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IFN-γ-mediated inhibition of JAK/STAT signaling via nano-scutellarin treatment is an efficient strategy for ameliorating liver fibrosis

Journal of Translational Medicine volume 23, Article number: 195 (2025) Cite this article

Abstract

Background

Metabolic dysfunction-associated steatohepatitis (MASH) is a large group of metabolic diseases that are hazardous to human health. Endothelial-to-mesenchymal transition (EndMT) mediated myofibroblast activation is an important factor that aggravates the development of liver fibrosis during MASH. However, the limited understanding of the underlying molecular mechanisms that drive EndMT in MASH has hindered the development of molecularly targeted therapies specifically targeting this pathological process.

Methods

We employed wild-type and ifn-γ-deficient mice, MASH models were induced repeated CCl4 injections and a high-fat diet to verify the significance of IFN-γ role in vivo and its impact in EndMT. Male mice models of MASH were used to further analyze the effect of Scutellarin@BSA on the improvement of liver fibrosis during MASH in vivo and HUVECs were used to assess IFN-γ effect on EndMT and its interaction with JAK signaling pathway in vitro.

Results

The results showed that IFN-γ is revealed as a key regulator of EndMT during MASH, as evidenced by the significantly lower levels of EndMT and reduced pathological damage in the livers of ifn-γ knockout mice. Furthermore, our research has led to the development of Scutellarin@BSA therapy, which targets and mitigates IFN-γ-driven EndMT, which showed excellent therapeutic effects on EndMT and liver fibrosis in vivo and in vitro during MASH. Mechanistically, IFN-γ can directly bind to the JAK protein and activate downstream STAT1 transcription factors, exerting transcriptional activity and further driving the expression of EndMT-associated proteins. Notably, Scutellarin@BSA treatment effectively diminishes the hallmarks of liver fibrosis by modulating the canonical JAK/STAT1 signaling pathway.

Conclusions

IFN-γ was identified as a key regulator of EndMT, and Scutellarin@BSA, as an emerging treatment, has been found to effectively inhibit EndMT by directly targeting the regulatory influence of the IFN-γ signaling. This result demonstrates significant therapeutic efficacy in alleviating hepatic fibrosis during MASH, highlighting its great potential as an innovative liver fibrosis treatment.

Graphical abstract

Introduction

Metabolic dysfunction-associated steatohepatopathy (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD) or metabolism-associated fatty liver disease (MAFLD), has a current global prevalence of approximately 32.4% [1, 2], is a rising cause of clinical liver diseases paralleling a global increase in diabetes and metabolic syndrome [3]. Metabolic dysfunction-associated steatohepatitis (MASH) is considered a progressive form of MASLD characterized by steatosis, inflammation, and hepatocellular injury (appearance of hepatocellular ballooning) with various degrees of fibrosis. Resulting liver fibrosis and cirrhosis are major health burdens worldwide that frequently lead to systemic complications and comorbidities, and effective antifibrotic treatments are lacking [4, 5]. Liver fibrosis is a wound-healing process associated with excessive extracellular matrix (ECM) deposition and scar formation, with the former representing the most important cause of the disease; thus, blocking ECM production is an important objective for preventing and treating liver fibrosis [6,7,8].

The major source of ECM production during liver fibrosis is myofibroblasts, however, the exact source of these activated cells remains a topic of ongoing discussion and controversy [9]. Extensive research has established that hepatic stellate cells (HSCs) serve as the primary precursors for the development of myofibroblasts, which are responsible for the synthesis of ECM, this conclusion has been firmly validated through the use of genetic lineage tracing experiments [10, 11]. Furthermore, recent investigations have illuminated a potential role for endothelial cells (ECs) in liver fibrosis progression, as they may contribute to the generation of activated myofibroblasts via a process known as EndMT [12, 13].During EndMT, ECs lose and down-regulate the expression of endothelial cell markers such as CD31, VE-cadherin, and Vimentin [14]. EndMT-transformed cells are able to migrate to plaque-prone areas. During migration, these transformed cells can further differentiate into smooth muscle-like cells or fibroblasts, thereby respectively promoting neointimal formation or fibrous cap thickening [15, 16]. EndMT has been identified as being important in liver fibrosis during MASH. Nevertheless, the lack of insight into the molecular triggers of EndMT in MASH patients has hampered the creation of precision molecular therapies that could effectively target this process.

Interferon (IFN) signaling, a pivotal regulator of innate immune responses, has come to prominence as a critical factor influencing hepatic inflammation by modulating the function of liver macrophages [17]. IFN-γ is central to effective innate and adaptive immune reactions, and previous research underscores its harmful contribution to the initiation and/or perpetuation of proinflammatory activation, which drives the progression of obesity-linked insulin resistance and steatohepatitis [18,19,20]. Furthermore, prior investigations have highlighted the pivotal role of IFN signaling, in conjunction with IFN-modulated molecules like interferon regulatory factors (IRFs) and stimulator of interferon genes (STING), in regulating immune activation throughout the progression of MASH [21]. The activation of the innate immune system plays a pivotal role in facilitating the progression from MASLD to MASH. STING can induce inflammation in MASLD by activating NF-κB signaling in kupffer cells, and targeted intervention with STING can block the progression of MASLD to MASH [22, 23]. In addition, IFN-γ may exacerbate the progression of MASLD by regulating the accumulation of CD8+ T cell subsets associated with hepatic insulin sensitivity and gluconeogenesis during MASLD [24]. Targeting aberrant IFN-γ-associated activity, particularly those linked to inflammatory cascades and abnormal immune system stimulation, could potentially facilitate liver regeneration processes while concurrently inhibiting the progression of fibrosis, thereby enhancing the overall efficacy of liver repair. However, the regulatory role of IFN signaling in vascular endothelial-associated cells during MASH remains unclear.

In China, herbal medicine has a long-standing tradition of being clinically prescribed as a therapeutic modality for the treatment of various diseases, including chronic liver diseases [25]. Scutellarin, a flavonoid mainly found in Erigeron karvinskianus of the Asteraceae family, possesses broad pharmacological properties, including antioxidant, anti-inflammatory, cardioprotective, vasorelaxant, antineoplastic, and antibacterial actions [26,27,28]. Recently, scutellarin was shown to regulate lipid metabolism and reduce blood lipid levels in C57BL/6J mice [29], and has demonstrated therapeutic effects on MAFLD rats by reducing oxidative stress [30]. In addition, scutellarin can inhibit the proliferation and metastasis of liver cancer cells by inhibiting the activation of the PI3K/Akt/NF-kappaB signaling pathway [31]. Studies on Scutellarin’s direct impact on the IFN-JAK1 pathway are limited, but its potential role in immune and inflammatory responses suggests it may modulate IFN-γ signaling [32, 33]. It has been shown that baicalin regulates macrophage conversion from M1-type to M2-type and attenuates myocardial injury through inhibition of the phosphorylation of JAK2 and STAT3 [34]. Although the subject of the study was baicalin, its structure is similar to that of Scutellarin and may provide some reference value. These findings suggest that Scutellarin may play a role in immune and inflammatory responses, which is linked to the modulation of the IFN-γ signaling pathway and provide a new perspective on our understanding of the role of Scutellarin in disease models. However, the comprehensive understanding of scutellarin’s role in the therapeutic management of MASH remains elusive, especially its regulatory effect on EndMT in MASH, which has not been fully studied.

In this study, we found that EndMT was significantly alleviated after the deletion of the ifn-γ gene in vivo during MASH, and we identified Scutellarin@BSA as one of the most promising candidates for blocking IFN-γ-induced EndMT. Our findings reveal that Scutellarin@BSA can block the direct binding of IFN-γ to JAK proteins, consequently inhibiting the activation of downstream STAT1 transcription factors, which results in the suppression of EndMT-related protein expression. In conclusion, we have identified for the first time that IFN-γ is a key regulator of EndMT during liver fibrosis, and the identification of Scutellarin@BSA as a potential therapeutic agent that reduces the hallmark features of liver fibrosis via blocking IFN-γ-mediated EndMT offers a novel approach to counteract the progression of liver fibrosis.

Materials and methods

Mouse models

WT male mice and ifn-γ knockout (ifn-γ−/−, Strain NO. T012669) mice were purchased from GemPharmatech Co, Ltd. All mice used in this study were C57BL/6J male mice (20–25 g), in the age range of 6 to 8 weeks old. Prior to the trials, animals were given 7 days to acclimatize to the laboratory environment before the start of the experiment. The study was conducted meticulously, with strict adherence to the highest ethical standards, scientific protocols, and guidelines on the ethical handling and utilization of laboratory animals. The protocol was formally approved by the Animal Care and Use Committee of Southwest Medical University, ensuring compliance with the highest ethical requirements. Upon completion of the animal experiments, the mice were fasted overnight and then anaesthetized with a 2.5% concentration of isoflurane. After anaesthesia, mice underwent ocular bleeding via the orbital sinus, and liver samples were carefully collected post-mortem following cervical dislocation for further analysis.

Induction of experimental MASH and treatments

Following a week of acclimatization to the feeding regimen, the mice were randomly allocated to three distinct experimental groups: the Negative Control (NC) group, the MASH-Model (MO) group, and the Scutellarin@BSA treatment group. For the induction of the MASH model, mice were subjected to a Western-style diet enriched with 21.1% fat, 41% sucrose, and 1.25% cholesterol (w/w), along with a sugary beverage consisting of 18.9 g/L d-glucose and 23.1 g/L d-fructose, for a period of four weeks. In addition, mice received intraperitoneal injections of CCl4 (diluted to 20% in corn oil), at a dose of 0.32 g/kg body weight for every third day, up to a total of seven injections. Corn oil served as the vehicle for the control group, administered in a similar manner. Following the final injection, the mice were euthanized the subsequent day, and comprehensive liver tissue samples for follow-up experiments. The entire experimental protocol involving mice was granted approval by the Animal Experiment Administration Committee of Southwest Medical University (No. 20240043), ensuring compliance with ethical standards and regulatory guidelines.

Human samples

Liver biopsy samples were obtained from a cohort of five individuals diagnosed with advanced clinical fibrosis stages (F3-F4) of MASH, as well as from five control subjects without MASH. The sampling methodology adhered to previously established protocols, and liver transplant tissue specimens derived from patients were embedded in an optimal cutting temperature (OCT) medium, facilitating their use in immunofluorescence analyses. The Ethical Review Committee of the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University (No. KY2020025-XZ01) authorized the use of these human liver samples to ensure compliance with ethical principles.

Chemicals and reagents

Scutellarin was purchased from MedChemExpress (CAT: HY-N0751), and recombinant human IFN-γ was purchased from Shanghai Acmec Biochemical Technology Co., Ltd. (CAT: AC13071). In addition to the aforementioned materials, all other chemicals and solvents utilized in this study met the standards of analytical grade quality. Details of the antibodies used in this study are shown in Table 1.

Table 1 Antibodies used in this study
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Serum biochemical analysis

The concentrations of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum samples were quantitatively assessed utilizing a highly automated biochemical analyzer system (Cobas 8000 modular analyzer series, Roche Diagnostics GmbH, Mannheim, Germany).

Histopathological analysis

Immediately after obtaining the fresh liver tissues (n = 4 mice/group), we divided them into three parts: one piece was fixed in 4% paraformaldehyde, another was embedded in OCT gel for cryosectioning, and the last piece was used for Western blotting. To preserve the native cellular structure and morphology of the tissue, we immersed it in 4% paraformaldehyde for stability. Subsequently, the fresh tissue was embedded in OCT gel for ease of handling and quickly immersed in liquid nitrogen to achieve quick freezing. This rapid cooling process helps to maintain the antigenic reactivity of the tissue, thus facilitating subsequent immunofluorescence staining procedures. After 72 h of fixation in 4% paraformaldehyde, the tissues were dehydrated through graded ethanol solutions and then the paraffin sections were rehydrated with a reverse ethanol series prior to pathological staining. These sections were then subjected to hematoxylin-eosin (H&E), Masson’s trichrome staining and Sirius red staining to assess the improvement of liver fibrosis.

Immunofluorescence staining

For immunofluorescence staining, frozen liver tissues (n = 4 mice/group) were sectioned into 4 μm thick sections and immersed in 4% paraformaldehyde for fixation. Initially, the slides were blocked with a 5% solution of goat serum (Beyotime Biotechnology, C0265), and then incubated overnight at 4 °C with primary antibody to promote specific antibody-antigen interactions. After rinsing with PBS, the sections were exposed to secondary antibody for 1 h to amplify the signal. Subsequently, the sections were counterstained with DAPI (Beyotime Biotechnology, C1002) to visualize nuclei, rinsed again with PBS and prior to imaging, samples were sealed with a solution containing 20% glycerol.

Western blotting

Proteins were extracted using ice-cold RIPA lysis buffer (Beyotime, CAT: P0013B), and the amount of protein was determined by the bicinchoninic acid (BCA) assay. The proteins were separated by 10% SDS-PAGE and transferred onto PVDF membranes (Merck Millipore, CAT: IPVH00010). Following incubation in a solution of 5% bovine serum albumin (BSA) at 37 °C for 2 h, the membranes were subjected to overnight incubation at 4 °C with primary antibodies, including Collagen-I (1:1000), STAT1 (1:1000), p-STAT1 (1:1000), α-SMA (1:1000), CD31 (1:1000), FSP1 (1:1000), VE-cadherin (1:1000), and GAPDH (1:2000). After washing, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at 37 °C. Protein bands were then visualized using an ECL blotting technique (Beyotime, CAT: P0018S).

In vitro cell culture and EndMT stimulation

Primary human umbilical vein endothelial cells (HUVECs), sourced from iCell Biosciences, were initially maintained in 1X F-12 K medium (manufactured by Sperikon Life Sciences and Biotechnology Ltd., CAT: SP03020500). These cells were propagated until they reached 50% confluency and subsequently subjected to a starvation period of 12 h in a medium enriched with 3% fetal bovine serum (FBS). Following this starvation, the HUVECs were treated with interferon-gamma (IFN-γ) at a concentration of 100 ng/mL for a duration of 24 h.

Molecular docking

The interaction between scutellarin and JAK was investigated through molecular docking employing the software tools Autodock vina1.2.0, open babel3.1.1, and pymol2.3.1. The JAK crystal structure (PDB: 5wo4) was retrieved from the RCSB Protein Data Bank website (https://www.rcsb.org/) for this analysis. The molecular framework of scutellarin (CAS No. 27740-01-8), was obtained from the Traditional Chinese Medicine System Pharmacology (TCMSP) platform (https://tcmsp-e.com/tcmsp.php) and subsequently converted to a 3D model using OpenBabel. Molecular docking simulations were performed using Autodock and the most favourable orientations of the docked complexes were visually inspected and analysed in PyMOL.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9 software and results are reported as arithmetic mean ± standard error of the mean for clarity and accuracy. To compare values across groups, we used statistical methods including two-tailed Student’s t-tests to assess differences between the two groups; for comparisons that included more than two groups, we used one-way analysis of variance (ANOVA) with subsequent post-hoc tests. Grubbs’ test was used to detect outliers. p < 0.05 and 0.01 were deemed significant and highly significant, respectively, and are denoted as *p < 0.05 and **p < 0.01.

Results

IFN-γ levels are increased in liver ECs from MASH patients, and the deletion of ifn-γ ameliorates liver fibrosis symptoms and EndMT during MASH

It has been suggested that IFN-γ is a key factor in the progression of MASH [35]. In human liver samples exhibiting MASH, we detected significantly elevated levels of IFN-γ expression compared to that in normal liver tissue (Fig. 1B & Fig. S1A). Interestingly, the immunofluorescence staining results showed that IFN-γ overexpressed during MASH had a high degree of colocalization with the endothelial marker CD31, this observation suggests that IFN-γ may play a critical role in regulating the physiological functions of endothelial cells during MASH (Fig. 1B). To delve deeper into the potential therapeutic implications of IFN-γ in addressing MASH, we conducted an investigation focusing on the consequences of knocking out the ifn-γ gene in vivo in a MASH mouse model. Liver tissues from model mice exhibit distinct MASH phenotypes such as steatosis, inflammatory cell infiltration and significantly increased collagen deposition. Histological assessment using H&E, Sirius red and Masson’s trichrome staining (Fig. 1C) demonstrated the presence of steatosis, hepatocyte ballooning, inflammatory cell infiltration and fibrosis in the livers of high-fat diet (HFD) mice. Notably, these pathological features were attenuated in ifn-γ−/− mice. The serum levels of ALT and AST were elevated in HFD-fed mice; however, ifn-γ−/− mice decreased the serum levels of ALT and AST in HFD-fed mice (Fig. 1D, E). Moreover, the immunofluorescence staining results indicated that ifn-γ−/− mice significantly reduced collagen deposition during liver fibrosis, which was reflected by downregulated of Fibronectin (Fig. 1F). The presence of CD31 and mesenchymal markers like α-SMA and FSP1 in liver tissue, as detected by immunofluorescence imaging, clearly demonstrated that the proportion of endothelial cells undergoing EndMT was significantly lower in ifn-γ−/− mice as compared with controls (Fig. 1G, H). Taken together, these results show that ifn-γ gene deletion inhibits EndMT and attenuates liver fibrosis, which highlights the important role of IFN-γ as a key regulator in EndMT during liver fibrosis.

Fig. 1
figure 1

IFN-γ plays a key regulatory role in mediating EndMT during liver fibrosis. (A) Animal experiment design flow chart. (B) Immunofluorescence staining of human liver sections from control and MASH cases was performed to visualize IFN-γ (green) and CD31 (red), while nuclei were counterstained with DAPI (blue). Scale bar, 100 μm. (C) H&E, Sirius Red, and Masson staining was performed on liver sections from MASH mice (n = 4 mice/group). Scale bar, 100 μm. (D, E) ALT or AST enzyme activities were measured in designated groups of mice (n = 4 mice/group). (F) Immunofluorescence staining of mouse liver sections for Fibronectin (green) after induction of chronic liver injury by HFD-fed (n = 4 mice/group). Scale bar, 100 μm. (G, H) Co-localization of the endothelial marker CD31 with the mesenchymal markers α-SMA and FSP1 was detected in liver sections from mice exhibiting MASH (n = 4 mice/group). Scale bar, 100 μm. Protein expressions were qualified with densitometry analysis using ImageJ. Statistical analyses were performed using GraphPad Prism 9 software and values across groups were compared using one-way ANOVA. Outliers were detected using Grubbs’ test. Data are expressed as the mean ± S.D. *p < 0.05; **p < 0.01

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Scutellarin is a candidate for blocking IFN-γ-driven EndMT during MASH

IFN-γ is an important regulator of EndMT and represents an important target for MASH therapy; thus, the search for drug candidates that block IFN-γ-driven EndMT is highly important for MASH therapy. The typical signaling pathway of IFN-γ includes IFN-γ receptors 1 and 2 (IFNGR1 and IFNGR2), Janus kinases 1 and 2 (JAK1 and JAK2), signal transducers and activators of transcription 1, 2, and 3 (STAT1, STAT2, and STAT3), and interferon regulatory factor 1 (IRF1), all of which play an indispensable role in the transduction process [36]. Upon binding to IFN-γ, the receptor subunits IFNGR1 and IFNGR2 oligomerize and activate the Janus kinases JAK1 and JAK2, which leads to STAT1 phosphorylation and facilitates the formation of STAT1 dimers, which are subsequently translocated to the nucleus [36, 37]. Therefore, it is highly important to search for targeted drugs to inhibit JAK to block the pathogenicity of IFN-γ during MASH. Scutellarin is a small molecule drug with strong immunomodulatory activity (Fig. 2A, B).

To further evaluate the therapeutic potential of scutellarin in regulating IFN-γ-driven EndMT, we simulated the interaction between scutellarin and the JAK1 protein using molecular docking. In general, the stronger the binding affinity of a ligand to its receptor, the lower the associated binding energy. Specifically, binding energies ≤ -5.0 kcal/mol indicate strong binding, while ≤ -7.0 kcal/mol indicates very strong or tight binding. The binding energy of scutellarin to JAK1 was − 8.4 kcal/mol, which indicated a strong binding affinity between scutellarin and JAK1. In order to gain a deeper understanding of the potential interactions between the active components of scutellarin and JAK1, we analyzed their binding sites and binding affinities in depth (Fig. 2C). This analysis suggested that scutellarin has the potential to regulate the pathological process driven by IFN-γ.

To prolong the blood circulation and improve metabolic stability of scutellarin, we encapsulated scutellarin into BSA to prepare nanometer sized Scutellarin@BSA nanoparticles. The size distribution of Scutellarin@BSA nanoparticles was determined by dynamic light scattering (DLS) (Fig. 2D). The average hydrodynamic diameter of Scutellarin@BSA particles was determined to be 167.7 nm and have a narrow size distribution with a PDI value of 0.612. Later, both the particle size, PDI and ζ-potential of Scutellarin@BSA remained in a relatively stable state during 12 days of storage (Fig. 2D, E and F). These results indicate that scutellarin and BSA can form relatively stable nanoparticles.

Fig. 2
figure 2

(A, B) Illustration and chemical structure of scutellarin. (C) Visual representation of the docked conformation as well as a 3D plot showing the interaction between scutellarin and JAK1 protein. (D) Particle size and (E) PDI and (F) ζ-potential of Scutellarin@BSA during the 12-day storage period. All data are presented as the mean ± S.D. (n = 3)

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Scutellarin@BSA attenuates liver fibrosis during MASH in mice

Using the MASH mouse model, we further analyzed the effect of Scutellarin@BSA on ameliorating liver fibrosis during MASH. Scutellarin@BSA demonstrated low organ toxicity (Fig. S2), which lays the foundation for its potential use in the treatment of liver fibrosis. We then evaluated the therapeutic effect of Scutellarin@BSA on the MASH model (Fig. 3A). Administration of Scutellarin@BSA reversed pre-existing hepatic fibrosis compared to the untreated group. In addition, serum levels of AST and ALT were significantly reduced, indicating a reduction in liver injury in the Scutellarin@BSA treatment group (Fig. 3B, C). In comparison to the model group, the Scutellarin@BSA-treated group exhibited significant improvements in liver architecture, accompanied by a reduction in the deposition of collagenous fibers, suggesting that Scutellarin@BSA could reverse liver fibrosis (Fig. 3D, F, G). Moreover, the immunofluorescence staining results indicated that Scutellarin@BSA significantly reduced myofibroblast activation and collagen deposition during liver fibrosis, which was reflected by the downregulation of collagen protein (Collagen I/III) and Fibronectin expression (Fig. 3E, I-K). In addition, Western blot (WB) analysis of key markers, such as Collagen-I, was performed to assess the extent of liver fibrosis. The results demonstrated that the expression levels of these markers were downregulated in the Scutellarin@BSA treated group compared to the model group, highlighting the efficacy of Scutellarin@BSA in attenuating liver fibrosis in vivo (Fig. 3H, L).

Fig. 3
figure 3

Scutellarin@BSA attenuated chronic liver injury-associated MASH. (A) Illustration of mice livers. (B, C) Measurement of serum ALT and AST enzyme activities in designated groups of mice (n = 4 mice/group). (D, F, G) H&E, Sirius red and Masson’s trichrome staining were used to detect connective tissue components in mouse liver sections (n = 4 mice/group). Scale bar, 100 μm. (E, I-K) Immunofluorescence staining for Collagen-I (green), Collagen-III (green), and Fibronectin (green) in liver sections from MASH mice (n = 4 mice/group). Scale bar, 100 μm. (H, L) The expression of Collagen-I was detected using WB (n = 4 mice/group). Protein expressions were qualified with densitometry analysis using ImageJ. Statistical analyses were performed using GraphPad Prism 9 software and values across groups were compared using one-way ANOVA. Outliers were detected using Grubbs’ test. Data are expressed as the mean ± S.D. *p < 0.05; **p < 0.01

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Scutellarin@BSA inhibits the development of EndMT during MASH by inhibiting JAK1-STAT1 signaling in vivo

To assess the potential effects of Scutellarin@BSA on EndMT, we performed double staining assays against CD31 along with α-SMA and FSP1 in mouse liver tissues. Enhanced expression of α-SMA/FSP1 was observed in regions positive for CD31 expression relative to control specimens (Fig. 4A-D). Furthermore, treatment with Scutellarin@BSA led to a significant downregulation of α-SMA/FSP1 expression within the CD31-positive areas (Fig. 4A-D). These findings indicate that Scutellarin@BSA could ameliorate liver fibrosis by inhibiting EndMT. The activation of JAK-STAT1 signaling driven by IFN-γ is the key to the development of EndMT during MASH (Fig. 1). In this study, we demonstrated that scutellarin exhibits a strong affinity for binding JAK1, suggesting that scutellarin is a potential candidate drug for targeting JAK1 (Fig. 2). To further evaluate the inhibitory effect of scutellarin on the activation of STAT1, we performed WB analysis to assess the activation of STAT1 in the liver tissues of MASH mice. The experimental results showed that the activation of STAT1 was inhibited by Scutellarin@BSA during MASH (Fig. 4E-H). Overall, the reversal of EndMT paralleled the inhibition of JAK/STAT1 signaling, which indicates that the anti-EndMT effect of Scutellarin@BSA depends on the inhibition of JAK/STAT1 signaling.

Fig. 4
figure 4

Scutellarin@BSA inhibited EndMT by inhibiting JAK1-STAT1 signaling. (A-D) To assess changes in endothelial and mesenchymal cells, liver sections from control mice and mice from the MASH model group treated with Scutellarin@BSA were stained and analyzed for the endothelial cell marker CD31 (red) and the mesenchymal cell markers α-SMA (green) and FSP1 (green). After staining, these markers were assessed quantitatively (n = 4 mice/group). Scale bar, 100 μm. (E-H) Scutellarin@BSA effectively inhibited the activation of the STAT1 protein in liver tissue. Liver tissue extracts were subjected to various therapeutic evaluated using WB (n = 4 mice/group). Protein expressions were qualified with densitometry analysis using ImageJ. Statistical analyses were performed using GraphPad Prism 9 software and values across groups were compared using one-way ANOVA. Data are expressed as the mean ± S.D. *p < 0.05; **p < 0.01

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Scutellarin@BSA inhibits the development of IFN-γ-induced EndMT by inhibiting JAK1-STAT1 signaling in HUVECs

In this study, additional validation was conducted to confirm the ability of Scutellarin@BSA to inhibit EndMT in HUVECs. To further explore the regulatory effect of IFN-γ on endothelial cells during MASH, we used IFN-γ to simulate the EndMT process during MASH in vitro. After exposing HUVECs to IFN-γ for 48 h, we consistently observed changes in CD31 and α-SMA levels that were comparable to those we observed in our in vivo model. In addition, IFN-γ led to a downregulation of CD31 and VE-cadherin expression and was accompanied by an upregulation of α-SMA, Collagen-I, and FSP1 expression, as compared with controls (Fig. 5A-F). However, the introduction of Scutellarin@BSA effectively mitigated the IFN-γ-induced changes in the above factors initially (Fig. 5A-F). After treating HUVECs with Scutellarin@BSA for 48 h, the activation of STAT1 was inhibited, and the degree of inhibition of STAT1 activation was consistent with the degree of inhibition of EndMT (Fig. 5G-I). Overall, the reversal of EndMT paralleled the inhibition of JAK/STAT1 signaling, which indicates that the anti-EndMT effect of Scutellarin@BSA depends on the inhibition of JAK/STAT1 signaling.

Fig. 5
figure 5

Scutellarin@BSA inhibited EndMT by inhibiting JAK1-STAT1 signaling in HUVECs. (A-F) The expression levels of α-SMA, CD31, Collagen-I, FSP1, and VE-cadherin were assessed using WB. (G-I) The expression levels of STAT1 and its phosphorylated form p-STAT1 were assessed by WB analysis. Protein expressions were qualified with densitometry analysis using ImageJ. Statistical analyses were performed using GraphPad Prism 9 software and values across groups were compared using one-way ANOVA. Data are expressed as the mean ± S.D. *p < 0.05; **p < 0.01

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Discussion

Activation of myofibroblasts is the cornerstone of the pathogenesis of tissue fibrosis [38]. The EndMT, largely contributes to the expansion of myofibroblasts and the development of hepatic fibrosis [9, 39]. Endothelial cells are capable of undergoing EndMT, a process in which they adopt a mesenchymal phenotype by losing the expression and functions of endothelial markers while acquiring the expression and functions of mesenchymal cell markers [40]. During endogenous mesenchymal formation, cells previously expressing markers such as VE-cadherin transform into a mesenchymal or myofibroblast phenotype. This transformation is marked by the appearance of specific products, including α-SMA and Collagen I. Vascular endothelium can be viewed as a specialized form of epithelial tissue. EndMT leads to stratification of endothelial cell-derived mesenchymal stromal cells, which subsequently migrate to neighboring tissue layers [13, 41]. From a histological perspective, EndMT disrupts the integrity of the endothelium, resulting in a phenotype characterized by migration, invasion, and proliferation of the endothelial-derived cells [42]. EndMT should be viewed as a dynamic and potentially reversible process in which mesenchymal cells with acquired phenotypes can recover endothelial characteristics in response to specific stimuli.

In a previous study, throughout the development of hepatitis, IFN-γ not only played an important role in triggering hepatic inflammation but was also involved in hepatocyte injury during the progression of steatohepatitis [43]. In this study, based on human MASH liver tissues, we were surprised to find that IFN-γ is present in vascular endothelial cells. Based on this finding, we first demonstrated in this study that ifn-γ knockout mice within a CCl4 and high-sugar with HFD-fed induced MASH model, the liver pathological damage and liver function levels were significantly lower than those of WT mice, thus verifying that IFN-γ plays a key role in the pathogenic remodeling of the MASH. A limited number of reports have confirmed that the ifn-γ gene stimulator acts as a ubiquitous receptor that recognizes extracellular DNA and subsequently initiates an innate immune response [44], and that the involvement of the ifn-γ gene in signaling pathways and its role in the production of pro-inflammatory cytokines is critical in the pathogenesis of MASH [45]. Our previous study showed that genetic deletion of ifn-γ in endothelial cells inhibit the emergence of EndMT and improves liver fibrosis in a CCl4 mouse model.

JAK/STAT signaling is central to the production of many cytokines. The STAT family was originally thought to be key mediators in the integration of cytokine signals during transcription [46]. However, recent studies have shown that obesity exacerbates the development of MASH through STAT1-mediated pathways and promotes STAT3-driven HCC [47]. IFNs were initially known as key regulators of macrophage-activating factors, primarily triggering a signal transduction cascade through the JAK/STAT pathway [48]. In type II IFNs, IFN-γ triggers STAT1 activation mainly by phosphorylation two specific amino acid residues, leading to homodimerization of the STAT1 molecule, this homodimer then binds to the IFN-γ activation site, initiating downstream signaling and translocate to the nucleus to induce target gene transcriptions and expressions [49]. Our findings highlight the role of IFN-γ in promoting hepatic inflammation in MASH induced by the HFD-fed, mediated via the JAK/STAT1 signaling pathway. This establishes a firm foundation for future investigations into the regulatory effects of IFN-γ on JAK and the crucial underlying mechanisms that drive MASH.

Due to the excellent biocompatibility and non-toxicity of BSA, it has been widely designed as a drug delivery system for chemical drugs, traditional medicines, and other pharmaceuticals [50]. We encapsulated scutellarin into BSA to prepare nanoscale Scutellarin@BSA. This nanoformulation has been proven to prolong the circulation time of drugs in the blood and increase the possibility of binding to target tissue or cell receptors [51]. Using this drug delivery system, we have successfully improved the stability of scutellarin, thereby paving new avenues for its application in contemporary pharmaceutical systems.

Conclusion

We demonstrated for the first time that IFN-γ is a key regulator of EndMT, that the regulatory effect of IFN-γ on EndMT is dependent on the JAK1-STAT1 pathway, and that targeted blockade of IFN-γ-mediated EndMT can significantly reduce the symptoms of liver fibrosis during MASH. Furthermore, we found that scutellarin can effectively block the effects of IFN-γ on EndMT by inhibiting JAK1 to ameliorate liver fibrosis in vitro and in vivo. Our future efforts will focus on applying Scutellarin@BSA for the treatment of fibrosis in other organs, such as myocardial fibrosis and pulmonary fibrosis, and on exploring the role of IFN-γ in these biological processes.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

NC:

Negative Control

MO:

MASH-Model

MASH:

Metabolic dysfunction-associated steatohepatitis

EndMT:

Endothelial-to-mesenchymal transition

MASLD:

Metabolic dysfunction-associated steatohepatopathy

NAFLD:

Nonalcoholic fatty liver disease

MAFLD:

Metabolism-associated fatty liver disease

ECM:

Extracellular matrix

HSCs:

Hepatic stellate cells

ECs:

Endothelial cells

IFN:

Interferon

IRFs:

Interferon regulatory factors

STING:

Stimulator of interferon genes

OCT:

Optimal cutting temperature

ALT:

Alanine aminotransferase

AST:

Aspartate aminotransferase

H&E:

Hematoxylin-eosin

HUVECs:

Human umbilical vein endothelial cells

BCA:

Bicinchoninic acid

HRP:

Horseradish peroxidase

IFN-γ:

Interferon-gamma

FBS:

Fetal bovine serum

TCMSP:

Traditional Chinese Medicine System Pharmacology

HFD:

High-fat diet

JAK:

Janus kinases

STAT:

Signal transducers and activators of transcriptiona

IRF1:

Interferon regulatory factor 1

BSA:

Bovine serum albumin

DLS:

Dynamic light scattering

WB:

Western blot

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Acknowledgements

Not applicable.

Funding

This work was financially supported by the Luzhou Science and Technology Bureau (No. 2021LZXNYD-Z09, 2023SYF106, 2022-JYJ-154); Sichuan Provincial Science and Technology Department (No. 2024NSFSC2101); Sichuan Provincial Administration of Traditional Chinese Medicine (No. 2024MS515); A Research Project on the Integration of Traditional Chinese and Western Medicine Conducted at Southwest Medical University (No. 2024ZXYZX06).

Author information

Author notes

  1. Ting Wang and Bangguo Liu contributed equally to this work.

Authors and Affiliations

  1. Drug Research Center of Integrated Traditional Chinese and Western Medicine, The Affiliated Traditional Chinese Medicine Hospital, Southwest Medical University, Luzhou, 646000, Sichuan, China

    Ting Wang, Bangguo Liu, Juan Huang, Qixin Zhao, Hongping Shen, Tao Bi, Zengjin Liu & Qin Sun

  2. Institute of Integrated Chinese and Western Medicine, Southwest Medical University, Luzhou, 646000, Sichuan, China

    Ting Wang, Bangguo Liu, Juan Huang, Qixin Zhao, Hongping Shen, Tao Bi, Zengjin Liu & Qin Sun

  3. Sichuan Police College, Luzhou, 646000, Sichuan, China

    Yong Dai

  4. State Key Laboratories for Quality Research in Chinese Medicines, Faculty of Chinese Medicine, Macau University of Science and Technology, Macau, 853, China

    Tao Bi

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  1. Ting Wang
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Contributions

Ting Wang: Methodology, Investigation. Bangguo Liu: Investigation, Preparation, Methodology, Data curation. Juan Huang: Writing original draft, Methodology, Investigation, Supervision. Qixin Zhao: Methodology, Investigation. Hongping Shen: Funding acquisition. Tao Bi: Writing review & editing. Zengjin Liu: Funding acquisition, Supervision. Yong Dai: Methodology, Investigation. Qin Sun: Conceptualization, Funding acquisition, Supervision, Writing review & editing.

Corresponding authors

Correspondence to Zengjin Liu, Yong Dai or Qin Sun.

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Ethics approval and consent to participate

The experimental protocol involving mice was formally approved by the Animal Care and Use Committee of Southwest Medical University to ensure strict adherence to the highest ethical principles and regulatory guidelines. The use of human liver samples was authorized by the Ethical Review Committee of the Affiliated Hospital of Traditional Chinese Medicine of Southwest Medical University, ensuring strict adherence to all ethical principles and guidelines.

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Wang, T., Liu, B., Huang, J. et al. IFN-γ-mediated inhibition of JAK/STAT signaling via nano-scutellarin treatment is an efficient strategy for ameliorating liver fibrosis. J Transl Med 23, 195 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12967-025-06155-5

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Journal of Translational Medicine

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