Open Veterinary Journal, (2025), Vol. 15(10): 4936-4948

Research Article

10.5455/OVJ.2025.v15.i10.13

Therapeutic efficacy of liposomal caffeic acid in high-fat diet-induced obesity in rats: Targeting hedgehog signalling pathway, adipokines, and insulin resistance

Wafa S. Alansari¹, Mahran Mohamed Abd El-Emam^2*, Rawan Altalhi³, Safaa I. Khater², Tarek Khamis⁴, Hoda S. Sherkawy⁵, Abdelhamid M. Fares⁶, Marwa M. Lotfy², Ola Mohammed Youssef⁷ and Engy Mohamed Mohamed Yassin²

¹Biochemistry Department, Faculty of Science, University of Jeddah, Jeddah, Saudi Arabia

²Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt

³Department of Biological Sciences, College of Science, University of Jeddah, Jeddah, Saudi Arabia

⁴Department of Pharmacology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt

⁵Medical Biochemistry and Molecular Biology Department, Faculty of Medicine, Aswan University, Aswan, Egypt

⁶Department of Pathology, Faculty of Veterinary Medicine, University of Sadat City, Monofiya, Egypt

⁷Department of Human Anatomy and Embryology, Faculty of Medicine, Mansoura University, Mansoura, Egypt

*Corresponding Author: Mahran Mohamed Abd El-Emam. Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt. Email: MMMahran [at] vet.zu.edu.eg

Submitted: 16/06/2025 Revised: 24/08/2025 Accepted: 03/09/2025 Published: 31/10/2025

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Abstract

Background: Obesity spurred by a high-fat diet (HFD) has emerged as a significant worldwide health issue. Caffeic acid (CA) is a plant-derived phenolic compound found in various foods such as coffee, wine, and tea. It exhibits antioxidant, anti-inflammatory, and anticarcinogenic properties. However, the therapeutic potential of this substance is hindered by its poor water solubility and low oral bioavailability.

Aim: In this study, CA was loaded into liposomes Caffeic Acid -Loaded Liposomes (CA-Lip) , and the potential role of CA-Lip in mitigating HFD-induced obesity in rats was examined.

Methods: Forty male Wistar rats were used in this investigation. They were allocated into four groups: the first was fed a regular diet; the second received a normal diet plus CA-Lip; the third received an HFD; and the fourth received both an HFD and CA-Lip.

Results: The developed CA-Lip showed a particle size of 125 nm, zeta potential of −15 mV, and entrapment efficiency of 82%. The HFD+CA-Lip group's body weight was considerably lower than that of the HFD group. The HFD+CA-Lip group also showed a significant improvement in their serum lipid profiles. CA-Lip lowered levels of adipokines, which encourage inflammation, including leptin, interleukin-6, and tumor necrosis factor-α. It also reduced the size of the adipocytes when compared to the HFD control group. Furthermore, it reversed the HFD-induced upregulation of Hh genes [protein patched homolog 1 (PTCH1), Hh-interacting protein (HHIP), glioma-associated oncogene homolog 1 (Gli1), and smoothened (Smo) receptor] in adipose tissue.

Conclusion: In summary, CA-Lip may reduce obesity and related inflammation by activating the Hh signaling pathway.

Keywords: Caffeic acid, Liposomes, Obesity, Lipid, Adipokines, Hhog pathway.

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Introduction

Obesity is a multifaceted health concern impacted by various factors such as genetics, lifestyle choices, and dietary habits (McCafferty et al., 2020). Nowadays, the rates of obesity have doubled in many countries (WHO, 2023). By 2035, over half of the global population is predicted to be overweight due to various lipid problems. This condition is marked by various lipid issues, including high triglyceride (TG) and total cholesterol (TC) levels, low high-density lipoprotein cholesterol (LDL-C) levels, and irregular LDL-C composition (Vekic et al., 2019).

Moreover, consuming a high-fat diet (HFD) regularly leads to the body accumulating extra energy, which is stored as TGs in white adipose tissue (Bastías-Pérez et al., 2020). Furthermore, Obesity leads to malfunctioning white adipose tissue, lipid overflow, metabolic problems, and ectopic fat accumulation in muscle and liver (Cioffi et al., 2022). Additionally, abnormal metabolic processes are intimately associated with dementia, cancer, heart disease, and type 2 diabetes (Yan et al., 2023). As well, obesity impairs organ and system physiological processes, leading to increased mortality rates and a 5–7-year decrease in human lifespan (Tam et al., 2020). It significantly impacts public health, making prevention and treatment crucial yet still a significant challenge.

The Hedgehog (Hh) signaling pathway is a conserved regulator of tissue development and cellular function. Activation of this pathway inhibits adipocyte differentiation in various cell models, including 3T3-L1 and NIH-3T3 (Shi and Long, 2017). The Hh ligand binds to the Patched (PTC1/PTC2) receptor complex, relieving inhibition of the Smoothened (Smo) receptor, which then transduces signals to Glibenclamide transcription factors in the nucleus, modulating genes involved in cell fate, polarity, and proliferation (Sigafoos et al., 2021). Notably, Hh activation suppresses adipogenesis, whereas its inhibition promotes fat cell formation, suggesting a potential therapeutic role for Hh signaling modulation in metabolic diseases such as type 2 diabetes and obesity (Wei et al., 2019; Chou et al., 2021).

There are several approaches to managing obesity and/or overweight, such as medication, exercise, and nutrition (“Expert Panel Report: Guidelines (2013) for the management of overweight and obesity in adults,” 2014). Orlistat and sibutramine are anti-obesity drugs with modest clinical efficacy, but they also have serious side effects (Tak and Lee, 2021). Natural products, such as caffeic acid (CA), are often associated with minimal side effects, which enhances their appeal as alternative therapeutic options (Wang et al., 2023). CA, also known as 3,4-dihydroxycinnamic acid, is a type of phenolic acid characterized by a hydroxybenzene acrylic acid structure. It is a catechol-based secondary metabolite found in plants like Melissa officinalis, various fruits, and certain traditional Chinese medicinal herbs (Chen et al., 2019). CA and its analogs exhibit various pharmacological activities such as anti-inflammatory, anti-cancer, anti-oxidant, anti-mutagenic, and anti-virus properties (Bounegru and Apetrei, 2020). Additionally, CA can enhance lipid metabolism and help reduce obesity (Kim et al., 2018). However, CA's therapeutic effects in vivo are hindered by its limited solubility, absorption, bioavailability, instability, lack of specificity, and high systemic clearance (Stasiłowicz-Krzemień et al., 2023).

Drug delivery systems (DDSs) help address limitations by protecting compounds from degradation and improving their physicochemical properties (Chen et al., 2024). Liposomes are nanocarriers with phospholipid bilayers, accommodating hydrophilic agents and lipophilic molecules due to their amphiphilic nature (Ramalho et al., 2018). Liposomes offer excellent biocompatibility and bioavailability, enabling controlled, sustained drug release. This allows for effective treatment with lower doses, reducing potential side effects (Chelliah et al., 2025). This investigation aims to develop and evaluate a novel liposomal CA formulation Caffeic Acid -Loaded Liposomes (CA-Lip) as a targeted anti-obesity therapy, investigating its multifaceted effects on metabolic health—specifically glycaemic status, lipid profile, adipokine secretion, and the molecular regulation of the Hh signaling pathway in adipose tissue of HFD-induced obese rats.

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Materials and Methods

Materials

CA (98% purity) and cholesterol were supplied by Sigma–Aldrich (St. Louis, MO). Saturated phosphatidylcholine from soybean was provided by Lipoid GmbH (Ludwigshafen, Germany). Furthermore, the study employed only the highest-quality reagents and other compounds.

Preparation of liposomal characterization

CA-Lip has been developed using the ethanol injection method, as outlined previously (Alaaeldin et al., 2021). In this approach, a mixture of saturated phosphatidylcholine from soybean, cholesterol, and CA, in a molar ratio of 10:1:1, was dissolved in a minimal volume of anhydrous alcohol to form the organic phase, which was subsequently heated to 60°C–70°C. In parallel, phosphate-buffered saline (PBS) at pH 7.4 was maintained at the same temperature and agitated at 1,000 rpm within a closed system. The heated organic phase was then injected into the PBS solution through a 23G syringe, resulting in the formation of a liposomal suspension. This mixture was continuously stirred at 60°C–70°C until any residual ethyl alcohol was completely evaporated. After the completion of the process, the CA-Lip was stored at 4°C for later analysis and experimentation. Blank niosomal systems were prepared following the same protocol without CA. The liposomal systems were characterized by evaluating various parameters, including entrapment efficiency, drug loading efficiency, mean particle size, and zeta potential, as described in earlier work (Abd El-Emam et al., 2023).

Experimental animals

Forty male Sprague–Dawley rats weighing 250 ± 5 g were acquired from the research animal farm located at Zagazig University's Faculty of Veterinary Medicine. Each rat was housed in a stainless-steel cage that was maintained at a temperature between 21°C and 24°C, offering a clean, pathogen-free environment. To achieve ideal living circumstances, the rats were exposed to a 12-hour light-dark cycle and a 60% relative humidity. Commercial food pellets and unlimited water were provided to the rats. The standard rat diet and the HFD were crafted based on the research conducted by Lasker and colleagues in their study (Lasker et al., 2019). The composition of the HFD used in this study is based on protocols reported in previous studies (Salem et al., 2023).

Animals and experimental design

Four groups of 10 rats each were randomly assigned based on their body weights. The first one is the control group, which received a 20-week standard rat diet. The second group, referred to as the CA-Lip group, was maintained on a normal diet for the entire 20-week period. During the final 8 weeks, the animals received CA-Lip (1.8 mg/kg body weight) via oral gavage once daily while continuing on the same diet (Shahin et al., 2022). The third one was the HFD-fed group, which was given an HFD for 12 weeks for the induction of obesity and fed with a standard rat diet for the last 8 weeks. During the final 8 weeks, the animals also received normal saline via oral gavage once daily. The fourth group was fed an HFD for the initial 12 weeks, followed by a standard rat diet for the final 8 weeks. During the final 8 weeks, the animals also received CA-Lip (1.8 mg/kg body weight) via oral gavage once daily (Shahin et al., 2022).

Body weight measurement

Body weight was measured at the beginning of the experiment to ensure proper randomization and equal distribution of animals into experimental groups. Weight measurements relevant to the treatment protocol were then initiated at week 12, following the obesity induction phase, and final body weights were recorded at the end of the 20-week study.

Sampling

Upon the conclusion of the experimental period, the rats were anesthetized, and blood was drawn from the medial canthus. Part of the blood was put in test tubes without anticoagulants, which were centrifuged at 3,000 rpm for 15 minutes at room temperature, and a serum sample was used for biochemical analysis. The remaining portion was resuspended in tubes containing sodium fluoride, centrifuged at 3,000 rpm for 15 minutes, and then used for blood glucose and insulin analysis. The adipose tissue sample was dissected into two parts for gene expression [real-time quantitative PCR (RT-qPCR)] and histopathological examination.

Biochemical analysis

The bioMed diagnostic kit from Egypt was used to detect blood glucose and insulin levels, and Randox assay kits were used to evaluate the serum lipid profile following the manufacturer's instructions. Furthermore, Thermofisher Scientific CO's KRC3012 and ERA31RB ELISA kits were used to measure the levels of TNF-α and IL-6 in serum. The serum's adiponectin and leptin levels were ascertained using an ELISA kit from Millipore in the US.

RT-qPCR analysis

Previous documentation of the real-time analysis was done by Mansour et al., 2025. At the beginning, we utilized the Trizol Reagent from Thermo Fisher Scientific in Massachusetts to extract total RNA from adipose tissue. The concentration of the extracted RNA was then measured using the nanoDrop^® ND-1000 Spectrophotometer from NanoDrop Technologies in North Carolina. Subsequently, first-strand cDNA synthesis was conducted using the HiSenScriptTM RH (-) cDNA Synthesis Kits from iNtRON Biotechnology in the Republic of Korea. This process was performed in a Veriti 96-well heat cycler using the cDNA synthesis kit from Applied Biosystems in California. The primer sequences are presented in Table 1. The real-time PCR amplification was performed following the established protocol devised by Livak and Schmittgen (2001).

Table 1. The sequence of primers used for RT-PCR.

Histopathological examination

The adipose tissue was excised and handled with caution to minimize any damage to the tissue using a 21-gauge needle before immersion in modified Davidson’s solution for 24 hours (Bancroft and Layton, 2013). Next, the adipose tissue was processed for paraffin infiltration and embedding, and 4–5 µm thick transverse tissue sections from the middle portion of the testicles were prepared, stained with hematoxylin and eosin (Erfan, 2017), and examined microscopically.

Statistical analysis

We utilized GraphPad INSTAT software (Version 2) to conduct the statistical analysis. The statistical assessment involved applying a one-way analysis of variance and Tukey's multiple range test. The results comprise the presentation of SE and SEM differences. Significance was determined by considering a p-value of 0.05 or less.

Ethical approval

The ethics committee at Zagazig University's Faculty of Veterinary Medicine in Egypt carefully reviewed and gave its approval to the research protocol. All steps of the study were conducted by the relevant laws and regulations (approval number: ZU-IACUC/2/F/187/2022). The research was also carried out following the guidelines set forth by ARRIVE to ensure the highest standards of research conduct.

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Results

Characterization of CA-Lip

The ethanol injection method was employed to fabricate CA-Lip, which was subsequently characterized using several analytical techniques, including particle size analysis, polydispersity index (PDI), zeta potential measurement, and encapsulation efficiency. The average particle size of the CA-Lip, determined via Zetasizer Nano, was found to be 125.1 ± 12.6 nm (Fig. 1A). The PDI of 0.27 indicated a relatively narrow size distribution, suggesting a uniform dispersion of the liposomal particles. The zeta potential of the niosomal systems was measured at −15 ± 0.84 mV (Fig. 1B), reflecting a moderate negative surface charge, which is indicative of the stability of the formulations. The encapsulation efficiency of the CA-Lip was determined to be 82% ± 3.6%, indicating a substantial entrapment of the active compound within the vesicles.

Fig. 1. Characterization of CA-Liposomes: (A) Particle size (125.1 ± 12.6 nm), and (B) zeta potential (−15 ± 0.84 mV). Data are presented as mean ± SEM.

CA-Lip administration induces body weight loss in HFD-fed rats

We examined the effect of HFD on body weight to assess the impact of CA-Lip. There was no significant difference in body weight among the experimental groups at the start of the experiment (Day 0) (Fig. 1A). On the other hand, HFD significantly increased body weight compared to the control group. However, there was no significant difference in body weight between the CA-Lip-treated group and the control group, nor between the HFD group and the HFD + CA-Lip group at week 12. Conversely, HFD markedly increased body weight by 44% compared to the control group at the end of the experiment (week 20th). HFD-fed rats’ body weight significantly dropped by 26% after CA-Lip injection. Also, there was a decrease in body weight in the CA-Lip group compared to the control group (Fig. 1C).

CA-Lip restores glucose and insulin levels, as well as Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) scores in HFD-fed rats

The impact of CA-Lip was assessed by monitoring the blood levels of fasting glucose and insulin, as well as the HOMA-IR scores. Compared to the control group, it was observed that there was a significant increase of 76% in fasting glucose levels, 46% in insulin levels, and 158% in HOMA-IR after consuming HFD. However, when the HFD was accompanied by the administration of CA-Lip, there was a reduction of 30% in fasting glucose levels, 15% in insulin levels, and 40% in HOMA-IR. The CA-Lip group exhibited similar results to the control group (see Fig. 2A–C).

Fig. 2. Effect of CA-Lip on the body weight in HFD-fed rats. Data are presented as the mean ± SEM (n=10). ****p < 0.0001, *p < 0.05, ^####p < 0.0001 versus HFD group, ns indicates nonsignificant.

CA-Lip modulates changes in serum levels of lipid profile in HFD-fed rats

Rats fed an HFT showed a notable increase in serum TC, TG, very low-density lipoprotein (VLDL-C), and LDL-C levels by 41%, 45%, 82%, and 92%, respectively, and a significant decrease in high-density lipoprotein (HDL) levels by 44% in comparison to the control group. CA-Lip injection induced a significant decline in TC, TG, VLDL-C, and LDL-C levels by 22%, 21%, 22%, and 34%, and an elevation in HDL by 51% in CA-Lip-fed HFD rats versus the HFD group. Serum levels of lipid profile were insignificantly different in the CA-Lip group compared to the control rats (Fig. 3A–E).

Fig. 3. Influence of CA-Lip on fasting glucose, insulin, and HOMA-IR levels in HFD-fed rats. The levels of fasting glucose (A), insulin (B), and the scores for HOMA-IR (C) were measured for the different groups. ****p < 0.0001, ***p < 0.001, **p < 0.01, ^#p < 0.05 versus HFD group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n=10).

Effect of CA-Lip on adipokines changes in HFD-fed rats

When compared to the negative control, HFD dramatically decreased the levels of blood adiponectin by 61% and increased serum leptin by 75%. CA-Lip administration significantly decreased serum leptin levels by 34% and significantly inhibited serum leptin levels by 100% in the HFD-fed group. CA-Lip group showed almost similar effects on adipokine levels compared to the control rats (Fig. 4A and B).

Fig. 4. Influence of CA-Lip on serum lipid profile. The levels of TC (A), TGs (B), HDL (C), VLDL-C (D), and LDL-C (E) were measured for the different groups. ****p < 0.0001, ***p < 0.001, ^####p < 0.0001 versus HFD group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n=10).

CA-Lip suppresses proinflammatory cytokines in HFD-fed rats

The present research identified several significant proinflammatory cytokines associated with obesity. The pro-inflammatory cytokines TNF-α and IL-6 had significantly higher (p < 0.05) levels in rats on an HFD. They were significantly suppressed (p < 0.05) by CA-Lip administration (Fig. 5A and B). These findings imply that CA-Lip could reduce systemic inflammation driven by obesity.

Fig. 5. Effect of CA-Lip on leptin and adiponectin in HFD-fed rats. Data are presented as the mean ± SEM (n=10).****p < 0.0001, **p < 0.0, ^####p < 0.0001, ^##p < 0.01 versus HFD group, ns indicates nonsignificant.

CA-Lip regulates the Hh pathway in adipose tissue of HFD-fed rats

Protein patched homolog 1 (PTCH1), Smo, glioma-associated oncogene homolog 1 (Gli1), and Hh-interacting protein (HHIP) relative mRNA expression were significantly upregulated in obese rats by 470%, 450%, 580%, and 725%, respectively, compared to the control group. The treated rats showed a considerable decrease in PTCH-1, Smo, Gli-1, and HHIP expression by 57%, 73%, 64%, and 60%, correlated with the obese group. There was no obvious statistically significant difference between the CA-Lip group and the control group. The CA-Lip group showed a decrease in PTCH-1, Smo, Gli-1, and HHIP expression by 10%, 11%, 6%, and 2%, compared to the control group (Fig. 7 A–D).

Fig. 6. Effects of CA-Lip on serum inflammation (n=10 per group). (a) TNF-α and (b) IL-6. ****p < 0.0001, ^####p < 0.0001 versus HFD group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n=10).

Fig. 7. Effect of CA-Lip on the mRNA expression of (A) PTCH1, (B) Smo, (C) GLI-1, and (D) HHIP genes in adipose tissue of the HFD group. ****p < 0.0001, ^####p < 0.0001 versus HFD group, ns indicates nonsignificant. Data are presented as the mean ± SEM (n=10).

Histopathological findings

Normal histological structures of adipocytes, represented by clear cytoplasm, peripherally located nuclei, and intact cell membranes, were noticed in both the control (Fig. 8A) and the CA-Lip group alone (Fig. 8B) of abdominal fat. However, increased volumes of fat cells due to more lipid deposits in adipocytes were seen in the HFD group (Fig. 8C) when compared to the control group. On the other hand, there are reductions in the sizes of adipocytes in most examined sections in the treated obese group by CA-Lip (Fig. 8D) in comparison with the obese rats’ group. Representative figures from three sections per mouse (n=5 rats) were examined to determine average adipocyte sizes by counting 50 cells using ImageJ software (Fig. 8E).

Fig. 8. Representative photomicrographs of H&E-stained sections from abdominal fat (Scale bar 20 μm) in rats (A–D) and a representative graph illustrating average adipocyte sizes among different groups (E).

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Discussion

The global obesity epidemic, influenced by genetics, fast food, and urban decline, is causing limited mainstream remedies due to side effects and restrictions, prompting an urgent need for a safe, effective natural alternative (Payab et al., 2020; Aron-Wisnewsky et al., 2021). This research investigates the potential of CA-Lip in combating obesity and promoting a healthy lifestyle.

When rats are fed HFD, their body fat percentage increases linearly with their body weight. Our findings indicated that HFD significantly raises body weight levels. The results obtained matched those of Huang et al. (2023), who found a rise in the body weight of HFD-induced obese recipient mice. However, the administration of CA-Lip to obese rats resulted in a notable enhancement in weight loss, suggesting a promising therapeutic avenue for obesity management. A study by Xu et al. (2020) found that administrated CFA at 50 mg/kg BW by daily gavage for 12 weeks significantly reduced body weight in HFD mice. The bioavailability of CA is increased considerably when delivered in liposomal form, allowing for more pronounced biological effects at lower doses. Our results were supported by histopathological examinations, which revealed that CA-Lip could effectively suppress the HFD-associated increase in abdominal fat accumulation and adipocyte size. These findings imply that consuming CA-Lip may be a viable approach for managing and preventing overweight and obesity.

Glucose homeostasis encompasses all metabolic processes that help regulate blood sugar levels. Obesity can lead to insulin resistance (IR) due to chronic hyperinsulinemia and inflammatory mediator interference with insulin signaling molecules (Rohm et al., 2022). HFD-fed rats exhibited a rise in fasting blood glucose, insulin, and HOMA-IR. The same findings were found by Huang et al. (2024). Fasting blood glucose, insulin, and HOMA-IR were markedly decreased in CA-Lip in HFD-fed rats. The reduction in HOMA-IR suggests an improvement in insulin sensitivity. CA treatment in chronic restraint stress-induced insulin-resistance mice reduced fasting blood sugar, oxidative stress, and improved insulin sensitivity (Choudhary et al., 2016). Castro et al. (2021) found that treatment with CA (50 mg/kg) managed to lower glucose levels in diabetic rats. These findings support the high therapeutic potential of nanoliposome-encapsulated CA in managing obesity-related hyperglycemia and IR.

HFD-induced hyperlipidemia is a suitable model due to its ability to mimic pathological conditions in humans and rats. Feeding with an HFD caused a significant elevation of serum TC, TG, VLDL-C, and LDL-C levels and a marked decrease in HDL-C levels compared to rats fed a normal diet. According to Eynde et al. (2023) , feeding rats HFD results in hyperlipidemia, as demonstrated by a considerable drop in serum HDL-C levels compared to the normal group and a significant increase in serum levels of TC, LDL-C, and TG. HFD can alter fat composition and impact fat breakdown, absorption, and stomach enzyme activation. Elevated TC and LDL-C levels increase the risk of coronary heart disease, while higher HDL-C promotes cholesterol removal from the liver (Saravanan and Ponmurugan, 2012). Dietary cholesterol increases TG levels, which reduces fatty acid oxidation, thereby increasing hepatic and plasma TG levels (Huang et al., 2024). The administration of CA-Lip to HFD rats restored the increase in TG, TC, and LDL-C levels and the drop in HDL-C levels. The observed lipid-lowering effects are consistent with the established role of polyphenolic substances, such as CA, in modifying lipid profiles through inhibiting lipid peroxidation, reducing lipid accumulation, and promoting adipocytes' white-to-brown transition (Mariana et al., 2018). CA down-regulates lipogenesis gene expression of sterol regulatory element-binding protein-1 and target genes, fatty acid synthase, while promoting lipolysis through up-regulation of AMP-activated kinase (Liao et al., 2014).

Obesity caused by an HFD is well-known for altering the levels of adipokines in the body. Leptin and adiponectin are types of adipocytokines that originate from fat tissue. Thus, controlling these substances is crucial in preventing metabolic diseases such as obesity (Masuyama et al., 2016). Adiponectin, a hormone produced by adipocytes, has anti-inflammatory, anti-obesity, and antidiabetic properties, while leptin regulates satiety and energy homeostasis. Obesity is linked to lower levels of adiponectin and higher levels of leptin (Clemente-Suárez et al., 2023). Furthermore, HFD-induced mice displayed elevated leptin levels and hyperleptinemia compared to control mice (Yeh et al., 2017). Rats given CA-Lip as a supplement had lower leptin levels than the non-supplemented HFD group, but their adiponectin levels were higher. Specifically, CA-Lip was found to reduce the release of abnormal adipokines by controlling the levels of leptin and adiponectin in the body. Research showed that CA increased the levels of adiponectin receptors like AdipoR1 in the liver of rats (Tian et al., 2025). Additionally, Obesity increases the body's production of pro-inflammatory adipokines, disrupting the balance between pro-inflammatory and anti-inflammatory reactions (Kawai et al., 2021). This is evidenced by the rise in serum pro-inflammatory cytokines such as TNF-α and IL-6 in rats on an HFD. Tarbiah et al. (2024) found that rats given CA (0.8/100 g diet) resulted in significant adjustments of serum proinflammatory cytokines, including IFN-γ, TNF-α, IL-6, and NF-kB in rats fed HFD. Similar results were observed in our study as oral supplementation with CA-Lip alleviated the inflammatory condition, as evidenced by the marked reduction of proinflammatory cytokines. Our findings indicated that the liposomal encapsulation likely enhanced the bioavailability and stability of CA, contributing to its effectiveness at a relatively low dose.

The Hh signaling pathway is crucial in adipose tissue regulation, inhibiting adipocyte growth in vivo and in vitro. It strongly regulates the adipogenesis of white and brown adipose tissue (Zhang et al., 2020 a). The Hh signaling pathway modifies cellular metabolism through an unconventional pathway, encouraging a glycolytic state resembling the Warburg effect (Teperino et al., 2012). The Gli-1 target genes are HHIP and PTCH-1. Both can lessen Hh signaling. SMO is constitutively depressed by PTCH, and HHI interacts with the Hh ligand to prevent it from binding to PTCH, hence blocking the activation of SMO (Sigafoos et al., 2021). The activation of Hh signaling leads to the inhibition of BAT production and suppression of brown preadipocyte differentiation (Zhang et al., 2020 b). The research findings showed a notable rise in the mRNA levels of HHIP, Gli-1, Ptch-1, and Smo in obese rats compared to the group of rats in a healthy weight range. These results were consistent with other findings (Khater et al., 2023). CA-Lip administration in our study can reduce obesity by inhibiting the activation of the Hh pathway. These findings align with prior research indicating that attenuation of Hh signaling blocked early brown-preadipocyte differentiation, inhibits BAT formation in vivo, and results in the replacement of neck BAT with poorly differentiated skeletal muscle (Nosavanh et al., 2015), highlighting the potential of CA-Lip as a novel therapeutic approach in obesity-related metabolic dysfunction. Several prior studies have reported similar mechanisms involving phenolic compounds. In a study by Aslam et al. (2022), quercetin could ameliorate liver fibrosis by antagonizing the key mediators of the Hh pathway and inflammation [Sonic Hedgehog (Shh), Indian hedgehog (Ihh), patched homolog 1 (Ptch-1), Smo, Hh-interacting protein (Hhip), Gli-3]. Additionally, CA-Lip group showed a decrease in expression of HHIP, Gli-1, Ptch-1, and Smo compared to the control group, suggesting that CA-Lip has regulatory effects even under standard diet conditions. In contrast, high fat exhibited a significant increase, reinforcing the limited impact of a standard diet alone without therapeutic intervention. These comparisons support the conclusion that the observed molecular effects are primarily driven by the CA-Lip treatment.

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Conclusion

These findings support the potential application of CA-Lip as a promising nanotherapeutic strategy for the prevention and treatment of obesity and its related metabolic disorders. CA-Lip treatment significantly reduced body weight, improved insulin sensitivity, normalized lipid profiles, modulated adipokines, and suppressed systemic inflammation. It also downregulated key genes in the Hh signaling pathway and ameliorated adipocyte hypertrophy. Further research is needed to optimize the use of CA-Lip as an anti-obesity agent. Future studies are required to establish a full dose–response profile to determine the minimal effective dose, evaluate the potential for greater therapeutic benefit at higher doses, and assess any associated toxicity. Furthermore, the efficacy and safety of CA-Lip need to be confirmed through preclinical safety assessments and pharmacokinetic analyses, with well-designed human trials required to validate its potential for obesity treatment.

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Acknowledgments

Not applicable.

Conflict of interest

The authors declare no conflict of interest.

Funding

Not applicable.

Authors' contributions

Each author contributed to the study's conception; W.S.A., S.I.K., and T.K. contributed to the design, performed the experiments, and analyzed the data. O.M.Y., H. S. S., M. M. L., and E. M. M. Y. performed the experiments, data acquisition, and data analysis. M.M.A. and A.M.F. performed statistical analysis, data analysis, wrote, reviewed, and edited the manuscript. The final paper was read and approved by all authors.

Data availability

The corresponding author can provide the datasets used and/or analyzed during the current investigation upon reasonable request.

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