Protective effects of kaempferol against diet-induced metabolic disorders

Abstract

The study examines the effects of kaempferol on obesity and steatotic liver disease in C57BL/6J mice using an Indian diet-mimicking experimental animal model. Obesity and metabolic dysfunction-associated steatotic liver disease prevalence is rising in India due to high-calorie dietary habits. The condition starts with simple hepatic fat accumulation and may progress to fibrosis and cirrhosis. An Indian high-fat diet significantly induced the macrovesicular as well as microvesicular steatosis in mice with increased serum transforming growth factor beta level. Kaempferol, a natural flavonoid, is generally known for its lipid-lowering potential. In this study, kaempferol treatment for four weeks reduced body weight, steatosis, and hepatic inflammation induced by the Indian high-fat diet. Moreover, kaempferol stabilized the liver injury markers and reduced the triglyceride levels. In conclusion, the study demonstrated the potential of kaempferol as a therapeutic agent for managing metabolic dysfunction-associated steatotic liver disease associated with high-calorie region-specific dietary patterns.

Key Words: Fibrosis; Indian high-fat diet; Kaempferol; Liver; Metabolic-dysfunction-associated steatotic liver disease; Transforming growth factor beta

Core Tip: The increasing prevalence of obesity and metabolic dysfunction-associated steatotic liver disease in the Indian population is closely linked to high-fat, high-sugar dietary patterns and a sedentary lifestyle. Developing region-specific dietary models can aid in effective preclinical screening of therapeutic agents. In this context, Kaempferol, a natural flavonoid with antioxidant, anti-inflammatory, and lipid-regulating properties, shows promising potential as a nutraceutical strategy for the management of metabolic dysfunction-associated steatotic liver disease.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a progressive liver disease that is closely linked with type 2 diabetes, obesity, and other metabolic disorders[1]. Due to the high global prevalence of obesity and type 2 diabetes in recent decades, MASLD, which affects 38% of the global population, is now thought to be the most common cause of chronic liver disease worldwide. By 2040, the prevalence of MASLD is predicted to rise to 55.2% globally[2,3]. Furthermore, the majority of liver-related clinical outcomes in patients with the progressive form of MASLD, also referred to as metabolic dysfunction-associated steatohepatitis (MASH), are thought to affect 5% of the population globally[4]. The progression of MASH is multifactorial and can be affected by genetic predisposition, metabolic state, and other environmental factors. However, research has shown that the development and progression of hepatic fibrosis in patients with MASLD-MASH are primarily driven by insulin resistance and type 2 diabetes[5,6]. Insulin resistance upregulates the hepatic lipid metabolism, thereby elevating the fatty acid flux to the liver, promoting lipogenesis and hepatic fat accumulation[7,8]. The fat buildup within hepatocytes leads to the production of harmful free radicals that trigger the release of pro-inflammatory cytokines such as transforming growth factor beta1 (TGF-β1), tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1[9,10]. Oxidative stress and inflammation lead to the activation of hepatic stellate cells (HSCs). HSCs activation produces extracellular matrix (ECM) proteins such as collagens I and III, resulting in hepatic fibrosis[11,12]. Numerous clinical studies are being conducted to assess the range of potential treatments for MASLD. Recently, resmetirom (Rezdiffra) and semaglutide (Wegovy) were the first drugs approved by the United States Food and Drug Administration for MASH and moderate-to-advanced fibrosis[13-15]. However, these medications are only recommended for the chosen fibrotic patients. Currently, the most effective clinical strategy relies on dietary modifications and lifestyle management.

Natural molecules have drawn significant attention for the treatment of MASLD because of their multifactorial mode of action, in contrast to the single-targeted approach of synthetic drugs[16]. Kaempferol, a flavonoid, is widely present in green vegetables, fruits, and tea. It is generally recognized as a safe molecule and well known for its lipid-lowering, anti-inflammatory, and antioxidant properties[17,18]. Despite increasing evidence on the advantages of natural flavonoids, their efficacy in MASLD models that account for region-specific dietary patterns remains poorly understood. The study by Nair et al[19] fills this gap by evaluating the efficacy of kaempferol against obesity and hepatic steatosis in C57BL/6J mice fed with an Indian diet-mimicking regimen. The findings provide key insights into the potential of kaempferol for the management of MASLD, particularly among populations following Indian dietary patterns.

DIET-INDUCED MASLD MODEL MIMICKING THE INDIAN DIET

The shift in diet and lifestyle due to rapid urbanization and development led to a severe nutrition transition in India, leading to a drastically increasing prevalence of MASLD. Unbalanced dietary composition and habitual eating patterns are recognized as the major contributors to MASLD-related risk[20]. Many clinical and preclinical studies have shown that high-fat and carbohydrate intake, particularly fructose, promote hepatic de novo lipogenesis, leading to hepatic steatosis[21]. Evidence from Indian and broader Asian populations highlights that the increased prevalence of liver-related diseases is strongly related to the region-specific dietary patterns. Western diets are typically dominated by animal fat, processed meat, and fructose-rich sugary beverages, which strongly promote MASLD and obesity[22,23]. However, Indian high-fat dietary patterns typically include a mix of refined carbohydrates, vegetable oils, ghee, and junk foods, often with moderate or lower protein intake[24,25]. A rapid nutritional transition marked by an increase in the consumption of processed foods, refined carbohydrates, and high-energy diets is reported by many region-specific studies in South Asia. Notably, even non-obese individuals in these populations have been found to have a high chance of MASLD, highlighting the significance of creating specific diet-based models and interventions tailored to the needs of these populations[26,27]. Therefore, the Indian diet-mimicking regimen fed to the dietary model is metabolically relevant and may more accurately reflect the pathophysiological aspects of MASLD in the Indian population with regional nutritional characteristics.

The pattern and course of hepatic fat buildup, inflammatory responses, and related metabolic abnormalities are usually influenced by dietary composition[28]. The study by Nair et al[19] provides a useful platform for preclinical screening of therapeutic agents, especially those targeting MASLD, which is influenced by Indian dietary patterns. Triglyceride buildup in hepatocytes is the primary characteristic of MASLD. Hepatic triglyceride accumulation can lead to oxidative stress, organelle dysfunction, and other pathophysiological alterations, thereby promoting the progression of MASLD to MASH and liver fibrosis[29-31]. Genetic predisposition, dietary practices, and socioeconomic status all contribute to the prevalence of MASLD, which shows significant variation across Indian regions[32]. Compared to rural areas, the urban population has a higher prevalence of MASLD, largely due to sedentary lifestyles and high-calorie diets. However, rapid dietary and lifestyle changes, such as increased consumption of processed foods and decreased physical activity, are also occurring in rural communities, which may be contributing to the increasing prevalence of metabolic disorders[29,30,33]. The reduced consumption of a traditional fiber-rich diet and an increase in ultra-processed foods, high in unhealthy fats and refined carbohydrates, has been linked to the increase of MASLD in Indian[34]. These dietary alterations and lifestyle modifications are further linked to the metabolic disturbance that leads to disease progression. The altered metabolism of free fatty acids, which are substrates for triglyceride formation, is particularly significant, leading to the production of reactive oxygen species and endoplasmic reticulum stress[35]. Beyond causing direct macromolecular damage, reactive oxygen species are potent inflammatory second messengers that activate resident Kupffer cells and recruit peripheral immune cells[36].

The increased secretion of pro-inflammatory cytokines, such as TNF-α and IL-6, activates the resulting inflammatory cascade, which not only exacerbates hepatocellular damage but also upregulates the fibrogenic response[37]. A profibrotic cytokine, TGF-β1, promotes the progression of inflammation and fibrosis from simple steatosis. One of the primary mechanisms by which TGF-β1 induces histological damage is through the activation of HSCs. TGF-β1 triggers quiescent HSCs to undergo trans differentiation into cells resembling myofibroblasts in response to liver damage[38-40]. These activated cells became the main source of fibrotic markers, including collagen I and III, fibronectin, and other ECM proteins. In the meantime, TGF-β1 suppresses the upregulation of matrix metalloproteinases while elevating tissue inhibitors of metalloproteinases, thereby reducing the ECM degradation. These alterations, along with ECM breakdown and synthesis, lead to fibrotic scar formation[41,42]. Nair et al[19] reported significantly increased serum TGF-β1 levels in Indian high-fat diet (HFD)-fed mice compared with the normal group. The histopathological analysis in liver tissue revealed the presence of fat vacuoles along with small inflammatory cell clusters, and the presence of inflammatory loci was observed in HFD-fed mice.

HEPATOPROTECTIVE ROLE OF KAEMPFEROL IN INDIAN HFD-INDUCED STEATOSIS

Nair et al[19] revealed that kaempferol exerts hepatoprotective effects against the Indian HFD-induced steatosis model. Kaempferol treatment significantly reduced body weight gain and biochemical markers in HFD-fed mice. Histological analysis further revealed the substantial decrease in both macrovesicular and microvesicular steatosis, along with reduced inflammatory cell infiltration in the liver. Many studies have demonstrated the protective effect of kaempferol against liver diseases. For their lipid-lowering potential, kaempferol treatment is known to activate the sirtuin 1 and adenosine monophosphate-activated protein kinase, which further shows the significant improvement in the process of fatty acid oxidation and also downregulates the gene involved in de novo lipogenesis (Figure 1)[43,44]. In preclinical studies, kaempferol treatment significantly reduced the levels of liver injury markers in CCl₄-induced liver injury and improved acetaminophen-induced hepatotoxicity, via activation of Sirtuin 1, which further showed anti-inflammatory, antioxidant, and anti-apoptotic effects by reducing the acetylation of downstream markers, such as p53, nuclear factor kappa-light-chain-enhancer of activated B cells, and Forkhead Box O1[45-47]. It also mitigates liver injury and inflammation by reducing M1 macrophage activation via regulation of mitogen-activated protein kinase/nuclear factor kappa-light-chain-enhancer of activated B cells signaling pathways[48,49]. Beyond its anti-inflammatory response, kaempferol also showed its anti-fibrotic role by suppression of fibrotic markers such as acid-sensitive ion channel 1a, vascular endothelial growth factor, α-smooth muscle actin, TGF-β, and collagen-I in CCl₄-induced liver fibrosis. These effects were linked to the suppression of intracellular Ca²⁺ influx, as well as reduced ER-associated Ca²⁺ accumulation, which further downregulates the expression of elF2α, activating transcription factor 4, and vascular endothelial growth factor, thereby inhibiting the HSCs activation[50,51] (Figure 1).

Figure 1 Protective role of kaempferol on the pathogenic mechanism of high-calorie diet-induced metabolic dysfunction-associated steatohepatitis. Excess calorie intake increases blood glucose levels and promotes insulin resistance, which stimulates hepatic de novo lipogenesis. This leads to triglyceride accumulation in hepatocytes, contributing to hepatic steatosis. Simultaneously, impaired β-oxidation and mitochondrial dysfunction increase the production of reactive oxygen species, resulting in oxidative stress, which further upregulates inflammatory pathways, including nuclear factor kappa-light-chain-enhancer of activated B cells, leading to elevated pro-inflammatory cytokines such as interleukin-1β and interleukin-6. Chronic inflammation and oxidative damage promote hepatic stellate cell activation, which stimulates the transforming growth factor beta/suppressor of mother against decapentaplegic signaling pathway, ultimately resulting in liver fibrosis and progression toward metabolic dysfunction-associated steatohepatitis. Kaempferol may exert hepatoprotective effects by reducing de novo lipogenesis, improving mitochondrial function and β-oxidation, decreasing oxidative stress and inflammatory signaling, and inhibiting hepatic stellate cell activation and fibrogenesis. ROS: Reactive oxygen species; MASH: Metabolic dysfunction-associated steatohepatitis; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; IL6: Interleukin 6; IL1β: Interleukin 1 beta; TGFβ: Transforming growth factor beta; SMAD: Suppressor of mother against decapentaplegic; HSC: Hepatic stellate cell.

In alcohol injury models, kaempferol has been shown to decrease cytochrome P450 family 2 subfamily E member 1 expression, which potentiates the antioxidant defence system. It further showed cytotoxicity towards hepatocellular carcinoma cells, lipopolysaccharide-induced liver damage, and antifibrotic potential via downregulation of TGF-β/Smads signaling pathways[52,53]. Importantly, in the context of MASLD-induced disruption of the gut-liver axis, kaempferol has been shown to restore gut microbiota and increase the concentration of beneficial bacteria, including short-chain fatty acid-producing bacteria, thereby contributing to reduced hepatic inflammation as well as oxidative stress[54]. Overall, these findings provide a translationally relevant preclinical platform for the MASLD field (Table 1). In addition, Nair et al[19] showed that kaempferol significantly reduced liver damage severity induced by the Indian HFD diet via modulation of profibrotic mediators, such as TGF-β, reinforcing its broad-spectrum hepatoprotective potential. The plausible mechanism of action of kaempferol is depicted in Figure 1.

Table 1 Therapeutic potential of kaempferol across various experimental models.

Ref.	Model	Route	Findings
Baghaei et al[42], 2022	HepG2 cells		Enhanced fatty acid oxidation and reduced lipogenesis
BinMowyna and AlFaris[44], 2021	Mice	Orally	Anti-diabetic effect via activating the AMPK pathway
Park et al[43], 2025	Rats	Orally (250 mg/kg BW)	Decreased the acetylation of all SIRT1 targets, including PARP1, NF-κB, FOXO-1 and p53 that mediate antioxidant, anti-inflammatory and anti-apoptotic effects
Aodah et al[45], 2024	HepG2 C8 cells		Upregulation of antioxidant response elements (ARE)-mediated antioxidative enzymes, such as heme oxygenase, catalase and superoxide dismutases under the control of Nrf2 signaling pathways
Saw et al[46], 2014	Mice	Orally (25, 50, 100 mg/kg BW)	Reduced CCl4-induced liver damage via restoring gut microbiota diversity, increasing beneficial genera (e.g., Lactobacillus), and activating Nrf2 signaling
Alkandahri et al[52], 2023	Mice	Intraperitoneal (2.5, 5, 10, 20, 40 mg/kg BW)	Increased the expression of Grp78, decreased the expression of CHOP, and protected hepatocytes from ER stress-induced apoptosis
Shi et al[58], 2025	Rats	50 mg/kg BW	kaempferol-loaded nanoparticles (KFP-NPs) improved the antioxidant defense, evidenced by increased levels of SOD, GPx, and Nrf2 in SD rats
Qu et al[60], 2025	Raw 264.7 macrophage	24 μmol/L drugs	Kaempferol-loaded fibroin nanoparticles downregulate the expression of TNF-α and eliminate the intracellular reactive oxygen in raw macrophages

AMPK: Adenosine 5’-monophosphate-activated protein kinase; BW: Body weight; SIRT1: Sirtuin 1; PARP1: Poly ADP-ribose polymerase 1; NF-κB: Nuclear factor kappa-light-chain-enhancer of activated B cells; FOXO-1: Forkhead box O 1; NrF2: Nuclear factor erythroid 2-related factor 2; Grp78: Glucose-regulated/binding immunoglobulin protein 78; CHOP: C/EBP-homologous protein; ER: Endoplasmic reticulum; KFP-NPs: Kelp fucoidan polysaccharide nanoparticles; SOD: Superoxide dismutase; GPx: Glutathione peroxidase; SD: Sprague-Dawley; TNF-α: Tumor necrosis factor-alpha.

KEY CHALLENGES AND RECENT ADVANCES

Despite growing research interest, several debates and controversies persist in the management of MASLD. Currently, lifestyle modification remains the cornerstone of treatment; however, it is often limited by poor patient adherence and reduced effectiveness in advanced stages of the disease[55]. Pharmacological options are also restricted, with no widely approved therapies in many regions, and the use of off-label medications raises safety concerns. Furthermore, the complex and multifactorial nature of MASLD presents a significant barrier to the development of universally effective treatments[56]. However, for kaempferol, one of the major limitations is its low oral bioavailability, mainly due to extensive first-pass metabolism, which minimizes its therapeutic efficiency and may require higher doses, which further increase the possibility of side effects. The compound also exhibits a short half-life (2-8 hours), requiring frequent dosing and thereby affecting patient compliance[39,57]. A further challenge is the lack of standardized formulations, as kaempferol content varies depending on source, extraction, and manufacturing processes, emphasizing the need for standard analytical methods.

Recent advances in kaempferol research have focused on addressing its limitations, particularly poor solubility and low bioavailability, through innovative therapeutic strategies and structural modifications. Nanoformulation approaches, including nanomatrices, nanoemulsions, and gold nanoparticles, have demonstrated significant potential in enhancing targeted delivery, solubility, and overall bioavailability of kaempferol[58]. Supporting this, Kazmi et al[59] developed kaempferol-loaded nanoparticles to assess their hepatoprotective and antioxidant effects in a cadmium chloride (CdCl₂)-induced hepatocellular carcinoma model in male Sprague Dawley rats. Their findings indicated a marked improvement in antioxidant defense, evidenced by increased levels of superoxide dismutase, glutathione peroxidase, and nuclear factor erythroid 2-related factor. Additionally, food-grade delivery systems have been explored to overcome bioavailability challenges further. For instance, zein-pectin nanoemulsions were synthesized by a novel dual-frequency pulsed ultrasound technology to improve the loading effect of kaempferol. Dual-frequency pulsed ultrasound treatment in Caco-2 significantly improved the intracellular absorption rate, transport rate and bioavailability of kaempferol by 7.67%, 9.96% and 14.67%, respectively, which was attributed to the significant downregulation of mRNA expression levels of tight junction protein occludin and efflux proteins multidrug resistance protein 1 and breast cancer resistance protein by 21.27%, 51.05%, and 62.26%, thereby enhancing intracellular transport capacity of kaempferol[60]. Furthermore, silk fibroin-based nanoparticles have emerged as promising carriers due to their unique physicochemical properties, showing excellent biocompatibility, efficient cellular uptake, and the ability to reduce TNF-α expression and intracellular reactive oxygen species[61]. Future studies should focus on the development of advanced delivery systems, including mitochondria-targeted, stimuli-responsive, and co-delivery approaches, as promising strategies to enhance therapeutic efficacy. These innovative platforms may enable precise modulation of lipid metabolism, oxidative stress, and inflammation, thereby facilitating the advancement of precision phytochemical-based interventions.

STRENGTHS AND LIMITATIONS

Nair et al[19] addressed the increasing prevalence of MASLD in India, which is linked to a diet containing high fat, calories, and refined carbohydrates. The study is closely related to the dietary patterns and metabolic traits frequently seen in South Asian populations. This approach strengthens the relevance and translation value of the study, as it represents the metabolic and nutritional context contributing to MASLD development in the Indian population. Further research revealed the therapeutic potential of kaempferol, a naturally occurring flavonoid, in a mouse model. A few limitations of the present study should be acknowledged. First, the study was conducted as a pilot experiment with a very small sample size (n = 3), which limits statistical power and the generalizability of the findings. The study mainly focused on phenotypic, biochemical, and histological outcomes, while detailed molecular mechanisms underlying the protective effects of kaempferol were not extensively explored. Further, interventions are required to address the poor bioavailability of kaempferol through formulation strategies.

CONCLUSION

In conclusion, a region-specific Indian HFD was developed that closely resembles the dietary exposures and metabolic alterations leading to MASLD in South Asian populations. The study demonstrated that kaempferol treatment significantly improved the hepatic steatosis and histopathological alterations. The adoption of an Indian-HFD dietary model enhances the clinical relevance of these findings, as it mimics the real-world nutritional patterns associated with the rising burden of liver disease in the region. The study identifies kaempferol as an effective dietary phyto molecule for disease modulation. Therefore, comprehensive mechanistic investigations, formulation strategies, and well-designed large-scale studies are warranted to substantiate its therapeutic potential and future clinical translation.