Bone marrow mesenchymal stem cells promote uterine healing by activating the PI3K/AKT pathway and modulating inflammation in rat models

Abstract

BACKGROUND

Uterine injury can cause uterine scarring, leading to a series of complications that threaten women’s health. Uterine healing is a complex process, and there are currently no effective treatments. Although our previous studies have shown that bone marrow mesenchymal stem cells (BMSCs) promote uterine damage repair, the underlying mechanisms remain unclear. However, exploring the specific regulatory roles of BMSCs in uterine injury treatment is crucial for further understanding their functions and enhancing therapeutic efficacy.

AIM

To investigate the underlying mechanism by which BMSCs promote the process of uterine healing.

METHODS

In in vivo experiments, we established a model of full-thickness uterine injury and injected BMSCs into the uterine wound. Transcriptome sequencing was performed to determine the enrichment of differentially expressed genes at the wound site. In in vitro experiments, we isolated rat uterine smooth muscle cells (USMCs) and cocultured them with BMSCs to observe the interaction between BMSCs and USMCs in the microenvironment.

RESULTS

We found that the differentially expressed genes were mainly related to cell growth, tissue repair, and angiogenesis, while the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway was highly enriched. Quantitative reverse-transcription polymerase chain reaction was used to validate differentially expressed genes, and the results demonstrated that BMSCs can upregulate genes related to regeneration and downregulate genes related to inflammation. Coculturing BMSCs promoted the migration and proliferation of USMCs, and the USMC microenvironment promoted the myogenic differentiation of BMSCs. Finally, we validated the PI3K/AKT pathway in tissues and cells and showed that BMSCs activate the PI3K/AKT pathway to promote the regeneration of uterine smooth muscle both in vivo and in vitro.

CONCLUSION

BMSCs upregulated uterine wound regeneration and anti-inflammatory factors and enhanced uterine smooth muscle proliferation through the PI3K/AKT pathway both in vivo and in vitro.

Key Words: Uterine injury; Bone marrow mesenchymal stem cells; Uterine smooth muscle cells; Phosphoinositide 3-kinase/protein kinase B pathway; Cell-cell interactions; Cell proliferation; Immune regulation; Wound regeneration

Core Tip: We identified differentially genes in uterine wound tissues through transcriptome sequencing. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that regeneration and anti-inflammatory factors were upregulated in bone mesenchymal stem cell (BMSCs) groups and that the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin pathway was highly enriched. This study also demonstrated that BMSCs significantly promoted the proliferation and migration of uterine smooth muscle cells (USMCs) and that USMCs enhanced the myogenic differentiation of BMSCs. In vivo and in vitro experiments demonstrate that BMSCs activate the phosphoinositide 3-kinase/protein kinase B pathway in wound tissues and USMCs.

INTRODUCTION

A uterine scar refers to the formation of scars in the uterus due to disease or trauma, which increases the risk of several conditions, such as placenta accreta, uterine rupture, postpartum hemorrhage, scar diverticulum, and incisional pregnancy[1]. Lin et al[2] investigated the natural healing process of the uterus by creating mechanical full-layer injuries in the uterus, resulting in extensive defects in the endometrium and smooth muscle tissue. They reported that there are differences in the repair process of each layer of the wound. The repair of uterine injury involves both the endometrium and the myometrium. Owing to its robust regenerative capacity, the endometrium typically achieves complete repair. In contrast, the regenerative capacity of uterine smooth muscle is limited, resulting in a longer repair time and often failure to achieve complete repair. Incomplete repair of the myometrium is the primary cause of complications associated with a scarred uterus. Defective healing after myometrial injury is the pathological basis for its complications[3]. However, effective treatment methods for repairing the myometrium are currently lacking. Thus, finding therapeutic strategies to promote myometrial repair is of paramount importance.

Bone marrow mesenchymal stem cells (BMSCs) are a group of multipotent stem cells derived from the mesoderm that have the potential for self-renewal and multidirectional differentiation[4]. These stem cells are among the few types of stem cells approved by regulatory organizations, such as the Food and Drug Administration, for clinical research[5,6] because they are easy to obtain, rapidly amplify, are suitable for autologous transplantation, and are nontumorigenic. Previous studies have demonstrated that BMSCs exhibit favorable therapeutic efficacy in treating cardiac injury, inflammatory bowel diseases, and pelvic floor dysfunction disorders[7-9].

Mesenchymal stem cells (MSCs) promote the proliferation and regeneration of smooth muscle cells through autocrine and paracrine effects[10-12]. Our previous studies established a full-thickness uterine injury model using Sprague-Dawley (SD) rats and demonstrated that BMSCs promote regeneration of the myometrium, alleviate collagen formation, and reduce scar formation[13]. However, the specific mechanisms underlying these therapeutic effects remain unclear, as research has been limited to phenotypic observations.

To elucidate the underlying mechanism, whole-transcriptome sequencing was performed to identify differentially expressed genes in different wound tissues. Gene Ontology (GO) and Kyoto Encyclopedia of Genes (KEGG) enrichment analyses revealed that pro-regenerative genes and genes inhibiting inflammation were upregulated but that proinflammatory genes were downregulated, and the phosphoinositide 3-kinase/protein kinase B/mechanistic target of rapamycin (PI3K/AKT/mTOR) pathway was highly enriched. This led us to further investigate the highly enriched PI3K/AKT pathway, which is closely related to cell growth. The PI3K/AKT pathway is a key regulator of cell proliferation and growth, which is important in development, tumorigenesis, and also involved in various types of MSC-mediated tissue repair, including thermal skin injury, diabetic wound healing, lung injury, and nerve injury repair[14-17]. The role of BMSC transplantation in uterine injury repair closely aligns with the present study, further validating the relevance of the transcriptome data. Considering the regenerative effect of BMSCs on the myometrium, uterine smooth muscle cells (USMCs) were isolated and cocultured with BMSCs. BMSCs promoted the proliferation and inhibited apoptosis of USMCs through the PI3K/AKT/mTOR pathway. These findings provide an in-depth understanding of the mechanisms by which BMSCs promote myometrial regeneration and support their potential clinical application.

MATERIALS AND METHODS

Animals

All animals were purchased from the Experimental Animal Centre of Chongqing Medical University [license number: SCXK (Chongqing) 2022-0010]. All animals were handled according to the guidelines of the Ethics Committee of Chongqing Medical University and Guizhou Provincial People’s Hospital. Twenty female SD rats (aged 8-10 weeks, sexually mature, nulliparous, and weighing 240-260 g) were used to construct the model. USMCs and BMSCs were extracted from 10 SD rats aged 3-4 weeks. All materials were specific pathogen-free (SPF) grade, and the experimental animals were kept in an SPF animal room (23 °C, 12 h/12 h light/dark photoperiod, 50% humidity, and ad libitum access to food and water). All animals were preoperatively anesthetized with pentobarbital sodium injections (intraperitoneal injection, 50 mg/kg) and euthanized by overdose (intravenous injection, 150 mg/kg pentobarbital sodium) for tissue collection.

Extraction and culture of BMSCs

In the present study, our protocol for stem cells were extracted as described previously[18]. After euthanasia, the bilateral femurs and tibias were removed under sterile conditions, and the surrounding muscles and tissues were removed and soaked in phosphate buffered saline (PBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, United States). A No. 4 needle was used to puncture the epiphysis, and the bone marrow cavity was washed with DMEM/F12 (HyClone Laboratories, Logan, UT, United States) culture medium. A single-cell suspension was made by mixing the media and cells. After centrifugation, the supernatant was discarded. DMEM/F12 complete culture medium containing 100 mL/L fetal bovine serum (Gibco) and 10 mL/L antibiotics (penicillin/streptomycin; Gibco), was added, and the cells were inoculated into sterile T25 cm culture bottles and placed in a 37 °C incubator with 50 mL/L CO₂. The medium was changed every 24 h for the first 3 d and then every 3 d following. The cell growth status was observed and recorded. When the cells reached 80%-90% confluence, they were passaged, and cells from the third to sixth passages with good growth were used for experiments.

Identification of BMSCs

Flow cytometry was used to detect stem cell markers and assess multipotent differentiation potential through induction. For flow cytometry analysis, passage 3 BMSCs were digested to prepare a single-cell suspension, and the supernatant was discarded after centrifugation. The cell density was adjusted to 1 × 10⁷/mL, and the following FITC-conjugated primary antibodies (Invitrogen, Thermo Fisher Scientific) were added to 100 μL of cell suspension (1 × 10⁶ cells per sample) and incubated at 4 °C for 20 minutes: CD31-FITC (1 μg/sample), CD34-FITC (3 μg/sample), CD44-FITC (1 μg/sample), CD90-FITC (0.1 μg/sample), CD105-FITC (20 μL/sample), and CD45-FITC (0.25 μg/sample). Then, 1 mL PBS was added to resuspend the cells, which were subsequently centrifuged at 300 × g for 5 min. The supernatant was discarded and the wash step was repeated once. The cell suspensions were collected in flow tubes for flow cytometry analysis.

For the identification of cell differentiation potential, adipogenic or osteogenic differentiation was induced for 21 d according to the manufacturer’s instructions. The differentiation potential of stem cells was assessed via Oil Red O staining (OriCell, RAXMX-90031; Cyagen, Santa Clara, CA, United States) for adipogenesis and alkaline phosphatase staining (OriCell, RAXMX-90021; Cyagen) for osteogenesis. To determine the safety of BMSCs, the heart, liver, brain, kidney, and spleen were collected from the model group at 90 days after stem cell transplantation. Hematoxylin and eosin (HE) staining was performed to assess tumor infiltration in the major organs.

Establishment of a rat model of full uterine layer injury

SD rats received 7 d of adapted feeding. Using a random number system, the rats were divided into the BMSC group and natural repair group (NR group), and each group included five rats. For the surgical method, the rats were fixed in the supine position on a surgical board after anesthesia. The lower abdomen was sterilized, and a midline longitudinal incision was made approximately 2 cm above the symphysis pubis to expose the uterus. At a point 1 cm away from the “Y”-shaped uterus towards the ovary, the uterine mesentery on the opposite side was longitudinally incised 0.5 cm from the edge of the uterine incision. The incision ends were marked with 5-0 silk sutures, and the incision was closed with 6-0 absorbable sutures. After surgery, an intramuscular injection of penicillin (160000 U/rat/d) was administered for 3 d to prevent infection.

In accordance with the experimental requirements, the BMSC group and the NR group were established. P3 BMSCs were harvested and conventionally digested, and PBS was used to resuspend the cells to produce single-cell suspensions, which were counted. The cell concentration was adjusted to 5 × 10⁶/mL. In the BMSC group, 200 μL of cell suspension (1 × 10⁶ cells) was injected along the edge of the muscle layer on each side of the uterine incision. In the NR group, 200 μL of physiological saline was injected along the edge of the muscle layer on each side of the uterine incision. The surgery time was recorded.

Transcriptome sequencing and differential gene clustering analysis

The rats were maintained in an SPF-grade environment postoperatively. On day 30 post-surgery, the rats were euthanized, and the tissue labeled with suture marks was cut from the wound site. The tissue was quickly washed with PBS prepared with RNase-free water (Roche, Basel, Switzerland) at 2-6 °C to prevent bloodstains and then dried with lint-free paper towels before being placed into EP tubes. Total RNA from the uterine wound site of the BMSC group and NR group was extracted via TRIzol (Invitrogen). Sequencing and subsequent data analysis were completed by Shanghai Chenpu Technology Co., Ltd (China). Differentially expressed genes were subjected to GO and KEGG analyses. Differential gene analysis between samples was conducted via the edgeR package in R, and GO terms with corrected P values < 0.05 were considered significantly enriched for differentially expressed genes. The enriched differentially expressed genes were subjected to a PubMed literature search, which focused on regeneration-, wound healing-, and inflammation-related differential genes, and a heatmap was generated.

Pathological changes in uterine histopathology were detected by HE staining

On day 30 post-surgery, the rats were euthanized. Following removal from the abdomen, the uterus was dissected to remove visible blood and surrounding serosa, and it was then placed on ice. After dehydration, the uterus was fixed in 40 g/L paraformaldehyde solution, made transparent, and embedded in paraffin. Sections with a thickness of 5 μm were generated. HE staining and microscopic examination were performed to observe any abnormal changes in the tissues from each group.

Quantitative reverse-transcription polymerase chain reaction

The gene primers were synthesized by Shanghai Sangon Biotech, and the primer sequences are shown in Table 1. The tissues were ground in liquid nitrogen, and 1 mL TRIzol (TIANGEN, Beijing, China) was added to extract total cellular RNA. The extracted total RNA was treated with DNaseI (TaKaRa, Shiga, Japan) to degrade the DNA, and the concentration and quality of the RNA were measured via a UV spectrophotometer. The RNA was reverse transcribed into cDNA via reverse transcription reagents (TRT-101; TOYOBO, Osaka, Japan). The reaction conditions were as follows: 42 °C for 10 min, 30 °C for 20 min, 99 °C for 5 min, and 4 °C for 5 min. Two microlitres of cDNA was added to the corresponding primers for SYBR Green quantitative PCR (QPK-201). The reaction conditions were as follows: 40 cycles of 95 °C for 15 sec and 53 °C/54 °C for 1 min. Beta-actin was used as an internal reference, and the relative quantitative results were analyzed via the 2^-ΔΔCt method.

Table 1 Primer sequences.

Gene name	Primer	Sequence	Size
Prss23	Forward	5’-GACTCCTCATCCTTCTCC-3’	208 bp
	Reverse	5’-AGCGGTGTTCCCTTGTG-3’
Table 2	Forward	5’-TACTCAACAGCCCACAGG-3’	100 bp
	Reverse	5’-GATACCCAGCCAGAATGC-3’
Vegfα	Forward	5’-ACACACCCACCCACATAC-3’	243 bp
	Reverse	5’-AGGACGAAAGACCACACC-3’
IL4r	Forward	5’-GTGGAGGAGGAAGAGGA-3’	190 bp
	Reverse	5’-GCAGAAGGGAGGATGAC-3’
Tnfaip8	Forward	5’-CAGGGAAGTGGCTACAG-3’	97 bp
	Reverse	5’-GGTGGCGATGGATTTGG-3’
IL1r1	Forward	5’-CCCACGGAATGAGACGA-3’	247 bp
	Reverse	5’-GCAGATGAACGGATAGCG-3’
IL1α	Forward	5’-GAGTGCTCAGGGAGAAGAC-3’	135 bp
	Reverse	5’-GCTGCGGATGTGAAGTAG-3’
IL1β	Forward	5’-TCTCACAGCAGCATCTCG-3’	184 bp
	Reverse	5’-AGGTCGTCATCATCCCAC-3’
IL4	Forward	5’-CCACGGATGTAACGACAG-3’	205 bp
	Reverse	5’-GTTCTTCAAGCACGGAGG-3’
TNFα	Forward	5’-CGAGATGTGGAACTGGCA-3’	197 bp
	Reverse	5’-GAACTGATGAGAGGGAGC-3’
TGFβ	Forward	5’-CCACTCCCGTGGCTTCTAGT-3’	103 bp
	Reverse	5’-CTTCGATGCGCTTCCGTTTC-3’
FGF	Forward	5’-TTTGCAGCCCTGACCGAGAG-3’	100 bp
	Reverse	5’-GAAGAATCCTCAAGAAGTGGCC-3’
β-actin	Forward	5’-ACCCCGTGCTGCTGACCGAG-3’	250 bp
	Reverse	5’-TCCCGGCCAGCCAGGTCCA-3’

Relative mRNA expression levels were analyzed using the 2^-∆∆Ct method with β-actin as internal references. Prss23: Serine protease 23; Table 2: Transforming growth factor-beta activated kinase 1 (MAP3K7) binding protein 2; Vegfα: Vascular endothelial growth factor alpha; IL4r: Interleukin 4 receptor; Tnfaip8: Tumor necrosis factor alpha induced protein 8; IL1r1: Interleukin 1 receptor type 1; IL1α: Interleukin 1 alpha; IL1β: Interleukin 1 beta; IL4: Interleukin 4; TNFα: Tumor necrosis factor alpha; TGFβ: Transforming growth factor beta; FGF: Fibroblast growth factor.

Isolation and culture of primary USMCs from rats

Following euthanasia, the uterus of each rat was excised. The outer membrane and endometrium were removed under a dissecting microscope. The tissue was immersed in PBS containing penicillin and streptomycin (HyClone) for 5 min and then transferred to 1.5 mL EP tubes. The uterine muscle strips were minced with ophthalmic scissors (less than 1 mm³) and centrifuged at 1500 rpm for 5 min. The tissue was transferred to small conical tubes, and 2 mL of 2 mg/mL collagenase II solution (Sigma-Aldrich, St Louis, MO, United States) was added, followed by incubation for 45-60 min. After adding 2 mL HG-DMEM (Gibco), the mixture was filtered through a 200-mesh cell sieve, and the filtrate was collected and transferred to centrifuge tubes. After centrifugation, the cells were seeded in T25 culture flasks and incubated at 37 °C in a 50 mL/L CO₂ incubator until they reached confluence.

Identification of USMCs

The USMCs were seeded onto slides, fixed, and incubated with primary antibodies against alpha-smooth muscle actin (α-SMA) (Affinity Biosciences, Cincinnati, OH, United States), calponin (Proteintech, Rosemont, IL, United States), and estrogen receptor (Proteintech) at a 1:200 dilution at 4 °C overnight. Then, FITC-conjugated goat anti-mouse IgG (ZSGB-BIO, Evanston, IL, United States) was added at a 1:100 dilution, followed by incubation at room temperature in the dark for 1 h. The cell nuclei were counterstained with DAPI (Boster Bio, Pleasanton, CA, United States) at room temperature for 10 min. A microscope (Life Evos, Thermo Fisher Scientific) was used for fluorescence imaging.

Establishment of the BMSC/USMC coculture system

USMCs were seeded in 6-well plates (1 × 10⁶ cells/well), and BMSCs were seeded on Transwell inserts with 6-well plate compatibility (0.6 × 10⁶ cells/insert, 3 μm pore size; Labselect, Anhui, China). The cells were cultured at 37 °C with 50 mL/L CO₂. After 24 h, the cells were washed with PBS, and the inserts containing BMSCs were placed into six-well plates containing USMCs for coculture. Phenotypic changes in the USMCs and BMSCs were observed at different coculture times.

In vitro scratch assay

A scratch assay was used to evaluate the effect of the BMSC microenvironment on the migratory capacity of USMCs. Following the above method, USMCs and BMSCs were seeded. When the USMCs reached more than 90% confluence, the cell monolayers were scratched across the center of the well using a 200 μL pipette tip. After washing one to two times with PBS to remove cellular debris, the Transwell chambers containing BMSCs were placed into the scratched wells. Images of USMCs were captured at 2 h, 24 h, and 48 h via a microscope. The remaining scratch area was measured via ImageJ software (National Institutes of Health, Bethesda, MD, United States).

5-ethynyl-2’-deoxyuridine cell proliferation assay

BMSCs and USMCs were cocultured for 24 h. The Transwell inserts containing the BMSCs were removed, and the USMCs were subjected to 5-ethynyl-2’-deoxyuridine (EdU) staining according to the manufacturer’s instructions (Cell-Light EdU Apollo567 In Vitro Kit; Ribobio, Guangzhou, China). Images were acquired under a microscope. The number of EdU-positive cells was determined via ImageJ software, and the positivity rate was calculated.

Immunofluorescence detection of BMSC differentiation

BMSCs were seeded in wells from a coculture system and then incubated at 37 °C with 50 mL/L CO₂ for 24 h. Immunofluorescence staining was performed following the aforementioned method to observe the expression of α-SMA and calponin in BMSCs, and untreated BMSCs served as the negative control.

Western blot analysis

Total protein was extracted from cocultured BMSCs, USMCs, and uterine wound tissue. The samples (60 μg protein) were separated on sodium-dodecyl sulfate gel electrophoresis gels and transferred onto polyvinylidene fluoride membranes. The membranes were then incubated overnight at 4 °C with primary antibodies against α-SMA (1:1000, rabbit monoclonal; Affinity), calponin (1:1000, rabbit monoclonal; Proteintech), Ki67 (1:500, rabbit monoclonal; Affinity), PI3KCA (1:1000, rabbit monoclonal; Invitrogen), p-PI3KCA (1:1000, rabbit monoclonal; MedchemExpress, Monmouth Junction, NJ, United States), AKT (1:1000, rabbit monoclonal; Invitrogen), p-AKT (1:1000, rabbit polyclonal; Invitrogen), mTOR (1:1000, mouse monoclonal; Proteintech), p-mTOR (1:1000, rabbit polyclonal; Invitrogen), GAPDH (1:1000, rabbit monoclonal; Santa Cruz Biotechnology, Dallas, TX, United States), and LY294002 (25 μM; MedchemExpress). After being washed with Tris-buffered saline with Tween, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse secondary antibodies (ZSGB-BIO) for 1 h. Following three washes with Tris-buffered saline with Tween, the membranes were incubated with ECL ultrasensitive luminescent reagent AB solution at a 1:1 ratio in a dark room and then visualized via a ChemiDoc XRS imaging system. ImageJ software was used for grayscale analysis and image quantification.

Statistical analysis

ImageJ software was used to detect the residual area, percentage of EdU-positive cells, and grayscale band of the western blot. The data are expressed as the mean ± SD. Single-factor analysis of variance (ANOVA) was used for single-variable analysis among multiple groups, whereas two-factor ANOVA was used for multivariate analysis. Tukey’s post hoc test was used for multiple comparisons. A t test was used for comparisons between two groups. Statistical significance was defined as P < 0.05.

RESULTS

BMSC identification

Flow cytometry analysis revealed that the BMSCs expressed CD90 (99.5%), CD105 (99.4%), and CD44 (97.9%) mesenchymal cell markers but that the BMSCs did not express CD31 (0.18%), CD45 (0.15%), or CD34 (2.01%) (Figure 1A). The former three markers represent mesenchymal cell origins. CD31 is an endothelial cell marker, and CD45 and CD34 are hematopoietic cell markers. BMSCs exhibited rapid adhesion, with a fibroblast-like cell morphology under the microscope and whirlpool-like growth at high density (Figure 1B); these cells rapidly proliferated and were easily digestible. The BMSCs possessed multiple differentiation abilities (Figure 1C). With respect to the safety of the stem cells, the growth of all the transplant models was observed for 90 d. Tumor formation was not observed by either gross morphology or in histological sections of major organs (Figure 1D).

Figure 1 Characterization, morphology, differentiation capacity, and safety of bone marrow mesenchymal stem cells. A: Flow cytometric analysis of CD90, CD44, CD105, CD34, CD45, and CD31 expression in bone marrow mesenchymal stem cells (BMSCs). The black lines represent isotype controls, whereas the green lines represent the levels of surface markers; B: Representative optical microscopy image of passage 0 BMSCs cultured for 5 d in plastic cell culture flasks (scale bar = 100 μm); C: Multilineage differentiation potential of BMSCs. BMSCs differentiate into mature adipocytes and osteoblasts upon induction. Lipid droplets and bone trabecular structures were observed under a light microscope and stained with Oil Red O and Alizarin Red to detect adipocytes and osteoblasts derived from BMSCs (scale bar = 100 μm); D: Gross appearance images of primary organs in rat models after BMSC transplantation, as assessed via hematoxylin and eosin staining to examine tissue structures (scale bar = 100 μm).

Transcriptomic cues reveal an anti-inflammatory and pro-regenerative phenotype in the BMSC group

We performed transcriptome sequencing on the uterine wound tissues of rats at 30 d post-surgery and compared the differentially expressed genes in the uterine wounds between the different treatment groups (BMSC group and NR group, n = 3). Compared with those in the NR group, 604 genes were upregulated in the BMSC group, and 523 genes were downregulated in the BMSC group (Figure 2A). The volcano plot in Figure 2B shows genes with fold changes greater than 2 and a P value less than 0.05. GO enrichment revealed that the differentially expressed genes were enriched in the following areas: Biological functions, such as cell growth, tissue repair, and vascular formation; cellular components, including extracellular vesicles and extracellular regions; and molecular functions, including protein binding and GTPase activity (Figure 2C). The enriched KEGG pathways were enriched mainly in cell signaling transduction, cell adhesion molecules, and pathways related to proliferation and apoptosis, such as the PI3K/AKT pathway (Figure 2D). The top differentially expressed genes are listed and enriched in the heatmap (Figure 2E). The functions of the enriched differential genes were analyzed via the NCBI Gene and UniProt databases combined with literature reports from PubMed. Moreover, the expression of regeneration-related genes, such as Apoe, Thbs1, and protease 23 (prss23)[19-21], was elevated in the BMSC group. In addition, genes related to cell regulation and wound healing, including platelet-derived growth factor receptor beta (PDGFRβ), transforming growth factor beta (TGF-β), fibroblast growth factor (FGF), and vascular endothelial growth factor alpha (VEGFα), were identified[22-25]. The expression of tumor necrosis factor alpha (TNFα) and interleukin (IL)-1 inflammatory cytokines[26,27] was downregulated, whereas that of IL-4[28] was upregulated. Twelve of these genes were further validated via quantitative reverse-transcription polymerase chain reaction, which confirmed the transcriptome data (Figure 2F). These results suggested that BMSCs promote uterine tissue healing by regulating cell proliferation and immune modulation, leading to a transition from proinflammatory fibrosis to anti-inflammatory regeneration processes.

Figure 2 Transcriptome sequencing and differential gene clustering analysis. A: Bar graph illustrating significantly differentially expressed genes at the uterine wound site, with a total of 604 upregulated genes and 523 downregulated genes identified; B: Volcano plot and scatter plot of significantly differentially expressed genes; C: Gene Ontology analysis of the impact of bone marrow mesenchymal stem cells (BMSCs) on the uterine wound tissue transcriptome (bar graph); D: Kyoto Encyclopedia of Genes and Genomes analysis of significantly different pathways; E: Cluster analysis of differential gene expression changes. Red represents upregulated genes, and green represents downregulated genes; F: Quantitative reverse-transcription polymerase chain reaction validation of differential genes and their corresponding cytokine genes. The values are shown as the means ± SDs. ^aP < 0.05 vs the natural repair group, ^bP < 0.01 vs the natural repair group, ^cP < 0.001 vs the natural repair group, n = 3 per group. BMSCs: Bone marrow mesenchymal stem cells; BP: Biological process; CC: Cellular component; FC: Fold change; FGF: Fibroblast growth factor; IL-1α: Interleukin 1 alpha; IL-1β: Interleukin 1 beta; IL1r1: Interleukin 1 receptor type 1; IL-4: Interleukin 4; IL4r: Interleukin 4 receptor; MF: Molecular function; NR: Natural repair; Prss23: Protease 23; Tab2: Transforming growth factor-beta activated kinase 1 (MAP3K7) binding protein 2; TGF: Transforming growth factor; TNFα: Tumor necrosis factor alpha; Tnfaip8: Tumor necrosis factor alpha-induced protein 8; Vegfa: Vascular endothelial growth factor alpha.

Comparison of uterine histopathological changes in each group

Based on the transcriptomic results, we performed preliminary validation in the rat uterine injury model tissue. We observed that normal rat uterus consisted of the myometrium and endometrium, and the outer muscle layer was composed of bilateral uterine smooth muscle. USMCs had a distinct, orderly, and closely packed structure. The endometrial stroma was located within the tissue, and there was no edema or congestion in the interstitium. Small blood vessels were scattered within the interstitium, and there was no significant inflammatory cell infiltration (Figure 3A). The uterine tissue structure was disrupted in the BMSC group and NR group. The BMSC group exhibited significant proliferation of thin-walled small blood vessels in the mucosal lamina propria, proliferation of interstitial smooth muscle fibers, and scattered infiltration of a few inflammatory cells (Figure 3B). In the NR group, the lamina propria of the mucosa showed slight proliferation of thin-walled small blood vessels, and there was significant infiltration of inflammatory cells in the lamina propria, muscular layer, and serosal layer (Figure 3C). These results indicated that BMSCs promote the regeneration of damaged uterine vessels and muscle fibers, as well as reduce the inflammatory response.

Figure 3 Hematoxylin and eosin staining of the damaged uterus. A: Uterine tissue of rats in the sham surgery group; B: Uterine tissue of rats in the bone marrow mesenchymal stem cell (BMSC) group, red arrow: Thin-walled small blood vessels; C: Uterine tissue of rats in the natural repair (NR) group, black arrow: Inflammatory cells; D: The quantitative analysis of inflammatory cells and blood vessels in the BMSCs group and NR group. ^aP < 0.05 vs the NR group, ^bP < 0.01 vs the NR group, n = 5 per group.

Interaction between BMSCs and USMCs

In our previous study[13], we found that BMSCs promote the regeneration of the myometrium. Considering that myometrial repair may arise both from the proliferation of smooth muscle cells and potentially from the myogenic differentiation of BMSCs, we chose to co-culture BMSCs with USMCs. First, we isolated USMCs. Under inverted phase contrast microscopy, USMCs appeared elongated, with cells growing in parallel or whirlpool-like patterns (Figure 4A). Fluorescence microscopy revealed that USMCs expressed the α-SMA and calponin smooth muscle cell-specific markers but did not express the estrogen receptor (ER) endometrial cell marker (Figure 4B). USMCs were cocultured with BMSCs to observe the interaction between BMSCs and USMCs. Scratch experiments revealed that the BMSC microenvironment promoted USMC migration (Figure 4C and D). EdU cell proliferation assays demonstrated that the BMSC microenvironment promoted USMC proliferation (Figure 4E and F). The effect of the USMC microenvironment on BMSCs was evaluated by cell immunofluorescence and western blot analyses. The USMC microenvironment induced BMSC myogenic differentiation and promoted BMSC proliferation, and the extent of these effects was positively correlated with the coculture time (Figure 4G-I).

Figure 4 Influence of the microenvironment on uterine smooth muscle cells and bone marrow mesenchymal stem cells. A: Representative optical micrographs of P1 uterine smooth muscle cells (USMCs) cultured for 7 d (scale bar = 100 μm); B: Immunofluorescence staining for USMC markers, with green fluorescence indicating positivity (scale bar = 200 μm); C: Bright-field image showing USMC migration at 2 h, 24 h, and 48 h, with red dashed lines delineating migration contours (scale bar = 1000 μm); D: Quantification of the residual USMC area. ^bP < 0.01 vs the USMCs, ^cP < 0.001 vs the USMCs; E: Fluorescence images depicting the 5-ethynyl-2’-deoxyuridine (EdU) cell proliferation assay used to detect USMC proliferation, where red fluorescence indicates EdU-positive proliferating cells and blue fluorescence indicates Hoechst 33342-positive nuclei (scale bar = 400 μm); F: Percentage of EdU-positive USMCs. ^cP < 0.001 vs USMCs; G: Immunofluorescence staining of alpha-smooth muscle actin (α-SMA) and calponin in bone marrow mesenchymal stem cells (BMSCs). Green indicates positivity, and the fluorescence intensity reflects the expression level (scale bar = 200 μm); H: Western blot analysis of α-SMA, calponin, and the Kiel-67 marker of proliferation (Ki67) expression in BMSCs cocultured with USMCs for 1, 3, or 5 d; I: Statistical analysis of relative protein levels at different coculture times. ^aP < 0.05 vs the 3-d group, ^bP < 0.01 vs the 1-d group, ^cP < 0.001 vs the 1-d and 3-d groups, ^dP < 0.001 vs the 1-d group. ImageJ software was used to process the images. The data are presented as the mean ± SD, n = 3 per group. P values for D were determined via two-way ANOVA, and those for I were determined via one-way ANOVA. Comparisons between two groups in F were conducted via a t test. ER: Estrogen receptor.

BMSCs promote USMC proliferation through the PI3K/AKT pathway

BMSCs promote USMC proliferation through the PI3K/AKT pathway in vitro, and PI3K/AKT pathway activation was also observed in vivo. Based on the transcriptomic data, which indicated significant enrichment of the PI3K pathway, this signaling pathway may be involved in the biological effects of BMSCs on uterine wound healing. Therefore, we selected this pathway for further investigation and validated it both at the tissue and cellular levels. We validated the changes in the PI3K/AKT signaling pathway in uterine tissues of rats. We collected uterine wound samples from two groups of model rats (BMSC group and NR group, n = 5). Western blot analysis revealed that the phosphorylation of PI3K and AKT was greater in the BMSC-transplanted group than in the natural healing group (Figure 5A and B), confirming that the PI3K/AKT pathway is involved in BMSC-related uterine scar repair. To validate the effect of the PI3K/AKT pathway on the proliferation of USMCs, the PI3K-specific inhibitor LY294002 was used. BMSC coculture increased the PI3K and AKT phosphorylation, as well as Ki67 expression in USMCs, whereas LY294002 treatment inhibited the activation of the PI3K/AKT pathway (Figure 5C and D) and the proliferation of USMCs (Figure 5E and F). Western blot analysis confirmed that the BMSC microenvironment activated the PI3K/AKT/mTOR pathway in USMCs and promoted cell proliferation. The above regulatory effects were suppressed after treatment with the PI3K inhibitor (Figure 5C-F).

Figure 5 Bone marrow mesenchymal stromal cells regulate uterine smooth muscle cells through the phosphoinositide 3-kinase/protein kinase B signaling pathway in vitro and in vivo. A: Examination of the impact of bone marrow mesenchymal stromal cells (BMSCs) on phosphorylated phosphoinositide 3-kinase (p-PI3K)/phosphorylated protein kinase B (p-AKT) in uterine wound tissues; B: Comparison of the relative expression levels of p-PI3K/p-AKT in the wound tissues. ^aP < 0.05 vs the natural repair (NR) ; C: Detection of the Kiel67 marker of proliferation (Ki67), p-PI3K, p-AKT, and phosphorylated mechanistic target of rapamycin (p-mTOR) in uterine smooth muscle cells (USMCs) treated with complete culture medium in the presence or absence of 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), as well as in BMSCs cultured in the presence or absence of LY294002 (25 μM); D: Relative expression levels of Ki67, p-PI3K, p-AKT, and p-mTOR in USMCs under different treatment conditions; E: 5-ethynyl-2’-deoxyuridine assay for assessing the proliferation of USMCs. The treatment was the same as in C (scale bar = 400 μm); F: Percentage of 5-ethynyl-2’-deoxyuridine (EDU)-positive USMCs. The data are presented as mean ± SD, with n = 5 per group for A and n = 3 for C and E. One-way ANOVA was used for multiple comparisons, and a t test was used for comparisons between two groups. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001.

DISCUSSION

In China, the incidence of scarred uteri is high[29], and clinical treatment methods are currently lacking for scarred uteri. With the relaxation of the fertility policy, an increasing number of women are facing the threat of scarred uteri[30]. The present study demonstrated that BMSCs upregulated uterine wound regeneration and the expression of anti-inflammatory factors, including Apoe, Thbs1, prss23, PDGFrβ, TGF-β, FGF, and VEGFα, but that BMSCs downregulated the expression of the TNFα and IL-1 proinflammatory factors. IL-4, which is generally considered an anti-inflammatory immunomodulatory factor, was also upregulated. These findings were confirmed histologically. Additionally, the PI3K/AKT pathway, which controls cell growth and survival, was highly enriched in the BMSC transplantation group. Subsequent in vitro and in vivo experiments demonstrated that BMSCs activated the PI3K/AKT pathway. These findings indicated that BMSCs promote uterine wound repair through anti-inflammatory and pro-regenerative processes.

Several studies have shown that the primary function of transplanted MSCs lies in maintaining tissue homeostasis and integrity and enhancing tissue-resident cells, which may surpass MSC differentiation. Intra-bone marrow injection of MSCs significantly improves platelet and bone marrow regeneration in mice with leukemia, prolonging their survival time[31]. Injected MSCs can induce arginase 1+ macrophages, promoting M2 macrophage differentiation and facilitating tissue repair[32]. Some in vivo studies have indicated that growth factors released by stem cells, including epidermal growth factor, FGF, keratinocyte growth factor, VEGF, and PDGF, are crucial for directly mobilizing tissue-resident progenitor cells to repair injured tissues[33,34]. Currently, more attention is focused on the immunomodulatory response of MSCs to inflammation in injured tissues[35]. At inflamed tissue sites, MSCs restore immune homeostasis by directly affecting innate and adaptive immune cells, thereby blocking the cascade activity of immune responses[36,37]. In the present study, BMSCs reduced the apoptosis of USMCs and promoted smooth muscle proliferation. USMCs are important structural cells of the uterus, and BMSCs promote the integrity of uterine tissue. Consistent with the literature, the present study demonstrated upregulation of factors associated with regeneration and anti-inflammation (Apoe, Thbs1, prss23, PDGFrβ, TGF-β, FGF, VEGFα, and IL-4), as well as downregulation of proinflammatory factors (TNFα and IL-1).

GO and KEGG enrichment analyses are two commonly used methods in gene function studies, and they are employed to understand the biological processes, molecular functions, cellular components, and metabolic or signaling pathways involved in a gene list. By performing GO and KEGG enrichment analyses on differentially expressed genes, potential molecular mechanisms can be elucidated. The present results revealed that physiological processes were mainly enriched in cell growth and wound healing, whereas cellular components, such as extracellular vesicles, were related to intercellular interactions. In terms of molecular functions, protein binding was prominent, and the PI3K/AKT pathway was enriched. In addition, there was significant enrichment in cellular senescence. Tissue repair comprises four main stages, namely, hemostasis, inflammation, proliferation, and remodeling, all of which are influenced by senescence[38]. Cellular senescence promotes regeneration in the skin and liver[39,40]. These findings suggested that senescence is extensively involved in the tissue injury repair process; however, its specific role and mechanisms remain underexplored and require further investigation.

The coculture system is an ideal in vitro model for studying cell-cell interactions. BMSCs cocultured with USMCs affects both types of cells, with BMSCs effectively improving the function of USMCs. On the basis of transcriptomic sequencing analysis and a literature review, the present study focused on the PI3K/AKT pathway. The AKT serine/threonine kinase is an important component of the PI3K signaling pathway. Activated AKT regulates various cellular functions, including cell cycle, proliferation, and cell metabolism[41]. Our findings confirmed that BMSCs activate the PI3K/AKT pathway both in vitro and in vivo to promote uterine repair. Moreover, in vitro coculture experiments reflect the paracrine effect of cells; that is, BMSCs regulate USMCs through paracrine secretion. The secretome derived from mesenchymal stem cells is defined as a set of bioactive factors originating from stem cells, including soluble proteins, nucleic acids, lipids, and extracellular vesicles[42]. These bioactive molecules have anti-inflammatory and antiapoptotic effects, and they promote cell survival and tissue repair[43,44]. Thus, the factors secreted from BMSCs are speculated to be involved in the activation of the PI3K/AKT/mTOR pathway. To identify the secreted factors and their functional roles, subsequent studies will focus on the relationship between secretome components and the PI3K/AKT pathway, and future studies will determine if intervening in this pathway can further improve repair.

Tissue injury repair consists of four stages, namely, hemostasis, inflammation, proliferation, and remodeling, each involving dynamic changes in different cell populations. The present study had several limitations. The present study focused only on investigating the interaction between BMSCs and USMCs. The uterus is primarily composed of the endometrium and myometrium, which have complex cellular compositions. The repair process often involves the infiltration of various immune cells, further complicating the microenvironment at the injury site. Elucidating the interactions among various cells involved in tissue repair is essential to gain a comprehensive understanding of the role played by BMSCs and to uncover more underlying mechanisms. It is challenging to comprehensively study the effects of BMSC transplantation via conventional methods. In contrast, single-cell analysis, with its high throughput and cell-specific insights, offers an advantage in studying such processes. This approach allows for a more comprehensive understanding of cell composition, intercellular communication, and changes in cell differentiation and function during injury repair. Further studies in this area will be performed in the future.

CONCLUSION

The present study demonstrated the complex interaction between BMSCs and USMCs during uterine scar repair. BMSCs increase the proliferation of USMCs, whereas USMCs induce the myogenic differentiation of BMSCs, thereby contributing to regeneration of the myometrium. The PI3K/AKT pathway underlies the regulation of USMC proliferation by BMSCs, thus enhancing the present understanding of the mechanisms by which BMSCs promote the repair of injured uterine tissue. In addition, transcriptome sequencing revealed several factors associated with cell growth and anti-inflammatory responses that may serve as effective targets for improving the repair of scarred uterine tissue.

ACKNOWLEDGEMENTS

We would like to appreciate the support from the “111 program” of Ministry of Education of China and State Administration of Foreign Experts Affairs of China.