1I) and nuclear factor erythroid 2-related factor (NRF-2; Figure 1J). Analysed transcripts were decreased or remained unchanged in EPCs after β-LG treatment. This expression profile obtained for genes coding essential antioxidant enzymes was noted in standard culture conditions and under oxidative stress ( Figure 1G-J).
Overall, the obtained data showed that β-LG decreases the . The comparative analysis with statistics was performed on the results obtained from three cytometric measurements (E,F). The study also included the comparison of transcript levels for catalase (CAT; G), superoxide dismutase 1 (SOD -1; H) glutathione peroxidase (GPx; I) and nuclear factor erythroid 2-related factor (NRF-2; J). Comparative analysis was performed on results obtained from three independent tests. The data are presented as columns with bars mean ± SD. The significant difference was indicated with an asterisk, that is *p-value < 0.05 and ****p-value < 0.0001, while ns symbol refers to non-significant differences.
control conditions was not changed in response to β-LG stimulation. However, the accumulation of GPx transcripts was lowered simultaneously with increased mRNA levels for NRF-2. In turn, transcript levels for CAT were significantly increased under oxidative stress after β-LG treatment, while GPx and NRF-2 levels remained unchanged.

| β -LG regulates the viability of endometrial progenitor cells
We determined the distribution of EPCs based on Annexin V and 7-AAD staining to analyse the influence of β-LG on cellular viability. The levels of pro-apoptotic regulators, that is BAD ( Figure 3A) and BAX ( Figure 3B), were also decreased in EPCs treated with β-LG under standard conditions (CTRL) and H 2 O 2 -induced (OxS).
However, simultaneously the transcript levels of anti-apoptotic BCL-2 were significantly reduced in EPCs after β-LG treatment in standard culture conditions. Moreover, the mRNA levels for BCL-2 were significantly reduced in cells with induced oxidative stress, and β-LG treatment did not alter the BCL-2 expression ( Figure 3C).
The observed expression profile of BCL-2 also reflected the BAX/ BCL-2 ratio, indicating an increased amount of pro-apoptotic transcripts after β-LG treatment in standard conditions( Figure 3D).
Nevertheless, the BAX/BCL-2 ratio decreased significantly after β-LG in EPCs with induced oxidative stress ( Figure 3D). This result is in line with reduced levels of apoptosis noted after β-LG treatment ( Moreover, β-LG allows for maintaining functional growth structure and morphology of EPCs exposed to oxidative stress.

| β -LG modulates the expression of decidualization
Bearing in mind the influence of β-LG on mRNA levels for ITGB1 and ENPP3, we have decided to test the mRNA levels of decidualization markers, that is PRL and its receptor (rPRL), as well as insulin-like growth factor-binding protein 1 (IGFBP1) ( Figure 5A

| DISCUSS ION
Maintaining endometrium functionality is one of the main challenging aspects of regenerative human and animal medicine. 20,21 Endometrial progenitors are a unique population of cells that secretory activity and cellular plasticity contribute to endometrial regeneration and repair. 22,23 Recently, we have characterized equine progenitor cells (Eca EPCs), showing mesenchymal phenotype and multipotent character. 8 We demonstrated that Eca EPCs disturbed metabolism is associated with intracellular accumulation of ROS. Deteriorated oxidative status of Eca EPCs significantly affects their pro-regenerative function related to proliferation and multilineage differentiation properties. 8 Oxidative stress of stromal cells accompanied by tissue inflammation also underlie the pathophysiology of endometriosis. 24,25 Different agents were applied to target endometrial cell oxidative stress, improve endometrium regeneration and treat endometriosis. 24,26,27 In the present study, we have tested the potential antioxidant properties of β-LG using the model of equine EPCs. We have shown Nevertheless, β-LG role as a molecule of significant importance in the biology of progenitor cells derived from a unique tissue niche, that is endometrium, has not been studied so far.
Here, we have shown that β-LG reduces the accumulation of ROS in EPCs and may restore disturbed oxidative status. Obtained results are also in line with studies by Pepe et al., who showed that β-LG -derived peptide, that is BRP2 reduced ROS release in intestinal epithelial cells. BRP2 reduced ROS accumulation in cells in a concentration-dependent manner, exerting a cytoprotective effect against induced oxidative stress. 30 Pepe et al. also showed that BRP2 upregulates cytoprotective enzymes activating NRF2/ ARE pathway.
In our study, we have shown that mRNA expression for NRF2 increases after β-LG treatment, but significantly only under control conditions. NRF2 is a transcription factor regulating the expression of antioxidant enzyme genes, including CAT, SOD and GPx.
It is essential to protect against oxidants, increase cell survival responses and maintain cellular redox homeostasis by regulating ROS production. 31  The study included measurement of transcripts for ITGB1 (Integrin beta-1, (E) ENPP3 (ectonucleotide pyrophosphatase-phosphodiesterase 3, (F) as well as long non-coding RNA TUNAR (TCL1 upstream neural differentiation-associated RNA, LOC111770406, (G) and small non-coding RNA, that is miR-19b-3p (H). Columns with bars in comparative statistics represent mean ± SD. The significant difference was indicated with an asterisk, that is *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001 and ****pvalue < 0.0001, while ns symbol refers to non-significant differences. endometrial regenerative cells increases after pretreatment with stromal cell-derived factor-1 (SDF-1), resulting in the enhanced therapeutic potential of the cells. 31 Furthermore, silencing NRF2 expression in endothelial progenitor cells lowers their survival and resistance to oxidative stress injury. 32 Here, we also have demonstrated that mRNA levels for CAT, SOD-1 and GPx decrease in response to β-LG treatment. Such expression profiles of genes that code primary defence enzymes against oxidative stress may be associated with transcriptome dy- We also noted that EPCs treated with β-LG show increased mRNA expression for ENPP3. However, a significant difference was noted only in standard conditions, which also correlated with increased levels of miR-19b-3p. ENPP3 is a typical ectoenzyme localized to the cell surface and has a crucial role in metabolizing extracellular nucleotides and their derivatives. 37 However, due to its catalytic activity, ENPP3 also modulates the level of intracellular nucleotide sugars, and most recently, it was recognized as an enzyme modulating endometrial receptivity. 14,37 The levels of small non-coding RNA miR-19b-3p were not previously established for F I G U R E 5 The expression of decidualization markers regulated by β-lactoglobulin (β-LG) at protein (a-d) and transcriptional levels (e-i). The analysis was performed in control conditions (CTRL) and under oxidative stress (OxS). The prolactin (PRL; A,B,E) and its receptor (rPRL; A,C,F), as well as insulin-like growth factor-binding protein 1 (IGFBP1; A,D,G) were tested at protein and mRNA levels. Transcripts for long non-coding RNA MALAT-1 (metastasis-associated lung adenocarcinoma transcript 1; H) and small non-coding RNA miR-200b-3p (I) were also established. Columns with bars in comparative statistics represent mean ± SD. The quantitative statistic was performed from Western blots performed in triplicate. The significant difference was indicated with an asterisk, that is *p-value < 0.05, **p-value < 0.01, ***pvalue < 0.001 and ****p-value < 0.0001, while ns symbol refers to non-significant differences. endometrial progenitor cells. However, it was shown that miR-19b-3p promotes proliferation and reduces heterochromatinmediated senescence in male germline stem cells. 38 Moreover, the role of miR-19b-3p seems to be essential for the functional differentiation of stem/progenitor cells derived from bone marrow. miR-19b-3p may regulate the expression of transcripts with an important role in processes such as cell proliferation, migration and differentiation, including insulin-like growth factor 1 (IGF-1) and lncRNA H19. 39 Furthermore, increased accumulation of ENPP3 transcripts in EPCs after β-LG treatment correlated with the increased levels of long non-coding RNA TUNAR (i.e. TCL1 upstream neural differentiationassociated RNA; here: LOC111770406, an equine homologue).
LncRNA TUNAR stimulates the growth but also the proliferation of endometrial stromal cells and thus is a critical regulator of endometrium decidualization. In addition, lncRNA TUNAR was also found to modulate embryo attachment to the endometrial epithelium. 15 Given the expression profile of the ENPP3-TUNAR molecular axis characteristic for EPCs treated with β-LG, we were interested in whether β-LG modulates the expression of master regulators of endometrial progenitor decidualization, that is PRL, its receptor (PRLR) and insulin-like growth factor-binding protein 1 (IGFBP1). We noted decidualization of human endometrial stem cells (hESCs) via binding miR-498-3p and thus upregulating its target, that is histone deacetylase 4 (HDAC4). 16 MALAT1 also regulates the viability of hESCs. Its downregulation promoted apoptosis associated with increased BAX/ BCL-2 ratio and suppressed the growth of hESCs. 46 Nevertheless, MALAT-1 role in endometrium homeostasis is still elusive, and studies highlighting its role in the development of endometriosis are emerging. For instance, it was shown that MALAT-1 contributes to hypoxia-triggered protective autophagy, which is a process that promotes cell survival in endometriosis, reducing apoptosis. 47,48 To the best of our knowledge, this is the first report showing that β-LG exerts antioxidant properties and improves the vi- writing -original draft (supporting); writing -review and editing (equal). Agnieszka Śmieszek: Conceptualization (lead); data curation (equal); formal analysis (equal); funding acquisition (equal); investigation (equal); methodology (lead); project administration (equal); resources (lead); supervision (lead); validation (equal); visualization (equal); writing -original draft (lead); writing -review and editing (lead).

ACK N O WLE D G E M ENTS
The project was financed from an individual research project funded

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.