MELTF Might Regulate Ferroptosis, Pyroptosis, and Autophagy in Platelet-Rich Plasma-Mediated Endometrial Epithelium Regeneration

The endometrial basal layer is essential for endometrial regeneration, whose disruption leads to thin endometrium or intrauterine adhesion (IUA) with an unsatisfactory prognosis. Emerging data indicate that platelet-rich plasma (PRP) can promote endometrial proliferation, but the mechanism by which PRP regulates endometrial regeneration remains unclear. Herein, we investigated the therapeutic effects and possible mechanisms of PRP on endometrial regeneration. IUA animal model was generated by sham, mechanically damaging endometrium with or without PRP for 10 days. The uterine section in the model group showed degenerative changes with a narrow endometrial lumen, atrophic columnar epithelium, decreased number of endometrial glands, decreased endometrial thickness, and increased collagen deposition. The above disruption could be ameliorated by the PRP. Transcriptome sequencing analysis displayed that the retinol metabolism pathway and extracellular matrix (ECM) receptor interaction pathway were up-regulated and enriched in differential expression genes (DEGs). Melanotransferrin (MELTF) was the key up-regulated gene in PRP-induced endometrial regeneration, which was verified in vivo and in vitro. Ferroptosis, autophagy, and pyroptosis were down-regulated in PRP-treated Ishikawa cells. Conclusively, PRP promotes endometrium regeneration by up-regulating the retinol metabolism and ECM receptor interaction pathway with MELTF. Meanwhile, PRP could also inhibit endometrial epithelial cell death by regulating ferroptosis, autophagy, and pyroptosis.


Introduction
The endometrium is the inner lining of the uterus that undergoes complex regeneration and differentiation during the human menstrual cycle [1]. The endometrium contains the functional layer and the basal layer. The repeated curettage or endometritis to the endometrial basal layer will impair the regeneration of endometrial epithelium, even causing thin endometrium or IUA [2]. IUA is characterized by poor growth of the glandular epithelium with little stroma, intrauterine adhesions or fibrosis, and poor vascular development [3]. Women with damaged endometrium present various clinical symptoms, such as hypomenorrhea, amenorrhea, recurrent pregnancy loss, pregnancy complication [3], and even infertility [4]. Emerging data indicate that platelet-rich plasma (PRP) can promote endometrial proliferation. PRP is an autologous concentration of platelets in a small volume of plasma, containing more than 1,000,000 platelets per microliter [5]. The α-granules in platelets with many kinds of growth factors and cytokines are released during platelet activation at the site of injury or inflammation. These factors are critical for the activation of fibroblasts and the recruitment of leukocytes to the injury site, which regulate the proliferation and migration of smooth muscle cells and mesenchymal stem cells, promoting angiogenesis [6,7]. Although some studies [8,9] suggest that intrauterine infusion of autologous PRP has a positive effect on endometrial growth and pregnancy outcomes, whether PRP can promote endometrial proliferation is controversial [10,11]. To date, the mechanism by which PRP regulates endometrium regeneration remains unclear.
Herein, proliferative effects of PRP on Ishikawa and immortalized human endometrial stromal cell line (HESC) were validated by cell counting kit-8 (CCK-8) test in vitro. The animal model of damaged endometrium with or without PRP treatment was established to investigate the effects of PRP on endometrial regeneration. Transcriptome sequencing analysis and corresponding validation assays were performed to explore the underlying molecular mechanisms by which PRP orchestrated endometrial regeneration. Ferroptosis, autophagy, and pyroptosis were evaluated in Ishikawa and HESCs under the treatment of PRP. This study will provide a better understanding of PRP in endometrial regeneration, laying the foundation for identifying more therapeutic targets for IUA, even infertility.

Ethics
This study was approved by the medical Ethics Committee of the Zhongnan Hospital of Wuhan University (2022051 K). A total of 18 adult female Sprague-Dawley (SD) rats (9 weeks old; 230-250 g) were purchased from Wanqianjiaxing Co. (Wuhan, China). The rats were housed in a specific pathogen-free (SPF) lab in an environment with 22 ± 1 °C, relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. Sterilized water and food were ad libitum. All animal studies were done in compliance with the regulations and guidelines of the Experimental Animal Center of Zhongnan Hospital of Wuhan University.

Preparation of PRP
PRP was prepared from the blood of 2 healthy 35-yearold female donors by using a modified two-step centrifuge process. The donors were excluded from hepatitis B, tuberculosis, hepatitis C, syphilis, and HIV with the blood type of O and RH positive. A 100 ml peripheral venous blood was drawn in the tubes which contains 180 mg EDTA as a coagulant and were centrifuged immediately at 300 g for 10 min. The blood was divided into three layers: red blood cells at the bottom, cellular plasma in the supernatant, and a buffy coat layer between them. The plasma layer and buffy coat were collected to another tube and centrifuged again at 750 g for 15 min. The platelets were allowed to sediment at the bottom of the tube. Different volumes of plasma in the upper phase were added to create PRP with concentrations of about 1500 × 10 9 platelets per liter, which was checked by the Coulter counter. For activation, 0.1 ml 20% CaCl2 (C7250, Solarbio, Beijing, China) was added per ml of PRP, and 25 U thrombin (T8020, Solarbio, Beijing, China) was added per ml of the mixture. The mixture was incubated at  Lamb3  ATG ATG ACG GCA CTT TTC C  AAC GTT CTC CAC TCG GTG A  Lamc2  ATC AAC ATA GTC TCC GCC TC  CCC CAG ATT GTT TCGCA  Gp6  AAT GCT TCC TTT CCT CGG  CAG ATG GAC CTG GGA CTA A  Ugt1a7c  TTT CTC ACA CTC AGG AGG ATT  GGC AGG GCT AGT CAG TAG AG  Dhrs9  TCA GGC TTC GGA AAC TTA G  AGT CTC TCT GAG GTT TTG GC  Lrat  GGC ACA GGG AAG AAA CAT C  TCG CTG ATT CTG TCT GGC  CK18  AGA ACC TGG AAA CCG AGA A  AAA GTC ATC AGC GGC AAG  TGF-α  TAC GTG GGT GTT CGC TGT  ACT CAC AGT GCT TGC GGA  EGFR  GGA AAT AAC AGG GTT TTT GC  GGT CTT TTG ATT GGG CGT  GAPDH  CAA GTT CAA CGG CACAG  CCA GTA GAC TCC ACG ACA T  1 3 37 °C for 1 h and then at 4 °C for 12 h. After activation, the gel-like mixture was centrifuged at 5000 rpm for 30 min at 4 °C and the supernatant was aspirated and filtered through a 0.22-μm filter and then stored at − 80 °C.

In Vitro Assay of Cell Proliferation and Migration
Cell proliferation assays were performed with two different cell types: Ishikawa cells were human adenocarcinoma  Island, NY) with 10% FBS and 1% penicillin/streptomycin. CCK-8 cell proliferation assay was performed by cell counting kit-8 (Beyotime, Shanghai, China) according to the manufacturer's protocols. Cells were seeded and cultured at a density of 1 × 10 4 Ishikawa/well or 3 × 10 3 /well in 96-well microplates (Corning, USA). Cells were treated under complete medium with various concentrations of PRP (0% PRP, named as control group later, 1%, 2%, 5%, and 10%). After treatment for 24 h, 48 h, and 72 h, a complete medium with 10 uL of CCK-8 reagent was added to each well and then cultured for 2 h. All experiments were performed in triplicate. The absorbance was analyzed at 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA) using wells with complete medium but without cells as blanks. The proliferation of cells was expressed by the absorbance.
For the wound healing assay, HESCs were seeded in 6-well plates. The monolayer was then scratched with the use of a 20-μl pipette tip. After washing the wells with phosphate-buffered saline solution for removing detached cells, different concentration of PRP (0% named also as the control group, 1%, 2%, 5%, and 10%) was added to the medium. Cells were photographed at 0 and 24 h after scratching, and the wound area and wound distance were quantified by IPP6.0.
The optimal concentration of PRP was selected according to the result of the CCK-8 assay. Ishikawa Cells were inoculated in six-well plates and were cultured in a medium with 10% PRP for 48 h and the control group was cultured in medium without PRP. The HESCs were cultured in a medium with 2% PRP for 48 h and the control group was cultured in a medium without PRP. The cells were later used for western blot detection.

Animal Model
Eighteen rats were randomly assigned into three groups: sham-operated (sham) group, model group, and model + PRP group (named as the PRP group after here). The animals were treated as follows: (1) sham group: the rats were anesthetized by 1 ml/kg 3% sodium pentobarbital i.p. and an abdominal vertical incision (~ 25 mm) was performed with an intrauterine injection of 50 μl physiological saline; (2) model group: after anesthesia, a vertical incision (2-3 cm) was performed in the left uterine horn, the endometrium was scraped using a T10 scalpel blade until the internal-surface of the uterine horn was rough and bleeding leaving the uterine serosa intact. The uterine horn was subsequently washed with physiological saline. Finally, it was stitched using 6-0 absorbable sutures. The right uterine horn was treated in the same way; (3) PRP group: 50 μl PRP was injected immediately into each uterine horn just after modeling.
Seven days later, all the animals were treated with pregnant mare serum gonadotropin (PMSG) (P9970, Solarbio, China) i.m. at a dose of 10 U/100 g by weight. PMSG had been shown to induce ovulation at any stage of the estrous cycle in adult rats, and the estrous cycle was verified by daily vaginal smears within 3 days [12]. 10 days after modeling, uterine horns were immediately excised after the animals were sacrificed. The samples were taken from the modeled segments of the uterine horns or the same segments of the uterine horns for the sham group. The marginal portions of the upper and lower parts of the uterine horns were disregarded. Samples were separately kept for further research.

Hematoxylin-Eosin Staining
The biopsy specimens were fixed for 24 h in 4% paraformaldehyde, embedded in paraffin, cut into 3-μm-thick sections and stained with hematoxylin-eosin (H&E). Endometrial morphology was analyzed by H&E staining, and images were captured by Leica Application Suite imaging system. Images were captured in magnifications of × 40 and × 200. Each slide was analyzed in a double-blinded manner by two experts using IPP6.0 image software. The area of the endometrium (mm 2 ) were calculated as follows: (1) the basal endometrial zone was outlined using area selection tools; (2) the endometrial cavity was outlined using area selection tools; (3) the difference between the two areas was treated as the endometrial area. To determine the number of glands, 4 fields for each slice in a magnification of × 200 were selected for counting the number of glands. The average of the 4 values was treated as the number of glands. The endometrial

Masson Staining
Masson staining was used to detect fibrosis according to the manufacturer's protocol. To verify the area of fibrosis, six uterine cross sections were evaluated for each group, 4 fields for each slice were randomly chosen at a magnification of × 400 and subsequently, the images were analyzed in a double-blind manner by two experts using IPP6.0 image software. The blue area percentage for each image was calculated. The average of the 4 values was treated as a blue area percentage for the section.

Immunohistochemistry and Immunofluorescence Staining
The biopsy specimens were cut into 3-μm-thick sections. Antigen retrieval was performed with 1 mM TRIS-EDTA buffer solution under high pressure (125 ℃, 103 kPa) for 18 min. After inhibition of endogenous peroxidase for 10 min and protein block for 30 min at 37 °C, the slides were incubated with rabbit polyclonal antibodies to TGF-α (1:100, ab112030, Abcam, UK), or rabbit polyclonal antibodies to cytokeratin 18 (CK18) (1:500, ab133263, Abcam, UK), or rabbit polyclonal antibodies to EGFR at a dilution of (1:100, PAB30732, Bioswamp, Wuhan, China) or rabbit polyclonal antibodies to MELTF (1:150, Cat No.10428-1-AP, Proteintech, China) overnight at 4 °C, then incubated with secondary antibody for 1 h at 37 ℃. The primary antibody was replaced by PBS as a negative control. Quantification of immunoreactivity was performed by IPP 6.0, and 4 fields in the endometrial area were randomly selected at a magnification of × 400 from each slice to determine the mean intensity of optical density (MIOD). The average of the 4 values was considered the MIOD of the slice. We used immunofluorescence staining for the detection of the MELTF protein.

RNA Extraction
Total RNA was extracted from the samples reserved in the Invitrogen RNAlater® Stabilization Solution using the TRIzol reagent (15,596,026, Ambion, USA) and chloroform (10,006,818, Sinopharm Chemical Reagent Co., LTD, China) following manufacturer's protocols. The RNA quantity and concentration were assessed using Nano-300 (Aosheng, Hangzhou, China). Total RNA samples were stored at − 80 ℃ before further analysis.

Transcriptome Sequencing Analysis
Nine samples were selected for transcriptome sequencing. 3 samples from the sham group were named A1, A2, and A6. A total of 3 samples from the model group were named B2, B19, and B20. A total of 3 samples from the PRP group were named as D1, D2, and D4. For transcriptome sequencing, total RNA was extracted by the manufacturer's procedure and only samples with an RNA integrity number (RIN) value > 7.0 were used for the cDNA library construction. The cDNA libraries were constructed for each RNA sample according to the manufacturer's instructions. The PCR products were size selected and sequenced on the DNBSEQ platform. The sequencing read length was 150 bp.
Before data analysis, raw reads were filtered by SOAP2 [13] with a default parameter, so we could get the clean reads. The low-quality base sequence does not exceed 20%, and the end stain sequence does not exceed 5%. We used HI-SAT2 (V2.0.4) [14] to map clean reads to the Rattusnorvegicus reference genome sequence (GCF-0 00,001,895.-Rnor-6.0). The gene abundances were quantified by the RSEM (V1.212) [15]. The readings were normalized by DEseq2 (V1.4.5) [16]. The following criteria were used here: log2 fold change ≥ 1 and Q-value (adjusted P-value) < 0.05 were considered statistically significant. Heat map builder [17] was used to draw a sample clustering heat map. KEGG pathway and GO terms enrichment analysis were carried out by Cluster Profile V4.0 in R [18].

qRT-PCR Analysis
The cDNA synthesis was performed by PrimeScript II RTase (2690A, Takara, Japan) following the manufacturer's instructions. qRT-PCR was performed using the SYBR FAST qPCR Master Mix (KM410, KAPA Biosystems, USA) according to the manufacturer's instructions. Primers are listed in Table 1. Each sample was done in triplicate. Analysis of relative gene expressions was performed using 2 −ΔΔCT methods. Ratios of mRNA expressions were given as fold changes relative to the sham group after normalizing to the GAPDH gene.

Western Blot
The uterine horns and Ishikawa and HESCs were separately prepared using RIPA (G2002, Servicebio, China)

Statistical Analysis
All data are expressed as the mean ± standard deviation (SD) for continuous variables and as numbers or percentages for categorical variables. One-way analysis of variance (ANOVA) was used to analyze variables among groups when variances among groups were of homogeneity and LSD tests were performed for multiple comparisons when statistical significance was recognized. The Kruskal-Wallis test was used to compare the differences when variances among groups were heterogeneous. For statistical analysis, 3-5 replicates were used. *P < 0.05, **P < 0.01, and ***P < 0.001 were considered statistically significant differences. All statistical analyses were performed using IBM SPSS statistics software (version 19, IBM Corp., Armonk, NY, USA).

PRP Promoted Endometrial Cells Proliferation and Migration In Vitro
The effects of PRP on Ishikawa and HESC cell proliferation were evaluated using CCK-8 assays. As shown in Fig. 1A, PRP significantly increased the number of viable cells and showed positive concentration-dependent effects in Ishikawa cells. During the first 24 h, OD values in all groups with different concentrations of PRP were nearly identical but higher than the control group. OD values for 10% PRP were significantly higher than that for other concentration groups in 48 and 72 h (Fig. 1A). But in HESCs, 2% PRP and 5% PRP showed the best effect for cell proliferation (Fig. 1B ).
In the wound healing assay, 2% PRP showed the best effect on HESC migration. The wound healing percentages and wound distances for HESCs in medium with 2% PRP were significantly higher than that in the control group (Fig. 1C, D, E). Accordingly, all further experiments for Ishikawa were conducted with 10% PRP. All further experiments for HESCs were conducted with 2% PRP.

PRP Promoted the Endometrial Regeneration and Decreased the Endometrial Fibrosis
Thirty rats were randomly assigned into three groups: sham group, model group, and PRP group. A total of 10 days after mechanical damage, endometrial morphology, and tissue proliferation were checked. After modeling, the endometrium was damaged and degenerative changes with narrow endometrial lumen, atrophic columnar epithelium, decreased number of endometrial glands, decreased endometrial thickness, and increased collagen deposition were found. The endometrial thickness, endometrial area, endometrial glands, and endometrial lumen area were significantly decreased ( Fig. 2A, B) in the model group. The above disruption could be ameliorated by the PRP. The endometrial thickness, the number of endometrial glands, endometrial area, and endometrial gland increased significantly, and the endometrial lumen area had an increased tendency ( Fig. 2A, B) in the PRP group compared with the model group. The regeneration of endometrial epithelium was also evaluated by the expression of CK18. The expression of CK18 was mainly localized in the cytoplasm of the endometrial epithelial cells and endometrial glands (Fig. 2C). The expression of CK18 decreased in the model group compared with the sham group (Fig. 2D). The mRNA of CK18 had a slightly increased tendency and the protein expression of CK18 was increased significantly in the PRP group compared with the model group (Fig. 2D). Masson staining indicated that collagen deposition (the blue area) was increased significantly in the model group compared with the sham group, which was ameliorated by the PRP treatment (Fig. 2E, F). These data suggested that the PRP could promote endometrial regeneration and inhibit endometrial fibrosis (Fig. 2).

Profiles of Differential Expression Genes (DEGs) by Transcriptome Sequencing Analysis in PRP-Induced Endometrial Regeneration
The average output of each sample was 6.51G. A total of 18,762 genes were identified in the 9 samples from the 3 groups. The q20 values of clean data for 9 libraries were > 95%. The sample clustering heatmap and the boxplot revealed the DEGs expression mapping of each sample (Fig. 3A, B).
Transcriptome sequencing analysis showed 1109 mRNAs were up-regulated and 917 mRNAs were down-regulated in the model group compared with the sham group (Fig. 3C). KEGG pathway and GO terms enrichment analysis were performed to uncover the related functions and signaling pathways for the DEGs. The top 20 enriched KEGG pathways and GO terms for the up-regulated DEGs were listed in (Fig. 3D, E). The osteoclast differentiation pathway, cytokine-cytokine receptor interaction pathway, and hematopoietic cell lineage pathway were up-regulated in the model group. The up-regulated DEGs were significantly enriched in the inflammatory response, immune system response, neutrophil response, and immune response by Go terms enrichment analysis.
Transcriptome sequencing analysis showed 136 mRNAs were up-regulated and 21 mRNAs were down-regulated in the PRP group compared with the model group (Fig. 3F). KEGG pathway and GO terms enrichment analyses were performed to uncover the related functions and signaling pathways of the DEGs. The top 20 enriched KEGG pathways and GO terms for the up-regulated DEGs were listed in (Fig. 3G, H). The retinol metabolism pathway and the ECM receptor interaction pathway were remarkably enriched with up-regulated DEGs in the PRP group.

qRT-PCR Assays for Validations of DEGs and Up-Regulation of TGF-α and EGFR Expression in PRP-Induced Endometrial Regeneration
To further confirm the results of transcriptome sequencing analysis, we selected some DEGs from the retinol metabolism pathway and the ECM receptor interaction pathway, for qRT-PCR validation. Consequently, mRNA expression for the 7 genes had concurrent expression trends with the transcriptome sequencing analysis (Fig. 4A).
TGF-α was one of the DEGs and had an important effect on cell proliferation, and EGFR was the receptor of TGFα. mRNA relative expression of TGF-α was significantly lower in the model group compared with the PRP group and sham group (Fig. 4D). IHC staining showed that TGF-α was mainly localized in the cytomembrane, cytoplasm, and nucleus of the endometrial luminal epithelial cells and glandular epithelial cells. Protein expression of TGF-α was significantly lower in the model group compared with the PRP group and sham group (Fig. 4B,E). EGFR was mainly localized in the cytomembrane, cytoplasm, and nucleus of the endometrial luminal epithelial cells and glandular epithelial cells. Lower expression of EGFR could also be found in some endometrial stroma cells. Protein expression of EGFR was significantly lower in the model group compared with the PRP group and sham group (Fig. 4C,E). These data verified DEGs and up-regulation of TGF-α and EGFR expression in PRP-induced endometrial regeneration.

MELTF was Up-Regulated in PRP-Treated Endometrial Epithelial Cells Rather than Stromal Cells
The protein-protein interaction network (PPI) suggested that MELTF was a key gene in up-regulated DEGs in the PRP group compared with the model group (Fig. 5A). Immunofluorescence analysis showed that MELTF was mainly expressed in epithelial cells, including endometrial glandular epithelial cells and vascular epithelial cells, while it was barely expressed in endometrial stromal cells (Fig. 5B). Compared with the model group, MELTF protein expression was significantly increased in the PRP group (Fig. 5B,C,F). Western blot for MELTF in uterine horns showed the MELTF protein expression was also up-regulated in the PRP group compared with the model group (Fig. 5C,F). Furthermore, MELTF protein was up-regulated in Ishikawa after PRP treatment (Fig. 5D,G) but not expressed in the HESCs (Fig. 5E,H), which was consistent with the above research results (Fig. 5B).

MELTF Might Regulate Ferroptosis, Pyroptosis, and Autophagy in PRP-Inhibited Endometrial Cells Deaths
To further investigate the possible mechanisms by which PRP regulated endometrial regeneration, we explored the manners of cell death by testing ferroptosis, pyroptosis, and autophagy markers. The decreased expression of ACSL4 and the increased expression of GPX4 and FPN1 were observed in PRP-treated Ishikawa cells (Fig. 6A, C). GPX4 was a critical inhibitor of ferroptosis [19]. Our results showed that the PRP could inhibit the Ishikawa deaths by inhibiting ferroptosis. The NLRP3, as a regulator of pyroptosis [20], was decreased in the PRP group (Fig. 6A, C), suggesting that PRP could protect cells from pyroptosis. LC3B, as a marker of cell autophagy [21] was observed. The expression of LC3B-II and ratio of LC3B-II/I was decreased in PRPtreated Ishikawa (Fig. 6A, C), indicating that autophagy was Fig. 5 MELTF was up-regulated in PRP-treated endometrial epithelial cells rather than stromal cells. A Protein-protein interaction network (PPI) suggested that the MELTF gene was a key gene in upregulated DEGs. B Representative Immunofluorescence staining of MELTF (× 400). White arrows indicate MELTF positive. C-E Representative immunoblotting of MELTF in uterine horns, Ishikawa cells, and HESCs by Western blot. F-H Statistical analysis of representative immunoblotting of MELTF in uterine horns, Ishikawa cells, and HESCs. *P < 0.05 ◂ inhibited in PRP-treated Ishikawa. However, in the HESC group, the expression of LC3B-II and the ratio of LC3B-II/I were increased (Fig. 6B, D), implying that autophagy was activated in the PRP-treated HESCs. GPX4 protein did not express in the HESCs as well as the MELTF (Fig. 6B, D), suggesting that PRP did not affect the HESCs' apoptosis by regulating the ferroptosis.
RAS-selective lethal (RSL3) is a ferroptosis activator. RSL3 could irreversibly inactivate GPX4. After the addition of different concentrations of RSL3 (0.5 µM, 1 µM, 2 µM, 4 µM, and 8 µM) into the culture media with 10% PRR, the proliferation rate for Ishikawa had a decreased tendency. When Ishikawa was cultured with 8 µM RSL3 for 48 h, the proliferation rate for Ishikawa was significantly lower than that of the PRP group (P < 0.05) (Fig. 6E). The above results suggested that MELTF might regulate ferroptosis, pyroptosis, and autophagy in PRP-inhibited endometrial cell deaths.

Discussion
In this study, we found that PRP could promote endometrial proliferation and decrease endometrial fibrosis. Transcriptome sequencing analysis showed that the retinol metabolism pathway and ECM receptor interaction pathway were up-regulated, which was validated. MELTF, the key differentially expressed gene, was up-regulated in the PRP-induced endometrial proliferation. Furthermore, MELTF might regulate ferroptosis, pyroptosis, and autophagy in PRP-mediated endometrial epithelium regeneration.
In our study, we found that PRP could promote the Ishikawa and HESC proliferation in vitro, which was consistent with other research [22]. To further investigate the PRP on endometrial regeneration, we established the IUA animal model by mechanically damaging the endometrium. With the co-treatment of PRP, the damaged endometrium was repaired and the morphology of the endometrium was ameliorated. The protein expression of CK18 was significantly increased in the PRP group compared with the model group. These results showed that the PRP promoted endometrial epithelium regeneration, which was verified in some studies [5,7]. However, Javaher A et al. [10] deemed that the inactivated PRP could not promote endometrial regeneration. The controversial result was probably due to that the different states of PRP had different effects. The mRNA expression of CK18 had no significant difference between the model groups and the PRP group, although it had an increased tendency in the PRP group. The process from mRNA expression to protein expression was affected by many factors, which need further research.
Transcriptome sequencing analysis is a novel bioinformatics analysis method to test the differential expression levels of mRNA. In our research, the reliability of transcriptome sequencing was confirmed by qRT-PCR. We found that the up-regulated DEGs were enriched in the retinol metabolism pathway after PRP treatment. The two main bioactive metabolites of vitamin A are all-trans-retinoic acid (atRA) and 11-cis-retinaldehyde [23]. The atRA could promote cellular proliferation, differentiation, morphogenesis, cell survival, angiogenesis, wound recovery, and stem cell migration mediated by the retinoic acid receptors (RARs) [24,25]. The atRA could inhibit the fibrosis of the lung, liver, and rein [26][27][28]. The atRA could also inhibit endometrial fibrosis in a rat IUA model [29]. Therefore, PRP might promote epithelium proliferation and inhibit endometrial fibrosis by influencing the atRA synthesis through the retinol metabolism pathway.
We also found the ECM receptor interaction pathway was up-regulated in the PRP group. The ECM is the noncellular component in all tissues and organs, providing essential physical scaffolding for the cellular constituents and initiating crucial biochemical and biomechanical cues. Therefore, the ECM is required for tissue morphogenesis, differentiation, and homeostasis, promoting the recruitment of endogenous mesenchymal stem cells [30,31]. The extent of cell proliferation is highly dependent on cell-cell and cell-matrix interaction. Meanwhile, the decrease in ECM was also accompanied by cell apoptosis [32], which involved specific genes [33].
In our research, transcriptome sequencing analysis showed that TGF-α expression was decreased in the model group, which was reversed by PRP. TGF-α could promote the endometrial epithelial cells, stromal cells, and endometrial cancer cell proliferation [34,35]. The receptor of TGF-α was EGFR [36], which was known to drive cell migration and proliferation [37]. In our study, we verified that protein expression of EGFR was also up-regulated in the PRP group compared with the model group.
MELTF was the key up-regulated gene in PRP-induced endometrial epithelial proliferation. MELTF is a protein belonging to the transferrin (Tf) family [38]. MELTF was previously thought to contribute to iron transport, but it might not be as significant as transferrin [39]. MELTF hyper-expression was reported to increase cellular proliferation, whereas MELTF down-regulation resulted in the opposite effect [40]. Therefore, how MELTF works in endometrial regeneration needs further investigation.
Cell death has been reported in the form of autophagy, apoptosis, ferroptosis, and pyroptosis with new biological targets and pathophysiological characteristics. According to increasing evidence, excessive autophagy and lysosomal activity could promote iron-dependent ferroptosis through iron accumulation or lipid peroxidation. Autophagy works in concert with apoptosis to rebuild the endometrium during the menstrual cycle, and it has also been described as a potential molecular mechanism responsible for estrogen withdrawal-induced endometrium atrophy [21]. Autophagy was also impaired in the IUA model, which could affect endometrial regeneration [41]. However, how ferroptosis affected endometrium regeneration was unclear. Evidence showed that endometriotic stromal cells had a high affinity for iron, which might protect adjacent epithelial cells from iron-mediated ferroptosis and promote epithelial cell proliferation [42]. Pyroptosis is a type of inflammatory necrosis. Increasing pyroptosis could reduce endometrial proliferation, and suppression of pyroptosis could promote endometrial proliferation instead [43].
In our study, PRP could inhibit ferroptosis by increasing the formation of GPX4, which could suppress iron-catalyzed lipid peroxidation in membranes, protecting against cell death associated with lipid peroxidation and oxidative stress [19]. Cellular iron export is mediated by the unique ferrous iron exporter FPN1 [44]. We found that FPN1 was also up-regulated under PRP treatment in Ishikawa. ACSL4 is specifically required for ferroptosis upon GPX4 inhibition [44]. It is down-regulated in PRP-treated Ishikawa. These data indicated that PRP inhibited cell death by regulating ferroptosis. Our results showed that autophagy was downregulated in PRP-treated Ishikawa. Down-regulation of autophagy could also confer resistance to ferroptosis [19]. Whether PRP directly inhibits ferroptosis or is mediated by high expression of the MELTF gene is not clear at present, which needs further investigation.
There are several limitations to this study. Firstly, prolonging the time for observation of the fibrosis degree after modeling might be more appropriate because fibrosis takes time to observe; secondly, in order to maximize the effect of PRP, increasing the drug administration frequency might be better due to the rapid metabolic degradation of PRP; thirdly, further research on MELTF on endometrium regeneration is needed.
PRP might promote endometrium regeneration by upregulating the retinol metabolism pathway and ECM receptor interaction pathway with TGF-α, EGFR, and MELTF. Meanwhile, PRP could also inhibit endometrial epithelial cell death by inhibiting ferroptosis, autophagy, and pyroptosis. Conclusively, MELTF might regulate ferroptosis, pyroptosis, and autophagy in PRP-mediated endometrial epithelium regeneration. This study will provide a better understanding of PRP in endometrial regeneration, laying the foundation for identifying more therapeutic targets for IUA, even infertility.