Human menstrual blood-derived stem cells alleviate autoimmune hepatitis via JNK/MAPK signaling pathway in vivo and in vitro

Abstract

Autoimmune hepatitis (AIH) is a severe globally distributed liver disease that could occur at any age. Human menstrual blood-derived stem cells (MenSCs) have shown therapeutic effect in acute lung injury and liver failure. However, their role in the curative effect of AIH remains unclear. Here, a classic AIH mouse model was constructed through intravenous injection with concanavalin A (Con A). MenSCs were intravenously injected while Con A injection in the treatment groups. The results showed that the mortality by Con A injection was significantly decreased by MenSCs treatment and liver function tests and histological analysis were also ameliorated. The results of phosphoproteomic analysis and RNA-seq revealed that MenSCs improved AIH, mainly by apoptosis and c-Jun N-terminal kinase/mitogen-activated protein signaling pathways. Apoptosis analysis demonstrated that the protein expression of cleaved caspase 3 was increased by Con A injection and reduced by MenSCs transplantation, consistent with the TUNEL staining results. An AML12 co-culture system and JNK inhibitor (SP600125) were used to verify the JNK/MAPK and apoptosis signaling pathways. These findings suggested that MenSCs could be a promising strategy for AIH.

Keywords

autoimmune hepatitis (AIH) / concanavalin A (Con A) / human menstrual blood-derived stem cells (MenSCs) / apoptosis / mitogen-activated protein kinase (MAPK)

1 Introduction

Autoimmune hepatitis (AIH) is an inflammation of the liver parenchyma regulated by an autoimmune response in hepatocytes, and it could occur at all ages in genetically susceptible individuals [1,2]. Currently, prednisone and azathioprine are the main AIH treatment drugs. However, they require long-term medication and have several side effects. Besides, no apparent symptoms usually occur at AIH onset, thus causing late diagnosis. Therefore, effective treatment with fewer side effects is needed [1,3].

Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells first identified from bone marrow by Friedenstein et al [4]. MSCs could be obtained from other adult tissues, such as umbilical cord blood. They could also differentiate into mesodermal lineage [5], migrate to injured tissues to promote survival [6], and take effect by paracrine [7]. Previous studies showed mouse bone-marrow-derived MSCs (BM-MSCs) and exosomes derived from mouse BM-MSCs could rescue AIH mouse models [8–10]. No human-derived MSCs (hMSCs) in AIH have been studied, thus limiting the clinical usages of MSCs therapy. Currently, several clinical trials were conducted using hMSCs in areas of trauma, neurology, cardiology, and immunology, showing positive effects [11]. Human menstrual blood-derived stem cells (MenSCs) are newly used hMSCs with practical clinical applications because they are easily accessible, resourceful, and require minimal ethical regulations [12]. Besides, MenSCs have anti-inflammatory effects [13] and anti-fibrotic effects [5] as other MSCs, such as BM-MSCs [7], placentas, and umbilical cord-derived MSCs (UC-MSCs) [14].

Concanavalin A (Con A)-induced mouse model is the most common used immune hepatitis mouse model of AIH by far [15], and its pathogenesis and pathological changes are similar to those of AIH [16]. Herein, a Con A mouse model was used to imitate AIH. Con A-induced liver injury is activated by T cells and macrophages [17], and apoptosis plays a critical role during the pathogenesis [18,19]. Besides, the c-Jun N-terminal kinase (JNK) signaling pathway could regulate apoptosis in Con A mice [16].

Apoptosis is an important form of programmed cell death; it involves the activation of catabolic enzymes in signaling cascades. The morphology of apoptosis always demonstrates cellular shrinkage with nuclear chromatin condensation and nuclear fragmentation [20]. The mitogen-activated protein kinase (MAPK) signaling pathways regulate multiple biological processes, such as apoptosis, act dually depending on the cell type and the stimulus [21]. Among MAPKs, JNK cascade mediates the pro-apoptosis process. JNK could regulate the transcription factor AP-1, which contains c-Jun, and it is associated with apoptotic scenarios. Besides, JNK could induce caspase 8 and caspase 3 to stimulate apoptosis [22]. SP600125, a reversible and ATP-competitive JNK inhibitor, could inhibit c-Jun phosphorylation and block lipopolysaccharide-induced TNF-α [23].

In this study, the autoimmune hepatitis curative effects of MenSCs therapy and the mechanisms underlying the process in vivo and in vitro were discussed.

2 Materials and methods

2.1 MenSCs culture and identification

MenSCs were kindly provided by Charlie Xiang’s laboratory. MenSCs from three young healthy female volunteers (aged under 30 years) were collected, as approved by the Ethics Committee of the First Affiliated Hospital, Collage of Medicine, Zhejiang University (permit number: 2017-623). In brief, menstrual blood was collected from the second or third day of menstrual cycle with DivaCup and separated using Ficoll-Paque to obtain the interlayer cells [12]. The cells were cultured with Chang medium to obtain adherent cells. Then, they were changed with DMEM/F12 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, USA) every 2–3 days and passaged at 80%–90% confluency. MenSCs from the third to the eighth passage were used in this study.

Flow cytometry was used to identify the MenSCs’ surface markers. In brief, MenSCs were collected by trypsinization and washed twice with phosphate-buffered saline (PBS). They were then incubated with Fc block (BD Biosciences, USA) and stained using the following APC-conjugated antibodies: anti-CD29, anti-CD73, anti-CD90, anti-CD34, antiCD45, anti-CD105, anti-CD117, and anti-human leukocyte antigen DR (HLA-DR). Meanwhile, IgG1 and IgG2a (BD Biosciences, USA) were used as the isotype control for antibodies. The stained cells were washed and resuspended with Pharmingen Stain Buffer (BSA, BD Biosciences, USA) and analyzed using CytoFLEX LX (Beckman Coulter, USA) and FlowJo software (BD Biosciences, USA) [5].

The fourth passage of MenSCs was cultured in six-well plates and 15 mL centrifuge tubes. Then, they were treated with human mesenchymal stem cells differentiation kits (Cyagen, China) to differentiate into osteogenic, chondrogenic, and adipogenic. After differentiation was performed, the cells were fixed with 4% paraformaldehyde and stained with Oil Red O for 30 min for adipogenic differentiation. The cells were then fixed and stained with Alizarin red for 5 min for osteogenic differentiation. Similarly, the cells were sliced after embedding in paraffin wax and stained with Alcian blue for 30 min for chondrogenic differentiation [24]. A Leica DMi1 microscope was used to obtain images of adipogenic and osteogenic differentiation, while NanoZoomer 2.0-RS scanners were used to obtain images of chondrogenic differentiation sections.

2.2 Animals

The Animal Care Committee of the Animal Experimental Ethical Inspection of the First Affiliated Hospital, College of Medicine, Zhejiang University approved the animal experiment protocols (permit number: 2020-1505). C57BL/6 mice (4–6 weeks old, male) were obtained from the Experimental Animal Center of Zhejiang Academy of Medical Sciences. Food and water were freely provided to the mice. They were housed in the Laboratory of Animal Center of the First Affiliated Hospital, College of Medicine, Zhejiang University, at standard conditions in a 12-h photoperiod.

2.3 Con A-induced hepatitis and MenSCs transplantation

All mice were acclimatized at the Laboratory Animal Center for 1 week. Mice weighing 20–25 g were intravenously injected with Con A (15 or 20 mg/kg) to establish the AIH model (Sigma–Aldrich, USA) [25]. A total of 5 × 10⁵ MenSCs in 0.2 mL PBS (Con A + MenSCs group) or an equal volume of PBS (Con A group) was injected through the tail veins to evaluate the therapeutic effect of MenSCs in AIH mouse model. The control group was injected with only PBS (NC group) or MenSCs (NC + MenSCs group). The mice were anesthetized using isoflurane and sacrificed. The serum and liver tissues in the Con A group (Con A dosage: 15 mg/kg) were harvested at 6, 12, 24 (n = 7 per group), 48, 72, and 96 h (n = 4 per group). The serum and liver tissues from the Con A + MenSCs group (Con A dosage: 15 mg/kg) were collected at 6, 12, and 24 h (n = 7 per group). The tissues from the NC group, which was only injected with PBS, were harvested at 24 h (n = 7).

2.4 Evaluation of liver injury

The serum samples of the Con A and Con A + MenSCs groups were diluted 10–20 times with PBS before use. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed with ALT and AST assay kits (Nanjing Jiancheng Bioengineering Institute, China).

For hematoxylin and eosin (H&E) analysis, liver lobes were fixed in 4% paraformaldehyde overnight and embedded in paraffin. Then, 3–5 µm-thick slices were used for H&E staining, and NanoZoomer 2.0-RS was used to scan the sections.

2.5 Cell labeling and imaging

XenoLight DiR (DiR; PerkinElmer, USA) stock solution was prepared by dissolving 25 mg of DiR in 3 mL of ethanol. The working solution (320 µg/mL) was prepared by diluting 199 µL of stock solution in 5 mL PBS. MenSCs were trypsin-digested and incubated with 320 μg/mL DiR. After 30 min of incubation, the cells were centrifuged at 1000 rpm for 3 min at 4 °C, washed twice with PBS, and injected intravenously (5 × 10⁵ cells/mouse). After 12 h, the mice were sacrificed to obtain lung, liver, heart, spleen, and kidney for MenSCs imaging. The organs were captured with the Lumina LT III Small Animal In Vivo Imager (PerkinElmer, USA) equipped with a cooled CCD camera.

2.6 Phosphoproteomic analysis

About 100 mg of liver tissue was lysed by 1 mL of 8 M urea with 150 mM NaCl, 50 mM Tris-HCl, protease and phosphorylase inhibitor. After centrifugation was performed at 15 000 g at 4 °C for 15 min, the supernatant was reduced by dithiothreitol and stopped by iodoacetamide. Then, the protein was desalted by Zeba Spin Columns (Thermo Fisher Scientific, USA) and digested with trypsin (Sigma, USA) for 14 h. The peptides were acidified with trifluoroacetic acid and desalted with C18 Vac cartridges (Waters, USA). Phosphopeptide enrichment was applied with High-Select Fe-NTA kit (Thermo Fisher Scientific, USA). Ten samples were combined by 10-TMT kit (Thermo Fisher Scientific, USA) and dried by vacuum centrifugation (Beijing Giam Technology, China) prior to LC-MS/MS analysis. The peptide sample was separated by LC-MS/MS and analyzed using Q Exactive HF-X (Thermo Fisher Scientific, USA). MaxQuant was used to identify the proteins, and those containing phospho-sites with fold change ≥ 2 were selected for enrichment analysis.

2.7 RNA-seq and bioinformatics analysis

Total RNA was extracted from liver tissues by TRIzol (Invitrogen, USA). NanoDrop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, USA) were used to quantify the total RNA. Oligo(dT)-attached magnetic beads were then used to collect and fragment purified mRNA. The first-strand cDNA and a second-strand cDNA were synthesized and incubated for end repair, amplified, and purified, and then the quality was validated [26]. Single-strand cDNA was synthesized from double-stranded cDNA products to obtain the final library and amplified to make DNA nanoballs analyzable by the MGI2000 platform (TSINGKE, China). MGISEQ-2000 was used to obtain raw data by base calling, and the filtered raw data were compared with the reference sequence to quantitative analysis of gene expression.

On the basis of the gene expression in different sample groups, the differentially expressed genes (DEGs) with fold change ≥ 2 and FDR < 0.01 were chosen. KOBAS software [27] was used to detect the KEGG pathway [28].

2.8 TUNEL staining

TUNEL assay (Vazyme, China) was used for analyzing the apoptosis of liver tissues. In brief, liver paraffin sections were deparaffinized and rehydrated, and then 20 μg/mL of protease K solution was added at room temperature (RT) for 20 min. The sections were washed thrice by using PBS and then immersed in equilibration buffer for 30 min before incubating with TUNEL reaction admixture in a humidified chamber at 37 °C for 60 min. The reactions were stopped, and then the sections were stained using DAPI at RT for 5 min. PANORAMIC 250 Flash series digital scanners were used to scan the slides. Five visual fields (40× magnification) were randomly selected in each slide, and ImageJ software was used to determine the index of apoptotic cell rate.

2.9 Western blot analysis

The whole protein of the liver was obtained via mechanical lapping using radioimmunoprecipitation assay with protease and phosphatase inhibitor cocktail (Beyotime Biotechnology, China). About 20 μg of protein samples was separated using 4%–20% precast mini polyacrylamide gels (Genscript, China) and transferred to polyvinylidene fluoride membranes (Bio-Rad, USA). The membranes were incubated with blocking buffer (Beyotime, China) at RT for 30–60 min and incubated with primary antibodies at 4 °C for 12–16 h. The membranes were washed thrice, incubated with secondary antibodies for 60–120 min, and then washed thrice. Chemiluminescence reagents (Beyotime, China) were used for result detection, and ImageJ was used for analysis. The following antibodies were used: anti-AKT (4691), anti-phospho-proteinkinase B (p-AKT) (4060), anti-JNK (9252), anti-phospho-JNK (p-JNK) (4668), anti-phospho-c-Jun (p-c-Jun ser73) (3270), p-c-Jun ser63 (2361), anti-caspase 3 (9662), anti-caspase 8 (4790), anti-PARP (9532), anti-Bcl-xL (2764) anti-GAPDH (2118) (1:1000, Cell Signaling Technology, USA), anti-Fas (sc-74540), and anti-Bad (sc-8044, 1:500, Santa Cruz, China).

2.10 Transwell coculture assay

The mouse hepatocytes cell line AML12 (Procell, China) were cultured in DMEM medium (Gibco, USA). A Transwell chamber system (0.4 μm pore size, Corning, USA) was used. AML12 cells were seeded in the lower chamber, whereas MenSCs were cultured in the upper chamber. After 24 h of cell adhesion, the medium was changed with serum free medium with or without Con A. The AML12 cells were collected by protein lysis after 24 h of co-culturing for apoptosis and Western blot analyses. The apoptosis was also analyzed as follows: the AML12 cells were washed twice with cold PBS and incubated with 5 µL of FITC Annexin V and 5 µL PI (BD Pharmingen, USA) for 15 min in the dark and analyzed via CytoFLEX LX. The cellular supernatants of AML12 were collected for ALT and AST analysis.

2.11 SP600125 inhibition assay

Vehicle control (6% DMSO in normal saline) or SP600125 (SP) was administered via intraperitoneal injection (15 mg/kg) 10 min before Con A injection. The serum and liver tissues were collected after 12 h of Con A injection.

The total RNA of the liver tissue was extracted using TRIzol reagent (Invitrogen Life Technologies, USA) as follows: 50 mg of liver tissue was homogenized with 1 mL TRIzol regent and incubated at RT for 5 min, 0.2 mL chloroform was added and shaken vigorously for 15 s; the mixture was centrifuged at 12 000× g for 15 min to collect the colorless aqueous phase; 0.5 mL of isopropanol was added, let stand for 10 min at RT, and centrifuged to obtain the precipitate; and the RNA pellet was washed with 1 mL 75% ethanol twice, and the RNA samples were lysed with RNase-free water. The RNA samples were measured by NanoDrop (Thermo Fisher Scientific, USA). cDNA was generated using the PrimeScrip RT Master Mix kit (Takara, Japan). In brief, 500 ng RNA and 2 μL 5× PrimeScript RT Master Mix were added with RNase-free water to an amount of 10 μL volume. The mixture was incubated at 37 °C for 15 min and 85 °C for 5 s to obtain cDNA. Subsequently, qPCR was performed using the TB Green Premix Ex Taq II kit (Takara, Japan). The reaction mixtures consisted of 5 μL TB Green, 0.2 μL ROX Reference Dye (50×), 0.4 μL PCR forward primer (10 mM), 0.4 μL PCR reverse primer (10 mM), 0.5 μL cDNA, and 3.5 μL RNase-free water. Cycling procedure was performed using the QuantStudio Design & Analysis Software version 1.4.3 as follows: 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The primer sequences used for the PCR reactions were as follows: FasL, forward 5′-GCAGAGGCACAGAGAAAGA-3′ and reverse 5′-AAGTAGACCCACCCTGGAA-3′; IL-6, forward 5′-ACAGAAGGAGTGGCTAAGGA-3′ and reverse 5′-AGGCATAACGCACTAGGTTT-3′; TNF-α, forward 5′-GGTCAGGTTGCCTCTGTCTC-3′ and reverse 5′-TGCACCTCAGGGAAGAATCTG-3′; GAPDH, forward 5′-CAGTGGCAAAGTGGAGATTGTTG-3′; and reverse 5′-TCGCTCCTGGAAGATGGTGAT-3′. The serum TNF-α (Beijing Dakwei Biotechnology, China) and IL-6 (R&D Systems, USA) concentrations were processed with commercial ELISA kits, and the OD₄₅₀ values were measured with a microplate reader (Bio-Rad, USA).

2.12 Statistics

R version 4.1.0 and GraphPad Prism 8 (GraphPad, USA) were used to draw graphs and data analysis. Two-tailed unpaired t-test was used to compare differences between two groups, and one-way ANOVA was used in more than two groups. Log-rank (Mantel–Cox) test was used to compare the differences in survival rates between two groups. Statistical significance was set as follows: *P < 0.05 and **P < 0.01.

3 Results

3.1 Identification of MenSCs

Flow cytometry was used to identify the phenotype of MenSCs. The results showed that MenSCs were significantly positive for CD29, CD73, CD90, and CD105 compared with the isotype control and negative for CD34, CD45, CD117, and HLA-DR (Fig. S1A and S1B). Therefore, MenSCs are not hematopoietic and have low immunogenicity. Besides, MenSCs could differentiate into adipocytes, chondroblasts, and osteoblasts after induction with the corresponding differentiation medium (Fig. S1C).

3.2 Establishment of Con A-induced AIH model

C57BL/6 mice with more Th1-like T helper cells are more sensitive to Con A than other mouse strains and require 15–20 mg/kg body weight to take effect [29]. First, 15 and 20 mg/kg dosages were examined, and the results found that 20 mg/kg dosage could cause 10 deaths among 12 mice, whereas 15 mg/kg dosage did not cause mouse death (Fig. S1A). Besides, 15 mg/kg Con A exhibited lower Knodell score and lighter liver morphology in the liver than 20 mg/kg Con A (Fig.1 and 1B, S2B). Thus, 15 mg/kg Con A was selected for further study.

The serum ALT and AST values were determined at 6, 12, 24, 48, 72 h, and 96 h after Con A administration and at 24 h in the NC group. The serum ALT decreased after 6 h of Con A administration, while that of AST decreased after 12 h (Fig.1 and 1D). The H&E staining of Con A mouse liver sections showed changes in liver injury. At 24 h timepoint, the liver injury area was the largest, and immune cell infiltration increased at 48 h time point (Fig.1). Thus, the Con A mouse model was confirmed, and the 24 h timepoint was selected as the endpoint.

3.3 MenSCs transplantation ameliorates Con A-induced hepatitis

Con A-induced hepatitis was used as an AIH model to test whether MenSCs have therapeutic effects. First, the ALT and AST levels of the NC + MenSCs and NC groups were examined to check the MenSCs’ safety (Fig. S3A and S3B). The results showed that the distribution of MenSCs in the NC group was similar to that in the Con A group, mainly in the lung, liver, and spleen (Fig. S3C). MenSCs significantly reduced the mortality of Con A-induced mice (20 mg/kg), and the 15 mg/kg Con A did not cause mouse death at 36 h (P < 0.01, Fig.2). H&E staining also showed that the hepatocyte injury in the Con A + MenSCs group was lighter than that in the Con A group (Fig.2 and 2C). The serum ALT and AST levels of the Con A group and Con A + MenSCs group were measured at 6, 12, and 24 h. The Con A group had higher ALT at 12 and 24 h (P < 0.01) and higher AST at 6 (P < 0.05), 12, and 24 h (P < 0.01) than the Con A + MenSCs group (Fig.2 and 2E). Given that MenSCs could rapidly improve Con A-induced hepatitis at 12 h, it was chosen as the most important timepoint.

3.4 Phosphoproteomics and RNA-seq revealing the underlying mechanisms

Principal component analysis showed that the three groups had good homogeneity (Fig.3 and S4A). A total of 188 proteins with 2538 phosphorylation sites among the NC, Con A, and Con A + MenSCs groups at 12 h (Fig.3). GO enrichment analysis of the differentially expressed sites revealed that the biological process (BP) terms were mainly enriched in mRNA processing and regulation (Fig. S4B). Nuclear speck, heterochromatin, and actin cytoskeleton were mostly concerned in cellular component (CC) terms (Fig. S4C), and transcription coregulator activity, actin binding, and mRNA binding were chiefly in molecular function (MF) part (Fig. S4D). For further analysis, Con A/NC differential sites were combined with Con A + MenSCs/NC indifference sites, and 180 overlapped sites were found. Then, the 180 sites were placed into STRING and Cytoscape to construct a protein-protein interaction (PPI) network (Fig.3). Besides, the KEGG enrichment analysis showed that Hippo and NOD-like receptor signaling pathways were mainly involved (Fig.3). Meanwhile, the GO enrichment analysis showed that the BP term was mainly enriched in spindle organization (Fig.3), the MF term was mainly enriched in protein kinase A binding (Fig.3), and the CC term was mainly enriched in actin cytoskeleton (Fig.3). Fig.3 displays an overview of the differentially expressed proteins and sites between two groups. The differentially expressed sites between Con A/NC and Con A + MenSCs/Con A groups were compared to determine the MenSCs’ roles in Con A-induced hepatitis, and seven sites were found. Moreover, Jun was found to be an important part of the JNK/MAPK pathway (Fig.3).

Then, RNA-seq was performed among the three groups. The volcano map showed the DEGs between the Con A group at 12 h and the NC group (Fig.4). The KEGG enrichment analysis found that MAPK and PI3-AKT signaling pathways were the most important (Fig.4). The DEGs between Con A and Con A + MenSCs groups at 12 h were further compared (Fig.4). The apoptosis, cell cycle, MAPK, and PI3-AKT signaling pathways were mainly enriched (Fig.4 and Table S1). Combination of the results of phosphoproteomics and RNA-seq revealed that the apoptosis and JNK/MAPK signaling pathway may play the most important function during MenSCs treatment.

3.5 Improvement of Con A-induced hepatitis by MenSCs transplantation through inhibiting apoptosis

The TUNEL staining results showed that Con A could promote hepatocyte apoptosis, and MenSCs could significantly reduce Con A-induced apoptosis (P < 0.01; Fig.5 and 5B). For further verification, the cleaved caspase 3, cleaved caspase 8, cleaved PARP, Bad, and Bcl-xL protein levels of the liver tissue lysed in NC, Con A, and Con A + MenSCs groups at 12 h were detected (Fig.5). The levels of cleaved caspase 3, cleaved caspase 8, cleaved PARP, and Bad proteins in the Con A group significantly increased, indicating that Con A injection could promote hepatocyte apoptosis and MenSCs could attenuate this process (Fig.5–5G). Although the level of Bcl-xL protein in the Con A group was higher than that in the NC group, MenSCs could further improve it (Fig.5).

3.6 Suppression of Con A-induced apoptosis by MenSCs transplantation through JNK/MAPK pathway

First, the expression of p-c-Jun ser63 was verified (Fig.6 and 6B). Then, the JNK/MAPK and AKT signaling pathways were examined. The Con A + MenSCs group had a significantly lower protein expression at 12 h (p-JNK, p-c-Jun ser73, and Fas) and higher p-AKT protein expression than the Con A group (Fig.6 and 6C–6F).

The AML12 cell coculture system was used to further clarify the function of MenSCs in the inhibition of hepatocyte apoptosis (Fig.7). The apoptosis among the three groups was detected by flow cytometry. The early apoptotic rate and the total apoptotic rate in the Con A group were notably the highest, but no difference was found in the end-stage apoptosis and death rate (Fig.7–7E). Then, the ALT and AST levels of the cell supernatant were determined. The Con A group showed the highest level among the three groups (Fig.7 and 7G). It also displayed higher cleaved caspase 3, p-JNK, p-c-Jun ser63, and p-c-Jun ser73 protein expression (Fig.7–7L).

Then, a JNK inhibitor (SP600125) was used to further verify the JNK/MAPK signaling pathway. The results of H&E staining showed the Con A + SP group had lighter liver injury than the Con A group (Fig.8 and 8B). The serum ALT and AST levels were tested, and the Con A group showed the highest level among the three groups (Fig.8 and 8D). The protein levels of p-JNK, p-c-Jun ser63, p-c-Jun ser73, and cleaved caspase 3 were significantly inhibited by SP600125 treatment (Fig.8–8I). Besides, the FasL, IL-6, and TNF-α mRNA expression and the serum IL-6 and TNF-α levels were inhibited by SP600125 (Fig.8–8N).

4 Discussion

The specialized angiogenesis that occurs during endometrial formation allows for the extraction of specialized stem cell populations. Meng et al. observed that menstrual blood cells could expand up to 68-fold without losing karyotype normality or the potential to develop into tumors [30]. Besides, Chen et al. found that few differences between different age sources and passages may cause senescence of MenSCs [31]. Since 2007, scientists have successively extracted and identified MSCs from menstrual blood and discovered their multi-directional differentiation potential [32]. MSCs have been extensively investigated, and they own great promise for clinical application as cell therapy [7]. MenSCs as novel MSCs have the advantages in collection and isolation. Previous reports found that MenSCs had more powerful immunomodulatory properties in inhibiting T cell proliferation than UC-MSCs [33] and BM-MSCs [34]. In this study, MenSCs could rescue the Con A-induced AIH model through the JNK/MAPK signaling pathway. Moreover, the JNK inhibitor SP600125 further verified that MenSCs could inhibit the JNK pathway to inhibit hepatocyte apoptosis.

The results of phosphoproteomics showed that Hippo and NOD-like receptor signaling pathways played an important function (Fig.3). Besides, RNA-seq showed the importance of Hippo and NOD-like receptor signaling pathways. The Hippo signaling pathway [35,36] and NOD-like receptor signaling pathway [37] could activate the MAPK signaling pathway to mediate cell death. Activated caspase 3 cleaves PARP, which is important in DNA repair, thus promoting apoptosis [38,39]. Anti-apoptotic members, including Bcl-xL, promote cell survival, and the PI3K-AKT pathway is a strong pro-survival pathway [40]. Once Akt is activated, it could regulate cellular processes, such as apoptosis, protein synthesis, metabolism, and cell cycle, by phosphorylation [41]. The JNK signaling pathway is an evolutionarily conserved kinase cascade, known for its role in stress-induced apoptosis and tumor progression [21,42]. In the present study, MenSCs mainly suppressed JNK phosphorylation and c-Jun phosphorylation induced by Con A, and they could stimulate AKT phosphorylation (Fig.6). This finding clarified that MenSCs ameliorated liver parenchymal apoptosis by mediating the JNK/MAPK pathway [43,44]. In the AML12 cell coculture system, MenSCs could secrete substances to help AML12 cells survive from Con A. Previous articles reported that key paracrine factors, such as stem cell factor (SCF) [45], fibroblast growth factor 21 (FGF21) [46], TGF-β1/2/3, IL-10, and MCP-1 [47], could be important candidates for MSCs against cell injury and suppressing immune reactions. SP600125, an inhibitor of JNK [23], could induce G2/M-phase arrest, endoreduplication, and apoptosis [48,49]. Combination of the data shown in Fig.8 indicated that MenSCs treatment ameliorated AIH by inhibiting the JNK/MAPK pathway to inhibit caspase 3-related apoptosis.

Though the Con A-induced mouse model is the most used AIH mouse model by far, it is representative of an acute inflammatory state and may not adequately address the chronic nature of AIH. MenSCs take time to migrate and perform immunomodulation, which could hinder the clinical application in acute diseases. Considering MenSCs have preponderance in sources compared with most MSCs, additional research is needed for the clinical applications of MenSCs in the future. Besides, induced pluripotent stem cell-derived MSCs (iPSC-derived MSCs) could be a great source of MSCs due to their high proliferative capacity without obvious losing self-renewal potential [50]. Bloor et al. conducted a good manufacturing practice of iPSC-derived MSCs in refractory graft-versus-host-disease in a clinical trial and found it was safe and well tolerated [51]. MenSCs-based therapy requires further research and verification for clinical usage, including age of donor, appropriate dose, selection of optimal transplantation routes, systematic study of various diseases, and long-term monitoring of MenSCs [52].

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