Obesity, resulting from chronic energy imbalance due to excessive caloric intake and insufficient physical activity, is a major public health concern that triggers widespread metabolic disturbances. These include systemic inflammation, dyslipidemia, and insulin resistance[1], primarily driven by the secretion of pro-inflammatory cytokines and dysfunctional adipocyte metabolism. Such disruptions foster the development of numerous chronic diseases, among which non-alcoholic fatty liver disease (NAFLD) has emerged as a leading concern[2]. Over the past two decades, the prevalence of NAFLD within the spectrum of chronic liver diseases has risen significantly from 47% to 75% underscoring its expanding global impact[3].

NAFLD represents a continuum of hepatic disorders, beginning with simple steatosis or non-alcoholic fatty liver (NAFL), characterized by lipid accumulation in hepatocytes without significant inflammation. In some individuals, this condition progresses to non-alcoholic steatohepatitis (NASH), a more severe phenotype marked by hepatocellular injury, inflammation, and fibrotic remodeling. Approximately 20% of NAFLD patients develop NASH, which markedly elevates the risk of advanced complications, including cirrhosis and hepatocellular carcinoma [4, 5]. Central to this pathological transition is persistent hepatic inflammation, which activates hepatic stellate cells (HSCs) the primary fibrogenic cells in the liver[6]. Once activated, HSCs differentiate into myofibroblast-like cells and secrete excess extracellular matrix (ECM) proteins, contributing to liver fibrosis[7]. Fibrosis severity remains the most robust histological predictor of liver-related mortality in NASH, yet effective anti-fibrotic therapies remain elusive[8, 9]. The severity of fibrosis is the sole histologic characteristic that predicts liver-related mortality in patients with NASH, and it reflects the course of the illness[10]. Treatment options for liver fibrosis are limited. Therefore, new anti-fibrotic medications are desperately needed to stop the disease's progression and effects.

Amid this therapeutic gap, growing attention has turned to naturally occurring compounds with anti-inflammatory and metabolic regulatory properties. Apigenin (5, 7-dihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one), a flavonoid found abundantly in chamomile, parsley, celery, and citrus fruits, has demonstrated considerable promise[11]. 1 Known for its antioxidant, anti-inflammatory, and lipid-lowering effects, apigenin has shown efficacy in ameliorating obesity-related metabolic dysfunctions. Preclinical studies suggest that apigenin improves hepatic lipid profiles by downregulating lipogenesis-related enzymes such as SREBP-1c and ACC, while promoting fatty acid oxidation through the activation of PPARα and CPT1[11]. It further attenuates oxidative stress by enhancing the Nrf2-mediated expression of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), and by reducing lipid peroxidation through the inhibition of 5-lipoxygenase (5-LOX). In addition, apigenin plays a role in preserving mitochondrial function, maintaining electron transport chain stability, and supporting cellular energy metabolism [12]. Furthermore, recent research has shown that apigenin has anti-obesity and anti-diabetic properties[13]. In mice given an HFD, it also enhanced hepatic lipid metabolism, glucose tolerance, and glucose homeostasis[14].

To complement biochemical and molecular evaluations, dielectric spectroscopy (DS) has emerged as a novel, non-invasive modality for assessing tissue integrity and pathology. By measuring the frequency-dependent electrical properties of biological tissues, DS provides critical insights into membrane stability, hydration status, and cellular composition. In the context of obesity-induced liver dysfunction[15]. DS has proven capable of detecting early pathological changes such as lipid accumulation, fibrotic remodeling, and ion transport impairment. Alterations in dielectric properties specifically increased impedance, reduced conductivity, and a diminished dielectric constant are indicative of compromised liver tissue, often linked to oxidative stress and ECM deposition[16]. Particularly, shifts in β-dispersion frequencies (1 kHz–10 MHz) reflect membrane degradation and fibrosis progression[17]. Notably, the therapeutic modulation of these parameters by apigenin further reinforces its role in restoring hepatic bioelectrical and functional homeostasis[18].

Despite the compelling biochemical and physiological benefits associated with apigenin, its specific influence on liver fibrosis in the context of obesity remains underexplored. Therefore, this study aims to investigate the potential of apigenin as a therapeutic agent against obesity-induced liver fibrosis. By integrating biochemical assays, oxidative stress profiling, and dielectric spectroscopy, the research seeks to elucidate the mechanisms through which apigenin may mitigate hepatic injury and restore metabolic and bioelectrical balance. This approach not only advances understanding of apigenin’s multifaceted therapeutic effects but also highlights its potential as a viable strategy for combating obesity-related liver pathology.

Obese rats exhibited a significant increase in body weight, with a 96.4% elevation relative to the normal control group. Oral administration of apigenin (APG) at doses of 25 mg/kg and 50 mg/kg resulted in reductions in body weight by 8.0% and 10.8%, respectively, compared to untreated obese rats. However, neither dose fully restored body weight to normal levels. Similarly, the body mass index (BMI) increased by 64.6% in obese rats relative to controls. Treatment with APG at 25 mg/kg and 50 mg/kg led to reductions in BMI by 8.9% and 24.7%, respectively, with the higher dose yielding a significantly greater effect than the lower dose (p < 0.05). These findings are illustrated in Fig. 1.Morover, waist circumference increased in all treatment groups, and no significant differences were observed between obese group and O + APG of both doses.

Obesity markedly impaired liver function, as evidenced by substantial elevations in serum glutamic pyruvic transaminase (SGPT) and serum glutamic oxaloacetic transaminase (SGOT) levels—by 452% and 140.9%, respectively—accompanied by a 60.7% reduction in serum albumin levels compared to the control group. Treatment with apigenin (APG) at 25 mg/kg resulted in decreases in SGPT and SGOT levels by 45.3% and 22.9%, respectively. The higher dose of 50 mg/kg produced more pronounced effects, with reductions of 71.1% and 47.4% in SGPT and SGOT, respectively. Additionally, albumin levels increased by 17.9% with 25 mg/kg APG and by 76% with the 50 mg/kg dose. The 50 mg/kg treatment significantly outperformed the lower dose in improving all measured liver function parameters (p < 0.05), with albumin levels approaching those of the control group. These findings are illustrated in Fig. 2.

The bars represent the mean ± standard error of the mean (n = 6). Statistical analysis was conducted using one-way ANOVA, followed by Tukey–Kramer multiple comparisons within the designated time intervals, with significant levels indicated on the horizontal bars of pairwise comparisons.

Obesity triggered pronounced oxidative stress and systemic inflammation, as evidenced by a 163% increase in monocyte chemoattractant protein-1 (MCP-1) and a 471% elevation in malondialdehyde (MDA) levels, alongside marked reductions in antioxidant defenses—superoxide dismutase (SOD) and glutathione (GSH) levels decreased by 73.9% and 73.3%, respectively, compared to the control group. Administration of apigenin (APG) at 25 mg/kg attenuated these effects, reducing MCP-1 and MDA by 19.4% and 37%, respectively, while enhancing SOD and GSH levels by 93.8% and 97.3%. A more pronounced therapeutic effect was observed with the 50 mg/kg dose, which reduced MCP-1 and MDA by 32.7% and 49.1%, and elevated SOD and GSH levels by 169.2% and 137.7%, respectively. The higher dose of APG was significantly more effective than the lower dose (p < 0.05), although neither treatment fully restored these parameters to baseline levels (Fig. 3). Furthermore, apigenin significantly mitigated the obesity-induced elevations in pro-inflammatory cytokines, including interleukin-6 (IL-6), C-reactive protein (CRP), and tumor necrosis factor-alpha (TNF-α), as detailed in Table 1.

Obesity markedly activated the hepatic STAT3 signaling pathway, with total STAT3 and phosphorylated STAT3 (p-STAT3) levels increasing by 198.5% and 377%, respectively, compared to normal controls. Concurrently, nuclear factor erythroid 2–related factor 2 (NRF2), a key regulator of cellular antioxidant defense, was suppressed by 60.5%. Treatment with apigenin (APG) at a dose of 25 mg/kg resulted in moderate reductions in STAT3 and p-STAT3 levels by 30.4% and 21%, respectively, and enhanced NRF2 expression by 53.3%. The higher dose of 50 mg/kg APG elicited a more pronounced response, reducing STAT3 and p-STAT3 levels by 41% and 50%, respectively, and elevating NRF2 levels by 98.4%. The 50 mg/kg dose produced significantly greater improvements across all parameters (p < 0.05), although full normalization of these markers was not achieved (Fig. 4).

Histological examination of liver tissues revealed normal hepatic architecture in the negative control group. The liver parenchyma was organized into classical hepatic lobules composed of hepatocyte cords radiating from a central vein toward the periphery, with intact sinusoidal architecture (Fig. 5A). In contrast, the obese positive control group exhibited pronounced histopathological alterations, including distorted lobular architecture, dilated central veins, leukocytic infiltration, scattered lipid droplets, markedly dilated sinusoidal spaces—some with hemorrhagic foci—and hepatocellular degeneration, such as hydropic changes (Fig. 5B). Treatment with apigenin at a dose of 25 mg/kg resulted in notable histological improvement. Liver sections displayed mild sinusoidal dilatation, fewer lipid-laden cells, and largely preserved hepatocyte morphology (Fig. 5C). The group treated with 50 mg/kg apigenin demonstrated near-normal hepatic architecture, with minimal fat accumulation and restoration of tissue integrity (Fig. 5D).

Fibrosis assessment using Masson’s Trichrome staining and graded according to the Ishak scoring system further supported these findings. The negative control group exhibited no fibrosis (F0) (Fig. 6A). The obese group showed severe fibrosis, particularly pericentral and periportal (F4) (Fig. 6B). Rats treated with 25 mg/kg apigenin exhibited moderate fibrosis (F2) (Fig. 6C), while those receiving 50 mg/kg showed only minimal fibrotic changes localized around the central vein (F1) (Fig. 6D). The Fig. 7 illustrated the fibrosis scoring in different treatment groups.The treatment of obese rats with with 25 mg or 50mg/kg apigenin reduced the fibrosis scoring significantly.

Figure (5): A: photomicrograph of Normal Control group showing normal structure of the classical hepatic lobule. B:photomicrograph of hepatic tissue of Obese group showing marked dilated congested central vein (star), hepatocytes with multiple scattered lipid droplets (steatosis) (orange arrow), scattered hepatocytes hydropic degeneration (yellow arrow), dilated sinusoidal spaces with hemorrhage (black arrow) and scattered inflammatory cells (red arrow).C: photomicrograph of hepatic tissue of Obese + APG (25 mg/kg) group showing dilated central vein with minimal steatosis orange arrow), and mild dilated sinusoidal spaces D: photomicrograph of hepatic tissue of Obese + APG (50 mg/kg) group showed normal hepatic tissue with minimal fat cells deposits (orange arrow) (H&E, x200).

Figure (7): Effect of APG on obese liver. Values are expressed as mean ± SD. Statistical analysis was carried out by one way ANOVA followed by Tukey’s multiple comparisons test against control group. APG-25 (Apigenin 25 mg/kg), APG-50 (Apigenin 50 mg/kg).

Figure 8 presents the variation in real impedance (Zs′) as a function of frequency across the experimental groups. Impedance decreased with increasing frequency across all groups, consistent with the expected electrical behavior of biological tissues, where ionic conduction dominates at lower frequencies, while capacitive effects become more prominent at higher frequencies. The Obese group exhibits the highest impedance at all frequencies, particularly at lower frequencies, where values exceeded 2.5×10⁵ Ohms. The Control group demonstrated the lowest impedance values across all frequencies. The Low-dose APG group displayed a moderate reduction in impedance relative to the Obese group but remained slightly elevated compared to the Control group. The High-dose APG group demonstrates a further reduction in impedance, approaching control values.

Figure 9 illustrates the real conductivity (σ′) of liver tissues as a function of frequency. Conductivity increased with frequency in all groups, following a characteristic power-law behavior. The Control group exhibits the highest real conductivity across all frequencies. The Obese group shows a significant increase in conductivity compared to the control group, particularly at lower frequencies. The Low-dose APG group exhibits the lowest conductivity at all frequencies, while the High-dose APG group displays intermediate conductivity values between the control and obese groups. At higher frequencies, conductivity in all groups approached a steady-state saturation point.

Figure 10 displays the variation in the dielectric constant (k) as a function of frequency. At low frequencies (< 10³ Hz), the dielectric constant was highest across all groups, indicating strong polarization effects. As frequency increased, the dielectric constant decreased for all groups. The control group shows a moderate dielectric response, maintaining a higher dielectric constant than the obese group but lower than the treated groups. The obese group showed a consistently lower dielectric constant compared to the control group across all frequencies. Low-dose APG group exhibited an increased dielectric constant compared to both the control and obese groups while the High-dose APG group demonstrated the highest dielectric constant values across all frequencies.

Figure 11 depicts the dielectric loss (ε'') as a function of frequency, representing the energy dissipation within liver tissues due to resistive and polarization effects. Dielectric loss was highest at low frequencies (< 10³ Hz and progressively decreased with increasing frequency across all groups. The control group exhibited the highest dielectric loss at low frequencies, reaching approximately 1.8 × 10⁸ at 10⁻¹ Hz. The obese group showed significantly lower dielectric loss than the control group across all frequencies. Low-dose APG group presented a higher dielectric loss than the obese group but remained lower than the control group at low frequencies. High-dose APG group exhibited a further increase in dielectric loss, closely approaching control values.

Figure 12 highlights the Cole-Cole plot (Nyquist plot) for liver tissues across the experimental groups, illustrating the dielectric relaxation behavior of cellular structures. The plots exhibit a characteristic semicircular profile, indicative of biological tissue impedance properties. The peak of each semicircle corresponds to the maximum reactance (Zs′′), reflecting capacitive behavior and tissue polarization effects. The Control group displayed the smallest semicircle, signifying the lowest impedance. The Obese group exhibited the largest semicircle, indicating the highest impedance values among all groups. The Low-dose and High-dose APG groups displayed intermediate behaviors, with the High-dose group showing a more pronounced reduction in impedance compared to the Low-dose group, reinforcing the dose-dependent therapeutic effect of APG.

This study underscores the multifaceted therapeutic efficacy of apigenin (APG) in ameliorating obesity-induced hepatic dysfunction, oxidative stress, inflammation, and fibrosis. Through the integration of biochemical analyses, histopathological evaluations, and dielectric spectroscopy, a comprehensive assessment of APG’s hepatotherapeutic potential was achieved. The observed dose-dependent enhancements in metabolic indices, antioxidant defenses, and inflammatory profiles were closely mirrored by substantial histological improvement and normalization of liver bioelectrical parameters. Notably, dielectric spectroscopy revealed distinct alterations in membrane polarization, conductivity, and impedance biophysical changes that strongly correlated with underlying molecular and cellular recovery. These findings highlight APG’s ability to simultaneously modulate metabolic, structural, and electrophysiological disruptions characteristic of nonalcoholic fatty liver disease (NAFLD), while positioning dielectric spectroscopy as a valuable, non-invasive tool for monitoring liver pathology and evaluating therapeutic responses[21, 22].

Body weight and body mass index (BMI) were significantly elevated in the obese control group, reflecting hallmark anthropometric changes typically associated with obesity. These results are in agreement with prior studies that documented comparable increases in BMI and body weight in both diet-induced obese animal subjects[19, 23]. Notably, apigenin (APG) treatment resulted in a pronounced reduction in both parameters, suggesting a potential anti-obesity effect. This observation aligns with earlier research demonstrating the efficacy of APG in mitigating weight gain and improving anthropometric indices in experimental models of obesity[24, 25].

Obesity induced by a high-fat, high-sucrose diet triggered significant hepatic oxidative stress, as evidenced by elevated malondialdehyde (MDA) levels and depleted antioxidant defenses, including glutathione (GSH) and superoxide dismutase (SOD)[26]. These biochemical alterations compromise membrane integrity through lipid peroxidation, which disrupts the phospholipid bilayer, causing ion leakage and altered transmembrane potential[27, 28]. This was reflected in the dielectric spectroscopy data, where obese rats demonstrated elevated real impedance (Zs′) and conductivity (σ′), particularly at low frequencies, suggesting extracellular ion accumulation due to membrane rupture. Conversely, increased impedance at higher frequencies indicated impaired capacitive storage, likely resulting from damage to intracellular organelles such as mitochondria and the endoplasmic reticulum[29, 30].

In this study, rats fed a high-fat diet (HFD) showed significant downregulation of hepatic NRF2, indicating impaired antioxidant defenses[31]. An observation consistent with prior findings linking reduced NRF2 activity to obesity-induced liver injury[32]. Apigenin (APG) supplementation dose-dependently restored NRF2 expression, aligning with previous studies, including Aldayel et al. that highlight APG’s antioxidant role via NRF2 activation[33].

Concurrently, Table 1 illustrates that the levels of inflammatory markers IL-6, TNF-α, and CRP were significantly elevated in the obese group, underscoring their pivotal role in the pathogenesis of nonalcoholic steatohepatitis (NASH). Apigenin (APG) treatment elicited a marked, dose-dependent suppression of these pro-inflammatory cytokines. Specifically, administration of 25 mg/kg APG resulted in moderate reductions in IL-6, TNF-α, and CRP levels, while the higher dose of 50 mg/kg effectively normalized these markers to values approaching those of the control group. This potent anti-inflammatory effect was accompanied by a concurrent decrease in phosphorylated STAT3 levels, a key transducer in the IL-6 signaling cascade that drives hepatic fibrogenesis. These observations align with previous research demonstrating APG’s efficacy in attenuating inflammatory signaling and mitigating liver fibrosis[20, 34].

These molecular and biochemical improvements were mirrored in dielectric outcomes, with the 50 mg/kg APG group showing near-normalization of Zs′ and σ′ and significant recovery of ε′ and ε″ (Figs. 8–11). Correspondingly, histological analysis confirmed a reduction in hepatic fibrosis (Figs. 5, 6), further supporting the restoration of membrane and extracellular matrix conductivity[29]. Collectively, these findings highlight a strong correlation between APG’s redox-modulatory actions and the restoration of hepatic bioelectrical function[35]. Moreover, dielectric spectroscopy emerges as a sensitive, non-invasive biophysical modality capable of capturing subtle structural and functional changes in liver tissue during disease progression and therapeutic intervention.

The dielectric constant (ε′) and dielectric loss (ε″) serve as indicators of membrane polarization capacity and energy dissipation, respectively. In this study, obesity induced a marked decline in both ε′ and ε″, suggesting reduced membrane fluidity, impaired dipolar relaxation, and intracellular edema. These disruptions reflect underlying oxidative damage and structural degradation in hepatocyte membranes[36]. Apigenin (APG) administration, particularly at the 50 mg/kg dose, significantly reversed these changes. Restoration of ε′ and ε″ in treated groups indicates improved membrane lipid bilayer organization, facilitating charge separation and lowering resistive energy losses. These changes suggest that APG contributes to stabilizing membrane dynamics, potentially by reinforcing phospholipid packing, a mechanism observed in other bioactive flavonoids such as quercetin[37].

Nyquist plots further support these findings by visualizing the frequency-dependent capacitive behavior of liver tissue. Obese rats displayed flattened, enlarged semicircular arcs hallmarks of poor charge storage and increased resistivity. APG-treated groups exhibited more compact semicircles, reflecting improved capacitive responses and ionic mobility. These improvements were consistent with restored membrane function and reduced fibrotic interference, as observed histologically[38, 39].

Mechanistically, APG-mediated improvements in dielectric parameters paralleled molecular and biochemical corrections. The high-dose APG group demonstrated significant reductions in malondialdehyde (MDA) and concurrent elevations in key antioxidant enzymes such as glutathione (GSH) and superoxide dismutase (SOD). These changes reflect restored redox balance, further supported by the upregulation of nuclear factor erythroid 2–related factor 2 (NRF2) and downregulation of phosphorylated STAT3 (p-STAT3), mitigating inflammatory and fibrotic signaling cascades[40]. This dielectric normalization coincided with histological improvements, as fibrosis regressed from Ishak score F4 to near-normal lobular structure (F1), emphasizing the interdependence of structural integrity and membrane bioelectrical behavior.

Importantly, dielectric improvements were also evident in the 25 mg/kg APG group, which exhibited only moderate fibrosis histologically (F2)[41]. This suggests that dielectric spectroscopy may be capable of detecting early therapeutic responses, identifying subcellular and ionic corrections before macroscopic histological restoration becomes evident. These observations position dielectric spectroscopy as a sensitive, non-invasive tool for staging NAFLD and evaluating pharmacological intervention[42].

Recent investigations have confirmed that reduced dielectric constant (ε′) is strongly associated with lipid droplet accumulation and compromised membrane polarization, while increased low-frequency conductivity (σ′) corresponds with extracellular sodium leakage caused by membrane rupture and edema[43–45]. APG’s capacity to restore these dielectric properties, in parallel with reduced liver enzymes and improved BMI (Figs. 1, 2), reinforces its systemic hepatoprotective efficacy in obesity-associated liver disease[21, 46–48].

Furthermore, frequency-resolved dielectric profiles offer mechanistic insight into the progression of hepatic recovery. Restoration of low-frequency conductivity indicates early extracellular matrix repair and improved ion channel functionality, potentially linked to collagen breakdown and fibrosis attenuation. In contrast, normalization of high-frequency parameters reflects intracellular repair processes, including the functional recovery of mitochondrial and endoplasmic reticulum membranes, both crucial for cellular electrochemical stability[49]. This dual-phase recovery underscores the multifactorial actions of APG in correcting oxidative, inflammatory, and structural abnormalities in NAFLD[26, 50, 51].

It appears that apigenin combats obesity-induced liver injury through simultaneous modulation of oxidative stress, inflammatory signaling pathways, and the preservation of bioelectrical membrane integrity. Its dual regulatory mechanism upregulating NRF2-mediated antioxidant defenses while suppressing STAT3-driven inflammatory responses was evidenced by normalization of biochemical markers, attenuation of hepatic fibrosis, restoration of liver histoarchitecture, and correction of dielectric properties. These comprehensive findings highlight apigenin’s potential as a natural therapeutic agent for managing (NAFLD) and its fibrotic complications. Ultimately, this study underscores the utility of dielectric spectroscopy as an innovative, non-invasive modality capable of detecting early bioelectrical and structural alterations in hepatic tissue, effectively bridging membrane polarization, impedance, and conductivity measurements with histopathological and molecular parameters.