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Bone marrow mesenchymal stem cells alleviate liver fibrosis after rat liver transplantation through JAK1/STAT5 pathway

Stem Cell Research & Therapy volume 16, Article number: 217 (2025) Cite this article

Abstract

Objective

The effectiveness of bone marrow mesenchymal stem cells (BMSCs) in post-transplantation liver fibrosis has not been studied. The aim of this study was to investigate the effect of BMSCs on liver fibrosis and their role in the Janus-activated kinase (JAK) 1/ signal transducer and activator of transcription (STAT) 5 pathway after liver transplantation (LT).

Methods

A rat model of post-LT liver fibrosis induced by cold ischemia injury was successfully established. BMSCs were injected into the rats through the portal vein. Hepatic stellate cell (HSC)-T6 were co-cultured with BMSCs in vitro after hypoxia–reoxygenation. JAK1 inhibitor Abrocitinib and JAK1 agonist RO8191 were used to study the JAK1/STAT5 signaling pathway.

Results

BMSCs significantly alleviated liver fibrosis caused by cold ischemia–reperfusion injury after rat LT in vivo. After BMSCs transplantation, the levels of JAK1 and p-STAT5 in rat liver were significantly reduced. After using Abrocitinib, the stage of liver fibrosis and the levels of collagen type I alpha 1 chain (COL1A1) and actin alpha 2 (ACTA2) decreased. After using RO8191, the stage of liver fibrosis and the levels of COL1A1 and ACTA2 increased. BMSCs significantly reduced the activation of HSC-T6 after hypoxia–reoxygenation in vitro. After co-culturing with BMSCs after HSC-T6 hypoxia–reoxygenation, the levels of JAK1 and p-STAT5 were significantly reduced. After the addition of Abrocitinib, the levels of COL1A1 and ACTA2 decreased in HSC-T6; in contrast, after adding RO8191, the levels of COL1A1 and ACTA2 increased in HSC-T6 after hypoxia–reoxygenation. After using anti-IL7 antibody or anti-IL7Rα in vivo and in vitro, the stage of liver fibrosis and the levels of COL1A1 and ACTA2 decreased as well as the phosphorylation level of STAT5.

Conclusions

BMSCs alleviate hepatic cell damage, reduce hepatic cell-derived IL7, downregulate IL7R/JAK1/STAT5 in HSCs, thereby reducing HSCs’ activation and ultimately alleviating liver fibrosis after liver transplantation.

Highlights

BMSCs can alleviate liver fibrosis after rat liver transplantation in vivo.

BMSCs can reduce activation of HSCs after hepoxia-reoxygenation in vitro.

BMSCs inhibit activation of HSCs after injury through the JAK1/STAT5 pathway, thereby reducing liver fibrosis after liver transplantation.

Introduction

Liver transplantation (LT) is an effective treatment for end-stage liver disease. Multiple studies on long-term surveillance liver biopsy after pediatric LT (pLT) have shown that the incidence of liver fibrosis is 25–100% [1]. If liver fibrosis is not treated early in a timely manner, it may ultimately lead to irreversible cirrhosis, causing graft loss and reduced survival rate in the recipients [2,3,4]. Therefore, exploring the formation mechanism of liver fibrosis after LT and seeking methods to prevent liver fibrosis after LT are essential for improving the long-term survival rate of liver transplant recipients.

The activation of hepatic stellate cells (HSCs) is considered the central event in the occurrence and development of liver fibrosis [5]. Under normal circumstances, HSCs are in a quiescent state. When the liver is subjected to inflammation, mechanical stimulation, or ischemia–reperfusion injury, HSCs are activated and convert into myofibroblasts, which express actin alpha 2 (ACTA2) and cytoglobin and secrete a large amount of extracellular matrix, especially type I and III collagen fibers [6,7,8,9,10]. Activated HSCs can also inhibit the degradation of collagen fibers by overexpressing tissue inhibitor of metalloproteinase 1 (TIMP1), which can bind to matrix metalloproteinase 9 (MMP9) and inactivate it. The imbalance between the synthesis and degradation of fibrin ultimately leads to the formation of liver fibrosis [11]. In addition, activated HSCs can continue to be activated through the secretion of cytokines such as transforming growth factor beta 1 (TGFB1), platelet-derived growth factor, and endothelin. Hence, even after removing the primary factor, liver fibrosis continues to develop [12, 13].

It has been suggested that bone marrow mesenchymal stem cells (BMSCs) have a protective effect in liver ischemia–reperfusion injury. Due to their homing property, BMSCs can migrate to damaged liver tissue under stimulation from factors such as hepatocyte growth factor, fibroblast growth factor, and epidermal growth factor, and proliferate and differentiate into hepatocyte-like cells to alleviate liver damage [14]. Similar to their antifibrotic activity demonstrated in other fibrosis settings, including the radiation-induced colorectal fibrosis model, mesenchymal stem cells (MSCs) have been shown to reverse fibrosis by modulating several pathways, including extracellular matrix turnover mediated by hepatocyte growth factor and tumor necrosis factor-stimulated gene 6 [15]. Therefore, the interaction between BMSCs and HSCs was one of the focuses of this research.

Multiple cytokines and growth factors, including the interferon family, interleukin (IL) 10 family, gp130 family, γC family (including IL7), and single-chain families, can activate the Janus-activated kinase (JAK)/ signal transducer and activator of transcription (STAT) signaling pathway [16,17,18,19,20]. Studies have shown that IL7/IL7 receptor (IL7R) is associated with liver fibrosis [21]. Hepatocytes are the main source of IL7 in the liver, and most of the IL7 secreted by hepatocytes can only function within the liver [22]. An association of IL7 levels with fibrosis stage has been reported in an adult non-alcoholic steatohepatitis cohort but does not seem to have been investigated in pediatric non-alcoholic steatohepatitis [23]. As a receptor for IL7, IL7R can activate various intracellular signaling pathways, such as JAK/STAT, phosphoinositide 3-kinases, and mitogen activated protein kinases. After liver injury, various subpopulations of cells located in the liver (Kupffer cells, HSCs, hepatocytes, natural killer cells, dendritic cells, and T cells) produce a series of cytokines with inflammatory or hepatoprotective potential, ultimately leading to the activation of the JAK/STAT signaling pathway and the formation of liver fibrosis [24]. The role of STAT5 in liver fibrosis is controversial. To be specific, STAT5 showed hepatoprotective and antifibrotic effects in a mouse model of cholestasis [25]. After carbon tetrachloride (CCl4) intervention, the absence of STAT5 in liver cells enhanced growth hormone–induced STAT3 activity and increased TGFB1 levels. However, in the acute CCl4 administration model, reducing the expression of STAT5 in liver cells reduced liver inflammation [26]. Thus, it is meaningful to study the role of the JAK1/STAT5 pathway in BMSCs-related alleviation of liver fibrosis after LT. Specifically, in this study, we aimed to investigate the effect of BMSCs on liver fibrosis after LT and their role in the JAK1/STAT5 pathway in liver fibrosis.

Methods

Animals

All animal procedures used in this study were approved by the Experimental Animal Ethics Committee of Tianjin First Central Hospital and complied with the “Guide for the Care and Use of Laboratory Animals”. Clinical trial number: not applicable. Animals were randomly assigned to the experimental group, and all experiments were conducted on male animals. Sprague–Dawley (SD) rats aged 7–8 weeks were housed in a specific pathogen–free facility at the Tianjin Organ Transplantation Key Laboratory. During the study, the animals were exposed to a 12-hour light and 12-hour dark cycle, and freely received food and water. The work has been reported in line with the ARRIVE guidelines 2.0.

Rat model of post-LT liver fibrosis induced by prolonged cold ischemia time (CIT) of donor liver

Anesthesia method during rat liver transplantation using isoflurane inhalation anesthesia. The concentration during induction anesthesia is 4–5%, and the oxygen flow rate is controlled at 1 L/min. The concentration during maintenance anesthesia is 2–3%, and the oxygen flow rate is controlled at 0.2–0.4 L/min. The concentration during the anhepatic phase is 0.5-1%, and the oxygen flow rate is controlled at 0.4–0.6 L/min. After obtaining the SD rats’ livers, each liver was perfused through the portal vein with 10 mL of precooled Sodium Lactate Ringer’s Injection. Subsequently, the donor liver was transferred to a sterile bag containing Sodium Lactate Ringer’s Injection to ensure complete immersion, and any air was expelled from the bag. Then, the bag was placed into a foam box with ice water mixture and stored in a refrigerator at 4 °C. Maintaining a continuous supply of ice in the mixture of ice and water was crucial to ensure proper organ preservation. According to the experimental requirements, the donor liver had to be implanted in situ into another rat after 4 h. We observed and recorded the recovery time of consciousness and activity ability of the recipients after LT, and collected and analyzed blood and liver pathology results and recorded the survival status on postoperative day 28.

Animal euthanasia methods

The rats were sacrificed by inhaling CO2, in accordance with the Canadian Council on Animal Care guidelines on euthanasia of animals used in science. We placed the rats in a pre-filled CO₂ chamber (20–30% displacement rate) and maintained exposure for ≥ 5 min after respiratory arrest. Then we confirmed death via bilateral thoracotomy.

BMSCs extraction and characterization

The femur and tibia were removed aseptically after the rats were euthanized by inhaling CO2. The marrow cavity was rinsed with DMEM/F12 (1:1) containing 10% fetal bovine serum; the cell suspension was inoculated into T75 culture flasks, and cultured at 37 °C with 5% CO2. Well-grown passage 3 cells were resuspended for detection and backup, labeled with fluorescent antibodies: anti-CD34-FITC (0.25 µg/106 cells, Cat# sc-7324, Santa Cruz, CA, USA), anti-CD90-FITC (0.25 µg/106 cells, Cat# 202503, BioLegend, CA, USA), anti-CD45-PE (0.25 µg/106 cells, Cat# 202224, BioLegend, CA, USA), anti-CD29-PE (0.25 µg/106 cells, Cat# 102207, BioLegend, CA, USA), anti-RT1A-PE (0.25 µg/106 cells, Cat# 205208, BioLegend, USA), and anti-RT1B-FITC (0.25 µg/106 cells, Cat# 205305, BioLegend, CA, USA), and incubated for 30 min in the dark for flow cytometry.

Animal experiment grouping design

The animals were randomly divided into the Sham group, LT group, LT + BMSC group, LT + Abrocitinib group, LT + BMSC + RO8191 group, LT + anti-IL7 group, and LT + anti-IL7Rα group, with 6 animals in each group. The LT group was subjected to a transplantation of the liver with a cold storage time of 4 h. The LT + BMSC group received 5 × 106 BMSCs by injection through the portal vein after blood flow opening during LT. In addition to LT, the LT + Abrocitinib group received Abrocitinib (40 mg/kg of body weight, per os, daily) for 10 days. The LT + BMSC + RO8191 group received 5 × 106 BMSCs via portal vein injection with open blood flow after LT, and also received RO8191 (20 mg/kg of body weight, per os, daily) for 10 days. The LT + anti-IL7 group received anti-IL7 antibody (M25; Cat# HY-P990210, MCE, Shanghai, China) (250 µg/kg of body weight, per ip, once a week) for 2 weeks after LT. The LT + anti-IL7Rα group received anti-IL7Rα antibody (A7R34; Cat# HY-P990209, MCE, Shanghai, China) (250 µg/kg of body weight, per ip, once a week) for 2 weeks after LT. The Sham group and the LT group did not receive intervention. All of the rats were euthanized at 28 days after LT, and their livers and blood were obtained for subsequent experiments.

Construction of cellular hypoxia–reoxygenation model and co-culture model

Normal rat HSC cell line (HSC-T6), normal rat hepatic cell line (IAR20) and BMSCs were cultured in suitable DMEM or RPMI-1640 medium, respectively, in a normal 37 °C, 5% CO2 cell culture incubator. When the cells grew and fused to around 90% density in the culture dish, trypsin digestion was used to prepare a cell suspension. The cells in HSC-T6 or IAR20 cell suspension were counted and inoculated in a six-well plate (106 cells). Complete culture medium was replaced with low-glucose culture medium when the cells have grown and fused to a density of around 50%. Then, the medium was placed in a cell hypoxia–reoxygenation incubator and incubated under 99% N2 mixture for 12 h. After hypoxia, the low-glucose medium was replaced with complete medium and cultured in a normal 37 °C, 5% CO2 cell incubator. Cell counting was performed on BMSC suspension, and the cells were inoculated in a Transwell chamber with a pore size of 0.4 μm in a six-well plate (106 cells). We cultivated the cells in a normal 37 °C, 5% CO2 cell culture incubator using complete culture medium. If co-culture was required, we moved the chamber to the six-well plate that had just been deprived of oxygen in the previous step, replaced the complete culture medium, and placed it in a normal 37 °C, 5% CO2 cell culture incubator for cultivation.

RNA-sequencing analysis

Total RNA from the livers was extracted using the TRIzol reagent (Invitrogen, Waltham, MA, USA), and RNA quantification was based on the NanoDrop2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA). Then, libraries were constructed using VAHTS Universal V6 RNA-seq Library Prep Kit in accordance with the manufacturer’s instructions. The RNA libraries were sequenced on an Illumina NovaseqTM6000 platform (OE Biotech, Shanghai, China). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), Reactome, and WikiPathways enrichment analyses of differentially expressed genes were performed to screen the significantly enriched terms using R (v 3.2.0). R (v 3.2.0) was used to draw the column diagram, chord diagram, and bubble diagram of the significantly enriched terms.

Quantitative real-time polymerase chain reaction (RT-qPCR)

For RT-qPCR detection, total RNA was extracted from the liver tissue samples or cultured cells using TRIzol reagent. NanoDrop 2000 (Thermo Fisher, Waltham, MA, USA) and gel electrophoresis were used to quantitatively and qualitatively evaluate the separated RNA. Subsequently, in accordance with the manufacturer’s instructions, 1 µg of RNA was reverse-transcribed into cDNA using HiScript II Q RT SuperMix for qPCR. PCR amplification products were quantified using PowerUp SYBR Green Master Mix in line with standard procedures (95 °C for 15 s, 60 °C for 30 s, 72 °C for 30 s; 40 cycles). Table 1 lists the primer pairs used in this study.

Table 1 The primer sequences of genes in RT-qPCR assays
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Western blot (WB) analysis and antibodies

We separated the total protein extract of the liver tissue or cells using lysis buffer and quantified it using bicinchonic acid protein analysis kit (KeyGEN, Nanjing, Jiangsu, China). The protein samples were denatured by heating at 100 °C for 10 min. The protein lysate was separated on 10% sodium dodecyl sulfate–polyacrylamide gel and transferred onto a polyvinylidene difluoride membrane. We first sealed the membrane with 5% bovine serum albumin in Tris-buffered saline/0.1% Tween-20 for 2 h, and then incubated it with the following specific antibodies at 4 °C for 12 h: ACTA2 (Cat# ab124964, Abcam, Cambridge, MA, USA), CASP3 (Cat# ab184787, Abcam, Cambridge, MA, USA), COL1A11 (Cat# ab270993, Abcam, Cambridge, MA, USA), DES (Cat# ab32362, Abcam, Cambridge, MA, USA), IL7 (Cat# A1650, ABclonal, Wuhan, Hubei, China), IL7R (Cat# A11678, ABclonal, Wuhan, Hubei, China), JAK1 (Cat# A5534, ABclonal, Wuhan, Hubei, China), MMP9 (Cat# ab76003, Abcam, Cambridge, MA, USA), STAT1 (Cat# 66545-1-Ig, Proteintech, Wuhan, Hubei, China), p-STAT1 (Cat# GB115605-100, Servicebio, Wuhan, Hubei, China), STAT3 (Cat# 60199-1-Ig, Proteintech, Wuhan, Hubei, China), p-STAT3 (Cat# 60479-1-Ig, Proteintech, Wuhan, Hubei, China), STAT5 (Cat# A5029, ABclonal, Wuhan, Hubei, China), p-STAT5 (Cat# AP0758, ABclonal, Wuhan, Hubei, China), TGFB1 (Cat# ab215715, Abcam, Cambridge, MA, USA), TIMP1 (Cat# sc-21734, Santa Cruz, CA, USA), and TUBA1A (Cat# ab7291, Abcam, Cambridge, MA, USA). The antibodies were diluted in line with the instructions. We captured the membrane as a digital image on the ChemiDoc immunoblot detection system (Bio-Rad, Hercules, CA, USA). ImageJ software (National Institutes of Health, SA, USA) was used to quantify the bands.

Serum measurement

The blood samples were centrifuged at 1500 rpm for 10 min to obtain serum, which was rapidly frozen for further analysis. Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin (TBil) were measured using an automated biochemical analyzer (Abbott Laboratories, USA).

Liver histopathology

After harvesting, the rat liver tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sliced into 5-µm-thick sections. The tissue sections were stained with hematoxylin–eosin (HE), Sirius red, and Masson trichrome dyes, and inflammation, fibrosis, and structural changes were observed under a Nikon optical microscope.

Statistical analysis

All statistical analyses in this study were conducted using GraphPad Prism 10.0, and unless otherwise specified, data are presented as mean ± standard deviation. We calculated the statistical difference between two groups using a two-sided Student t-test, and performed one-way analysis of variance for comparisons more than two groups. Statistical differences with P values less than 0.05 were considered significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Detailed statistical information for each experiment is provided in the corresponding legend. Unless otherwise specified in the legend, all in vitro experiments were conducted in triplicate.

Results

Extraction and identification of primary BMSCs from rats and their survival time in rat liver

As shown in Fig. 1A, we extracted primary BMSCs from the femur and tibia of 4-week-old rats and conducted culture and osteogenic and adipogenic induction experiments. Using the Alizarin Red staining kit to stain osteogenic induced cells, we found that after the osteogenic induction experiment, the cytoplasm of the cells mostly turned into light brown to dark brown cell particles or clumps, which indicated the color of calcium nodules combined with Alizarin Red dye, consistent with the characteristics of osteoblasts. Using the Oil Red O staining kit to stain adipogenic induced cells, we found that after the adipogenic induction experiment, multiple circular lipid droplets, stained orange red, appeared in the cytoplasm of the cells. This color indicated the combination of lipid droplets and Oil Red O dye. We collected primary cells from the femur and tibia of rats and conducted flow cytometry for phenotype identification. The surface molecules of this group of cells, compared with those of the negative control, were CD34- and CD45-negative, but CD90- and CD29-positive (Fig. 1B). In addition, the obtained cells were stained with CM-Dil dye and injected into the portal vein of rats in a quantity of 107. Frozen sections showed that the cells were able to survive in the rat liver for 7–14 days or more (Fig. 1C).

Fig. 1
figure 1

Extraction and identification of primary BMSCs from rats and their survival time in rat liver. (A) Cultivation and induced differentiation experiments of primary cells extracted from rat femur and tibia. (B) Phenotype identification of primary cells extracted from rat femur and tibia. (C) The in vivo survival time of cells injected through the portal vein of rats. BMSCs, bone mesenchymal stem cells

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BMSCs alleviate liver fibrosis in rats after LT

Figure 2A shows the flowchart of BMSCs alleviating fibrosis after LT in rats. In the HE staining, the livers of the LT group (CIT 4 h) showed greater hepatic lobular structural disorder, fibrous connective tissue proliferation, and inflammatory cell infiltration than those of the Sham group. Masson staining and Sirius Red staining showed more severe fibrosis, with a larger ACTA2–stained area and a higher Metavir fibrosis stage. In addition, the serum ALT and AST levels in the LT group increased, while there was no difference in TBil. In the HE staining, the livers of the LT + BMSC group showed less hepatic lobular structural disorder, fibrous connective tissue proliferation, and inflammatory cell infiltration than those of the LT group. Masson staining and Sirius Red staining showed lighter fibrosis, with a smaller ACTA2–stained area and a lower Metavir fibrosis stage. In addition, the serum ALT and AST levels were reduced in the LT + BMSC group, while there was no difference in TBil levels (Fig. 2B).

Fig. 2
figure 2

BMSCs alleviate liver fibrosis in rats after liver transplantation. (A) Flowchart of BMSCs alleviating fibrosis after LT in rats. (B) Microscopic images of each group stained with HE, Masson, Sirius Red, and ACTA2 (magnification: ×100, n = 6). Area stained with Masson, Sirius Red, and ACTA2, as well as Metavir fibrosis staging. The levels of serum ALT, AST, and TBil in each group. (C) Western blot analyses of COL1A1, MMP9, DES, TGFB1, ACTA2, and TIMP1 in liver tissues. TUBA1A was used as the loading control. Col1a1, Mmp9, Des, Tgfb1, Acta2, and Timp1 mRNA levels in liver tissues. Full-length blots/gels are presented in Supplementary Fig. 1. The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSCs, bone mesenchymal stem cells; COL1A1, collagen type I alpha 1 chain; DES, desmin; LT, liver transplantation; MMP9, matrix metalloproteinase 9; TGFB1, transforming growth factor beta 1; TIMP1, tissue inhibitor of metalloproteinase 1; TBil, total bilirubin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

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Total RNA and protein were extracted from the livers of the Sham group, LT group, and LT + BMSC group 28 days after surgery, and COL1A1, MMP9, DES, TGFB1, ACTA2, and TIMP1 were detected by RT-qPCR and WB. The results showed that, compared with the livers of Sham group, those of the LT group showed a significant increase in the RNA levels of Col1a1, Des, Tgfb1, Acta2, and Timp1, while Mmp9 was significantly reduced. The trend of protein changes was similar to that of RNA. Compared with the livers of the LT group, those of the LT + BMSC group showed a significant decrease in the RNA levels of Col1a1, Des, Tgfb1, Acta2, and Timp1, while Mmp9 significantly increased. The trend of protein changes was similar to that of RNA (Fig. 2C). Full-length blots/gels are presented in Supplementary Fig. 1. The samples were derived from the same experiment and processed in parallel.

These results demonstrate that extending the cold storage time can cause fibrosis after LT in rats, induce activation of HSCs, promote collagen formation, and reduce matrix degradation. BMSC can alleviate fibrosis after LT in rats and reverse the abovementioned process.

BMSCs alleviate activation of HSCs after hypoxia–reoxygenation and reduce apoptosis, but do not affect proliferation

Figure 3A shows a flowchart of co-culturing BMSCs with HSC-T6 cells after hypoxia–reoxygenation. Total RNA and protein were extracted from the HSC-T6 group, HSC-T6 + IR group, and HSC-T6 + IR + BMSC group, and COL1A1, MMP9, DES, TGFB1, ACTA2, and TIMP1 were detected by RT-qPCR and WB. The results showed that, compared with the HSC-T6 group, the HSC-T6 + IR group showed a significant increase in the RNA levels of Col1a1, Des, Tgfb1, Acta2, and Timp1, while Mmp9 significantly decreased. The trend of protein changes was similar to that of RNA. Compared with the HSC-T6 + IR group, the HSC-T6 + IR + BMSC group showed a significant reduction in the RNA levels of Col1a1, Des, Tgfb1, Acta2, and Timp1, while Mmp9 significantly increased. The trend of protein changes was similar to that of RNA (Fig. 3B). Full-length blots/gels are presented in Supplementary Fig. 2. The samples were derived from the same experiment and processed in parallel.

Fig. 3
figure 3

BMSCs alleviate the activation of HSCs after hypoxia–reoxygenation and reduce apoptosis, but do not affect their proliferation. (A) Flowchart of BMSCs co-culturing with HSC-T6 cells after hypoxia–reoxygenation. (B) Western blot analyses of COL1A1, MMP9, DES, TGFB1, ACTA2, and TIMP1 in HSC-T6 cells. TUBA1A was used as the loading control. Col1a1, Mmp9, Des, Tgfb1, Acta2, and Timp1 mRNA levels in HSC-T6 cells. (C) Microscopic images of each group stained with EDU staining (magnification: ×200, n = 3). (D) Apoptosis results of flow cytometry in each group. Cell cycle results of flow cytometry in each group. Full-length blots/gels are presented in Supplementary Fig. 2. The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; BMSCs, bone mesenchymal stem cells; COL1A1, collagen type I alpha 1 chain; DES, desmin; EDU, 5-Ethynyl-2’-deoxyuridine; HSCs, hepatic stellate cells; MMP9, matrix metalloproteinase 9; TGFB1, transforming growth factor beta 1; TIMP1, tissue inhibitor of metalloproteinase 1. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

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The EDU staining experiment results showed that BMSCs did not affect the proliferation of HSC-T6 cells after hypoxia–reoxygenation (Fig. 3C). The results of the flow-cytometry apoptosis experiment showed that, compared with the HSC-T6 group, the HSC-T6 + IR group showed a significant increase in the proportion of apoptotic cells. Compared with the HSC-T6 + IR group, the HSC-T6 + IR + BMSC group showed a significant decrease in the proportion of apoptotic cells. The results of flow cytometry cell cycle experiments showed that BMSCs did not affect the proportion of S-phase cells among HSC-T6 cells after hypoxia–reoxygenation (Fig. 3D).

These results indicate that hypoxia–reoxygenation activates HSC-T6 cells and affects their apoptosis, while BMSCs alleviate the activation and apoptosis of HSC-T6 cells after hypoxia-induced enrichment, without affecting their proliferation.

BMSCs May alleviate liver fibrosis after LT through the JAK1/STAT5 pathway

To investigate the molecular mechanism of BMSC involvement in inhibiting liver fibrosis after LT, RNA sequencing was performed on normal liver (Group A), rat liver with fibrosis after LT (Group B), and BMSCs-treated liver after LT (Group C) to analyze differential genes and pathways. As shown in Fig. 4A, principal component analysis (PCA) clustering was significant among the three groups. Compared with Group A, Group B had 645 upregulated and 349 downregulated genes. Compared with Group B, Group C had 224 upregulated and 346 downregulated genes. The Venn graph displays the interrelationships between the three sets of DEGs. Overall, the expression pattern of RNA in the livers after BMSC treatment was different from that in the livers without BMSC treatment. GO and KEGG analyses were conducted to predict the potential functions of these RNAs. KEGG enrichment analysis showed that differentially expressed RNAs were mainly involved in the IL7, IL7R, and JAK/STAT signaling pathways.

Fig. 4
figure 4

BMSCs may alleviate liver fibrosis after liver transplantation through the JAK1/STAT5 pathway. (A) Box plot for FPKM values. PCA analysis. Bar plot of the statistic of differentially expressed genes. Venn graph of the differentially expressed genes. Volcano plot of the differentially expressed genes. Heat map of RNA differential expression. GO analysis reveals the potential impact of the differentially expressed RNAs on functions. KEGG analysis reveals the potential impact of the differentially expressed RNA on pathways. KEGG map of the JAK/STAT pathway. (B) Western blot analyses of JAK1, p-STAT5, STAT5, p-STAT1, STAT1, p-STAT3, STAT3, IL7R, and IL7 in liver tissue. TUBA1A was used as the loading control except for p-STAT5, p-STAT1, and p-STAT3. STAT5, STAT1, and STAT3 were used as the loading control for p-STAT5, p-STAT1, and p-STAT3, respectively. Jak1, Il7r, and Il7 mRNA levels in liver tissue. (C) Microscopic images of each group stained with HE, Masson, Sirius Red, and ACTA2 (magnification: ×100, n = 6). Area stained with Masson, Sirius Red, and ACTA2, as well as Metavir fibrosis staging. The levels of serum ALT, AST, and TBil in each group. (D) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5, ACTA2, and CASP3 in liver tissues. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Full-length blots/gels are presented in Supplementary Fig. 3. The samples were derived from the same experiment and processed in parallel. Col1a1, Jak1, Acta2, and Casp3 mRNA levels in liver tissues. ACTA2, actin alpha 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSCs, bone mesenchymal stem cells; CASP3, caspase 3; COL1A1, collagen type I alpha 1 chain; GO, Gene Ontology; IL, interleukin; JAK, Janus-activated kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; LT, liver transplantation; STAT, signal transducer and activator of transcription; TBil, total bilirubin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

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Through RT-qPCR and WB, we showed an increase in JAK1 expression and STAT5 phosphorylation levels in liver fibrosis, but the STAT1 and STAT3 phosphorylation levels were not changed (Fig. 4B). To verify that BMSCs alleviate post-LT liver fibrosis in rats through the JAK1/STAT5 pathway, we administered the JAK1 inhibitor Abrocitinib orally to the rats after LT. Compared with the livers of the LT group, those of the LT + Abrocitinib group showed reduced hepatic lobular structural disorder, fibrous connective tissue proliferation, and inflammatory cell infiltration in the HE staining. Masson staining and Sirius Red staining showed lighter fibrosis, with a smaller ACTA2–stained area and a lower Metavir fibrosis stage. In addition, the serum ALT and AST levels were reduced in the LT + Abrocitinib group, while there was no difference in TBil levels (Fig. 4C).

Total RNA and protein were extracted from the livers of the Sham group, LT group, and LT + Abrocitinib group 28 days after surgery. The results showed that, compared with the livers of the LT group, those of the LT + Abrocitinib group showed a significant decrease in the RNA levels of Col1a1, Jak1, Acta2, and Casp3. The protein levels of COL1A1, JAK1, ACTA2, and CASP3 were significantly reduced in the LT + Abrocitinib group, and the phosphorylation level of STAT5 was also reduced (Fig. 4D). Full-length blots/gels are presented in Supplementary Fig. 3. The samples were derived from the same experiment and processed in parallel.

These results indicate that BMSCs may alleviate postoperative fibrosis in rats after LT by inhibiting the JAK1/STAT5 pathway.

BMSCs may inhibit the activation of HSC-T6 cells after hypoxia–reoxygenation and reduce their apoptosis through the JAK1/STAT5 pathway

Through RT-qPCR and WB, we showed that after HSC-T6 cell hypoxia–reoxygenation, JAK1, and IL7R expression levels increased, STAT5 phosphorylation levels increased, and STAT1 and STAT3 phosphorylation levels were not changed (Fig. 5A). To verify that BMSCs alleviate the activation after HSC-T6 cell hypoxia–reoxygenation through the JAK1/STAT5 pathway, we added the JAK1 inhibitor Abrocitinib after HSC-T6 cell hypoxia–reoxygenation. The results showed that, compared with the HSC-T6 + IR group, the HSC-T6 + Abrocitinib group demonstrated a significant decrease in the RNA levels of Col1a1, Jak1 and Acta2. The protein levels of COL1A1, JAK1, and ACTA2 were significantly reduced in the HSC-T6 + Abrocitinib group, and the phosphorylation level of STAT5 was also reduced (Fig. 5B). Compared with the HSC-T6 + IR group, the HSC-T6 + IR + Abrocitinib group showed a significant decrease in the proportion of apoptotic cells (Fig. 5C). Full-length blots/gels are presented in Supplementary Fig. 4. The samples were derived from the same experiment and processed in parallel.

Fig. 5
figure 5

BMSCs may inhibit the activation of HSC-T6 cells after hypoxia–reoxygenation and reduce their apoptosis through the JAK1/STAT5 pathway. (A) Western blot analyses of JAK1, p-STAT5, STAT5, p-STAT1, STAT1, p-STAT3, STAT3, IL7R, and IL7 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5, p-STAT1, and p-STAT3. STAT5, STAT1, and STAT3 were used as the loading control for p-STAT5, p-STAT1, and p-STAT3, respectively. Jak1 and Il7r mRNA levels in HSC-T6 cells. (B) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5, and ACTA2 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1, Jak1 and Acta2 mRNA levels in HSC- T6 cells. (C) Apoptosis results of flow cytometry in each group. Full-length blots/gels are presented in Supplementary Fig. 4. The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; BMSCs, bone mesenchymal stem cells; COL1A1, collagen type I alpha 1 chain; IL, interleukin; JAK, Janus-activated kinase; STAT, signal transducer and activator of transcription. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Full size image

These results indicate that BMSCs may alleviate the activation and reduce apoptosis of HSC-T6 cells after hypoxia–reoxygenation by inhibiting the JAK1/STAT5 pathway.

The JAK1 agonist RO8191 reverses the function of BMSCs in alleviating liver fibrosis and attenuating HSCs activation

We administered the JAK1 agonist RO8191 orally to the rats after LT. Compared with the livers of the LT + BMSC group, those of the LT + BMSC + RO8191 group showed high hepatic lobular structural disorder, fibrous connective tissue proliferation, and inflammatory cell infiltration in the HE staining. Masson staining and Sirius Red staining showed more severe fibrosis, with a larger ACTA2–stained area and a higher Metavir fibrosis stage. In addition, the serum ALT and AST levels increased in the LT + BMSC + RO8191 group, while there was no difference in TBil levels (Fig. 6A).

Fig. 6
figure 6

The JAK1 agonist RO8191 reverses the function of BMSCs in alleviating liver fibrosis and attenuating HSCs activation. (A) Microscopic images of each group stained with HE, Masson, Sirius Red, and ACTA2 (magnification: ×100, n = 6). Area stained with Masson, Sirius Red, and ACTA2, as well as Metavir fibrosis staging. The levels of serum ALT, AST, and TBil in each group. (B) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5, ACTA2, and CASP3 in liver tissues. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1, Jak1, Acta2, and Casp3 mRNA levels in liver tissues. (C) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5, ACTA2, and CASP3 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1, Jak1, Acta2, and Casp3 mRNA levels in HSC-T6 cells. (D) Apoptosis results of flow cytometry in each group. Full-length blots/gels are presented in Supplementary Fig. 5. The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSCs, bone mesenchymal stem cells; CASP3, caspase 3; COL1A1, collagen type I alpha 1 chain; IL, interleukin; JAK, Janus-activated kinase; LT, liver transplantation; STAT, signal transducer and activator of transcription; TBil, total bilirubin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Full size image

Total RNA and protein were extracted from the livers of the Sham group, LT group, LT + BMSC group, and LT + BMSC + RO8191 group 28 days after surgery. The results showed that, compared with the livers of the LT + BMSC group, those of the LT + BMSC + RO819 group showed a significant increase in the RNA levels of Col1a1, Jak1, Acta2 and Casp3. The protein levels of COL1A1, JAK1, ACTA2, and CASP3 significantly increased in the LT + BMSC + RO8191 group, and the phosphorylation level of STAT5 also increased (Fig. 6B).

We added the JAK1 agonist RO8191 after co-culturing BMSCs with HSC-T6 cells. The results showed that, compared with the HSC-T6 + IR + BMSC group, the HSC-T6 + IR + BMSC + RO8191 group showed a significant increase in the RNA levels of Col1a1, Jak1, Acta2 and Casp3. The protein levels of COL1A1, JAK1, ACTA2, and CASP3 significantly increased in the HSC-T6 + IR + BMSC + RO8191 group, and the phosphorylation level of STAT5 also increased (Fig. 6C). Compared with the HSC-T6 + IR + BMSC group, the HSC-T6 + IR + BMSC + RO8191 group showed a significant increase in the proportion of apoptotic cells (Fig. 6D). Full-length blots/gels are presented in Supplementary Fig. 5. The samples were derived from the same experiment and processed in parallel.

These results indicate that the JAK1 agonist RO8191 reverses the function of BMSCs in alleviating liver fibrosis and attenuating HSCs activation.

BMSCs regulate the IL7R/JAK1/STAT5 pathway in HSCs by modulating the secretion of IL7 by hepatic cells

We administered the anti-IL7 antibody and anti-IL7Rα to the rats through intraperitoneal injection after LT. Compared with the livers of the LT group, those of the LT + anti-IL7 group or LT + anti-IL7Rα group showed reduced hepatic lobular structural disorder, fibrous connective tissue proliferation, and inflammatory cell infiltration in the HE staining. Masson staining and Sirius Red staining showed lighter fibrosis, with a smaller ACTA2–stained area, lower p-STAT5/STAT5 ratio and a lower Metavir fibrosis stage. In addition, the serum ALT and AST levels were reduced in the LT + anti-IL7 group or LT + anti-IL7Rα group, while there was no difference in TBil levels (Fig. 7A).

Fig. 7
figure 7

BMSCs regulate the IL7R/JAK1/STAT5 pathway in HSCs by modulating the secretion of IL7 by hepatic cells. (A) Microscopic images of each group stained with HE, Masson, Sirius Red, ACTA2, p-STAT5 and STAT5 (magnification: ×100, n = 6). Area stained with Masson, Sirius Red, ACTA2, and p-STAT5/STAT5, as well as Metavir fibrosis staging. The levels of serum ALT, AST, and TBil in each group. (B) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5 and ACTA2 in liver tissue. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1, Jak1, and Acta2 mRNA levels in liver tissue. (C) Western blot analyses of IL7 in IAR20 cells. TUBA1A was used as the loading control. Il7 mRNA levels in IAR20 cells. (D) Western blot analyses of COL1A1, JAK1, p-STAT5, STAT5 and ACTA2 in HSC-T6 cells. TUBA1A was used as the loading control except for p-STAT5. STAT5 was used as the loading control for p-STAT5. Col1a1, Jak1 and Acta2 mRNA levels in HSC- T6 cells. Full-length blots/gels are presented in Supplementary Fig. 6. The samples were derived from the same experiment and processed in parallel. ACTA2, actin alpha 2; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BMSCs, bone mesenchymal stem cells; COL1A1, collagen type I alpha 1 chain; IL, interleukin; JAK, Janus-activated kinase; LT, liver transplantation; STAT, signal transducer and activator of transcription; TBil, total bilirubin. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Full size image

Total RNA and protein were extracted from the livers of the LT group, LT + anti-IL7 group and LT + anti-IL7Rα group 28days after surgery. The results showed that, compared with the livers of the LT group, those of the LT + anti-IL7 group or LT + anti-IL7Rα group showed a significant decrease in the RNA levels of Col1a1, Jak1 and Acta2. The protein levels of COL1A1, JAK1, and ACTA2 were significantly reduced in the LT + anti-IL7 group or LT + anti-IL7Rα group, and the phosphorylation level of STAT5 was also reduced (Fig. 7B).

Total RNA and protein were extracted from the IAR20 group, IAR20 + IR group, and IAR20 + IR + BMSC group, and IL7 was detected by RT-qPCR and WB. The results showed that, compared with the IAR20 group, the IAR20 + IR group showed a significant increase in the RNA levels of Il7. The trend of protein changes was similar to that of RNA. Compared with the IAR20 + IR group, the IAR20 + IR + BMSC group showed a significant reduction in the RNA levels of Il7. The trend of protein changes was similar to that of RNA (Fig. 7C).

Total RNA and protein were extracted from the livers of the HSC-T6 + IAR20 + IR group, HSC-T6 + IAR20 + IR + anti-IL7 group and HSC-T6 + IAR20 + IR + anti-IL7Rα group 28days after surgery. The results showed that, compared with the HSC-T6 + IAR20 + IR group, those of the HSC-T6 + IAR20 + IR + anti-IL7 group or HSC-T6 + IAR20 + IR + anti-IL7Rα group showed a significant decrease in the RNA levels of Col1a1, Jak1 and Acta2. The protein levels of COL1A1, JAK1, and ACTA2 were significantly reduced in the HSC-T6 + IAR20 + IR + anti-IL7 group or HSC-T6 + IAR20 + IR + anti-IL7Rα group, and the phosphorylation level of STAT5 was also reduced (Fig. 7D). Full-length blots/gels are presented in Supplementary Fig. 6. The samples were derived from the same experiment and processed in parallel.

These results indicate that BMSCs regulate the IL7R/JAK1/STAT5 pathway in HSCs by modulating the secretion of IL7 by hepatic cells.

Discussion

During the process of liver transplantation, organ recovery, low-temperature preservation, and opening of blood flow to the donor liver inevitably result in hot ischemia-reperfusion injury, cold ischemia-reperfusion injury, and warm ischemia-reperfusion injury. These ischemia-reperfusion injuries can all lead to impaired graft function, acute and chronic rejection reactions, and liver fibrosis. In fact, ischemia-reperfusion injury can lead to a shortage of available donor organs, which is one of the most challenging issues in transplantation [27,28,29,30]. Among them, CIT and cold ischemia-reperfusion injury during liver transplantation are of great concern. Studies have shown that liver fibrosis one year after pediatric liver transplantation is associated with prolonged CIT [31]. At the same time, liver fibrosis 10 years after pediatric liver transplantation is associated with prolonged CIT, younger age at transplantation, older donor/recipient age, and the use of partial transplants, but not significantly correlated with rejection, chronic hepatitis, or immunosuppressive treatment regimens [32]. Even the occurrence and development of advanced liver fibrosis are associated with CIT during early liver transplantation [13, 32, 33]. Therefore, there is an urgent need for new treatment methods in clinical practice to address cold ischemia-reperfusion injury, improve liver transplant outcomes, and expand the donor organ pool. However, the mechanisms of fibrosis/cirrhosis and chronic graft dysfunction mediated by CIT and cold ischemia-reperfusion injury remain to be determined, and there is an urgent need to find a method that can alleviate liver fibrosis after liver transplantation.

Since Kamada proposed the “double sleeve method” for rat liver transplantation in 1979 [34], the duration of the anhepatic phase of rat liver transplantation has been greatly shortened, and the survival rate of rats after surgery has been significantly improved. In traditional research on liver fibrosis, drugs such as thioacetamide or CCl4 are commonly used, or physical methods such as bile duct ligation are used to induce bile stasis and liver fibrosis [35,36,37]. However, the causes, severity, and intervention methods of liver fibrosis caused by these methods are significantly different from those of liver fibrosis after liver transplantation. At present, research has begun to focus on the special disease of liver fibrosis after liver transplantation, and multiple center studies have found that CIT is an important risk factor affecting liver fibrosis after liver transplantation [13, 32, 33, 38]. However, in the establishment of animal models, in situ clamping models are still mostly used to construct short-term liver hot ischemia-reperfusion injury models [38], and long-term observation of the degree of liver fibrosis cannot be carried out. Obviously, the hot ischemia-reperfusion injury model cannot simulate the liver immune microenvironment after liver transplantation, which is precisely the key link. Using liver transplantation models to study liver fibrosis after liver transplantation is necessary to obtain more accurate conclusions.

The number of BMSCs injected into the portal vein of rats and the survival time of BMSCs in the rat body are the primary issues we considered. In other non-LT liver fibrosis models, studies have used single-dose portal vein injection regimens of 2 × 106, 4 × 106, or 1 × 107 BMSCs, as well as double-dose injection regimens [39, 40], but none of the available reports have tracked the injected BMSCs. After entering the rat body, BMSCs quickly circulate throughout the body, with the main early sites of residence being the lungs and the spleen. Afterward, most BMSCs are cleared. Due to the homing property of BMSCs, uncleared BMSCs tend to accumulate in the liver [41]. Our study demonstrates that after a single portal vein injection of 5 × 106 BMSCs, a large number of BMSCs still exist in the liver on the 14th day after surgery, which means a single portal vein injection of 5 × 106 BMSCs after LT in rats can meet the treatment effect of postoperative liver fibrosis.

In this study, we first demonstrated that extending the CIT can cause liver fibrosis after liver transplantation in rats. After postoperative injection of 5 × 106 BMSCs through the portal vein, liver fibrosis caused by CIT after liver transplantation can be significantly alleviated. As the central role in the occurrence and development of liver fibrosis, HSCs are significantly enhanced in activation after hypoxia-reoxygenation, while their activation is significantly weakened when co-cultured with BMSCs. Afterwards, we performed transcriptomic sequencing on the livers of normal rats, rats with liver fibrosis after liver transplantation, and rats injected with BMSCs via portal vein. We found that the JAK/STAT pathway was significantly upregulated in the liver fibrosis group, but significantly downregulated after intervention with BMSCs. Through further experiments, we further confirmed the role of the JAK1/STAT5 pathway in liver fibrosis after liver transplantation and the intervention of BMSCs in liver fibrosis. We concluded that BMSCs downregulate the activation of the JAK1/STAT5 pathway in HSCs, thereby reducing HSCs activation and alleviating liver fibrosis after liver transplantation.

Whether BMSCs alleviate the activation of HSCs directly or indirectly is a question that we need to further explore. Through the above transcriptome sequencing, we found that while the JAK/STAT pathway was altered, the upstream cytokines IL7 and cytokine receptor IL7R were also altered. IL7 is mainly secreted by hepatic cells in the liver and plays a key role in regulating liver damage, inflammation, fibrosis, and regeneration. The occurrence and development of liver fibrosis are controlled by various factors, including liver injury, inflammatory cells, inflammatory mediators, and cytokines. In addition to some classic cytokines such as interferon, IL1 family, IL6 family, and IL20 family cytokines that play important roles in regulating liver injury, inflammation, fibrosis, and regeneration, hepatic cells can also produce certain cytokines on their own, such as IL7, IL11, and IL33 [42]. Hepatic cells are the main parenchymal cells in the liver, producing various innate immune proteins and play a critical role in innate immunity [43]. IL7 plays an important role in controlling liver damage, repair, and inflammation in liver diseases [44]. IL7R consists of two chains, the IL7R α-chain (IL7Rα; also known as CD127) and the common cytokine-receptor γ-chain (IL7Rγ; also known as CD132), the intracellular domains of which are bound to the tyrosine kinases JAK1 and JAK3, respectively [45]. IL7R plays an important role in intracellular signaling processes. When IL7 binds to IL7R, it triggers phosphorylation of JAK1 and JAK3, which in turn activates downstream signaling pathways such as STAT5, thereby regulating gene expression and cellular function [45]. Studies have shown that BMSCs can alleviate acute or chronic liver inflammation and subsequent liver cell damage by regulating immune cell function, creating a liver protective environment through paracrine and/or intercellular interactions [46,47,48,49].

Through the analysis of liver fibrosis rats and BMSCs intervention after liver transplantation, as well as experiments on hypoxia-reoxygenation of normal rat hepatic cells and co-culture with BMSCs, we found that BMSCs can significantly reduce IL7 secretion after hepatic cells’ injury both in vivo and in vitro. By intervening with anti-IL7 and anti-IL7Rα in both in vivo and in vitro experiments, we further demonstrated that BMSCs downregulate the IL7R/JAK1/STAT5 pathway in HSCs by reducing hepatic cell secretion of IL7, ultimately alleviating liver fibrosis.

This study confirmed both in vivo and in vitro that BMSCs alleviate HSCs activation by inhibiting the IL7/IL7R/JAK1/STAT5 pathway, thereby reducing fibrosis after LT. Obviously, this study is only a preliminary observation and exploration of the mechanism by which BMSCs alleviate liver fibrosis. There are still many shortcomings in this study. Firstly, it is still unknown how BMSCs alleviate the secretion of IL7 by hepatic cells, such as through direct contact or secretion of extracellular vesicles. Secondly, this study only provided a simple exclusion proof for the role of other STATs, and its rigor needs further investigation. Thirdly, a comprehensive exploration of the effects of BMSCs on the genome, transcriptome, proteome, and other metabolic processes, such as the impact on natural killer cells, neutrophils, or other immune cell subpopulations, is worth further exploration.

Conclusions

This study focused on the role of BMSCs in alleviating liver fibrosis and HSCs activation after rat LT, and specifically investigated the role of the IL7/IL7R/JAK1/STAT5 pathway in this process. For the first time, we demonstrated that BMSCs alleviate hepatic cell damage, reduce hepatic cell-derived IL7, downregulate IL7R/JAK1/STAT5 in HSCs, thereby reducing HSCs’ activation and ultimately alleviating liver fibrosis after liver transplantation. This means that BMSCs are a potential means for treating liver fibrosis after LT, laying an experimental foundation for future exploration of BMSCs as an anti-fibrosis method in clinical applications.

Data availability

All additional files are included in the manuscript.

Abbreviations

ACTA2:

Actin alpha 2

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

BMSCs:

Bone marrow mesenchymal stem cells

CCl4 :

Carbon tetrachloride

COL1A1:

Collagen type I alpha 1 chain

CIT:

Cold ischemia time

DES:

Desmin

EDU:

5-ethynyl-2’-deoxyuridine

GO:

Gene ontology

HE:

Hematoxylin–eosin

HSCs:

Hepatic stellate cells

IL:

Interleukin

JAK:

Janus-activated kinase

KEGG:

Kyoto encyclopedia of genes and genomes

LT:

Liver transplantation

MMP9:

Matrix metalloproteinase 9

MSCs:

Marrow mesenchymal stem cells

PCA:

Principal component analysis

pLT:

Pediatric liver transplantation

RT-qPCR:

Quantitative real-time polymerase chain reaction

SD:

Sprague–dawley

STAT:

Signal transducer and activator of transcription

TBil:

Total bilirubin

TGFB1:

Transforming growth factor beta 1

TIMP1:

Tissue inhibitor of metalloproteinase 1

TUBA1A:

Tubulin alpha 1a

WB:

Western blot

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Acknowledgements

We thank LetPub (www.letpub.com.cn) for its linguistic assistance during the preparation of this manuscript. We thank the Shanghai OE Biological Co., Ltd. for providing technical support for transcriptome sequencing. The authors declare that they have not use AI-generated work in this manuscript.

Funding

This study is funded by the Tianjin Health Research Project (grant number TJWJ2022XK017) and National Natural Science Foundation of China (grant number: 82170672).

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  1. Zhuyuan Si, Shengqiao Zhao and Zhixin Zhang contributed equally to this work.

Authors and Affiliations

  1. Department of Organ Transplantation, Qilu Hospital of Shandong University, Jinan, China

    Zhuyuan Si

  2. Department of Liver Transplantation, Organ Transplantation Center, Tianjin First Central Hospital, Tianjin, China

    Zhuyuan Si, Shengqiao Zhao, Zhixin Zhang, Tianran Chen, Ruofan Wang, Chong Dong, Kai Wang, Chao Sun, Zhuolun Song, Zhongyang Shen & Wei Gao

  3. Tianjin Key Laboratory of Organ Transplantation, Tianjin First Central Hospital, Tianjin, China

    Zhuolun Song, Zhongyang Shen & Wei Gao

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Contributions

Wei Gao and Zhongyang Shen contributed to conception and design of the work; Zhuyuan Si, Shengqiao Zhao, Zhixin Zhang, Tianran Chen, Ruofan Wang, Chong Dong, Kai Wang, Chao Sun and Zhuolun Song contributed to acquiring data, drafting and approving the final content of the manuscript. Wei Gao, Zhuolun Song and Zhongyang Shen contributed to critical revision of the article.

Corresponding authors

Correspondence to Zhongyang Shen or Wei Gao.

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(1) Title of the approved project: The role and mechanism of marrow mesenchymal stem cells in liver fibrosis after pediatric liver transplantation; (2) Name of the institutional approval committee or unit: The Experimental Animal Ethics Committee of Tianjin First Central Hospital; (3) Approval number: 2022N140KY; (4) Date of approval: 2022/07/01.

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Si, Z., Zhao, S., Zhang, Z. et al. Bone marrow mesenchymal stem cells alleviate liver fibrosis after rat liver transplantation through JAK1/STAT5 pathway. Stem Cell Res Ther 16, 217 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04353-y

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512