Abstract

Background

   Liver fibrosis is a common pathological process of chronic liver
   disease, characterized by excessive deposition of extracellular matrix
   (ECM). Mesenchymal stem cells (MSCs) have been found to have potential
   therapy effect on liver fibrosis, but the mechanism involved was still
   unclear. The objective of this study is to investigate the therapeutic
   efficacy of adipose-derived mesenchymal stem cells (ADMSCs) on the
   treatment of liver fibrosis, with particular emphasis on elucidating
   the underlying mechanism of action through which ADMSCs inhibit the
   activation of hepatic stellate cells (HSCs).

Methods

   ADMSCs were isolated from adipose tissue and injected intravenously
   into hepatic fibrosis model of rats. The histopathological changes,
   liver function, collagen deposition, the expression of fibroin and
   Hippo pathway were evaluated. In vitro, ADMSCs were co-cultured with
   HSCs activated by transforming growth factor beta [1] (TGF-β[1]), and
   the inhibitor of Hippo pathway was used to evaluate the therapeutic
   mechanism of ADMSCs transplantation.

Results

   The results showed that after the transplantation of ADMSCs, the liver
   function of rats was improved, the degree of liver fibrosis and
   collagen deposition were reduced, and the Hippo signaling pathway was
   activated. In vitro, ADMSCs can effectively inhibit the proliferation
   and activation of HSCs induced by TGF-β[1] treatment. However, the
   inhibitory effect of ADMSCs was weakened after blocking the Hippo
   signaling pathway.

Conclusions

   ADMSCs inhibit HSCs activation by regulating YAP/TAZ, thereby promoting
   functional recovery after liver fibrosis. These findings lay a
   foundation for further investigation into the precise mechanism by
   which ADMSCs alleviate liver fibrosis.

   Keywords: Mesenchymal stem cells, Therapeutics, Hepatic stellate cells,
   Liver fibrosis, Hippo Pathway

Background

   Chronic liver diseases pose a significant challenge to global health,
   with an estimated 2 million annual deaths worldwide [[44]1]. The
   etiology of chronic liver disease encompasses diverse factors, such as
   viral infections (such as HBV: Hepatitis B, HCV: Hepatitis C),
   alcoholic or non-alcoholic steatohepatitis, autoimmune disorders, and
   genetic conditions [[45]2]. Liver fibrosis represents a pathological
   alteration that occurs in the majority of chronic liver diseases
   characterized by an excessive deposition and abnormal distribution of
   extracellular matrix [[46]3]. Without timely treatment, liver fibrosis
   may progress to cirrhosis and even liver cancer [[47]4]. The activation
   of hepatic stellate cells (HSCs) by fibrogenic mediators and cytokines
   is a crucial aspect of the liver fibrosis process [[48]5, [49]6]. This
   activation results in the increased expression of α-SMA and collagen,
   which consequently culminates in the accumulation of extracellular
   matrix and the subsequent formation of fibrosis [[50]7]. Therefore, the
   prevention of HSCs activation is of great significance in mitigating
   liver fibrosis, and inhibiting HSCs activation is considered as an
   appropriate strategy for antifibrosis therapy [[51]8, [52]9].

   Mesenchymal stem cells (MSCs), a type of multipotent stem cells, which
   can be obtained from various sources such as bone marrow, adipose
   tissue, umbilical cord, and other tissues [[53]10, [54]11]. Among these
   sources, adipose-derived mesenchymal stem cells (ADMSCs) have attracted
   considerable attention due to the abundance of their tissue sources and
   the ease with which they can be collected and isolated [[55]12,
   [56]13]. In recent years, there has been a growing body of evidence to
   suggest that stem cell transplantation therapy has the potential to be
   an effective treatment for a number of diseases and to repair tissue
   damage [[57]14, [58]15]. In the context of liver diseases, multiple
   animal studies have demonstrated the ability of MSCs to mitigate liver
   fibrosis and restore liver function [[59]16–[60]18]. Consequently,
   ongoing pre-clinical and clinical trials are being conducted to
   ascertain the therapeutic efficacy of MSCs-based therapy in liver
   diseases [[61]19].

   The primary role of the Hippo signaling pathway is to constrain tissue
   growth, meanwhile, it also governs cellular proliferation,
   differentiation, and migration within organs [[62]20]. The Hippo
   signaling pathway has been recognized as a pivotal regulatory pathway
   for the activation of HSCs in cases of acute liver injury [[63]21].
   Yes-associated protein (YAP)/ PDZ-binding motif (TAZ) represents the
   most significant effector within the Hippo pathway. A recent study has
   demonstrated that the expression of YAP in rat hepatocytes could result
   in the initiation of inflammatory responses, expansion of
   myofibroblasts, and the development of fibrosis in a rat liver fibrosis
   model induced by CCl[4] [[64]22]. It has also been reported that the
   expression of TAZ in hepatocytes is elevated in liver fibrosis caused
   by Nonalcoholic steatohepatitis (NASH) [[65]23]. Furthermore, the
   suppression of YAP/ TAZ expression in hepatocytes has been shown to
   alleviate CCl4-induced liver fibrosis [[66]24]. However, the precise
   involvement of the Hippo pathway in the mitigation of liver fibrosis by
   ADMSCs remains to be fully elucidated [[67]25]. Therefore, in this
   study, the regulatory effect of Hippo pathway in the treatment of liver
   fibrosis with ADMSCs was examined.

Materials and methods

ADMSCs isolation and characterization

   Adipose tissue was extracted from the abdominal wall and groin of
   Sprague-Dawley (SD) rats and subsequently rinsed with
   phosphate-buffered saline (PBS). The adipose tissue was then
   enzymatically digested to obtain cells with collagenase 1. All isolated
   cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
   supplemented with 10% fetal bovine serum (TransGen Biotech, Beijing,
   China) and 1% penicillin/streptomycin, and maintained in a CO[2]
   incubator with 5% humidity at 37 °C. The cells were subcultured when
   they reached approximately 70–80% confluency. The identification of
   MSCs was based on their adhesive properties and morphological
   characteristics, such as their elongated and spindle-like appearance or
   fibroblastic shapes, which were observed with an inverted microscope.
   MSCs were analyzed using flow cytometry and characterized based on the
   expression of Cluster of differentiation (CD)34, CD45, and CD105
   (TransGen Biotech).

Animals

   Male Sprague Dawley rats, aged 4 weeks, weighing 200–250 g, were from
   Chengdu Dashuo Company. The rats, fed in an environment with controlled
   temperature (22 ± 1 °C) and humidity (60 ± 10%) and 12-hour light /
   12-hour dark cycle, were given water and regular rat food. In this
   study, a total of 30 SD rats were used, among which 6 rats were
   randomly selected by random number generator for ADMSCs isolation, and
   the remaining 24 rats were used for the establishment of CCl[4]-induced
   liver fibrosis model. The weight, appearance, and behavior of each
   group of rats were monitored throughout the experiment. Our reporting
   of animal experiments adheres to the ARRIVE guidelines 2.0.

Animal model of liver fibrosis and transplantation of MSCs

   The rats were randomly divided into blank group (n = 6), CCl[4]
   group(n = 6), treatment control group (n = 6) and ADMSCs treatment
   group (n = 6) with a random number generator. Liver fibrosis was
   induced in the rats by administering CCl[4]/olive oil (Sangon,
   Shanghai, China) (ratio 4:6; 2 mL/kg intraperitoneally) twice per week
   for a duration of one month. The treatment control group received an
   equivalent volume of PBS.

   The ADMSCs were collected when passaged up to the third generation.
   Following cell counting, it was ensured that 1.0 × 10^6 cells were
   suspended in 0.5mL of PBS and intravenously administered to each rat
   using a 1mL injector on day 29. One week after ADMSCs treatment,
   anesthetized with pentobarbital sodium, the rats’ blood was collected
   from orbital vessels immediately after the resection of eyeballs for
   the analysis of liver function indicators. Subsequently, the rats were
   euthanized by cervical dislocation, and liver samples were collected.
   Tissue samples were frozen in liquid nitrogen and stored at -80 °C for
   subsequent experiments.

Detection of liver function biomarkers

   The primary indicators for evaluating the healing process, encompassing
   manifestations of disease and changes of body weight (with weekly
   weighing of the animals), were closely monitored. Furthermore, the
   overall condition of the liver was evaluated after euthanasia. The
   concentrations of alanine transaminase (ALT), aspartate
   aminotransferase (AST) and total bilirubin (TBIL) were measured by
   commercial reagent kits (C009-3-1, C010-3-1, E003-1-1, Nanjing
   Jiancheng Bioengineering Institut, China).

Histopathological and immunohistochemical studies

   The liver tissue was fixed in 10% paraformaldehyde solution, and then
   embedded in paraffin wax. Subsequently, sections were stained with
   Hematoxylin and eosin (H&E) and Masson’s Trichrome Staining to show
   histological features and collagen fiber deposition. In addition,
   immunohistochemistry (IHC) staining was performed with an antibody
   against α-SMA (cat No. A1011) and YAP (cat No. A21216, Abclone, Wuhan,
   China). The brown labeled area in immunohistochemical sections was
   analyzed using ImageJ software.

HSCs culture and treatment

   The human HSCs line LX-2 cells (YBIO, Shanghai, China) were obtained
   from the Cell Center of Shanghai Institutes for Biological Sciences,
   China. The cells were cultured in high glucose DMEM medium containing
   1% penicillin/streptomycin and 10% Fetal Bovine Serum (FBS). In order
   to induce activation, LX-2 cells were treated with 10 ng/mL of
   TGF-β[1](cat No. CH088-100HP, Chamot Biotechnology, Shanghan, China)
   for 24 h. Additionally, a group of cells was treated with 5 µM
   XMU-MP-1(a Hippo pathway kinase inhibitor) for 24 h.

MSCs/LX-2 co-cultures

   The impact of ADMSCs on LX-2 cells was assessed through a co-culture of
   activated LX-2 cells with ADMSCs using a Transwell chamber, and
   activated LX-2 cells were cultured separately as controls. ADMSCs
   cultured to 3–5 generations were digested and re-suspended, after which
   they were inoculated into the upper chamber of a Transwell. They were
   then co-cultured with LX-2 cells that were in the lower chamber using a
   high-sugar medium containing 10% FBS when the ADMSCs reached 70–80%
   confluency. LX-2 cells were harvested after 48 h.

Cell proliferation assays

   According to the manufacturer’s instructions, the proliferation of LX-2
   cells was measured using the Cell Counting Kit-8 (CCK-8). In short,
   after LX-2 cells were co-cultured with ADMSCs for 48 h, the upper
   compartment was removed, and CCK-8 reagent was added to the lower
   compartment containing LX-2 cells and incubated for 2 h. Subsequently,
   the optical densities at a wavelength of 450 nm were measured by a
   microplate reader.

Quantitative real-time PCR

   Total RNA was isolated from LX-2 cells or liver tissue with Trizol
   reagent and cDNA was synthesized. qPCR (Quantitative Real-Time PCR) was
   conducted in triplicate using the SYBR Premix Ex Taq on the ABI ViiA
   7Dx real-time PCR system. The expression levels of mRNA were determined
   using the comparative threshold cycle value (2^−ΔΔCt) method and were
   normalized against GADPH or LADH. All primers used in this study were
   synthesized by Sangon (Shanghai, China) (Table [68]1).

Table 1.

   Primer sequence of qRT-PCR
   Gene   Primer sequence (5‘-3’)
   Col1α1 F: 5’-GTGCGATGACGTGATCTGTGA-3’
          R: 5’-CGGTGGTTTCTTGGTCGGT-3’
   TGF-β1 F: 5’-TGC TTC CGC ATC ACC GT-3’
          R: 5’-TAG TAG ACG ATG GGC AGT GGC-3’
   α-SMA  F: 5’-GAG GAG CAT CCG ACC TTGC-3’
          R: 5’-TTT CTC CCG GTT GGC CTTA-3’
   IL-1β  F: 5’-TGT AAT GAA AGA CGG CAC ACC-3’
          R: 5’-TCT TCT TTG GGT ATT GCT TGG-3’
   IL-10  F: 5’-ACT GGC ATG AGG ATC AGC AG-3’
          R: 5’-CTC CTT GAT TTC TGG GCC AT-3’
   YAP    F: 5’-AGCCCAAGAACAGAAAGAACCT-3’
          R: 5’-TTGGACAAGTCCAGTGAGGC-3’
   [69]Open in a new tab

ELISA analysis

   ELISA kits (R&D Systems, Inc, Minneapolis, MN, USA) were used to detect
   fibrosis markers α-SMA and collagen 1 in the previously collected
   kidney and cell supernatant samples as well as the concentrations of
   p-YAP and TAZ in renal tissue and HSCs.

Bioinformatics analysis

   The data were aligned using HISAT2 and can be accessed at
   [70]http://daehwankimlab.github.io/hisat2/. The significance of
   differently-expressed genes (DEGs) was screened by P < 0.05 and
   |log2(foldchange)|> 1. Expression heatmaps and volcano plots were
   generated using R software. Based on the Kyoto Encyclopedia of Genes
   and Genomes (KEGG) data, available at
   [71]https://www.genome.jp/kegg/pathway.html/, is used to draw relevant
   pathway.

Statistical analysis

   Statistical analyses were carried out employing GraphPad Prism 9. The
   experimental data was presented as the mean ± SD. The determination of
   statistical significance involved the application of the two-tailed
   Student’s t-test for comparing the two groups, while for comparisons
   among multiple groups, ANOVA (oneway analysis of variance) was
   employed. A significance level of P < 0.05 was considered indicative of
   statistical difference.

Results

Identification and characterization of ADMSCs

   Liver fibrosis model was established by CCl[4] and ADMSCs were
   transplanted to observe the therapeutic effect (Fig. [72]1B). ADMSCs
   were spindle-shaped and attached to the culture flask. Consequently,
   after the third passage, only adherent Spindle cells remained in the
   flask. This population of ADMSCs was collected and employed for flow
   cytometry analysis. In order to examine the immunophenotype of ADMSCs,
   the cells were incubated with PE-conjugated antibodies targeting CD34,
   CD45, and CD105. Flow cytometry examination revealed that these primary
   rat ADMSCs expressed CD105 positively (+) and lacked expression of CD34
   (-) and CD45 (-) (Fig. [73]1A).

Fig. 1.

   [74]Fig. 1
   [75]Open in a new tab

   Identification of stem cells and the treatment of hepatic fibrosis. A:
   Flow cytometric analysis demonstrated positive expression of CD105 and
   negative expression of CD34 and CD45 in ADMSCs. B: A schematic
   representation of the in vivo experimental protocol is provided; C: The
   effect of ADMSCs transplantation on liver function was assessed by
   measuring ALT, AST and TBIL. *P < 0.05 compared in different groups;
   **P < 0.01, significant difference compared in different groups;
   ***P < 0.001, extremely significant difference compared in different
   groups. D: Morphological changes subsequent to ADMSCs transplantation.
   E: HE staining was employed to evaluate the pathological consequences
   of ADMSCs transplantation on liver. F: Masson’s trichrome staining was
   utilized to assess the impact of ADMSCs transplantation on the
   deposition of collagen fibers in the liver

The effect of ADMSCs on liver fibrosis in rats

   Macroscopic architecture of the liver was assessed. The livers procured
   from the CCl[4] treated cohort displayed a flattened, fractured surface
   and exhibited a light-red hue, whereas those from the control group
   showcased a smooth surface and a vibrant red coloration (Fig. [76]1D).
   The administration of ADMSCs yielded an amelioration of the overall
   state of the liver in comparison to the CCl[4] group. Analysis of serum
   biochemical markers indicated that the levels of ALT and AST expression
   in the CCl[4] group were significantly elevated beyond the established
   normal range (Fig. [77]1C). Compared to the CCl[4] group,
   ADMSCs-treated group exhibited lower expression levels of AST and ALT.
   Additionally, TBIL levels were assessed in the serum of each group, and
   the findings indicated that the TBIL changes in all groups fell within
   the normal range. Histological examination of the sections from the
   control group revealed the presence of characteristic architecture,
   which was characterized by a radial distribution of liver cells around
   the central vein, absence of portal inflammation, and a regular hepatic
   lobular structure. The hepatocyte steatosis was observed in CCl4 group,
   and a large number of inflammatory cells were infiltrated in the portal
   vein area, accompanied by distortion of hepatic lobular structure and
   central venous dilation. Conversely, the treatment group displayed a
   cellular architecture that closely resembled that of the PBS group,
   albeit with occasional detection of bi-nucleated hepatocytes. Notably,
   a substantial decrease in portal inflammation and a notable improvement
   in lobular structure were observed in comparison to the CCl[4] group
   (Fig. [78]1E).

   Masson’s Trichrome staining was employed for the evaluation of tissue
   fibrosis (Fig. [79]1F). The stained sections of both the control and
   treatment groups exhibited minimal presence of fibers surrounding the
   portal area, whereas the CCl[4] and PBS groups displayed extensive
   fibrous septa. In comparison to the CCl[4] group, the treatment group
   demonstrated a reduction in fibrous expansion around the portal area.

Effect of ADMSCs transplantation on liver fibrosis and Hippo pathway

   In order to assess the ECM component of the livers, qPCR was conducted
   to measure the mRNA expression levels of α-SMA, TGF-β[1], collagen 1,
   IL-1β, and IL-10 (Fig. [80]2A). The exposure to CCl[4] resulted in a
   significant increase in the gene expression of fibrotic markers
   compared to the control group. Notably, the treatment group exhibited a
   significant decrease in the mRNA expression of these fibrotic markers
   induced by CCl[4], indicating the potential efficacy of ADMSCs in
   mitigating liver fibrosis.

Fig. 2.

   [81]Fig. 2
   [82]Open in a new tab

   Effect of ADMSCs transplantation on liver fibrosis. A: The impact of
   transplantation of ADMSCs on the expression of α-SMA, collagen 1,
   TGF-β[1], IL-1β, and IL-10 genes in the liver tissue of rats with liver
   fibrosis. *P < 0.05 compared in different groups; **P < 0.01,
   significant difference compared in different group. B: Utilizing
   immunohistochemistry to detect the protein expression of α-SMA in liver
   tissue of a liver fibrosis model after ADMSCs transplantation; C:
   Employing ImageJ for quantitative analysis of immunohistochemistry
   results. *P < 0.05 compared in different groups; **P < 0.01,
   significant difference compared in different groups. D: ELISA detection
   of protein expression of α-SMA and collagen 1 in liver tissue of liver
   fibrosis model after ADMSCs transplantation. *P < 0.05 compared in
   different groups; **P < 0.01, significant difference compared in
   different groups

   Additionally, the protein levels of TGF-β[1], α-SMA, and collagen 1
   were assessed as they are crucial markers highly expressed during the
   fibrogenesis of liver tissue through IHC techniques (Fig. [83]2B, C)
   and ELISA (Fig. [84]2D). The protein expression of TGF-β[1], α-SMA, and
   collagen 1 generally paralleled the gene expression patterns observed
   in the various experimental groups. In comparison to the CCl[4] group,
   the administration of ADMSCs had been observed to result in a notable
   decrease in α-SMA, TGF-β[1], and collagen 1 protein levels.

   In order to reveal the specific mechanism of ADMSCs intervention in
   liver fibrosis, transcriptome sequencing was performed on the three
   experimental groups, and genes meeting |log2FC|>1 and P < 0.0.5 were
   defined as differentially expressed genes (Fig. [85]3A). KEGG pathway
   enrichment analysis was performed on the obtained differentially
   expressed genes, and significant changes in Hippo signaling pathway
   were found. The differentially expressed genes enriched in the Hippo
   signaling pathway were displayed in the form of heat maps, in which the
   expression of the key factor YAP was significantly different
   (Fig. [86]3B). The result of IHC analysis provided additional evidence
   that ADMSCs reduced YAP level (Fig. [87]3C, D). YAP gene (Fig. [88]3E)
   and protein expression (Fig. [89]3F) levels were evaluated
   subsequently. The results showed that mRNA level of YAP was
   significantly increased in CCL4-treated livers, accompanied by a
   corresponding increase in protein expression. In contrast,
   administration of ADMSCs resulted in a significant decrease in YAP mRNA
   level and protein expression in the liver. These results suggest that
   the treatment of ADMSCs is influenced by Hippo pathway.

Fig. 3.

   [90]Fig. 3
   [91]Open in a new tab

   Effect of ADMSCs transplantation on Hippo pathway in liver fibrosis
   model. A: Volcanic plot of differentially expressed genes in liver
   tissue after ADMSCs transplantation. B: Heatmap of differentially
   expressed genes enriched in the Hippo signaling pathway. C:
   Immunohistochemical detection of protein expression of YAP in liver
   tissue of liver fibrosis model after ADMSCs transplantation. *P < 0.05
   compared in different groups; **P < 0.01, significant difference
   compared in different groups; ***P < 0.001, extremely significant
   difference compared in different groups. D: Employing ImageJ for
   quantitative analysis of immunohistochemistry results. *P < 0.05
   compared in different groups; **P < 0.01, significant difference
   compared in different groups; ***P < 0.001, extremely significant
   difference compared in different groups. E: The impact of
   transplantation of ADMSCs on the expression of YAP gene in the liver
   tissue of rats with liver fibrosis. *P < 0.05 compared in different
   groups; **P < 0.01, significant difference compared in different
   groups; ***P < 0.001, extremely significant difference compared in
   different groups. F: ELISA detection of protein expression of YAP and
   collagen 1 in liver tissue of liver fibrosis model after ADMSCs
   transplantation

Effect of ADMSCs on HSC activation and Hippo pathway in vitro

   In order to investigate the potential impact of ADMSCs on liver
   fibrosis damage through modulation of HSCs activation, HSCs were
   subjected to serum deprivation for starvation and then stimulated with
   TGF-β[1]. Subsequently, a co-culture system with Transwell chambers was
   employed to co-culture HSCs with ADMSCs. The CCK-8 colorimetric method
   was employed to detect the proliferation of HSCs (Fig. [92]4A). The
   findings revealed that treatment with TGF-β[1] significantly enhanced
   the proliferation of HSCs, whereas ADMSCs effectively reversed this
   effect. In order to assess the activation effect of ADMSCs on HSCs,
   qPCR and ELISA analyses were conducted. The results demonstrated that
   the expression levels of activation markers α-SMA, collagen 1, and
   TGF-β[1] were significantly higher in HSCs treated with TGF-β[1]
   compared to those without TGF-β[1] treatment (Fig. [93]4B). These
   results indicated the successful activation of HSCs.

Fig. 4.

   [94]Fig. 4
   [95]Open in a new tab

   Effect of ADMSCs on HSC activation and Hippo pathway in vitro. A: The
   proliferation effect of activated HSCs co-cultured with ADMSC; B:
   Change of TGF-β, α-SMA and collagen 1 gene expression in activated HSCs
   and co-culture with ADMSCs. *P < 0.05 compared in different groups;
   **P < 0.01, significant difference compared in different groups;
   ***P < 0.001, extremely significant difference compared in different
   groups. C: Change of YAP and TAZ gene expression in activated HSCs and
   co-culture with ADMSCs. *P < 0.05 compared in different groups;
   **P < 0.01, significant difference compared in different groups;
   ***P < 0.001, extremely significant difference compared in different
   groups. D, E: ELISA detection of protein expression of α-SMA, collagen
   1, p-YAP and TAZ in activated HSCs and co-culture with ADMSCs.
   *P < 0.05 compared in different groups; **P < 0.01, significant
   difference compared in different groups; ***P < 0.001, extremely
   significant difference compared in different groups

   In this study, co-culturing with ADMSCs for a duration of 48 h resulted
   in a noticeable decrease in the expression of α-SMA, collagen 1 and
   TGF-β[1] mRNA, as determined through qPCR analysis. This downregulation
   was further confirmed through repeated ELISA analysis, indicating that
   ADMSCs inhibited the activation of HSCs induced by TGF-β[1]
   (Fig. [96]4D). In order to ascertain the role of the Hippo signaling
   pathway in the inhibitory effect of ADMSCs on HSCs activation, the mRNA
   and protein expression levels of key genes associated with the Hippo
   signaling pathway, namely YAP and TAZ, were assessed in HSCs treated
   with ADMSCs (Fig. [97]4C, E).

   The mRNA levels of YAP and TAZ exhibited a significant decrease in
   cells subsequent to ADMSCs treatment. Correspondingly, the protein
   expressions were also significantly diminished. These findings suggest
   that ADMSCs facilitate the inactivation of HSCs through Hippo pathway.

Loss of Hippo Pathway Activation attenuates the ADMSCs Induced inactivation
of HSCs

   To further ascertain the association between the Hippo signaling
   pathway and ADMSCs, the impact of ADMSCs on HSCs activation subsequent
   to the inhibition of the Hippo signaling pathway was investigated.
   XMU-MP-1 treatment had been found to have contrasting effects on YAP
   and TAZ expression in HSCs treated with TGF-β[1] and ADMSCs.
   Specifically, it leads to a decrease in YAP and TAZ expression in HSCs
   treated with TGF-β[1], while the increase in HSCs treated with ADMSCs
   (Fig. [98]5A, D). These findings suggested that XMU-MP-1 treatment can
   effectively block ADMSCs-induced inactivation of HSCs. Furthermore,
   analysis of gene and protein expression revealed that HSCs co-cultured
   with ADMSCs exhibited increased expression of α-SMA, collagen 1 and
   TGF-β[1] after XMU-MP-1 treatment (Fig. [99]5B, C). Additionally, the
   expression of TAZ was further increased, indicating that inhibition of
   the Hippo signaling pathway weakens the ability of ADMSCs to inhibit
   HSCs activation.

Fig. 5.

   [100]Fig. 5
   [101]Open in a new tab

   Loss of Hippo Pathway Activation Attenuates the ADMSCs Induced
   Inactivation of HSCs. A: Change of YAP and TAZ gene expression in
   activated HSCs and co-culture with ADMSCs, under the condition of
   applying XMU-MP-1 to block Hippo pathway. *P < 0.05 compared in
   different groups; **P < 0.01, significant difference compared in
   different groups; ***P < 0.001, extremely significant difference
   compared in different groups. B: Change of TGF-β, α-SMA and collagen 1
   gene expression in activated HSCs and co-culture with ADMSCs, under the
   condition of applying XMU-MP-1 to block Hippo pathway. *P < 0.05
   compared in different groups; **P < 0.01, significant difference
   compared in different groups; ***P < 0.001, extremely significant
   difference compared in different groups. C, D: ELISA detection of
   protein expression of α-SMA, collagen 1, p-YAP and TAZ in activated
   HSCs and co-culture with ADMSCs, under the condition of applying
   XMU-MP-1 to block Hippo pathway. *P < 0.05 compared in different
   groups; **P < 0.01, significant difference compared in different
   groups; ***P < 0.001, extremely significant difference compared in
   different groups

Discussion

   Liver fibrosis is a pathophysiological process characterized by
   abnormal hyperplasia of connective tissue in the liver, which is caused
   by the activation and secretion of a large amount of ECM by HSCs
   [[102]26]. Because of the association with an elevated risk of liver
   cancer, liver fibrosis is considered as a significant public health
   concern [[103]27]. Unfortunately, there is still a lack of widely
   accepted effective treatments currently, since the pathogenesis of
   liver fibrosis is intricate. MSCs possess robust tissue repair and
   immune regulation capabilities, making them a promising therapeutic
   option for liver diseases [[104]28]. Researches have shown that MSCs
   can effectively reduce ECM accumulation and enhance liver tissue repair
   ability, thereby mitigating liver fibrosis [[105]29, [106]30]. However,
   the specific mechanism by which MSCs treat liver fibrosis has not been
   fully elucidated. This study demonstrates that MSCs can inhibit HSCs
   activation by regulating YAP/TAZ expression in the Hippo pathway,
   subsequently reducing ECM deposition in the liver and effectively
   treating liver fibrosis.

   Our previous research has demonstrated that transplantation of ADMSCs
   can promote the cholesterol balance and the synthesis and secretion of
   bile acids in the body, reverse liver function impairment induced by
   CCl4 and lower serum TBIL levels [[107]31]. In this current study, the
   ADMSCs treatment group exhibited decreased levels of serum AST and ALT
   expressions compared to the CCl4 group, with a similar decreasing trend
   observed in TBIL levels, albeit the changes were minimal. Inflammatory
   factors are one of the key factors leading to liver fibrosis. Previous
   studies have shown that bone marrow-derived MSCs can reduce the
   expression of inflammatory factors that induce HSCs activation, thereby
   promoting the weakening of immune cell response and promoting the
   production of anti-inflammatory factors [[108]32]. Our results showed
   that after transplantation of ADMSCs, the expressions of inflammatory
   factors and fibrotic factors in liver tissue of rats with liver
   fibrosis were significantly down-regulated. In addition, the treatment
   group receiving ADMSCs displayed liver cell morphology similar to the
   control group, with decreased collagen fiber deposition and a
   significant improvement in lobular structure, aligning with the
   previous research by Abdelgwad [[109]33] et al. The above results
   suggest that the application of ADMSCs can reduce the deposition of ECM
   in the liver tissue of CCL4-induced liver fibrosis rats and regulate
   the inflammatory response, thereby alleviating the liver injury.

   The activation of HSCs is a crucial step in the development of liver
   fibrosis [[110]34]. Researches have indicated that human amniotic
   mesenchymal stem cells (hAMSCs) can suppress HSCs activation by
   inhibiting the Wnt/β-catenin signaling pathway, ultimately reducing
   liver fibrosis in mice [[111]35]. In this investigation, co-culturing
   TGF-β[1]-activated HSCs with ADMSCs led to a notable decrease in the
   proliferation of activated HSCs, suggesting a potential role of ADMSCs
   in inhibiting HSCs activation. To delve deeper into the mechanism of
   this inhibition, we performed transcriptome sequencing and discovered
   significant alterations in the Hippo signaling pathway during liver
   fibrosis intervention with ADMSCs. The Hippo signaling pathway has been
   shown to be a major player in damage repair of the liver [[112]36].
   Recent studies have demonstrated that the activation of the Hippo
   signaling pathway can reduce the activation of HSCs, leading to
   decreased ECM accumulation in the liver and contributing to the
   mitigation of CCl[4]-induced liver fibrosis [[113]37]. Upon further
   analysis, it was observed that the expression levels of YAP/TAZ in the
   Hippo signaling pathway exhibited significant changes. YAP/TAZ serves
   as a crucial effector in this pathway [[114]37]. Activation of the
   Hippo signaling pathway [[115]38] triggers upstream activation of
   mammalian Sterile 20-like kinase 1/2 (MST1/2) and large tumor
   suppressor 1 and 2 (LATS1/2), resulting in the phosphorylation of
   YAP/TAZ. Consequently, YAP/TAZ is hindered from translocating into the
   nucleus and interacting with TEA domain transcription factor 1–4
   (TEAD1-4) genes to modulate cell apoptosis and proliferation [[116]39].
   YAP/TAZ has been reported to play an important role in controlling the
   activation and aging of HSCs [[117]40]. Studies by Zhang K [[118]24]et
   al. have demonstrated that omega-3 polyunsaturated fatty acids (ω-3
   PUFA) can impede HSCs activation by facilitating the degradation of
   YAP/TAZ, thus offering a therapeutic approach for liver fibrosis. As a
   result, it is hypothesized that ADMSCs could suppress HSCs activation
   by modulating YAP/TAZ expression in the Hippo pathway, leading to a
   reduction in liver fibrosis. In order to demonstrate this, XMU-MP-1
   were used to block the Hippo signaling pathway. The findings showed a
   notable decrease in the ability of ADMSCs to suppress HSCs activation,
   confirming the hypothesis. The results of this study suggest that
   ADMSCs inhibit HSCs activation through the Hippo pathway. It is
   noteworthy that studies have demonstrated that YAP/TAZ engages in a
   crosstalk regulatory mechanism with a multitude of signaling pathways,
   including TGF-β-Smad [[119]41] and Notch [[120]42], which play a
   pivotal role in the process of liver fibrosis. It is therefore
   essential to conduct further research into the crosstalk regulatory
   mechanisms of YAP/TAZ and other signaling pathways in order to gain a
   comprehensive understanding of the mechanisms by which ADMSCs treat
   liver fibrosis.

Conclusions

   In summary, this study demonstrates that ADMSCs can inhibit the
   activation of HSCs to reduce liver fibrosis, and this ability of
   inhibition is achieved by regulating YAP/TAZ in the Hippo signaling
   pathway. These findings lay a foundation for future exploration of the
   exact mechanism of ADMSCs alleviating liver fibrosis and provide a
   basis for the treatment of liver fibrosis.

Acknowledgements