Isoliquiritigenin represses the pro-fibrotic TGF-β/smad2 signaling and nuclear enriched abundant transcript 1 (NEAT1) in oral submucous fibrosis

Abstract

Background/purpose

Isoliquiritigenin (ISL) has shown anti-fibrotic potential in several organ systems, but its effects on oral submucous fibrosis (OSF) remain unclear. This study investigated whether ISL attenuates myofibroblast activities in buccal mucosal fibroblasts (BMFs) from OSF patients by modulating TGF-β/Smad2 signaling and the long non-coding RNA NEAT1.

Materials and methods

Primary normal BMFs and fibrotic BMFs (fBMFs) were isolated from human buccal mucosa, and cell viability, collagen gel contraction, migration, and wound closure were examined after ISL treatment with or without arecoline. TGF-β1 secretion was quantified, while protein expression of phosphorylated Smad2, COL1A1, and α-SMA was analyzed, and NEAT1 expression was assessed with gain-of-function experiments.

Results

ISL showed selective cytotoxicity toward fBMFs compared with normal BMFs. ISL (5 and 10 μM) significantly reduced collagen gel contraction and migration capacity of fBMFs, and reversed arecoline-induced increases in contractility and migration. ISL also decreased TGF-β1 secretion and suppressed phosphorylated Smad2, COL1A1, and α-SMA expression. Furthermore, ISL downregulated NEAT1 in fBMFs, and NEAT1 overexpression enhanced collagen gel contraction, whereas ISL partially but significantly abrogated this pro-contractile effect, suggesting that NEAT1 mediates, at least in part, ISL’s anti-fibrotic actions.

Conclusion

In conclusion, ISL selectively targets OSF-derived fibroblasts and attenuates myofibroblast-like phenotypes by disrupting TGF-β1/Smad2 signaling and NEAT1-driven fibrogenic activity. These findings highlight ISL as a promising candidate for adjunctive therapy in OSF.

Introduction

The transforming growth factor-beta (TGF-β) signaling pathway serves as the canonical regulator of tissue homeostasis and fibrosis.1,2 Under pathological conditions, hyperactivation of this pathway, particularly through the Smad2/3 axis, drives the resident fibroblasts toward contractile myofibroblasts.3 These activated myofibroblasts, characterized by the expression of α-smooth muscle actin (α-SMA), are the central effector cells of fibrosis.4 They are responsible for the excessive synthesis and deposition of extracellular matrix (ECM) components, such as type I collagen (COL1A1), which leads to progressive tissue stiffening, organ dysfunction, and a higher risk for malignancy.5

Oral submucous fibrosis (OSF) is a debilitating and chronic disease that serves as a prime example of this TGF-β-driven pathology.1,6 Classified as a precancerous condition of the oral mucosa, OSF’s development is unequivocally linked to the widespread habit of areca nut chewing.7–9 Substantial evidence has demonstrated that constituents in the areca nut, such as arecoline, directly perpetuate the fibrogenic process by increasing the production and secretion of TGF-β.10,11 This creates a persistent pro-fibrotic loop, leading to the uncontrolled myofibroblast activation and profound collagen accumulation that defines OSF.

Recent studies have shown the critical role of long noncoding RNA (lncRNA) on the development of fibrotic disease. 12 Among OSF-associated lncRNAs, nuclear enriched abundant transcript 1 (NEAT1) has emerged as a pivotal regulator of myofibroblast activation. NEAT1 expression is significantly elevated in OSF tissues and arecoline-stimulated fibrotic buccal mucosal fibroblasts (fBMFs), and its knockdown markedly suppresses fBMF proliferation, migration, collagen gel contraction, and expression of α-SMA and COL1A1. Mechanistically, NEAT1 promotes myofibroblast activities in OSF at least in part through the miR-760/TPM1 axis, thereby amplifying pro-fibrotic cytoskeletal remodeling and matrix production.13 Beyond the oral mucosa, NEAT1 has been implicated as a conserved pro-fibrotic driver in multiple organs, further underscoring its central role in TGF-β-driven fibrosis. In cardiac and pulmonary fibrosis models, NEAT1 enhances TGF-β-induced fibroblast activation by recruiting chromatin modifiers such as EZH2 to repress the inhibitory Smad7, or by sponging anti-fibrotic miRNAs to upregulate Smad3, β-catenin and downstream collagen genes.14 NEAT1 inhibition consistently attenuates TGF-β/Smad2/3 phosphorylation, epithelial—mesenchymal transition, and ECM deposition, suggesting that NEAT1 acts as an upstream amplifier of canonical TGF-β/Smad signaling across diverse fibrotic contexts.15 Collectively, these findings position NEAT1 as a nodal integrator linking areca nut-induced stimuli, miRNA dysregulation and TGF-β/Smad-dependent myofibroblast activation in OSF. Targeting NEAT1 therefore represents a rational therapeutic strategy to disrupt the pro-fibrotic loop in OSF, and provides a mechanistic framework for evaluating small-molecule modulators that may concurrently inhibit TGF-β/Smad2 signaling and NEAT1-driven lncRNA networks.

Isoliquiritigenin (ISL), a bioactive chalcone from licorice root (Glycyrrhiza uralensis), exhibits potent anti-fibrotic properties by directly antagonizing TGF-β/Smad signaling and myofibroblast activation.16,17 In human lung fibroblasts, ISL inhibits TGF-β1-induced proliferation, migration and ECM production (α-SMA, COL1A1, fibronectin) by activating autophagy and suppressing the PI3K/AKT/mTOR pathway, with effects enhanced by autophagy activators.16 Similarly, in a diabetic renal fibrosis model, ISL blocks TGF-β1/Smad2/3 phosphorylation, upregulates matrix metalloproteinase-1 (MMP-1) while downregulating tissue inhibitor of MMP-2 (TIMP-2), and attenuates connective tissue growth factor (CTGF) expression.17

ISL’s ability to dampen lncRNA-amplified TGF-β responses in other fibrotic contexts via competing endogenous RNA networks involving miR-194-5p/NEAT1 suggests potential convergence on NEAT1-driven axes.18 However, ISL’s efficacy in OSF-specific fBMFs, particularly against arecoline-induced NEAT1 upregulation and TGF-β/Smad2 activation, remains untested, representing a critical knowledge gap. The present study addresses this by evaluating ISL’s impact on fBMF fibrogenic phenotypes by targeting the dysregulation of NEAT1 expression and the downstream signaling molecules.

Materials and methods

Cell culture

Primary BMFs were isolated from buccal mucosa tissues of OSF patients and healthy individuals undergoing oral surgery after written informed consent. Buccal mucosa specimens were immediately rinsed with ice-cold Hanks’ Balanced Salt Solution (Thermo Fisher Scientific, Waltham, MA, USA) finely minced, and subjected to enzymatic digestion with trypsin at 37 °C. Tissues fragments were then transferred to Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific) containing 10% fetal bovine serum (Thermo Fisher Scientific), and 1% penicillin-streptomycin for 14 days. Spindle-shaped cells migrated out from the tissues were considered BMFs and were further expanded. cells between passages 3 and 8 were used for all experiments to minimize phenotypic drift. All procedures involving human tissues were reviewed and approved by the Institutional Review Board of Chung Shan Medical University, Taichung, Taiwan (IRB approval number: to be provided).11 For the arecoline-induced fibrotic model, BMF-embedded collagen gels were overlaid with serum-free DMEM containing arecoline at 20 μM for 24 h, a condition previously shown to induce myofibroblast-like contraction and TGF-β1 activation in BMFs.19

Collagen gel contraction assay

fBMFs were resuspended in 500 μL of neutralized collagen solution (8 mg/mL bovine dermal collagen type I; Sigma—Aldrich, St. Louis, MO, USA) and plated in 24-well culture plates. The mixtures were incubated at 37 °C for 2 h to allow gel polymerization, after which 0.5 mL of serum-free DMEM containing arecoline with or without ISL (0, 5, or 10 μM) was gently overlaid. After 48 h incubation at 37 °C, gels were photographed, and contraction was quantified as the relative gel area using ImageJ software version 1.54 (National Institutes of Health, Bethesda, MD, USA), normalized to initial gel area.20

Transwell migration assays

The transwell migration assay was performed as described previously.21 fBMFs were suspended in 200 μL serum-free DMEM and seeded into the upper chamber of 8-μm pore transwell inserts (Corning, Acton, MA, USA). The lower chamber contained 600 μL DMEM supplemented with 10% FBS as chemoattractant, with or without arecoline and ISL (0, 5, or 10 μM). After 24 h incubation at 37 °C, nonmigratory cells on the upper surface were removed with a cotton swab, and cells on the underside of the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet for further quantification.

Western blot analysis

Whole-cell lysates were prepared from fBMFs, and 20 μg protein per sample was separated by 10–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride membranes (Millipore, Merck, Darmstadt, Germany). Membranes were blocked with 5% bovine serum albumin (BSA; Millipore) in TBST for 1 h at room temperature, then incubated overnight at 4 °C with primary antibodies against p-Smad2, total Smad2, COL1A1, α-SMA, NEAT1, or GAPDH (Cell Signaling Technology, Danvers, MA, USA; 1:1000 dilution). After washing with TBST, membranes were probed with horseradish peroxidase-conjugated secondary antibodies (1:5000) for 1 h at room temperature. Protein bands were visualized using enhanced chemiluminescence substrate (ECL; Millipore) and imaged with the LAS-1000 plus system (GE Healthcare, Chicago, IL, USA). Band intensities were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA) and normalized to GAPDH as loading control.22

Quantitative real-time PCR (qRT-PCR) analysis

Total RNA was extracted from fBMFs using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNase I to remove genomic DNA contamination, as detailed previously. Complementary DNA (cDNA) was synthesized from 1 μg total RNA using the SuperScript III First-Strand Synthesis System (Invitrogen Life Technologies) according to the manufacturer’s protocol. qRT-PCR was performed using Power SYBR Green Master Mix (Applied Biosystems, Waltham, MA, USA) on the ABI StepOnePlus Real-Time PCR System (Applied Biosystems). The specific primer sequences are listed as follows (5′-3′): NEAT1-F: 5′-GTTCCGTGCTTCCTCTTCTG-3′, NEAT1-R: 5′-GTGTCCTCCGACTTTACCAG-3′. The levels of NEAT1 were normalized using GAPDH and the 2-ΔCt method was applied.13

TGF-β1 enzyme-linked immunosorbent assay

TGF-β1 concentrations were quantified using a human TGF-β1 DuoSet enzyme-linked immunosorbent assay (ELISA) kit (DY240B; R&D Systems, Minneapolis, MN, USA) per the manufacturer’s instructions. Briefly, 96-well plates were coated with capture antibody overnight at 4 °C, blocked with reagent diluent, and incubated with 100 μL standards or samples or 2 h. After biotinylated detection antibody and streptavidin-HRP incubation, tetramethylbenzidine substrate was added, and absorbance was read at 450 nm on a microplate reader (BioTek, Winooski, VT, USA).

Statistical analysis

Data from ≥3 independent experiments (n = 3–6) are presented as mean ± SD. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test or unpaired Student’s t-test (GraphPad Prism 9). Differences were considered significant at P< 0.05.22

IRB Approval number

CSMUH No:CS1-24058.

Results

The chemical structure of ISL is shown in Fig. 1A. MTT assays revealed that ISL exhibited greater potency against patient-derived BMFs (IC50 27 ± 6.6 μM) compared to normal BMFs (IC50 101 ± 18.4 μM), indicating the selective anti-fibrotic activity (Fig. 1B). Since myofibroblasts exhibit enhanced contractility, hydrated collagen gels are commonly employed to model wound remodeling stages and assess intracellular tension on collagen substrates.23 To evaluate ISL’s therapeutic potential in OSF, we performed functional assays in fBMFs, which recapitulate the activated myofibroblast phenotype characteristic of this disease. 19 Co-treatment with ISL (5 and 10 μM) markedly attenuated these arecoline-enhanced contractile (Fig. 2A) and migratory responses (Fig. 2B), and wound healing capacity (Fig. 2C) compared to vehicle controls, confirming broad suppression of fibrogenic behaviors essential for OSF progression. To elucidate ISL’s impact on the TGF-β autocrine loop, a central driver of myofibroblast activation, TGF-β1 secretion was quantified by ELISA. ISL (5 and 10 μM) significantly reduced relative TGF-β1 levels in fBMFs supernatants (Fig. 3A), interrupting this key positive feedback mechanism. Western blot analysis was then conducted to examine canonical downstream effectors of TGF-β signaling. ISL downregulated protein expression of phosphorylated Smad2, COL1A1, and α-SMA, the hallmark contractile protein of myofibroblasts (Fig. 3B). These findings demonstrate that ISL targets multiple nodes of the TGF-β/Smad2 pathway, from ligand secretion to terminal differentiation markers.

Figure 1.

Cytotoxic effects of isoliquiritigenin (ISL) on the viability of buccal mucosal fibroblasts (BMFs) and fibrotic BMFs (fBMFs).

(A) Chemical structure of ISL. (B) Cell proliferation was examined utilizing MTT assay in response to various concentrations of ISL. IC50 values were calculated by GraphPad software.

Figure 2.

Inhibitory effects of isoliquiritigenin (ISL) on the collagen gel contraction, migration, and wound healing abilities of fibrotic buccal mucosal fibroblasts (fBMFs).

(A) Collagen gel contraction assay was performed to evaluate the contractility of fBMFs treated with various concentrations of ISL (0, 5, and 10 μM). The quantitative results of relative gel area (right) are shown. (B) Transwell migration assay was used to determine the migratory capacity of fBMFs. The percentage of migration ability relative to control (0 μM) are presented. (C) Wound healing assay was conducted to assess the lateral motility of fBMFs. Representative images captured at 0 and 48 h (top) and the quantitative analysis of wound healing ability (bottom) are shown. Data are mean ± SD (*P< 0.05 compared to the 0 μM Control group). Experiments were repeated three times.

Figure 3.

Isoliquiritigenin (ISL) attenuates TGF-β1 secretion and inhibits the TGF-β/Smad2 signaling pathway in fibrotic buccal mucosal fibroblasts (fBMFs).

(A) TGF-β1 secretion levels in the supernatants of fBMFs treated with indicated concentrations of ISL (0, 5, and 10 μM) were quantified by ELISA. (B) Protein expression levels of COL1A1, α-SMA, p-Smad2, and Smad2 in fBMFs were analyzed by Western blot following ISL treatment (0, 5, and 10 μM). GAPDH was used as an internal loading control for Western blot analysis. Data are presented as mean ± SD (*P< 0.05 compared to the 0 μM Control group).

Since arecoline is considered the primary etiological driver of OSF, BMFs were then co-treated with arecoline and ISL to examine whether ISL counteracts arecoline-induced fibrogenesis (Fig. 4). Arecoline significantly induced the contraction and mobile ability and enhanced the transwell migration ability in BMFs, recapitulating the hypercontractile and invasive phenotype observed in OSF lesions. ISL (5 and 10 μM) significantly attenuated arecoline-enhanced collagen gel contraction (Fig. 4A) and transwell migration (Fig. 4B), establishing ISL’s capacity to reverse areca nut-induced fibrotic remodeling.

Figure 4.

Isoliquiritigenin (ISL) suppresses arecoline-induced collagen gel contraction and buccal mucosal fibroblasts (BMFs).

(A) BMFs were treated with arecoline alone or co-treated with arecoline and indicated concentrations of ISL (5 and 10 μM) to evaluate cell contractility using the collagen gel contraction assay. The quantitative analysis of the relative gel area demonstrate that ISL dose-dependently inhibits arecoline-induced contraction. (B) Transwell migration assay was performed to assess the migratory capacity of BMFs under the same treatment conditions. The quantitative results indicate that ISL significantly reduces arecoline-enhanced migration. Data are expressed as mean ± SD. *P< 0.05 compared to the Control group. #P< 0.05 compared to the Arecoline-treated group.

Given the downregulation effect of ISL on the known pro-fibrotic regulator lncRNA NEAT1,18 we investigated whether NEAT1 overexpression could counteract ISL-mediated anti-fibrotic responses in OSF. BMFs were first treated with arecoline to establish fibrogenic state fBMFs. We then transfected fBMFs with NEAT1-overexpression vector prior to ISL treatment to dissect NEAT1’s functional role. qRT-PCR confirmed that NEAT1 expression was significantly reduced by ISL at 5 and 10 μM in fBMFs (Fig. 5A). As shown in Fig. 5B, NEAT1 overexpression markedly enhanced collagen gel contraction, whereas ISL treatment partially but significantly reversed this pro-contractile effect. These gain-of-function studies demonstrate that NEAT1 acts downstream of ISL to drive myofibroblast contractility, positioning NEAT1 as a critical mediator of ISL’s therapeutic effects in OSF.

Figure 5.

Isoliquiritigenin (ISL) suppresses NEAT1 expression, and NEAT1 overexpression rescues ISL-inhibited myofibroblast contractility in arecoline-induced fibrotic buccal mucosal fibroblasts (fBMFs).

(A) Relative NEAT1 expression levels in fBMFs treated with various concentrations of ISL (0, 5, and 10 μM) were determined by qRT-PCR. ISL significantly downregulated NEAT1 expression in a dose-dependent manner. (B) fBMFs were transfected with a NEAT1 overexpression vector or an empty control vector in the presence or absence of ISL to evaluate the functional role of NEAT1. Collagen gel contraction assay results (relative gel area) demonstrate that NEAT1 overexpression significantly reverses the inhibitory effect of ISL on fBMF-mediated gel contraction. Data are presented as mean ± SD. *P< 0.05 compared to the 0 μM or Control group. #P< 0.05 compared to the ISL + Vector group.

Discussion

This study shows that ISL preferentially inhibits the proliferation of patient-derived fBMFs over normal BMFs and suppresses collagen gel contraction and migration, indicating broad attenuation of myofibroblast-like functions critical for OSF progression. Similar inhibition of myofibroblast activities has been reported in our previous studies targeting other pro-fibrotic lncRNAs.11,21,22 For instance, silencing MIAT in arecoline-treated BMFs reduced contractility and motility, supporting the concept that targeting fibroblast activities is a rational therapeutic strategy in OSF.24 The ability of ISL to reverse arecoline-induced increases in contraction and migration further links its pharmacologic effects to the major etiological agent of OSF, strengthening its translational relevance.

Our data demonstrate that ISL suppresses TGF-β1 secretion, decreases Smad2 phosphorylation, and downregulates downstream effectors COL1A1 and α-SMA in fBMFs, indicating disruption of the canonical TGF-β/Smad fibrotic axis. These findings are consistent with previous work in high glucose-stimulated human mesangial cells, where ISL reduced TGF-β1 receptor expression, inhibited Smad2/4 activation, restored Smad7, and decreased CTGF and collagen production. Likewise, in TGF-β1-treated MRC-5 lung fibroblasts, ISL attenuated proliferation, migration, and expression of α-SMA, COL1A1, and fibronectin by suppressing PI3K/AKT/mTOR signaling and enhancing autophagy, further confirming its role as a negative regulator of TGF-β-driven fibrogenesis. 16,17 Taking together, ISL downregulates TGFB1, MMP9, EGFR, TNF, and MAPK3 in OSF-like models. These results position ISL as a multi-target inhibitor of TGF-β/Smad-dependent matrix remodeling in the oral mucosa.

Accumulating evidence suggests that NEAT1 is tightly intertwined with TGF-β/Smad signaling in fibrotic diseases. In cardiac and pulmonary fibrosis models, NEAT1 promotes TGF-β-driven fibroblast activation by recruiting EZH2 to repress Smad7 or by sponging anti-fibrotic miRNAs, thereby sustaining Smad2/3 phosphorylation and collagen gene expression.14,15 Together with our findings that ISL simultaneously decreases TGF-β1 secretion, Smad2 activation, and NEAT1 expression, and that NEAT1 overexpression partially rescues ISL-mediated inhibition of collagen contraction, these data support a model in which NEAT1 functions as a lncRNA amplifier downstream of TGF-β/Smad2 in OSF-derived fBMFs. Under this model, ISL would dampen the TGF-β autocrine loop and Smad2 phosphorylation, thereby constraining NEAT1-dependent myofibroblast contractility.13,18 However, whether NEAT1 is directly induced by TGF-β1 in fBMFs, or whether blockade of TGF-β receptors attenuates ISL-regulated NEAT1 expression, remains to be experimentally validated.

NEAT1 has been identified as a pivotal lncRNA promoting myofibroblast activities in OSF via miR-760/TPM1 and Wnt/β-catenin signaling, and its knockdown reduces α-SMA, COL1A1, collagen contraction, and transwell migration in fBMFs.13 Consistent with these reports, the current study shows that ISL downregulates NEAT1 expression in fBMFs and that NEAT1 overexpression enhances collagen gel contraction, recapitulating a pro-fibrotic phenotype. Importantly, ISL partially yet significantly reverses NEAT1-driven hypercontractility, suggesting that NEAT1 lies downstream, at least in part, of ISL’s anti-fibrotic signaling and may serve as a lncRNA target of phytochemical modulation. This is consistent with emerging evidence that phytochemicals can modulate lncRNA—miRNA—mRNA networks in fibrotic diseases and supports the broader concept of employing natural compounds to reprogram pathogenic lncRNA circuits.25,26

Emerging evidence indicates that lncRNAs extensively crosstalk with TGF-β/Smad signaling in fibrotic disorders, including OSF, where OSF-associated lncRNAs such as FENDRR,11 LINC00084,12 MIAT,24 XIST,27 and NEAT126 regulate myofibroblast activation by modulating TGF-β1 production, Smad phosphorylation, or Smad-dependent transcriptional programs. NEAT1 fits within this framework as a nodal regulator that integrates areca nut-derived stimuli with downstream cytoskeletal remodeling and ECM gene expression. In other organ systems, NEAT1 enhances TGF-β-driven fibrosis by recruiting chromatin modifiers to Smad-related promoters or by acting as a competing endogenous RNA for miRNAs targeting Smad3 and other profibrotic effectors, suggesting that lncRNA-Smad circuits are conserved across tissues.14,28 Our finding that ISL concomitantly attenuates TGF-β1/Smad2 signaling and NEAT1-driven contractility therefore suggests that phytochemical interventions may reprogram lncRNA-Smad networks to more effectively disrupt fibrotic pathways in OSF.

Current management of OSF mainly focuses on habit cessation and symptomatic relief, with limited options to directly reverse fibrosis or reduce malignant transformation risk.29 By simultaneously attenuating TGF-β1 autocrine signaling, Smad2 activation, ECM gene expression, and NEAT1-driven myofibroblast functions, ISL addresses several central nodes in OSF pathogenesis rather than acting on a single downstream marker. Given that TGF-β/Smad signaling and pro-fibrotic lncRNAs also contribute to epithelial—mesenchymal transition and progression toward oral squamous cell carcinoma,30 ISL may hold promise not only for mitigating fibrosis but also for modulating the carcinogenic microenvironment associated with areca nut chewing. However, in vivo validation in OSF animal or organoid models, pharmacokinetic profiling in oral tissues, and assessment of potential off-target effects will be essential before clinical translation.

Several limitations should be acknowledged in this study. First, the present work is confined to in vitro fBMF and normal BMF models and does not capture contributions from epithelial, immune, or endothelial cells known to participate in OSF pathogenesis. Second, although NEAT1 appears to mediate part of ISL’s anti-fibrotic effects, the precise upstream events, such as whether ISL directly regulates NEAT1 transcription, affects NEAT1 nuclear bodies, or modulates specific NEAT1—miRNA—mRNA axes, remain to be elucidated. Third, the relationship between ISL-induced changes in TGF-β/Smad2 and NEAT1 requires further dissection using combined knockdown/overexpression strategies and pathway inhibitors. Future studies should therefore include in vivo OSF models with areca nut exposure, evaluation of ISL in combination with current clinical interventions, and unbiased transcriptomic or lncRNA profiling to map the broader regulatory network targeted by ISL.

Funding Statement

This study was supported by grants from the Chung Shan Medical University and Changhua Christian Hospital (grant number: CSMU-CCH-114-02) in Taiwan.