Abstract

Background

   Metabolic dysfunction-associated fatty liver disease (MAFLD) is an
   increasing concern due to lifestyle changes, with metabolic
   dysfunction-associated steatohepatitis (MASH) leading to progressive
   liver damage, cirrhosis, and increased morbidity. The role of
   endoplasmic reticulum (ER) stress, particularly the unfolded protein
   response (UPR) pathway, in MASH progression remains unclear. Adipose
   tissue-derived stem cells (ADSCs) have shown promise in regenerative
   therapy; however, their mechanism for alleviating MASH-induced liver
   damage is not fully understood. In this study, we aimed to investigate
   the therapeutic mechanism of ADSCs in MASH, focusing on their
   modulation of ER stress in hepatocytes.

Methods

   C57BL/6J mice were fed either an atherogenic high-fat diet (AT + HF) or
   a high-fat diet (HFD-60) to induce MASH and simple steatosis (SS),
   respectively. Liver tissues were analyzed for gene expression, protein
   levels, and apoptotic markers using DNA microarray, quantitative PCR,
   western blotting, histological staining, and caspase activity assays.
   ADSCs were harvested, cultured, and treated to assess their effects on
   ER stress. In vitro experiments investigated palmitic acid-induced ER
   stress in hepatocytes and the effects of ADSCs on hepatic stellate
   cells and inflammatory markers.

Results

   The PERK arm of the UPR pathway was significantly upregulated in MASH
   liver tissues compared to SS tissues, correlating with increased
   apoptosis. ADSC administration reduced PERK activation, decreased
   apoptotic marker expression, and ameliorated hepatic fibrosis. However,
   ADSCs did not directly attenuate palmitic acid-induced ER stress in
   hepatocytes in vitro. Instead, they modulated the hepatic
   microenvironment by reducing hepatic stellate cell activation and
   IL-17-associated inflammation, indirectly mitigating ER stress and
   hepatocyte apoptosis.

Conclusions

   ADSCs alleviate MASH progression by modulating ER stress via
   immunomodulation rather than through directly rescuing hepatocytes.
   These findings highlight the potential of ADSCs as an immunomodulatory
   therapeutic strategy for MASH and support further investigation into
   their clinical application.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s13287-025-04482-4.

   Keywords: Metabolic Dysfunction-Associated steatohepatitis, Endoplasmic
   reticulum stress, Unfolded protein response, Adipose tissue-derived
   stem cells, Mesenchymal stem cells

Background

   Metabolic dysfunction-associated fatty liver disease (MAFLD) is an
   increasingly prevalent chronic liver condition associated with
   lifestyle changes [[54]1]. MAFLD encompasses a spectrum ranging from
   simple steatosis (SS), characterized by lipid accumulation in
   hepatocytes without associated inflammation, to metabolic
   dysfunction-associated steatohepatitis (MASH), a more advanced stage
   marked by hepatocellular injury, inflammation, and fibrosis, often
   progressing to cirrhosis [[55]2]. Clinically, cirrhosis in patients
   with MASH is associated with significantly poorer outcomes compared to
   other forms of chronic liver disease. A major challenge in developing
   effective treatments for MASH-related cirrhosis is the limited
   understanding of its underlying mechanisms. Hepatocellular damage,
   leading to lipid accumulation, is considered the “second hit” in the
   progression from SS to MASH. This process is driven by multiple
   pathological factors, including intestinal-derived lipopolysaccharide
   (LPS), which induces inflammation-induced hepatocyte injury, and
   oxidative and endoplasmic reticulum (ER) stress [[56]3]. The ER plays a
   central role in protein folding, lipid and carbohydrate metabolism, and
   the maintenance of cellular homeostasis [[57]4]. Accumulation of
   misfolded or denatured proteins in the ER induces ER stress and
   activates the unfolded protein response (UPR), a protective signaling
   network designed to restore homeostasis by reducing the protein load.
   If ER stress persists or becomes excessive, the UPR shifts from an
   adaptive to a pro-apoptotic response, ultimately leading to cell death
   [[58]5]. Recent evidence suggests that ER stress is a key contributor
   to MASH progression [[59]6], potentially acting downstream of LPS
   signaling and oxidative stress in the diseased liver. Nonetheless, the
   precise mechanisms by which ER stress contributes to MASH progression
   remain unclear.

   With advances in stem cell research, the application of stem cells in
   regenerative therapies for organ repair, including chronic liver
   disease, has garnered significant attention [[60]7]. Stem cells are
   classified into three main types: embryonic stem cells, induced
   pluripotent stem cells, and somatic stem cells. Among the latter,
   mesenchymal stem cells (MSCs), which can be isolated from autologous
   sources such as adipose tissue, bone marrow, and umbilical cord, are
   particularly promising due to their ease of collection and minimal
   ethical or technical concerns. In addition, MSCs are well recognized
   for their immunomodulatory properties, making them an attractive option
   for regenerative treatment strategies. We previously reported that the
   therapeutic effects of adipose tissue-derived stem cells (ADSCs), a
   type of MSC, are mediated through interactions with intrahepatic
   inflammatory cells in MASH [[61]8, [62]9]. However, the specific
   biological responses of hepatocytes and non-parenchymal cells to ADSC
   treatment in MASH remain poorly understood.

   In this study, we aimed to investigate the therapeutic mechanism of
   ADSCs in MASH by focusing on their modulation of ER stress in
   hepatocytes. Therefore, we employed a murine MASH model and used
   immortalized murine hepatocyte cell lines.

Methods

Mouse experiments

   This study was conducted in accordance with the ARRIVE guidelines for
   reporting animal research. All animal experiments were conducted in
   accordance with the experimental protocol approved by the Kanazawa
   University Animal Experiment Committee (approval no. AP21-019) and
   complied with Kanazawa University Animal Experiment Regulations at the
   Institute for Experimental Animals of Kanazawa University. Female
   C57BL/6J mice (8 weeks old) were purchased from Jackson Laboratory
   Japan and fed either an atherogenic high-fat diet (AT + HF) or a
   high-fat diet (HFD-60) to induce MASH and SS models, as described
   previously [[63]8, [64]9]. To compare liver tissue characteristics
   between MASH and SS mice, tissues were harvested at 3, 6, and 12 weeks
   after diet initiation for subsequent analyses or stored under
   appropriate conditions for future use. The number of mice was five per
   diet group at each time point, representing the minimum number required
   for statistical analysis. Each group was housed in a separate cage per
   diet. At eight and ten weeks after initiation of AT + HF feeding,
   2 × 10^5 ADSCs were injected into the splenic subcapsule to assess the
   treatment effect. At twelve weeks after the start of feeding, liver
   tissues were harvested for subsequent analysis. Mice were grouped
   randomly, although no special method of randomization was used. To
   minimize potential confounders, the order of treatments and
   measurements, as well as positions of animals and cages were
   randomized. At the time of surgery, medetomidine (0.3 mg/kg), midazolam
   (4 mg/kg), and butorphanol (5 mg/kg) were administered
   intraperitoneally as anesthesia. CO[2] gas was used for euthanasia.

Hepatocyte isolation

   Hepatocytes were isolated from liver tissue as previously described
   [[65]8]. Briefly, liver tissue was enzymatically digested at 37 °C for
   30 min using 0.25% collagenase type I (Wako, Osaka, Japan). Hepatocytes
   were isolated using 40% Percoll^® PLUS (GE Healthcare, Chicago, IL,
   USA) via centrifugation at 2000 rpm for 20 min at room temperature.

Acquisition of adipose tissue-derived mesenchymal stem cells

   ADSCs were isolated and cultured as previously described [[66]10].
   Briefly, inguinal subcutaneous adipose tissue was harvested from 10- to
   12-week-old male C57BL/6J mice. Tissue was enzymatically digested with
   0.15% type I collagenase (Worthington Biochemical Corporation,
   Lakewood, NJ, USA), followed by centrifugation at 620×g to isolate the
   stromal vascular fraction. Collected cells were then cultured and
   expanded in Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12
   (DMEM/F12) supplemented with 20% heat-inactivated fetal bovine serum
   (FBS) and 1% antibiotic–antimycotic solution (Life Technologies,
   Bedford, MA, USA). Cells at passages 5 to 6 were used for subsequent
   experiments.

Histological and immunohistochemical staining

   Liver tissue samples were fixed in IHC Zinc Fixative^® (BD Pharmingen,
   San Jose, CA, USA) and embedded in paraffin. Sections of 2 μm thickness
   were prepared for hematoxylin and eosin (HE), Azan, and
   immunohistochemical staining. For immunohistochemistry, 7 μm
   cryosections were prepared from frozen liver tissues embedded in OCT
   compound (Sakura Finetek, Tokyo, Japan). Endogenous peroxidase activity
   was blocked using Peroxidase Blocking Reagent (Agilent Technologies,
   Santa Clara, CA, USA), and non-specific protein binding was blocked
   using Protein Block (Agilent). Sections were incubated overnight at 4ºC
   with primary antibodies listed in the Table [67]S1A, followed by a
   30-min incubation at room temperature with Histofine^® Simple Stain™
   Mouse MAX PO, Rat, or Rabbit (Nichirei Bioscience, Tokyo, Japan). Color
   development was performed using the Histofine DAB Substrate Kit
   (Nichirei Bioscience), and sections were counterstained with
   hematoxylin (Nichirei Bioscience).

Cell lines

   The mouse hepatocyte cell line H2.35 and mouse Kupffer cell line KUP5
   were obtained from DS Pharma Biomedical (Osaka, Japan) and RIKEN BRC
   (RCB4627; Ibaraki, Japan), respectively [[68]11]. H2.35 cells were
   maintained in low-glucose D-MEM medium (Wako) supplemented with 4% FBS
   (Sigma-Aldrich) and 200 nM dexamethasone (Sigma-Aldrich). KUP5 cells
   were cultured in high-glucose D-MEM (Wako) containing 10% FBS
   (Sigma-Aldrich), 10 µg/mL bovine insulin, and 250 µM monothioglycerol.
   The IMS/N murine immortalized hepatic stellate cell line, a kind gift
   from Dr. Miura [[69]12], was cultured in DMEM (Wako) containing 10% FBS
   (Sigma-Aldrich), antibiotic-antimycotics (Gibco BRL/Life Technologies),
   and GlutaMAX (Gibco BRL/Life Technologies). Recombinant IL-17 A
   (PeproTech, Cranbury, NJ, USA) was added to IMS/N cultures at a final
   concentration of 100 ng/mL for activation as needed.

In vitro palmitic acid treatment

   H2.35 cells were seeded in 6-well plates, with 1 × 10^5 or 3 × 10^5
   cells per well. Palmitic acid (PA; Sigma-Aldrich), dissolved in 50%
   ethanol, was added at concentrations ranging from 0.1 mM to 0.4 mM to
   induce ER stress via lipotoxicity.

Gene expression analysis

   Total RNA was extracted from Liver tissue, isolated hepatocytes, H2.35,
   or IMS/N using the ISOSPIN Cell & Tissue RNA Kit (Nippon Gene, Tokyo,
   Japan), following the manufacturer’s protocol. As for the DNA
   microarray analysis, RNA obtained from hepatocytes or IMS/N was
   purified, amplified and labeled with Cy3 with the Quick Amp Labeling
   Kit (Agilent Technologies, Santa Clara, CA, USA), then complementary
   RNA was hybridized to the Whole Mouse Genome 4 × 44 K Array (Agilent
   Technologies), the Agilent G2505B Microarray Scanner used for the
   reading. BRB ArrayTools ([70]https://brb.nci.nih.gov/BRB-ArrayTools/)
   was used for the gene expression analysis. Filters were set to exclude
   spots with intensities below 1; quantile normalization was applied.
   Multiple probes recognizing were reduced to one per gene symbol by
   selecting the maximally expressed probe. Furthermore, a list of
   MetaCore pathway maps (MetaCore from Clarivate Analytics, Philadelphia,
   PA, USA) was obtained following the enrichment analysis of
   differentially expressing genes identified in the preliminary class
   comparisons. Microarray data comparing the gene expression of MASH
   hepatocytes and SS hepatocytes, shown in Fig. [71]1 were deposited in
   the NCBI Gene Expression Omnibus database as GSE ID: [72]GSE296715.
   Microarray data comparing the gene expression of IMS/N, IMS/M activated
   with IL-17 or palmitic acid, and co-cultured with ADSCs in each
   condition, were deposited in the NCBI Gene Expression Omnibus database
   as GSE IDs: [73]GSE296717, [74]GSE296718. As for the qRT-PCR, by using
   RNAs from liver tissue or H2.35, reverse transcription quantitative PCR
   was performed using the QuantStudio 12 K Flex Real-Time PCR system
   (Applied Biosystems, Foster City, CA, USA) with qPCR MasterMix Plus
   (Eurogentec, Seraing, Belgium) and TaqMan Gene Expression Assay probes
   described in the Table [75]S1B (Thermo Fisher scientific, Waltham, MA,
   USA).

Fig. 1.

   [76]Fig. 1
   [77]Open in a new tab

   Comparison of the characteristics of MASH and SS mice. For 12 weeks,
   mice from the MASH group were fed an atherogenic high-fat diet
   (AT + HF) while those from the simple steatosis (SS) group received a
   high-fat diet (HFD-60). For each timepoint, each group consisted of
   five mice. (A) Representative histological images of liver tissues
   stained with hematoxylin and eosin (HE) and Azan. Scale bars: 100 μm.
   (B-F) Gene expression analysis was performed by DNA microarray using
   three RNA samples randomly selected from isolated hepatocytes from
   liver tissue. (B) Hierarchical clustering analysis showing gene
   expression of hepatocytes. Heatmap of gene expression related to the
   PERK pathway (C), the Atf6 pathway (D), and the Ire1 pathway (E). (F)
   Quantitative real-time PCR (qRT-PCR) validation of UPR-related genes.
   (G) Western blot analysis of UPR-related proteins in liver tissues
   Full-length blots/gels are presented in Figure [78]S4. (H)
   Immunohistochemical staining of liver tissues with anti-PERK antibody.
   Scale bars: 100 μm. Data are presented as mean ± SEM (n = 5 for each
   group). *P < 0.05 indicates statistical significance between MASH and
   SS groups

Western blotting

   Proteins were extracted from hepatocyte cell lines and liver tissues
   using RIPA lysis buffer (Upstate Biotechnology, Boston, MA, USA)
   supplemented with PhosSTOP (Roche, Basel, Switzerland). Protein
   concentrations were determined using the Lowry method. After
   normalization, 20 µg of protein per 10 µL sample was loaded onto a
   5–20% SuperSep Ace polyacrylamide gel (13-well format; Wako) for sodium
   dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
   separation. Proteins were transferred onto Immobilon^®-P polyvinylidene
   fluoride (PVDF) membranes (Merck, Darmstadt, Germany), blocked with 5%
   skim milk prepared in TBS-T (25 mM Tris-HCl, pH 7.4; 138 mM NaCl; 2.7
   mM KCl; 0.5% Tween 20) at room temperature for 1 h. After washing with
   TBS-T, membranes were incubated with primary antibodies (dilutions
   listed in Table [79]S1C), followed by horseradish peroxidase
   (HRP)-conjugated rabbit anti-mouse IgG secondary antibody (Cell
   Signaling Technology, Danvers, MA, USA) diluted 1:1000 and incubated
   for 1 h at room temperature. Chemiluminescence signals were visualized
   using Clarity Western ECL Substrate (Bio-Rad Laboratories, Hercules,
   CA, USA).

Apoptosis assay

    1. TUNEL staining: Liver tissues from MASH model mice were fixed in
       IHC Zinc Fixative (BD Pharmingen), embedded in paraffin, and
       stained using the TUNEL Assay Kit-HRP-DAB (Abcam), according to the
       manufacturer’s instructions.
    2. Caspase assay: Liver tissue Sect. (20 μm per sample) from MASH
       model mice were homogenized in lysis buffer containing PhosSTOP
       (Roche) and 10x RIPA Lysis Buffer (Upstate). Resulting supernatants
       were analyzed for caspase-3/7 activity using the Caspase-Glo 3/7
       Assay System and GloMax Multi + Detection System (Promega),
       according to the manufacturer’s protocol.
    3. Flow cytometry: For in vitro apoptosis assessment, cells were
       stained with APC- or FITC-conjugated Annexin V (BD Pharmingen) and
       7-AAD for 15 min in the dark at room temperature, with gentle
       shaking. After washing, samples were analyzed using a FACSAccuri
       cytometer (BD Biosciences, Franklin Lakes, NJ, USA), and data were
       processed with FlowJo V10 software (Tree Star, Ashland, OR, USA).

Immunofluorescence staining

   IMS/N cells were seeded at 0.2 × 10^5 on BD Falcon CultureSlides
   (4-chamber, polystyrene, tissue-culture-treated glass slide). After
   24 h, 0.5 mM palmitic acid, IL-17, and ADSC-conditioned medium were
   added under specified conditions. After 48 h, cells were fixed with
   methanol, blocked with 1% BSA in PBS, and incubated overnight at 4 °C
   with anti-alpha smooth muscle actin (α-SMA) antibody (primary antibody,
   ab5694, Abcam) diluted 1:500. After washing, Alexa Fluor ^®
   594-conjugated goat anti-rabbit IgG H&L (secondary antibody, ab150080
   Abcam), diluted 1:500, was added and incubated in a dark place for
   30 min. Slides were mounted with VECTARSHIELD Mounting Medium (Vector
   Laboratories, Newark, CA, USA) and imaged using a fluorescence
   microscope (BZ-X800; KEYENCE, Japan).

Multiplex cytokine assay

   Recombinant IL-17 A (PeproTech) was added to IMS/N cultures at a final
   concentration of 100 ng/mL, followed by incubation for 24 h.
   Supernatants were collected and stored at -80 °C. Cytokine
   concentrations were quantified using the Bio-Plex Pro Mouse Cytokine
   23-plex Assay kit (Bio-Rad Laboratories) on the Bio-Plex Suspension
   Array System (Bio-Rad Laboratories), according to the manufacturer’s
   protocol.

Statistical analysis

   Statistical analysis was conducted using PRISM 9 (GraphPad Software,
   San Diego, CA, USA). Unpaired two-tailed Student’s t-tests were used
   for comparing reverse transcription quantitative PCR,
   immunohistochemistry, and cytokine assay data. P-values < 0.05 were
   considered statistically significant. Data analysis was performed by
   Dr. Seki (blinded to group assignment), while experimental allocation
   and animal testing were conducted by Dr. Ogawa and Dr. Inui (aware of
   group allocation).

Results

The PERK pathway is upregulated in the livers of MASH model mice

   MASH is characterized by hepatic inflammation and fibrosis in the
   context of MASLD and often coexists with SS. We established MASH and SS
   mouse models by feeding mice AT + HF and HFD-60 diets, respectively,
   for 12 weeks, as previously described [[80]9]. Both models demonstrated
   hepatic lipid accumulation (Fig. [81]1A, top). However, only MASH mice
   developed significant inflammatory cell infiltration and progressive
   hepatic fibrosis, distinguishing the MASH hepatic microenvironment,
   which consists of hepatocytes, non-parenchymal cells, and infiltrating
   immune cells, from that of SS (Fig. [82]1A, bottom).

   To investigate molecular differences, hepatocytes were isolated from
   MASH and SS mice, and gene expression profiles were compared using DNA
   microarray analysis. Hierarchical clustering of 3,363 genes revealed
   distinct transcriptional patterns between the two groups. Pathway
   enrichment analysis identified a marked upregulation of genes
   associated with the PERK branch of the UPR in hepatocytes from MASH
   mice, whereas the IRE1 and ATF6 pathways were not similarly activated
   (Fig. [83]1C, D, E). ER stress is mediated by three primary
   transducers–PERK, IRE1, and ATF6–each of which activates specific
   downstream pathways in response to the accumulation of unfolded
   proteins [[84]13]. The PERK pathway, in particular, is known to
   contribute to apoptosis in MASH livers [[85]14]. We validated the
   transcriptional activation of the PERK pathway by reverse transcription
   quantitative PCR, confirming elevated expression of Atf4 and Eif2ak3,
   key effectors of this pathway [[86]15]. In contrast, the expression of
   genes associated with the ATF6 and IRE1 arms of the UPR, including
   ATF6, Xbp1, Ubb, and Ern1, remained unchanged in MASH livers
   (Fig. [87]1F). Furthermore, PERK expression was significantly increased
   in the liver tissue of MASH mice after 12 weeks of AT + HF feeding
   (Fig. [88]1G). Immunohistochemical staining confirmed that PERK protein
   and UPR proteins involved in the PERK pathway, such as ATF4, were
   highly expressed in the liver of MASH model mice than in SS mice
   (Fig. [89]1H, [90]S1). These results collectively indicate that
   hepatocytes in MASH mice experience more severe ER stress than those
   from SS mice, primarily through selective activation of the PERK
   pathway.

ADSC administration ameliorates apoptosis and PERK expression in the livers
of MASH model mice

   Various factors contribute to the development and progression of MASH,
   with ER stress recently identified as a key pathological component
   [[91]16]. In our previous study, we confirmed that our cultured ADSCs
   expressed stem cell surface markers, including CD29, CD44, CD90, and
   CD105 [[92]17], and we demonstrated that ADSC treatment improved
   fibrosis and inflammation while enhancing tissue regeneration in MASH
   model mice [[93]9]. Building on these findings, we assessed whether the
   therapeutic effects of ADSCs were mediated through modulation of ER
   stress pathways. ADSCs were administered to MASH model mice, and
   changes in UPR signaling were evaluated. PERK proteins and gene
   expression in liver tissues were significantly reduced following ADSC
   treatment, whereas expression of IRE1 and ATF6 pathway components
   remained unchanged (Fig. [94]2A, B). Given that the PERK–ATF4 axis
   induces apoptosis [[95]18], we examined hepatocyte apoptosis using
   TUNEL staining. A marked reduction in TUNEL-positive hepatocytes was
   observed in MASH livers, which was significantly attenuated by ADSCs
   (Fig. [96]2C, D). Similarly, the activity of caspase 3/7, a key
   mediator of apoptosis, was increased in the liver of MASH model mice
   but significantly decreased after ADSC treatment (Fig. [97]2E). These
   results suggest that ADSCs reduce hepatocyte apoptosis by selectively
   suppressing the PERK pathway during ER stress in the MASH liver.

Fig. 2.

   [98]Fig. 2
   [99]Open in a new tab

   Therapeutic effect of ADSCs on MASH mice. Eight and ten weeks after the
   initiation of AT + HF feeding, ADSCs (2 × 10^5) were injected into the
   splenic subcapsule. Liver tissues were harvested 12 weeks after the
   initiation of feeding. Each group consisted of five mice. (A) Western
   blot analysis of UPR-related protein molecules in liver tissues.
   Full-length blots are presented in Figure [100]S5. (B) Quantitative
   real-time PCR (qRT-PCR) analysis of UPR-related gene expression. Data
   include mice treated with ADSCs or untreated controls (injected with
   PBS). (C) TUNEL staining of liver tissues to detect apoptotic cells.
   Magnification: ×200; scale bars: 100 μm (D). Quantification of
   TUNEL-positive cells. (E) Caspase 3/7 activity assay. Data are
   presented as mean ± SEM (n = 5 for each group). *P < 0.05 indicates
   statistical significance between groups

ADSCs do not directly attenuate ER stress in hepatocytes induced by fatty
acids in vitro

   Palmitic acid, the most abundant saturated fatty acid in the human body
   [[101]19], has been reported to induce ER stress and hepatocyte
   apoptosis in vitro [[102]20]. To investigate whether ADSCs directly
   mitigate ER stress, we conducted in vitro co-culture experiments using
   the immortalized mouse hepatocyte cell line H2.35 and PA [[103]21].
   First, we confirmed that PA treatment induces ER stress and apoptosis
   in H2.35 cells. When 0.4 mM PA was added to the culture medium, lipid
   accumulation was observed in the cytoplasm of H2.35 cells within 24 h
   (Fig. [104]3A). Dose-dependent increases in PA concentration led to an
   increase in PERK protein expression (Fig. [105]3B) and a reduction in
   H2.35 cell proliferation (Fig. [106]3C). To assess whether ADSCs could
   directly attenuate PA-induced ER stress, H2.35 cells were co-cultured
   with ADSCs in the presence of 0.4 mM PA (Fig. [107]3D). Co-culture with
   ADSCs did not restore lipid accumulation or cell viability of H2.35
   (Fig. [108]3A, E). Furthermore, while PA-treated H2.35 cells showed
   increased expression of Eif2ak3, a PERK gene, and Atf4, a gene involved
   in the PERK pathway, UPR-related gene expression remained unchanged in
   co-culture with ADSCs (Fig. [109]3F). These results suggest that ADSCs
   do not directly alleviate fatty acid-induced ER stress or apoptosis in
   hepatocytes under these in vitro conditions.

Fig. 3.

   [110]Fig. 3
   [111]Open in a new tab

   Steatohepatitis hepatocyte model using H2.35 with palmitate to assess
   the effects of ADSCs on the ER stress reduction. H2.35 hepatocytes were
   cultured with 0.4 mm palmitic acid (PA) and co-cultured with ADSCs
   using culture inserts. (A) Evaluation of lipid accumulation by Oil Red
   O staining (left: control (no PA), middle: PA, right: PA + ADSCs). (B)
   Western blot analysis of UPR-related proteins in the steatohepatitis
   hepatocyte model. Full-length blots are presented in Figure [112]S6.
   (C) Viable cell count using the trypan blue exclusion method, with
   counting of H2.35 cells in control and 0.4 mM PA conditions. (D-F) ADSC
   co-culture experiments in 0.4 mM PA-supplemented culture. H2.35 was
   seeded on 12-well plates at a concentration of 1 × 10^5 cells/well. (D)
   Schematic diagram of the experiment schedule. (E) Viable cell count
   using the trypan blue dye exclusion method between 0.4 mM PA and 0.4 mM
   PA + ADSCs conditions (n = 3 for each group). (F) Quantitative
   real-time PCR (qRT-PCR) analysis of UPR-related molecules for H2.35 in
   ADSC co-cultures under 0.4 mM PA-containing culture media or control,
   without PA and ADSC. The expression level was assessed compared to that
   of the β-actin reference gene (at least n = 5 for each group). Data are
   presented as mean ± SEM. *P < 0.05 indicates statistical significance
   between groups

ADSC treatment attenuates hepatic stellate cell activation and reduces
hepatocyte UPR gene expression

   Given that ADSCs did not directly attenuate fatty acid-induced ER
   stress in hepatocytes in vitro, we next explored whether their effects
   were mediated through interactions with non-parenchymal cells in the
   hepatic environment. Specifically, we evaluated the impact of ADSC
   treatment on hepatic stellate cells and immune cell infiltration in
   MASH model mouse livers using immunohistochemical staining. The area of
   α-SMA-positive staining, indicative of activated hepatic stellate
   cells, was markedly increased in MASH livers, but this activation was
   significantly attenuated following ADSC treatment (Fig. [113]4A, B).
   Furthermore, we observed increased infiltration of F4/80-, Gr-1-, CD4-,
   and CD8a-positive immune cells in MASH livers. ADSC treatment
   selectively reduced the number of Gr-1-positive cells, while the
   numbers of F4/80-, CD4-, and CD8a-positive inflammatory cells remained
   unchanged (Fig. [114]S2). α-SMA is a well-established marker of hepatic
   stellate cells, and these data suggest that ADSCs ameliorate the
   activation of hepatic stellate cells. Recent studies have shown that
   hepatic stellate cells can be activated by IL-17 [[115]22], and we
   previously reported that IL-17 and its related gene expression are
   elevated in intrahepatic immune cells (HICs) from MASH mice compared to
   SS controls [[116]9]. In the current study, IL-17 stimulation increased
   α-SMA expression in the IMS/N stellate cell line, confirming
   activation. However, this activation was markedly suppressed when IMS/N
   cells were co-cultured with ADSCs, as shown by immunofluorescence
   assessment (Fig. [117]4C). To determine whether activated stellate
   cells contribute to hepatocyte ER stress, we investigated the effect of
   conditioned media from IL-17-stimulated IMS/N cells on UPR gene
   expression in H2.35 hepatocytes. Exposure to the supernatant from
   IL-17-treated IMS/N cells increased UPR gene expression in H2.35 cells
   (Fig. [118]4D, E), but not with IL-17 alone or the supernatant from
   IL-17-treated KUP5 cells, which are immortalized Kupffer cells (Fig.
   [119]S3A, B). Notably, this effect was diminished when IMS/N cells were
   co-cultured with ADSCs before collecting the conditioned media
   (Fig. [120]4F, G). These results suggest that ADSCs indirectly suppress
   ER stress in hepatocytes by modulating the activation state of hepatic
   stellate cells in the MASH liver microenvironment.

Fig. 4.

   [121]Fig. 4
   [122]Open in a new tab

   ADSC treatment attenuates hepatic stellate cell activation and UPR gene
   expression in MASH mice. (A, B) At 8 and 10 weeks after the initiation
   of AT + HF feeding, ADSCs (2 × 10^5 cells) were administered into the
   splenic subcapsule. Liver tissues were harvested two weeks after the
   last injection. (A) Immunohistochemical staining of liver tissues with
   anti-α-SMA antibody. Each scale bar: 200 μm. (B) Quantification of
   α-SMA-positive cell number or area using ImageJ. Data represent
   mean ± SEM (n = 5). **P < 0.01, ****P < 0.001. (C) Immunofluorescence
   staining of α-SMA in IMS/N cells cultured with or without PA and
   co-cultured with or without ADSCs. Magnification: ×200; scale bars:
   100 μm. (D-E) IMS/N was cultured with an IL-17 supplement for 24 h.
   H2.35 was seeded in 12-well plates. Twenty-four hours after seeding,
   the medium was replaced with the IL-17-activated IMS/N supernatant. (D)
   Experimental timeline schedule. (E) Quantitative real-time PCR
   (qRT-PCR) analysis of UPR-related molecules in H2.35 cells cultured
   with IL-17-activated IMS/N supernatant. The expression level was
   assessed compared to that of the β-actin reference gene (n = 5 for each
   group). *P < 0.05, **P < 0.01. (F) Experimental timeline schematic for
   ADSC co-cultured with IL-17 activated IMS/N supernatant. (G) qRT-PCR
   analysis of UPR-related molecules in H2.35 cells co-cultured with ADSCs
   and IL-17-activated IMS/N supernatant. The expression level was
   assessed compared to that of the β-actin reference gene. Data represent
   mean ± SEM (n = 5 for each group). *P < 0.05, **P < 0.01

PA-stimulated IMS/N supernatant induces apoptosis and enhances UPR in H2.35
cells, with ADSCs mitigating these effects

   In addition to the ability of PA to induce ER stress and apoptosis in
   hepatocytes, we examined whether PA influences hepatic stellate cells
   in a manner that indirectly promotes ER stress in hepatocytes. H2.35
   hepatocytes were cultured with conditioned media from PA alone,
   untreated IMS/N cells, or IMS/N cells treated with PA. We assessed the
   impact of these treatments on unfolded UPR gene expression in
   hepatocytes. Compared to PA or IMS/N supernatant alone, the combination
   of PA and IMS/N supernatant significantly increased expression of Atf4
   and EifAK3, genes in the PERK pathway of the UPR, in H2.35 cells
   (Fig. [123]5A). Moreover, H2.35 cells cultured in this combined
   condition medium exhibited a significant increase in apoptosis, as
   indicated by annexin V and 7-AAD staining (Fig. [124]5B).
   Immunofluorescence analysis confirmed that PA treatment increased IMS/N
   activation, as evidenced by increased α-SMA levels. However, co-culture
   with ADSCs attenuated this activation (Fig. [125]5C). Furthermore,
   caspase-3 activity, a key indicator of apoptosis in H2.35 hepatocytes,
   was increased in response to conditioned media from IMS/N cells treated
   with IL-17 or the combination of IL-17 and PA. Notably, ADSC co-culture
   suppressed caspase-3 activation under both conditions (Fig. [126]5D).
   These results suggest that ADSCs mitigate ER stress and apoptosis in
   hepatocytes by modulating the activity of hepatic stellate cells.

Fig. 5.

   [127]Fig. 5
   [128]Open in a new tab

   ADSCs mitigate palmitic acid-activated IMS/N-induced apoptosis and UPR
   in H2.35 cells in vitro. H2.35 hepatocytes were cultured with
   supernatants from IMS/N stellate cells pre-treated with 0.4 mM palmitic
   acid (PA), with or without ADSC co-culture, to evaluate ER stress and
   apoptosis. (A) Gene expression analysis of UPR-related markers in H2.35
   cells cultured with PA-activated IMS/N supernatant was performed using
   qRT-PCR (n = 3 per group). **P < 0.01, ***P < 0.005, ****P < 0.001. (B)
   Apoptosis was assessed in H2.35 cells cultured with or without IMS/N
   supernatant by 7-AAD and Annexin V staining after 24 h of treatment
   (n = 5 per group). (C) Immunofluorescence staining of α-SMA in IMS/N
   cells cultured with or without PA and co-cultured with or without
   ADSCs. Magnification: ×200; scale bars: 100 μm. (D) Western blot
   analysis of cleaved caspase-3 in H2.35 cells cultured with the
   supernatant of IMS/N exposed to IL-17 or both IL-17 and PA, with or
   without ADSC co-culture. Full-length blots are presented in Figure
   [129]S7. (E) Cytokine production by IMS/N cells was measured by
   multiplex cytokine assay after IL-17 or PA stimulation, with or without
   ADSC co-culture. Data are expressed as mean ± SEM (n = 5 per group),
   *P < 0.05

   To further explore the mechanism underlying stellate cell-mediated ER
   stress, we analyzed gene expression profiles in IMS/N cells activated
   by IL-17 or PA and compared them to profiles from cells co-cultured
   with ADSCs. The addition of IL-17 or PA increased the expression of
   genes involved in interferon (IFN) signaling in IMS/N, whereas the
   expression of these gene groups decreased in co-culture with ADSCs. In
   addition to IFN pathway genes, expression of cytokine genes related to
   inflammasome signaling was also induced (Tables [130]S2–[131]S5). We
   then examined cytokine production in the culture supernatants of IMS/N
   and activated IMS/N cells. Activation with IL-17 or PA increased
   secretion of proinflammatory cytokines, including IFN-γ, IL-6, TNF-α,
   MCP-1, Eotaxin, and KC (Fig. [132]5E). These findings suggest that ER
   stress and apoptosis in hepatocytes may be induced by a combination of
   these inflammatory cytokines released from activated stellate cells.

Discussion

   This study introduces a novel therapeutic approach for MASH by
   employing ADSCs to modulate ER stress in hepatocytes. ER stress is a
   key contributor to MASH progression, interacting with oxidative stress,
   proinflammatory cytokines, microbiota-derived metabolites, and
   excessive lipid accumulation to exacerbate liver injury. Activation of
   the ER stress response, particularly the PERK pathway, leads to
   caspase-mediated apoptosis in hepatocytes [[133]23, [134]24]. In our
   MASH mouse model, we observed elevated hepatocyte apoptosis and
   caspase-3/7 activity. Importantly, ADSC treatment effectively reduced
   both apoptosis and caspase activation, suggesting a promising mechanism
   of action that may attenuate disease progression.

   Although ER stress is traditionally associated with lipotoxicity caused
   by saturated fatty acids such as PA, accumulating evidence highlights
   internal signals, particularly inflammatory cytokines including IFN-γ
   [[135]23] and IL-1β [[136]24], as critical drivers of ER stress in
   various organs. We used PA-treated immortalized hepatocytes (H2.35) to
   confirm that PA induces UPR activation and apoptosis. However,
   supernatants from PA-stimulated hepatic stellate cells (IMS/N) enhanced
   this effect, suggesting that stellate cells play a supportive role in
   ER stress propagation. These findings support a model in which
   hepatocyte ER stress is not only a result of direct lipotoxicity but is
   also amplified by cytokines and paracrine factors secreted by activated
   stellate cells. This mechanism is consistent with prior reports of
   stellate cells acting as immune modulators in the liver via secretion
   of IFN-γ [[137]25], IL-1β, or TNF-α [[138]26], which have been detected
   in the supernatant of activated immortalized hepatic stellate cells.

   ADSCs, which are readily obtained from subcutaneous adipose tissue, are
   valued for their multipotency, self-renewal capacity, and
   immunomodulatory properties. These characteristics make them suitable
   for use in regenerative medicine, including their approved use in
   Crohn’s disease-associated fistulas [[139]27]. MSCs derived from bone
   marrow have also demonstrated clinical efficacy in treating
   graft-versus-host disease [[140]28]. In the context of liver fibrosis,
   the presence of ADSCs at least two weeks after administration in MASH
   cirrhotic liver was confirmed using GFP transgenic mice [[141]8], and
   ADSCs have been shown to exert beneficial effects through the
   modulation of hepatic stellate cell function [[142]9]. IL-17, a
   cytokine known to be increased in MASH-associated cirrhosis, has been
   identified as a potent activator of hepatic stellate cells [[143]29].
   In our study, IL-17 stimulation increased α-SMA expression in IMS/N
   cells and hepatic stellate cells from MASH model mice. However, ADSC
   co-culture significantly attenuated this activation, indicating a key
   immunomodulatory role. These findings support the hypothesis that ADSCs
   mitigate ER stress in hepatocytes by altering the inflammatory and
   fibrogenic activity of hepatic stellate cells rather than through
   direct action on hepatocytes themselves.

   While this study provides several important insights into the
   pathogenesis of MASH and suggests a novel therapeutic mechanism
   involving ADSCs, it has a few limitations. First, it relied on a murine
   model, which may not fully replicate human MASH pathology. Second,
   while we demonstrated a correlation between cytokine signaling and ER
   stress, direct mechanistic inhibition (e.g., neutralizing antibodies or
   receptor knockdown) was not performed. Third, only immortalized cell
   lines were used for in vitro experiments, which may not entirely
   represent primary hepatocyte or stellate cell behavior. For example,
   immortalized cells may not share the same cell cycle, gene expression,
   and metabolic characteristics as primary cells due to several factors
   including genetic modification. This may result in differences in
   responses to lipotoxicity and ER stress. Finally, the long-term
   durability and functional impact of ADSC treatment on fibrosis
   resolution were not assessed.

   The results of the current study offer several key implications for
   future research and therapeutic development. First, it identifies
   hepatic stellate cells as key mediators of ER stress in hepatocytes,
   offering a new cellular target for anti-fibrotic therapies. Second, it
   suggests that regenerative cell therapies may be more effective when
   designed to modulate the liver immune niche rather than hepatocytes
   directly. Future studies should focus on validating these mechanisms in
   human tissues, as confirmation in clinically relevant samples is
   critical for translating preclinical insights into therapeutic
   strategies. Equally important is the thorough evaluation of the
   long-term effects of ADSC treatment, including sustained therapeutic
   efficacy over time and comprehensive assessments of safety.

Conclusions

   This study demonstrates that ER stress plays a central role in MASH
   progression and that ADSCs alleviate disease severity by suppressing
   hepatic stellate cell activation rather than acting directly on
   hepatocytes. Our findings highlight a novel, indirect mechanism by
   which ADSCs modulate the hepatic microenvironment to reduce ER stress
   and hepatocyte apoptosis, mediated through fatty acid- and
   cytokine-induced stellate cell activity. These results highlight the
   potential of ADSCs as an immunomodulatory therapeutic strategy for
   MASH, particularly in targeting stromal-immune interactions within the
   liver.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [144]Supplementary Material 1^ (23MB, docx)

Acknowledgements