Kaempferol attenuates diet-induced obesity and hepatic steatosis in C57BL/6J mice fed an Indian diet-mimicking regimen

Abstract

BACKGROUND

Metabolic dysfunction-associated steatotic liver disease (MASLD) ranges from simple steatosis to cirrhosis and liver cancer, with rising prevalence in India due to high fat, high sugar diets. Kaempferol, a generally recognized as safe-certified flavonoid found in fruits and vegetables with anti-inflammatory, antioxidant, and lipid-lowering properties. Despite its therapeutic promise, kaempferol’s efficacy remains unexplored in Indian diet-based obesity liver disease models. Developing such models could support targeted dietary interventions for the Indian population at elevated risk for MASLD progression.

AIM

To evaluate effect of kaempferol in C57BL/6J mice using an Indian diet-mimicking experimental animal model.

METHODS

Male C57BL/6J mice (6 weeks to 8 weeks, n = 3/group) were fed a normal diet or an Indian high fat diet (HFD) with fructose for 8.5 weeks, followed by kaempferol (50 mg/kg) or vitamin E (500 mg/kg) every other day for 4 weeks. Body weight, diet intake, liver function, and histopathology were evaluated. Liver samples were examined using hematoxylin and eosin staining. The non-alcoholic fatty liver disease score was assessed using the non-alcoholic steatohepatitis clinical research network criteria. The serum transforming growth factor beta 1 was detected using enzyme-linked immunosorbent assay kit. Additionally, the study also involves proximate and mineral composition of the diet for its validation. Moreover, the effect of kaempferol was further evaluated using an ex vivo precision-cut liver slice model.

RESULTS

We formulated a high calorie experimental HFD mimicking the Indian diet regimen (Indian HFD prepared and standardized in our lab and a provisional patent is filed), which shows an elevated crude fat and reduced crude fiber, ash, and moisture compared to the control feed. Mice fed this diet shows significant weight gain and hepatic steatosis, with macro-vesicular and micro-vesicular fat and lobular inflammation. Non-alcoholic fatty liver disease scoring confirms an increased steatosis and inflammation and also, transforming growth factor beta 1 levels were significantly elevated in the Indian HFD group. Kaempferol treatment significantly reduces body weight, steatosis, and inflammation. In ex vivo precision-cut liver slice models, kaempferol improved liver function enzymes and reduced hepatic triglycerides level.

CONCLUSION

Kaempferol improved hepatic steatosis and liver parameters in a diet-induced MASLD model mimicking the Indian diet, showing promising therapeutic potential.

Key Words: Metabolic dysfunction-associated steatotic liver disease; Indian high fat diet; High calorie diet; Natural product; Kaempferol; Hepatic steatosis

Core Tip: Metabolic dysfunction-associated steatotic liver disease spans from simple fat accumulation to cirrhosis and liver cancer, and its prevalence is steadily rising in India due to high fat, high sugar dietary habits. Kaempferol, a generally recognized as safe-approved flavonoid lipid-lowering effects, shows therapeutic potential but has not yet been evaluated in Indian diet-specific obesity driven liver disease models. This study demonstrates that kaempferol effectively attenuates diet-induced obesity and liver steatosis in C57BL/6J mice fed an Indian diet-mimicking regimen. The findings highlight kaempferol’s potential as a natural therapeutic agent for managing metabolic dysfunction and fatty liver disease associated with high calorie, region-specific dietary patterns.

INTRODUCTION

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly termed non-alcoholic fatty liver disease (NAFLD), has emerged as the most prevalent chronic liver condition worldwide, closely linked to obesity, type 2 diabetes, and other metabolic disorders[1,2]. The worldwide prevalence of MASLD continues to upsurge with an estimate rate of 24.2% in South Asian population. In India, the prevalence rate was found to be around 38.6% among adults and 35.4% among children[3,4]. Due to the lack of effective pharmacological therapies for MASLD/Metabolic dysfunction-associated steatohepatitis (MASH), lifestyle and dietary modifications remain the first-line treatment; however, poor long-term adherence limits their effectiveness[5]. Excessive free fatty acid (FFA) accumulation in hepatocytes induces lipotoxicity, promoting MASH development. During early onset of MASLD progression, trans-differentiated hepatic stellate cells produce transforming growth factor beta 1 (TGF-β1), a pro-fibrogenic marker. The FFA overload in hepatocytes generates toxic free radicals that activate necrotic pathways and elevate pro-inflammatory cytokines such as TGF-β1, tumor necrosis factor-α, interleukin-6, interleukin-8, and monocyte chemoattractant protein-1[6]. Persistent cytokine elevation drives chronic inflammation, activates quiescent hepatic stellate cells, and enhances extracellular matrix deposition, ultimately disrupting hepatic vascular architecture and liver structure[7].

Among the Indian population, rapid urbanization, sedentary lifestyle, and high intake of saturated fats and refined carbohydrates and sugars with very low dietary fiber have notably augmented the risk of obesity and MASLD[8]. The development of experimental animal model that accurately mimicking this dietary pattern is essential for understanding the intricate disease mechanisms and for screening therapeutic interventions.

In vivo animal models play a critical role in understanding the pathophysiological mechanisms of MASLD. Understanding the MASLD pathophysiology and its treatment approaches in humans is still limited due to lack of optimal animal models. An ideal animal model should closely replicate the intricate mechanism of human MASLD and reproduce the characteristic histological features observed in its various stages of disease progression[9]. Due to its complex and multidirectional nature of MASLD, no single animal model can fully replicate the entire disease spectrum within a practical experimental time[10]. Among the various experimental models, western-style diets are widely applied in MASLD research. However, these diets may not fully capture the unique nutritional risk factors observed in Indian populations, where diets are often rich in carbohydrates, vegetable oils, and calorie-dense foods[11]. To address this gap, we developed and validated an Indian diet-mimicking high fat diet (HFD) model in our lab that offers a more translationally relevant approach to study MASLD in the context of South Asian dietary patterns (filed provisional patent with No. TEMP/E-1/71179/2025-CHE) (Supplementary material).

Natural phytochemicals have gained increasing attention as potential therapeutic agents against metabolic diseases, owing to their diverse bioactive properties and favourable safety profiles[12]. Kaempferol, a dietary flavonoid abundantly found in tea, apples, berries, and leafy vegetables, possesses well-documented antioxidant, anti-inflammatory, and lipid-modulating effects[13,14]. Preclinical studies suggest that kaempferol can ameliorate obesity-related metabolic dysfunction by modulating adipogenesis[15], improving insulin sensitivity[16], and attenuating hepatic lipid accumulation[17,18]. However, its therapeutic efficacy in MASLD models that closely mimic Indian dietary habits has not been fully elucidated. The present study investigates the effects of kaempferol on diet-induced obesity and hepatic steatosis in C57BL/6J mice fed an Indian diet-mimicking regimen. By employing this regionally relevant dietary model, the study aims to: (1) Validate the translational potential of the Indian HFD for studying MASLD; and (2) Assess the protective effects of kaempferol against obesity-related metabolic and hepatic abnormalities. The findings are expected to provide valuable insights into the role of kaempferol as a potential nutraceutical intervention for MASLD, particularly within populations following Indian dietary patterns.

MATERIALS AND METHODS

Preparation of Indian HFD

The experimental Indian HFD was prepared using a standard pellet diet, cholesterol, and vanaspati ghee. Normal mice pellet was crushed and finely powdered using a mixer/blender. To the finely ground regular pellet diet, 25 g of vanaspati ghee, 2.5% cholesterol, and 15 mL to 20 mL palm oil were added and mixed properly to form a uniform dough-like mixture. The above mixture was placed in a silicone mold and froze for 1 hour to 2 hours to obtain solidified pellets with dimensions of 1 cm × 1 cm. Further, the pellets were stored in a clean and dry plastic container at 8 °C. The mice are fed with the in-house prepared diet for seven weeks. In addition, 25% fructose drinking water (100 mL daily) is given to animals along with the aforesaid high-fat diet.

Proximate analysis:

Moisture content: A clean, oven-dried petri dish was used for the experiment. Approximately 1 g to 2 g feed sample was placed in the petri dish and the initial wight was recorded. The dish with the sample was heated in the oven at 105 °C for 2 hours. Subsequently, the dish was reheated for another 1 hour until a steady result was obtained and the weight was noted. The drying procedure continued until a constant weight was achieved[19].

Ash content analysis: The ash content refers to the inorganic residue left over after the complete combustion of the organic matter. To determine ash content, a clean, empty platinum crucible was washed, dried and the initial weight was noted. Approximately 1 g to 2 g of feed sample was placed into the platinum crucible and incinerated in a muffle furnace at 550 °C for 3 hours. After burning, the sample was cooled in a desiccator and the weight was recorded[19].

Crude fat (Soxhlet fat extraction method): Approximately 5 g feed sample was wrapped in the filter paper and placed in the pre-dried Soxhlet (extraction) thimble. A pre-weighed round bottom flask containing 300 mL of petroleum ether (extraction solvent) was attached to the extraction thimble and refluxed for 6 hours to 8 hours. Following extraction, the solvent was recovered, and the flask with extracted fat was dried in an oven at 105 °C-110 °C for 1 hour, cooled in a desiccator and weighed. The crude fat was calculated from the weight gain in the flask[20].

Crude protein analysis: Crude protein in the rodent HFD was determined by the Kjeldahl method (AOAC 2001.11)[21]. About 1 g of sample was digested with concentrated H₂SO₄ and a H₂SO₄-CuSO₄ catalyst, followed by distillation and titration with 0.1 normality hydrochloric acid. Nitrogen content was calculated and converted to crude protein using the factor N × 6.25[22].

Mineral analysis: A feed sample weighing 0.5 g to 1 g was digested in 10 mL of concentrated nitric acid using a microwave digester (MPS 320™, PerkinElmer). From the digested solution, 1 mL was diluted to a final volume of 10 mL with 1% nitric acid. The prepared sample was then analyzed using an inductively coupled plasma optical emission spectrometer (Thermo Scientific iCAP 7000 Plus Series, MA, United states) to quantify 18 different mineral elements.

Animals

Male c57bl/6J mice (6 weeks to 8 weeks) were obtained from the Animal House facility, Amrita Institute of Medical Sciences, Kochi (No. IAEC/2020/1/7). The mice were housed in a temperature-controlled room on a 12-hour light-dark cycle. The mice were fed a regular diet for one week to acclimate to the environment.

Dietary intervention

In the first study for model standardization, animals were randomly divided into two groups fed with control diet (n = 1) and experimental diet (n = 3) respectively. The animals were monitored daily as part of the standardization protocol for evaluating daily consumption of murine diet and fructose solution. Body weights were recorded weekly. After 8.5 weeks of standardization protocol, the animals were sacrificed using CO₂ asphyxiation. Liver samples were collected, fixed in formalin, and paraffin-embedded sections were stained with hematoxylin and eosin (H&E) to validate the NAFLD scoring and to identify the histological features of NAFLD conditions.

In the second part of the study, following one week of acclimatization, the animals were randomly divided into four different groups (n = 3/group). The first group received normal pellet diet while the last three groups received experimental HFD for 8.5 weeks. The animals had free access to the in-house prepared murine diet and fructose solution for 8.5 weeks. After 8.5 weeks, the third group received kaempferol (50 mg dissolved in 0.5% carboxymethyl cellulose solution), and the fourth group received standard, vitamin E every other day (alternate days) for 4 consecutive weeks. The second group served as disease control group. Following the drug treatment, the animals were sacrificed using CO₂ asphyxiation. Liver samples were collected, fixed in formalin, and paraffin-embedded sections were stained with H&E to validate the NAFLD/NASH scoring and to identify the histological features of NAFLD/NASH conditions.

Histological analysis

Liver histology was examined using H&E staining. Fresh liver tissues were fixed in 10% formalin, paraffin-embedded and sectioned. The NAFLD activity score was assessed using the NASH clinical research network guidelines. Macro-vesicular steatosis and micro-vesicular steatosis were evaluated independently, and their severity was classified, based on the proportion of the total area affected as follows: 0 (< 5%), 1 (5% to 33%), 2 (> 34% to 66%) and 3 (> 66%). Similarly, inflammation was evaluated by counting the number of inflammatory foci per field using a 40 × magnification[23].

Enzyme-linked immunosorbent assay

The serum levels of TGF-β1 were measured using the Quantikine TGF-β1 enzyme-linked immunosorbent assay kit (R and D systems, MN, United States) according to the manufacturer’s instructions. The absorbance was detected at 450 nm on a microplate reader, and the concentration of cytokines was calculated based on a standard curve.

Ex-vivo study of NAFLD using murine PCL’s

Establishment of FFA-induced MASLD model in PCLs: Precision-cut liver slice (PCLs) was prepared from a single liver lobe using sterile scalpel and transferred into a 2 mL to 3 mL of Krebs-Ringer HEPES Buffer solution in a sealed beaker[24]. The PCLs were pre-incubated in a shaker water bath at 37 °C for 60 minutes and partially dried over a filter paper. The MASLD condition was induced by treating PCLs (200 mg) with PA (240 μm). Kaempferol (25 μm) was given before 24 hours of FFA incubation for both time-dependent and concentration-dependent studies.

Measurement of tissue biochemical parameters: Following the FFA incubation period, liver slices were homogenized in 0.25 molarity sucrose solution and centrifuged at 9000 × g for 30 minutes at 4 °C to obtain a clear supernatant solution. The supernatant was collected to estimate different liver function parameters like alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and triglyceride (TG) using a semi-auto analyzer (Stat Fax 3300, Awareness technology, RC, United States). Hepatic TG, ALP, ALT, and AST were measured using respective detection kit (Jeev diagnostics Pvt Ltd, Gerugambakkam, Chennai).

Statistical analysis

The data presented in the manuscript is expressed as mean ± SD. Statistical analysis was performed using GraphPad Prism software (version 8.2.1). The comparison between two groups was analysed by Student’s t test. A two-way analysis of variance was employed followed by Tukey’s Honest Significant Difference test with a threshold significance set at P value < 0.05.

RESULTS

Proximate composition of Indian HFD formulated to induce MASLD in mice

The proximate composition of the Indian HFD prepared in our lab is summarized in Table 1. The diet was formulated with high proportion of fat in order to replicate the MASLD condition in experimental animals. The analysis of the prepared Indian HFD reveals crude fat content was significantly higher and was found to be 98% that confirms that the dietary formulation successfully produced a HFD suitable for MASLD induction. The moisture content of the HFD was 11.67%, which is relatively low and implies good storage stability and low microbial risk. The ash content was found to be 5.75%, indicating the mineral fraction of the diet, not significantly altered formulation. The crude protein content was found to be relatively low (13.12%) ensuring the diet’s suitability for metabolic disease modeling.

Table 1 The proximate analysis of in-house prepared Indian high fat diet to induce metabolic dysfunction-associated steatotic liver disease in experimental animals, mean ± SD.

Proximate analysis of Indian high fat diet (%)
Moisture content	11.67 ± 3.33a
Ash content	98 ± 12.00a
Crude protein	13.12
Crude fat	98 ± 12.00a

^aP < 0.05.

Mineral composition of Indian HFD formulated to induce MASLD in mice

The mineral composition of the Indian HFD prepared in the laboratory is depicted in Table 2. The minerals present in feed play an important role in health and disease. Among the essential macronutrients calcium (1133.61 parts per million), magnesium (986.63 parts per million), and zinc (964.99 parts per million) were present in relatively higher concentration. Moderate amounts were recorded for molybdenum (140.69 parts per million), manganese (41.52 parts per million), nickel (23.25 parts per million), and copper (1.87 parts per million). In contrast, several other elements like arsenic, cadmium, cobalt, lithium, selenium, strontium, and vanadium were not detected in the sample.

Table 2 The mineral composition of in-house prepared Indian high fat diet to induce metabolic dysfunction-associated steatotic liver disease in experimental animals using inductively coupled plasma optical emission spectrometer.

Mineral composition of Indian HFD (ppm)
Arsenic	ND
Cadmium	ND
Calcium	1133.610
Cobalt	ND
Copper	1.879
Iron	591.813
Lithium	ND
Zinc	964.996
Magnesium	986.630
Molybdenum	140.692
Manganese	41.520
Nickel	23.253
Selenium	ND
Strontium	ND
Vanadium	ND

HFD: High fat diet; ND: Not detected.

The in-house prepared Indian HFD induced MASLD-like conditions experimental animals

Feeding animals with Indian HFD for 8.5 weeks successfully induced MASLD-like features. Animals receiving Indian HFD exhibited a gradual increase in the body weight compared to normal diet-fed animals, indicating diet induced obesity (Figure 1A). The animals fed with the Indian HFD exhibit an elevated serum TGF-β1 levels compared with the normal control group (Figure 1B). The histopathological examination further confirmed the establishment of MASLD pathology. The H&E staining depicts the presence of micro vesicular and macro vesicular steatosis along with small inflammatory cell aggregates, and presence of inflammatory loci were observed in Indian HFD fed mice, whereas the control liver retained normal liver physiology (Figure 1C). The presence of fat droplets in the hepatocytes is the key feature mimicking the human MASLD pathology. Further, to evaluate disease severity, NAFLD activity score was assessed using the NASH clinical research network guidelines. Macro-vesicular steatosis and micro-vesicular steatosis were evaluated independently, and their severity was classified, based on the proportion of the total area affected as follows: 0 (< 5%), 1 (5% to 33%), 2 (> 34% to 66%), and 3 (> 66%). Inflammation was quantified by counting the number of inflammatory foci per field under 40 × magnification (Figure 1D).

Figure 1 Optimization of in-house prepared Indian high fat diet in C57BL/6J mice. A: Change in body weight of C57BL/6J mice after consuming the in-house formulated Indian high fat diet (HFD); B: The concentration of transforming growth factor beta 1. Data represented in mean ± SD (n = 3), ^aP < 0.05 (control vs Indian HFD); C: Representative images of hematoxylin and eosin-stained liver sections of mice fed with normal pellet diet and Indian HFD after 8.5 weeks of induction. All photomicrographs: Hematoxylin and eosin stain; magnification 40 × (scale bar = 100 μm); D: Non-alcoholic fatty liver disease activity score conducted in liver samples of mice fed with normal pellet and Indian HFD group. Data represented in mean ± SD (n = 3), ^aP < 0.001 (control, micro-vesicular steatosis vs Indian HFD, micro-vesicular steatosis); ^bP < 0.001 (control, macro-vesicular steatosis vs Indian HFD, macro-vesicular steatosis); ^cP < 0.001 (control, lobular inflammation vs Indian HFD, lobular inflammation). HF: High fat; HFD: High fat diet; NAFLD: Non-alcoholic fatty liver disease; TGF-β1: Transforming growth factor beta 1.

Kaempferol improves Indian HFD-induced MASLD in C57BL/6J mice

Kaempferol administration markedly reduced the body weight gain in C57BL/6J mice in comparison with the positive control group (Figure 2A). In addition, kaempferol administration reduced hepatic fat deposition in mice fed with the Indian HFD. Histological examination revealed a marked decrease in the macro-vesicular steatosis (large lipid droplets) and micro-vesicular steatosis (small lipid droplets) within the hepatocytes with fewer inflammatory loci, in the kaempferol-treated groups and standard treatment groups (vitamin E) compared to Indian HFD control (Figure 2B-F).

Figure 2 Effect of kaempferol in Indian high fat diet model induced metabolic dysfunction-associated steatotic liver disease. A: Weekly body weight changes in mice fed the Indian high fat diet (HFD) for 8.5 weeks, followed by treatment with kaempferol or vitamin E on alternate days for 4 consecutive weeks. The kaempferol-treated group (blue line) demonstrates a marked reduction in body weight compared to the positive control group (green line); B: Non-alcoholic fatty liver disease activity score conducted in liver samples from control group, Indian HFD group, kaempferol group and vitamin E group confirms the development of metabolic dysfunction-associated steatotic liver disease in Indian HFD group and its alleviation in kaempferol treatment group; C-F: The histology images from control group, Indian HFD group, kaempferol group and vitamin E groups. All photomicrographs: Hematoxylin and eosin stain; magnification 40 × (scale bar = 100 μm). All data’s represented in mean ± SD (n = 3), ^aP < 0.001 (control, micro-vesicular steatosis vs Indian HFD, micro-vesicular steatosis); ^bP < 0.0001 (control, macro-vesicular steatosis vs Indian HFD, macro-vesicular steatosis); ^cP < 0.0001 (control, lobular inflammation vs Indian HFD, lobular inflammation); ^dP < 0.001 (control, hepatocellular hypertrophy vs Indian HFD, hepatocellular hypertrophy); ^eP < 0.0001 (Indian HFD, macro-vesicular steatosis vs kaempferol-treated, macro-vesicular steatosis); ^fP < 0.001 (Indian HFD, lobular inflammation vs kaempferol-treated, lobular inflammation); ^gP < 0.01 (Indian HFD, hepatocellular hypertrophy vs kaempferol-treated, hepatocellular hypertrophy). KMF: Kaempferol; Vit: Vitamin E.

Kaempferol alleviates the liver function enzymes and hepatic TG levels in PA induced MASLD in PCLs

The PA (240 μM) treated group exhibited a substantial rise in the level of ALT, AST, ALP, and TG levels in comparison with the control group. Co-incubation of kaempferol (25 μM and 50 μM) with PA treated PCLs for 24 hours markedly decreased the in the level of liver function parameters (Figure 3A-C) and hepatic TG levels in comparison with the PA-treated group (Figure 3D).

Figure 3 Kaempferol alleviates the liver function parameters and triglyceride levels in an ex-vivo precision-cut liver slice model. A: For alanine aminotransferase, data represented in mean ± SD (n = 3), ^aP < 0.0001 [control, vs palmitic acid (PA)-treated, 240 μM]; ^bP < 0.0001 (PA-treated, 240 μM vs kaempferol, 25 μM); ^cP < 0.0001 (PA-treated, 240 μM vs kaempferol, 50 μM); B: For aspartate aminotransferase, data represented in mean ± SD (n = 3), ^aP < 0.001 [control vs PA-treated, 240 μM]; ^bP < 0.001 (PA-treated, 240 μM vs kaempferol, 25 μM); ^cP < 0.001 (PA-treated, 240 μM vs kaempferol, 50 μM); C: For alkaline phosphatase, data represented in mean ± SD (n = 3), ^aP < 0.05 [control vs PA-treated, 240 μM]; ^bP < 0.05 (PA-treated, 240 μM vs kaempferol, 25 μM); ^cP < 0.05 (PA-treated, 240 μM vs kaempferol, 50 μM); D: For triglycerides, data represented in mean ± SD (n = 3), ^aP < 0.001 [control vs PA-treated, 240 μM]; ^bP < 0.001 (PA-treated, 240 μM vs kaempferol, 25 μM); ^cP < 0.001 (PA-treated, 240 μM vs kaempferol, 50 μM); ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; PA: Palmitic acid; KMF: Kaempferol.

DISCUSSION

The present study demonstrates that kaempferol supplementation effectively mitigates the Indian HFD-induced MASLD in C57BL/6J mice. We developed an Indian HFD mimicking regimen rich in saturated fats and sugar which closely reflects the dietary patterns associated with the rising prevalence of MASLD in Indian population[25]. The Indian HFD model used in this study differs from the several well-established western HFD and methionine choline deficient diets, particularly in its region-specific macronutrient distribution, proportion of saturated and unsaturated fats. These compositional differences may influence the pattern and rate of hepatic steatosis development, inflammation, and metabolic dysfunction[26]. While the Indian HFD developed in our lab directly reflects the dietary exposure in South Asian populations. Conventional western HFDs do not fully replicate the dietary patterns and metabolic responses observed in the Indian population[4]. Our lab optimized the composition of Indian HFD to reflect the Indian dietary pattern characterized with high intake of refined carbohydrates, saturated fats and sugars which provides a translationally significant model for studying the diet-induced MASLD in Indian context. From the validation and optimization, we observed micro-vesicular and macro-vesicular steatosis in experimental animals fed with Indian HFD, a hallmark feature of MASLD with significant increase in the bodyweight. As part of our standardization process, this phase of the work was designed as a pilot study, which utilizes very limited sample size of n = 3. Although a small cohort inherently limits statistical robustness and generalizability; however, similar exploratory studies often employ minimal sample numbers (n = 3) to optimize protocols, validate feasibility, and justify larger-scale experiments[27-29]. These findings establish the Indian HFD model, developed and validated in our laboratory, as a reliable preclinical platform for investigating MASLD and for screening potential therapeutic agents.

During MASLD progression the increased oxidative stress and the production of toxic free radicals activate several pro-inflammatory and inflammatory pathways. Among the several released inflammatory cytokines, TGF-β1 is found to have a potential role in worsening the steatotic condition into inflammatory MASH[30]. TGF-β1 serves as a pro-fibrotic cytokine which drives the disease from simple steatosis to inflammation and fibrosis. TGF-β1 signaling mechanisms play a central role in hepatic stellate cell activation and extracellular matrix production, which further contributes to the progression of MASLD. Excessive production of extracellular matrix production destroys tissue parenchyma and causes deterioration of vascular structure that subsequently results in the distortion of the structural framework of the liver[6]. During optimization, we assessed serum TGF-β1 levels in experimental animals and observed a significant increase in the Indian HFD group compared with the normal control group.

Flavonoids are reported to have multi-targeted effects in MASLD through various pharmacological actions like antioxidant, anti-inflammatory and may help protect against metabolic diseases like obesity and diabetes. Despite substantial preclinical evidence, the clinical translation of flavonoid molecules has been limited by variable efficacy outcomes and an incomplete understanding of their mechanistic roles in human metabolic regulation[31]. Flavonoids such as quercetin[32,33], naringenin[34], rutin[35], genistein[36] consistently show beneficial effects in MASLD/MASH models by improving lipid metabolism, antioxidant defense, and inflammatory modulation. Kaempferol is reported to modulate lipid metabolism, suppress oxidative stress, reduce inflammatory signaling, and improve insulin sensitivity[17,37,38]. It is a phase I-enrolled phyto-molecule abundantly found in leafy vegetables with diverse pharmacological activities against various chronic diseases. Despite its potent pharmacological actions, the clinical utility of kaempferol is limited by its poor bioavailability, with studies indicating that only about 2% bioavailability when ingested orally. To overcome these limitations, various formulation strategies have been employed to enhance its pharmacokinetic profile, including nanoformulations, liposomes and phospholipid complexes[39].

Our findings suggests that kaempferol, a naturally occurring flavonoid molecule and was found to significantly reduced body weight gain, improved biochemical markers of lipid metabolism in Indian HFD fed mice. Histopathological evaluation further confirmed kaempferol administration markedly decreased both macro-vesicular and micro-vesicular steatosis, along with reduced inflammatory cell infiltration in the liver. These results suggest that kaempferol exert beneficial effect in the context of diet-induced MASLD. Over all the results support kaempferol as a promising molecule for MASLD intervention and can be holds a potential as a dietary supplement. Further studies can be extended with large number of sample size to extrapolate its mechanistic evidence of kaempferol in MASLD progression preclinically and warrant its clinical translation.

CONCLUSION

In conclusion, the study first establishes and validates the Indian HFD model as a relevant dietary model for investigating MASLD. Secondly, it demonstrates the potential of kaempferol in reducing the liver steatosis using the Indian HFD model. The use of an India-specific high fat, high sugar dietary model further underscores the relevance of these findings to regional dietary patterns associated with metabolic disease. Taken together, these findings support the use of Indian HFD model to mimic the human MASLD features and highlight kaempferol as a promising dietary molecule in MASLD management. Further investigations are required to elucidate the mechanistic pathways involved and to assess its potential for clinical translation.

ACKNOWLEDGEMENTS

We acknowledge the inspirational guidance of our Chancellor, Sri Mata Amritanandamayi Devi, Amrita Vishwa Vidyapeetham. We are grateful to Amrita Animal Research facility for all the support in performing the animal experiments. We thank Dr Shantikumar. V. Nair, Associate Provost, Amrita Vishwa Vidyapeetham and Dr. Sabitha M, Principal, Amrita School of Pharmacy for the facilities provided. We acknowledge Dr. Deepa Ananth, Assistant Professor, and Dr. Deepthy Vijay, Assistant Professor, Kerala Veterinary and Animal Sciences University, Mannuthy, Thrissur, for helping us in conducting the crude protein estimation and the mineral analysis of Indian HFD. We acknowledge Dr. Greeshma Raju, iVET Labs, Kochi for histopathological evaluations and providing expert interpretation of the NAFLD scoring. We kindly acknowledge the language editing support by Ms. Devi S, Faculty, verbal and communication skills, Amrita School of Pharmacy.