Allyl isothiocyanate ameliorates metabolic dysfunction-associated steatotic liver disease via vitamin D receptors in hepatocytes

Abstract

BACKGROUND

Prior studies indicate that allyl isothiocyanate (AITC) alleviates metabolic dysfunction-associated steatotic liver disease (MASLD). The vitamin D receptor (VDR) is known to exert protective effects in MASLD; however, whether AITC alleviates MASLD through VDR remains unclear.

AIM

To clarify the function and underlying mechanisms of AITC in MASLD via VDR.

METHODS

AML-12 cells were exposed to 300 μM palmitate acid (PA) for 24 hours to establish an in vitro MASLD model, followed by treatment with AITC (20 μM). We quantified intracellular lipid content using oil red O staining and biochemical triglyceride assays, and measured the expression of key regulators of hepatic de novo lipogenesis, fatty-acid (FA) β-oxidation, and insulin-resistance-related signaling by immunoblotting and quantitative real-time polymerase chain reaction.

RESULTS

To establish an in vitro MASLD model, AML-12 cells were treated with 300 μM PA for 24 hours. In this model, AITC significantly reduced protein levels associated with lipid synthesis and insulin resistance while upregulating those involved in FA β-oxidation. AITC enhanced VDR expression and increased the expression of hepatocyte nuclear factor 4 alpha (HNF-4α) and the downstream targets microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (ApoB). These changes mitigated PA-induced lipid accumulation, alleviated insulin resistance, and stimulated FA β-oxidation. Additionally, vitamin D further enhanced the therapeutic effects of AITC on MASLD.

CONCLUSION

AITC provides a robust molecular basis for improving MASLD by activating hepatic VDR and driving the downstream HNF-4α/MTTP/ApoB signaling pathway. This pathway reduces hepatic lipid accumulation, promotes FA β-oxidation, and improves insulin resistance, establishing AITC as a promising treatment for MASLD.

Key Words: Allyl isothiocyanate; Metabolic dysfunction-associated steatotic liver disease; Vitamin D receptor; Fatty acid β-oxidation; Lipid synthesis

Core Tip: Metabolic dysfunction-associated steatotic liver disease (MASLD) denotes a continuum of disorders unified by hepatic steatosis and can affect approximately one quarter of the global population, reaching up to 75% among individuals with obesity. This work seeks to define the functional contribution and mechanism of allyl isothiocyanate (AITC) in mitigating MASLD via the vitamin D receptor (VDR). In an in vitro MASLD model, AITC activates VDR through hepatocyte nuclear factor 4 alpha/microsomal triglyceride transfer protein/apolipoprotein B signaling, thereby reducing lipid accumulation and insulin resistance while promoting fatty acid β-oxidation. Taken together, these results show the therapeutic promise of AITC in MASLD.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly termed nonalcoholic fatty liver disease[1], is a widespread chronic liver disorder marked by abnormal triglycerides (TGs) accumulation within hepatocytes. It represents a spectrum of disorders associated with hepatic fat deposition, ranging from simple steatosis to metabolic dysfunction-associated steatohepatitis, with the latter capable of progressing to liver fibrosis, cirrhosis, and hepatocellular carcinoma[2,3]. Globally, MASLD affects one in four individuals, with prevalence as high as 75% in individuals with obesity[4]. An estimated 20 million individuals are expected to be ultimately succumbing to MASLD-related liver diseases[5]. Beyond end-stage liver disease, MASLD markedly increases the risk of metabolic disorders such as diabetes and coronary heart disease[6].

The widely cited “two-hit” hypothesis explains the pathogenesis of MASLD[7]. Insulin resistance (IR) is recognized as the primary factor leading to hepatic steatosis; it affects the liver, skeletal muscle, and adipose tissue and is associated with reduced sensitivity to insulin. This results in abnormal lipid metabolism in hepatocytes, hyperinsulinaemia, and increased peripheral lipolysis, constituting the “first hit”. Subsequently, steatotic hepatocytes become more susceptible to exogenous and endogenous damages, leading to mitochondrial dysfunction, oxidative stress, and lipid peroxidation, which further promote inflammation, necrosis, and fibrosis, resulting in the formation of a “second hit”[8,9]. The more recent “multiple-hit” hypothesis builds on this framework, incorporating contributors, including lipotoxicity, endoplasmic reticulum stress, and oxidative stress[10]. Therefore, clarifying the molecular mechanisms that drive MASLD progression and pinpointing actionable therapeutic targets are essential for advancing prevention and treatment.

Allyl isothiocyanate (AITC), a widely prevalent natural isothiocyanate present in cruciferous vegetables, including horseradish[11]. As an activator of the transient receptor potential ankyrin 1 (TRPA1) channel[12], AITC exerts multiple biological effects, including anti-inflammatory, antitumor, and antidiabetic activities[13,14]. AITC also significantly reduces hepatic steatosis and improves IR in MASLD mouse models[15]. In addition, AITC increases expression of genes involved in fatty acid (FA) β-oxidation, such as peroxisome proliferator-activated receptor alpha (PPARα), PPAR gamma coactivator-1 alpha (PGC-1α), and carnitine palmitoyl transferase 1 alpha (CPT1α), while decreasing expression of genes associated with lipid synthesis, including sterol regulatory element-binding protein 1 (SREBP1), stearoyl-CoA desaturase 1 (SCD1), FA synthase (FAS), and acetyl-CoA carboxylase 1 (ACC1). Furthermore, AITC has been shown to reduce the levels of proinflammatory cytokines, including interleukin-1 beta, tumor necrosis factor-alpha and interleukin-6[16]. Although AITC shows promise for improving MASLD, the underlying mechanisms remain unclear.

The vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily, is widely expressed in gastrointestinal and endocrine tissues[17]. Transient receptor potential channels and vitamin D/VDR signaling have been shown to coordinate in the pathogenesis of various diseases. Recent studies highlight a protective role of VDR in MASLD[18,19]. VDR interacts with hepatocyte nuclear factor 4 alpha (HNF-4α) to promote HNF-4α transcription and expression. This upregulates HNF-4α target proteins, including microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (ApoB), thereby increasing FA β-oxidation, improving IR, and reducing lipid synthesis, which alleviates MASLD[20-22]. Based on these findings, we hypothesize that AITC may alleviate MASLD by modulating VDR expression, although the mechanisms remain undefined.

To determine whether AITC exerts beneficial effects on MASLD through VDR and to elucidate the underlying mechanisms, we conducted in vitro experiments examining FA β-oxidation, IR, and lipid synthesis. The results indicate that AITC promotes FA β-oxidation, improves IR, and reduces lipid synthesis via VDR. These findings support AITC as a potential therapeutic agent for MASLD.

MATERIALS AND METHODS

Cell culture and treatments

The immortalized AML-12 mouse hepatocyte cell line was acquired from the Typical Culture Preservation Center of the Chinese Academy of Sciences (Shanghai, China). AML-12 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. To establish an in vitro MASLD model, palmitate acid (PA) was dissolved in bovine serum albumin (BSA), and AML-12 cells were exposed to DMEM supplemented with PA (300 μM; Sigma-Aldrich) and BSA at a ratio of 2:1 for 24 hours. To evaluate the impact of AITC on lipid accumulation in vitro, PA-exposed AML-12 cells were incubated with AITC (20 μM; Sigma-Aldrich; 99.7%) or dimethyl sulfoxide under serum-free conditions for 24 hours. Previous studies assessed cytotoxicity in AML-12 cells after AITC treatment using the lactate dehydrogenase release assay and evaluated cell viability using the cell counting kit-8 method; based on those results, 20 μM was selected as the optimal AITC concentration, as it did not significantly affect cellular viability[16]. Accordingly, 20 μM was used as the working concentration of AITC in this study.

To activate VDR, AML-12 cells were treated with the VDR agonist 1α, 25-dihydroxyvitamin D3 (600 nM; Sigma-Aldrich) dissolved in anhydrous ethanol for 24 hours. Lipid accumulation was evaluated using TG quantification and oil red O staining, and indices of FA β-oxidation, IR, and lipid synthesis were assessed.

Cellular TG assay

Cellular TG levels were quantified using a commercial kit (Applygen Technologies Inc., Beijing, China) according to the manufacturer’s instructions. Each group of samples was set with n = 3 biological replicates, and each replicate was derived from an independent culture well.

Oil red O staining

Intracellular lipid deposition was evaluated by oil red O staining. Briefly, AML-12 cells were fixed with 4% paraformaldehyde, stained with freshly prepared oil red O working solution, and counterstained with hematoxylin. Lipid droplets were visualized under an optical microscope. For quantification, bound dye was eluted with isopropanol, and absorbance was measured at 500 nm, with results normalized to the control group. This approach enabled both qualitative and quantitative analysis of intracellular lipid deposition.

Western blot analysis

Protein expression related to lipid metabolism, FA β-oxidation, and IR was analyzed by Western blot. AML-12 cells were lysed in radio immunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Protein concentrations were quantified using a bicinchoninic acid assay kit (Beyotime Biotechnology, China). Proteins (20-50 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, United States), and blocked with 5% nonfat milk. Membranes were incubated overnight at 4 °C with primary antibodies against CPT1α (sc-393070; Santa Cruz), PPARα (sc-398394; Santa Cruz), ApoB (sc-393636; Santa Cruz), HNF-4α (sc-374229; Santa Cruz), phosphatidylinositol 3-kinase (PI3K) (4249; CST), acetyl-CoA carboxylase (3662; CST), SCD1 (ab236868; Abcam), FAS (sc-48357; Santa Cruz), VDR (ab3508; Abcam), SREBP1 (sc-17755; Santa Cruz), PGC-1α (sc-518025; Santa Cruz), and MTTP (sc-515742; Santa Cruz). After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (ABclonal) and visualized using an enhanced chemiluminescence kit (Servicebio, China). Band intensities were quantified with ImageJ and normalized to loading controls.

Quantitative real-time polymerase chain reaction

The messenger RNA (mRNA) expression of genes related to lipid metabolism, FA β-oxidation, and IR was analyzed by quantitative real-time polymerase chain reaction. Total RNA was extracted from AML-12 cells using the RNA Fast Extraction Reagent Kit (RN001-50Rxns, China), and complementary DNA was synthesized with the PrimeScript^® RT reagent kit (Takara, Japan). Quantitative polymerase chain reaction was performed with SYBR Green Master Mix (Promega, United States) on a LightCycler^® 480 system (Roche, Switzerland) using the following program: 95 °C for 5 minutes, followed by 45 cycles of 95 °C for 10 seconds and 60 °C for 30 seconds. Gene expression was quantified by the 2^-ΔΔCt method with glyceraldehyde-3-phosphate dehydrogenase as the reference gene, and results are reported as fold change relative to controls.

Statistical analysis

Statistical analyses were conducted in SPSS v22 (IBM, Chicago, IL, United States). Results are presented as mean ± SD. Independent-samples t-tests were used for group comparisons, with Welch’s correction applied in cases of unequal variances. Statistical significance was set at P < 0.05.

RESULTS

PA induces lipid accumulation through promoting hepatic lipogenesis and inhibiting FA β-oxidation in hepatocytes

To examine the effect of AITC on PA-induced lipid accumulation, AML-12 cells were treated with PA to establish a cellular model of MASLD. First, TG levels were measured in cultured hepatocytes. Exposure to 300 μM PA for 24 hours significantly increased intracellular TG level (Figure 1A). We then evaluated the expression of genes related to hepatic lipid metabolism and FA β-oxidation. As shown in Figure 1B, PA significantly upregulated the protein levels of ACC1 and FAS, which are lipogenesis-targeted genes[23-25]. In contrast, in an in vitro MASLD model, PA decreased the expression of PPARα and PGC1α (Figure 1C), which are involved in FA β-oxidation[26,27]. Collectively, these results indicate that the in vitro MASLD model was well established.

Figure 1 Palmitate acid induces lipid accumulation by promoting hepatic lipogenesis and inhibiting fatty acid β-oxidation in hepatocytes. AML-12 cells were treated with 300 μM palmitate acid (PA) for 24 hours to establish an in vitro model of metabolic dysfunction-associated steatotic liver disease. A: Intracellular triglyceride content in PA-stimulated AML-12 cells (n = 3/group); B: The protein expressions of lipogenesis target genes, fatty acid synthase, and acetyl-CoA carboxylase 1, were detected via Western blotting; C: The protein expressions of genes involved in fatty acid β-oxidation, such as peroxisome proliferator-activated receptor α and peroxisome proliferator-activated receptor gamma coactivator 1α, were detected via Western blotting. Data are presented as the mean ± SD. ^cP < 0.001. P value calculated between groups. TG: Triglyceride; PA: Palmitate acid; FA: Fatty acid; FAS: Fatty acid synthase; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; ACC1: Acetyl-CoA carboxylase 1; PPAR: Peroxisome proliferator-activated receptor; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1α.

AITC inhibits hepatic lipogenesis, activates FA β-oxidation, and protects against IR in hepatocytes

In AML-12 cells, 20 μmol/L AITC did not alter lipid accumulation (Supplementary Figure 1A) but inhibited lipogenesis by reducing the expression of SREBP1 target genes (Supplementary Figure 1B and C). AITC enhanced FA β-oxidation by increasing CPT1α and PPARα levels (Supplementary Figure 1D and E) and protected against IR by increasing PI3K expression (Supplementary Figure 1F).

AITC ameliorates hepatosteatosis by modulating lipid metabolism and IR in an in vitro MASLD model

The above results demonstrate that AITC exerts protective effects on lipogenesis, FA β-oxidation, and IR. We subsequently used PA-stimulated hepatocytes to assess the effect and process of AITC in the in vitro MASLD model; PA-stimulated AML-12 cells were treated with 20 μmol/L AITC or vehicle for 24 hours. First, as shown in Figure 2A and B, AITC reversed changes in intracellular TG levels and lipid accumulation in PA-stimulated AML-12 cells. Lipogenesis was subsequently assessed in the in vitro MASLD model, and AITC significantly decreased the mRNA and protein levels of SCD1, SREBP1, and ACC1 (Figure 2C and D). AITC also activated FA β-oxidation in PA-stimulated AML-12 cells, as demonstrated by significant upregulation of the mRNA and protein levels of CPT1α and PPARα (Figure 2E and F). Furthermore, AITC increased the protein level of PI3K in the in vitro MASLD model (Figure 2G), protecting against IR.

Figure 2 Allyl isothiocyanate ameliorates hepatosteatosis by modulating lipid metabolism and insulin resistance in an in vitro metabolic dysfunction-associated steatotic liver disease model. AML-12 cells were treated with 20 μmol/L allyl isothiocyanate or vehicle for 24 hours to stimulate palmitate acid. A: Intracellular triglyceride content in AML-12 cells (n = 3/group); B: Representative image of oil red O staining of AML-12 cells in the two groups; C: The messenger RNA (mRNA) levels of stearoyl-CoA desaturase 1 (SCD1), sterol regulatory element-binding protein (SREBP1), and acetyl-CoA carboxylase 1 (ACC1) were detected (n = 4/group); D: The protein expressions of SCD1, SREBP1, and ACC1 were determined by Western blot analysis; E: The mRNA levels of the fatty acid β-oxidation-related genes carnitine palmitoyl transferase 1 alpha (CPT1α) and peroxisome proliferator-activated receptor alpha (PPARα) were detected (n = 4/group); F: The protein expressions of CPT1α and PPARα were determined by Western blot analysis; G: The protein expression of the insulin resistance-related gene phosphatidylinositol 3-kinase was determined by Western blot analysis. The scale bar in the panel represents 50 μm. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. ^cP < 0.001. P calculated between groups. TG: Triglyceride; PA: Palmitate acid; AITC: Allyl isothiocyanate; ACC1: Acetyl-CoA carboxylase 1; mRNA: Messenger RNA; SCD1: Stearoyl-CoA desaturase 1; SREBP1: Sterol regulatory element-binding protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PPAR: Peroxisome proliferator-activated receptor; CPT1α: Carnitine palmitoyl transferase 1 alpha; FA: Fatty acid; PI3K: Phosphatidylinositol 3-kinase.

AITC activates VDRs in MASLD in vitro

The VDR gene is a member of the nuclear hormone receptor superfamily. Growing evidence indicates that VDR has a protective effect in MASLD[22,28]. Next, we investigated the role and mechanism of VDR in AITC-induced alleviation of MASLD. First, VDR expression was elevated in a compensatory manner in the in vitro MASLD model (Figure 3A and B). AITC increased the mRNA and protein levels of VDR in AML-12 cells (Figure 3C and D). VDR was also significantly increased by AITC in the in vitro MASLD model (Figure 3E and F). This data suggests that AITC enhances VDR expression.

Figure 3 Allyl isothiocyanate activates vitamin D receptors in metabolic dysfunction-associated steatotic liver disease in vitro. A: The messenger RNA (mRNA) level of the vitamin D receptor (VDR) was detected in palmitate acid-stimulated AML-12 cells (n = 4/group); B: The protein expression of VDR was determined by Western blot analysis; C: The mRNA level of VDR was detected in allyl isothiocyanate (AITC)-stimulated AML-12 cells (n = 4/group); D: The protein expression of VDR in AITC-stimulated AML-12 cells; E: The mRNA level of VDR was detected in an AITC-stimulated in vitro metabolic dysfunction-associated steatotic liver disease model; F: The protein expression of VDR was determined by Western blot analysis. Data are presented as the mean ± SD. ^aP < 0.05. ^cP < 0.001. P calculated between groups. PA: Palmitate acid; AITC: Allyl isothiocyanate; VDR: Vitamin D receptor; mRNA: Messenger RNA; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

AITC attenuates lipid accumulation by activating the HNF-4α/MTTP/ApoB signaling pathway

VDR can interact with HNF-4α to promote the transcription and expression of HNF-4α, MTTP, and ApoB, thereby reducing lipid synthesis and alleviating MASLD[22,29]. To investigate whether HNF-4α/MTTP/ApoB is involved in AITC-mediated amelioration of MASLD, the expression of HNF-4α/MTTP/ApoB signaling was assessed. As shown in Figure 4A, PA did not markedly affect HNF-4α expression but decreased the protein levels of MTTP and ApoB. In addition, AITC increased MTTP and ApoB levels in cultured hepatocytes (Figure 4B) and in a PA-stimulated MASLD model (Figure 4C). These data demonstrate that AITC alleviates lipid accumulation by activating the HNF-4α/MTTP/ApoB signaling pathway.

Figure 4 Allyl isothiocyanate attenuates lipid accumulation by activating the hepatocyte nuclear factor 4 alpha/microsomal triglyceride transfer protein/apolipoprotein B signaling pathway. A: The protein expressions of hepatocyte nuclear factor 4 alpha (HNF-4α), microsomal triglyceride transfer protein (MTTP) and apolipoprotein B (ApoB) in palmitate acid-stimulated AML-12 cells were determined by Western blot analysis; B: The protein expressions of HNF-4α, MTTP, and ApoB in allyl isothiocyanate (AITC)-stimulated AML-12 cells were detected by Western blot analysis; C: The protein expressions of HNF-4α, MTTP, and ApoB in the AITC-stimulated in vitro metabolic dysfunction-associated steatotic liver disease model were determined by Western blot analysis. PA: Palmitate acid; AITC: Allyl isothiocyanate; HNF-4α: Hepatocyte nuclear factor 4 alpha; MTTP: Microsomal triglyceride transfer protein; ApoB: Apolipoprotein B.

Activating VDR promotes the ability of AITC to alleviate MASLD through the HNF-4α/MTTP/ApoB signaling pathway

To determine whether AITC mitigates lipid deposition through VDR, we treated AML-12 cells with VD. VD increased VDR mRNA expression in AML-12 cells (Figure 5A). Moreover, VD enhanced the ability of AITC to ameliorate TG accumulation and lipid deposition in PA-treated AML-12 cells (Figure 5B and C). To investigate the mechanism by which AITC ameliorates lipid accumulation via VDR, we assessed hepatic lipogenesis, FA β-oxidation, and the HNF-4α/MTTP/ApoB signaling pathway. VD augmented the ability of AITC to decrease expression of the lipogenesis-related genes SREBP1, FAS, and ACC1 in PA-stimulated AML-12 cells (Figure 5D). VD also enhanced AITC-mediated upregulation of the FA β-oxidation-related genes PPARα and PGC-1α (Figure 5E). Furthermore, VD enhanced the AITC-induced increase in HNF-4α, MTTP, and ApoB expression (Figure 5F). These results indicate that AITC activates VDR to inhibit lipogenesis and accelerate FA β-oxidation through the HNF-4α/MTTP/ApoB signaling pathway.

Figure 5 Activating vitamin D receptors promotes the ability of allyl isothiocyanate to alleviate metabolic dysfunction-associated steatotic liver disease through the hepatocyte nuclear factor 4 alpha/microsomal triglyceride transfer protein/apolipoprotein B signaling pathway. AML-12 cells were treated with 600 nM vitamin D for 24 hours. A: The messenger RNA level of the vitamin D receptor was detected in AML-12 cells after vitamin D stimulation (n = 4/group); B: Intracellular triglyceride content in AML-12 cells was detected (n = 3/group); C: Oil red O staining of the vitamin D-stimulated in vitro metabolic dysfunction-associated steatotic liver disease model; D: The protein expressions of lipogenesis-related genes, including sterol regulatory element binding protein 1, fatty acid synthase and acetyl-CoA carboxylase 1, were detected via Western blotting; E: The protein expressions of fatty acid β-oxidation-related genes, including peroxisome proliferator-activated receptor α and peroxisome proliferator-activated receptor gamma coactivator 1α, were detected via Western blotting; F: The protein expressions of hepatocyte nuclear factor 4 alpha, microsomal triglyceride transfer protein, and apolipoprotein B were determined by Western blot analysis. The scale bar in the panel represents 50 μm. Data are presented as the mean ± SD. ^aP < 0.05. ^bP < 0.01. P calculated between groups. VDR: Vitamin D receptor; VD: Vitamin D; mRNA: Messenger RNA; TG: Triglyceride; PA: Palmitate acid; AITC: Allyl isothiocyanate; FAS: Fatty acid synthase; ACC1: Acetyl-CoA carboxylase 1; SREBP1: Sterol regulatory element-binding protein 1; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; PPAR: Peroxisome proliferator-activated receptor; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1α; HNF-4α: Hepatocyte nuclear factor 4 alpha; MTTP: Microsomal triglyceride transfer protein; ApoB: Apolipoprotein B.

DISCUSSION

This study investigated the impacts of AITC on MASLD and elucidated underlying mechanisms. Our findings demonstrate that AITC alleviates MASLD by activating VDR, which promotes FA β-oxidation, reduces lipid synthesis, and reduces IR through the HNF-4α/MTTP/ApoB signaling pathway (Figure 6).

Figure 6 Model by which allyl isothiocyanate alleviates metabolic dysfunction-associated steatotic liver disease. Allyl isothiocyanate activates vitamin D receptor to activate the hepatocyte nuclear factor 4 alpha/microsomal triglyceride transfer protein/apolipoprotein B signaling pathway, inhibits lipogenesis, promotes fatty acid β-oxidation, and improves insulin resistance, alleviating metabolic dysfunction-associated steatotic liver disease. AITC: Allyl isothiocyanate; HNF-4α: Hepatocyte nuclear factor 4 alpha; FA: Fatty acid; VDR: Vitamin D receptor; MTTP: Microsomal triglyceride transfer protein; ApoB: Apolipoprotein B.

Studies show that AITC markedly attenuates hepatic steatosis and inflammatory responses in MASLD through activation of the silent information regulator 1 (SIRT1)/adenosine 5’-monophosphate-activated protein kinase (AMPK) axis and suppression of nuclear factor kappa-B (NF-κB) signaling[16]. AITC also exerts hepatoprotective effects through the nuclear factor erythroid 2-related factor 2 (NRF2) pathway[14] and antioxidant mechanisms, as evidenced by protective effects on carbon tetrachloride induced acute liver injury and inhibition of liver fibrosis in a rat model[30]. Consistent with these findings, our data indicate that AITC mitigates PA-induced TG accumulation and lipid deposition in AML-12 cells, alleviating MASLD.

AITC has also been reported as a potential antidiabetic compound that significantly ameliorates IR and increases glucose uptake, mitochondrial activity, glycated hemoglobin levels, and glucose transporter type 4 translocation to the plasma membrane. Moreover, it protects against high-fat diet (HFD)-induced hepatic steatosis, weight gain, and lipid dysregulation[31]. AITC has been shown to prevent dysregulation of genes involved in hepatic lipid metabolism, such as FAS and SREBP, in HFD-fed mice[32]. SREBPs maintain lipid homeostasis by transcriptionally controlling genes that drive fatty-acid and cholesterol biosynthesis. In Huh-7 human liver cancer cells, AITC inhibits SREBP proteolytic maturation and downregulates their downstream targets, thereby curbing de novo biosynthesis of cholesterol and FAs[33]. Furthermore, AITC treatment downregulates lipogenesis-related gene expression and upregulates lipolysis-related genes such as HSL and LPL, thereby promoting lipolysis[34]. AITC has also been reported to improve glucose tolerance and insulin sensitivity by regulating mitochondrial dysfunction[15]. In our study, AITC effectively activated VDR to reduce lipid synthesis, promote FA β-oxidation, and alleviate IR via the HNF-4α/MTTP/ApoB signaling pathway in an in vitro MASLD model.

Benzyl isothiocyanate, AITC, phenylethyl isothiocyanate, and sulforaphane are prominent naturally occurring isothiocyanates[35]. Evidence indicates that sulforaphane may modulate gene expression indirectly through VDR-dependent mitogen-activated protein kinase/extracellular regulated protein kinase and NF-κB signaling pathways[36]. Further studies have established that sulforaphane acts as a natural epigenetic modulator of VDR, exerting synergistic effects in mitigating inflammatory oxidative stress[37]. Therefore, we propose that AITC may have the potential to ‘activate’ VDR via signal transduction dependent transcriptional upregulation that is, by indirectly enhancing VDR mRNA and protein levels.

According to current knowledge, several assumptions have been proposed regarding the mechanism of AITC and VDR. First, it has been established that AITC influences multiple signaling cascades. In cardiomyocytes, AITC, an agonist of TRPA1, enhances peak calcium transients and improves cardiac contractility by activating the CaMKII signaling pathway[38]. Notably, intracellular calcium ion fluctuations can modulate VDR expression and promote nuclear translocation, thereby influencing VDR transcriptional activity[39]. These findings suggest that the calcium ion peak triggered by AITC-mediated TRPA1 activation may facilitate VDR nuclear translocation and subsequently regulate VDR target genes. Second, in our previous study, AITC was shown to upregulate SIRT1 and activate AMPKα[16]. Similarly, in a model of diabetic nephropathy, empagliflozin has been shown to inhibit ferroptosis, potentially through AMPK-mediated activation of the NRF2 pathway[40]. In renal proximal tubular epithelial cells, VDR activation exerts antioxidant effects and suppresses ferroptosis via the NRF2/heme oxygenase-1 (HO-1) pathway[41]. Based on these observations, it is plausible that AMPK activation by AITC enhances Nrf2/HO-1 signaling, which may synergize with VDR-induced Nrf2/HO-1 activity to amplify antioxidant responses. Third, AITC has been reported to induce phosphorylation of serine/threonine protein kinase B (Akt) via TRPA1 activation[42]. In contrast, VDR activation has been shown to enhance the antitumor efficacy of chemotherapy in gastric cancer by inhibiting the PI3K/Akt pathway[43]. The concurrent influence of AITC and VDR on Akt signaling may result in either additive effects or negative feedback, depending on signal intensity and cellular context. Collectively, these studies suggest potential crosstalk between AITC-induced signaling and VDR activation.

VDR is broadly expressed in hepatic tissue and within inflammatory cell populations in individuals with chronic liver disease, and its expression adversely correlates with liver histology severity in patients with MASLD and chronic hepatitis C[44]. Recent studies report that higher serum vitamin D levels and VDR expression are associated with a lower risk of MASLD in the Chinese Han population[45]. Hepatic macrophage-specific deletion of VDR causes IR and promotes monocyte cholesterol transport, accelerating atherosclerosis in mice[28]. Conversely, activation of VDR in hepatic macrophages reduces hepatic inflammation, steatosis, and IR, whereas depletion of VDR leads to spontaneous hepatic inflammation in mice as early as six months of age[46]. In chronic hepatitis C, hepatic VDR expression inversely correlates with fibrosis and inflammation severity, suggesting that the vitamin D/VDR system may contribute to the progression of metabolic and viral chronic liver injury[47]. Vitamin D reduces lipid accumulation and IR in HFD-induced mouse models, suggesting therapeutic potential for vitamin D in MASLD[22]. Furthermore, studies have confirmed that VDR directly interacts with HNF-4α in a ligand-dependent manner, regulating expression of TG transport-related genes such as MTTP and ApoB. HNF-4α has also been reported to promote expression of MTTP and ApoB, which reduces lipid synthesis and alleviates MASLD[22]. Our findings demonstrate that AITC activates VDR and improves lipid metabolism and IR via the HNF-4α/MTTP/ApoB signaling pathway. Specifically, AITC treatment reduced expression of lipogenesis-related genes (SREBP1, SCD1, and FAS), increased expression of FA β-oxidation-related genes (PGC-1α, PPARα and CPT1α), and increased expression of the IR-related gene PI3K.

Despite these promising findings, our study has several limitations. First, no studies to date have directly examined the effects of AITC under VDR knockout or inhibition conditions. Therefore, it remains unclear whether AITC-induced upregulation of VDR and its downstream targets reflects a causal relationship or a correlative event. Although our data indicate a strong association between AITC treatment and VDR pathway activation, future studies must validate these findings in HFD-induced mouse models with VDR interference (e.g., small interfering RNA). Second, our results indicate that VDR interacts with HNF-4α to promote HNF-4α transcription and expression, leading to increased MTTP and ApoB expression. This relationship was verified only at the protein level. Additionally, AITC may have adverse effects on peripheral organs. For instance, irrespective of dose, AITC exacerbates hepatic, pancreatic, and thyroid status in diabetic rats, potentially because of severe lipid and hormonal metabolic disturbances in these animals. However, lower doses of AITC (2.5 mg and 5 mg) have been reported to improve glucose and insulin responses. Further studies are needed to clarify the role of AITC in improving IR and to establish its safety profile[32].

CONCLUSION

This study indicates that AITC alleviates hepatic steatosis and IR and enhances FA β-oxidation by activating VDR through the HNF-4α/MTTP/ApoB signaling pathway. These data support AITC as a promising therapeutic candidate for MASLD.

ACKNOWLEDGEMENTS

We extend our sincere gratitude to all investigators whose contributions made this work possible.