People with an unhealthy high-fat diet (HFD) lifestyle are at a greater risk of developing obesity and chronic systemic metabolic diseases (Organization and Organization, 2018). The impaired ability of the body to utilize fat, coupled with an imbalance between caloric intake and energy expenditure in HFD-fed animals, suggests that a significant portion of the increased energy intake is stored as fat, leading to an expansion of body fat stores (Cavaliere et al., 2018). The brain is a highly stress-sensitive organ, particularly vulnerable to key pro-inflammatory components from dietary sources, including saturated fatty acids, cholesterol, added sugars, and refined grains (Christ et al., 2019). Long-term consumption of excessive amounts of fat can disrupt the homeostasis of the entire body, including brain health, memory, and mood-related behavior (de Paula et al., 2021).

Neuroinflammation in the brain is a key risk factor for neurodegenerative disorders, with its impact varying based on the severity of these conditions (Kumar 2018). The impact of a high-fat diet (HFD) on brain function is attributed to several independent mechanisms, including oxidative stress, activation of inflammatory signaling, immune cell recruitment, mitochondrial dysfunction, increased permeability of the blood–brain barrier, and impaired synaptic plasticity (Carraro et al., 2018; Liu et al., 2015). Furthermore, eating a high-fat diet results in an imbalance between fatty acid uptake and utilization, leading to the accumulation of toxic lipid species, resulting in increased reactive oxygen species (ROS) production and inflammation. A high-fat diet increases oxidative stress in the brain and impairs mitochondrial function, although the underlying mechanisms are not yet fully understood (Cavaliere et al., 2019). Additionally, oxidative stress contributes to brain damage and induces cellular injury, impairing learning and memory (Hazzaa et al., 2020). The mechanisms by which high-fat consumption affects neural function and the potential role of these mechanisms in promoting exaggerated non-homeostatic eating are not yet fully understood.

Olive products are a valuable source of polyphenols, especially oleuropein and its derivatives, such as the hydroxytyrosol oleuropein, which destroy free radicals and hinder the chemical oxidation of lipoproteins (Chiou et al., 2007). Olive leaves (Olea europaea leaves) provide a distinctive opportunity to investigate the effects of their polyphenols on metabolic profiles and cardiovascular and hepatic structure and function, due to their rich polyphenol content and low levels of oleic acid (Manna et al., 2004). The extract of olive leaves contains secoiridoids, such as oleuropein, dimethyloleuropein, ligostroside, and oleoside, as well as flavonoids like kaempferol, apigenin, and luteolin, along with phenolic compounds such as tyrosol, caffeic acid, and hydroxytyrosol. Among the many polyphenols in olive leaf extract, oleuropein has garnered significant attention in the food industry for its potential in formulating functional products with improved nutritional properties or increased shelf-life, thanks to its antimicrobial properties and ability to prevent food oxidation (Nunes et al., 2016). Additionally, oleuropein exhibits a wide range of pharmacological functions, including anti-inflammatory effects, reduced blood pressure, inhibition of platelet aggregation and eicosanoid production, scavenging of free radicals, and inhibition of 5- and 12-lipoxygenases (Visioli et al., 2002). Polyphenolic compounds in olive leaf extract (OLE) may be a promising option for managing excess weight, reducing the risk and progression of chronic diseases, and preventing various aspects of neurodegenerative conditions associated with HFD-induced obesity (Amel et al., 2016).

The neuroprotective effect of OLE is mainly due to the antioxidant properties of its polyphenolics. Due to these advantages, olive leaf extract (OLE) has been extensively studied for its numerous health benefits, including support for brain and heart health, modulation of lipid metabolism, preservation of antioxidant stores, as well as its anti-inflammatory and anti-tumor properties (Jimenez-Lopez et al., 2020; Tsartsou et al., 2019). However, the use of bioactive compounds from olive leaf extract (OLE) faces several challenges due to their low solubility, sensitivity to environmental conditions (e.g., temperature, pH, and light), and often poor sensory characteristics (González-Ortega et al., 2021). To overcome these challenges, nano-encapsulation techniques have been developed to enhance the usability and bioavailability of these compounds (Vinceković et al., 2017). Liposomes (Lipo) are vesicles composed of phospholipid bilayers, offering characteristics valuable to the food industry, such as the ability to encapsulate both amphiphilic and water- or lipid-soluble compounds, enable controlled release, and support large-scale production (Pimentel-Moral et al., 2018). Moreover, the application of hydrogel-based technologies in combination with liposomes can further enhance their mechanical stability and membrane integrity (Ibrahim et al., 2017). From a pharmacological perspective, the use of antioxidant therapies alone to target neuroinflammation associated with HFD-induced obesity has proven largely ineffective. However, combining liposomal olive leaf extract encapsulated in hydrogel (Lipo-OLE-Hydrogel) may offer a more effective strategy for managing excess weight, reducing the risk and progression of chronic diseases, and preventing various aspects of neurodegenerative conditions associated with HFD-induced obesity (Amel, Wafa, Samia, Yousra, Issam, Cheraif, Attia, and Mohamed, 2016). The neuroprotective effect of Lipo-OLE-Hydrogel is primarily attributed to the antioxidant properties of its polyphenols. However, from a pharmacological perspective, using antioxidant therapies alone to target neuroinflammation associated with HFD-induced obesity has proven ineffective so far.

Recently, mesenchymal stem cells (MSCs) have garnered significant interest as promising cell-based therapeutic approaches due to their ability to migrate and promote tissue repair (Guo et al., 2020). MSCs are key adult multipotent cells that possess self-renewal capacity and differentiation potential. They have been explored in cell replacement therapies for repairing damaged mesenchymal or ectodermal tissues, including neural tissue (Bagher et al., 2016). Mesenchymal stem cells or their exosomes can be considered a promising therapeutic strategy to increase antioxidant capacity and neurotrophin expression, while inhibiting pro-inflammatory cytokine secretion, key factors in neurodegenerative pathologies (Angeloni et al., 2020). The contribution of MSCs as nanotherapeutic agents to brain tissue repair and functional recovery has gained widespread interest, due to their ability to participate in the complex intercellular communication system (Merino-González et al., 2016).

Therefore, considering all of the above factors, the current study aimed to provide novel insights by investigating the injection of MSC-derived exosomes (MSC-Exo) combined with a nutritional therapeutic approach, specifically the ingestion of Lipo-OLE-Hydrogel. This innovative combined therapy (Lipo-OLE-Hydrogel + MSC-Exo) seeks to reverse the detrimental effects and health risks associated with the progression of HFD-induced neuroinflammation. Consequently, the therapeutic impact of Lipo-OLE-Hydrogel + MSC-Exo on HFD-induced brain dysfunction in rats was evaluated by assessing its modulatory effects on behavior-related tasks, neurotransmitters, neuroinflammation, oxidative stress, endoplasmic reticulum markers, and immunostaining-related markers in brain tissues.

Obesity caused by prolonged consumption of HFD causes many life-threatening diseases, including insulin resistance, oxidative and endoplasmic reticulum (ER) stress, mitochondrial dysfunction, lipotoxicity, inflammation, hypertension, and cardiovascular mortality (Son et al., 2023). An HFD contributes to the increasing prevalence of obesity, which is associated with a heightened risk of impaired brain function, as well as behavioral and memory disturbances (de Paula et al., 2021). The impaired ability of HDF-fed animals to use fat as a metabolic fuel suggested that a large part of the higher energy intake is stored as fat, supporting findings from previous studies (Mollica et al., 2017; Cavaliere et al., 2018).

The brain is a highly stress-sensitive organ, particularly vulnerable to key pro-inflammatory constituents from dietary sources, including saturated fatty acids, cholesterol, added sugars, and refined grains (Christ et al., 2019). Changing nutritional lifestyle, aiming at weight loss and decreased fat accumulation, cannot provide sufficient treatment, especially when neurodegeneration occurs. Mesenchymal stem cells derived from exosome-based therapy (MSC-Exo) not only mediate communication among cells but can also be engineered to deliver specific substances. MSC-derived exosomes can mediate numerous cellular events, such as cell death, immune response, glucolipid metabolism, and oxidative stress, contributing to tissue repair (Yu et al., 2014; Li et al., 2021). Because exosomes can cross the blood–brain barrier (Kalluri and LeBleu, 2020), their implication in the treatment of neurodegenerative diseases has received recent attention (Soria et al., 2017). Adverse health consequences beyond HFD consumption include a higher incidence of brain oxidative stress and impairment of brain mitochondrial functions. However, the underlying mechanisms by which MSC-derived exosomes (MSC-Exo) exert their effects are not yet fully understood.

Olive leaf extract, with an abundant content of phenolic antioxidants, plays an important role in the treatment of obesity-related metabolic disorders via activating fatty acid oxidation and inhibiting lipogenesis (Mikami et al., 2021). However, due to its low solubility and sensitivity to environmental conditions, it needs an effective vehicle to enhance its usability and bioavailability (González-Ortega et al., 2021). In this sense, the development of effective novel evidence-based therapy, combining nanotechnology and phytotherapy, for neuroinflammation, and in particular, those with long-lasting effects, is urgently required. The generation of reactive oxygen species (ROS) is one of the most prevailing mechanisms for impeding MSCs’ stemness and triggering cellular senescence. Addressing oxidative stress or triggering antioxidant mechanisms via using phytotherapy of antioxidant power is one of the chosen approaches for improving the therapeutic efficacy of MSCs in further clinical applications. Achieving a therapeutic approach comprising the administration of LIPO-OLE-Hydrogel together with injection of MSC-Exo as a novel combined therapy (LIPO-OLE-Hydrogel + MSC-Exo) successfully reversed the progression of HFD-induced neuroinflammation.

The current study showed that an HFD increased body weight gain and body lipid levels, which is in accordance with Cavaliere et al. (2018) and Labban et al. (2020). Administration of LIPO-OLE-Hydrogel resulted in decreased body weight and fat mass in rats fed an HFD, which is in agreement with Djeziri et al. (2018). Oleanolic acid, a bioactive metabolite of olive leaf extract, causes a rise in β-oxidation and energy generation, resulting in fat reduction and anti-obesity effects (Watanabe et al., 2006). The response to HFD progression includes hyperglycemia and impairments in glucose clearance, which align with Dutheil et al. (2016). Dietary intervention with Lipo-OLE-Hydrogel may be an effective alternative for weight loss and weight management, decreasing the risk and progression of chronic diseases.

In vitro and animal studies suggest that the potential mechanisms of LIPO-OLE-Hydrogel for additional fat loss involve the phenols in LIPO-OLE-Hydrogel reducing adipocyte proliferation and increasing thermogenesis through the activation of brown adipose tissue (Melguizo Rodríguez et al., 2021). The delayed weight gain and reduced glucose tolerance supported the protective role of Lipo-OLE-Hydrogel + MSC-Exo against the metabolic syndrome associated with HDF-induced obesity. Additionally, the lowered level of glucose during the experimental period post-treatments with MSC-Exo could be attributed to the regulatory role of MSC-Exo on insulin sensitivity via modulating inflammation or by direct interaction with insulin-responsive organ mechanisms (Kumar et al., 2021). MSC-Exo facilitates the oxidation of fatty acids and inhibits the synthesis of fatty acids through lowering lipid metabolism-correlated proteins expression and thus inhibits lipid deposition in non-alcoholic fatty liver diseased rats (Cheng et al., 2021). Exosomes exert remarkable effects on lipid metabolism, including the synthesis, transport, and degradation of lipids (Wang et al., 2020). The delayed weight gain observed in rats fed a high-fat diet (HFD) and treated with the combined therapy of Lipo-OLE-Hydrogel and MSC-Exo could be related to higher energy expenditure associated with MSC-Exo injection (Castaño et al., 2022). The novel therapeutic approach involving the administration of Lipo-OLE-Hydrogel demonstrates protective effects on the progression of insulin resistance and reduces daily weight gain, likely due to its high polyphenolic content.

Supplementation with olive leaf extract improved insulin sensitivity by 15% (Haidari et al., 2021). Lipo-OLE-Hydrogel enhances insulin-stimulated glucose transport in adipocytes, suggesting that it may improve insulin sensitivity (Ryan et al., 2000). It has also been proposed that the anti-glycemic effects of OLE result from its ability to prevent starch digestion and glucose uptake, stimulate hepatic glycogen synthesis, and inhibit pancreatic α-amylase activity (Alshaer et al., 2025). Our interesting findings regarding improved glucose tolerance and insulin sensitivity after exosome therapy could be attributed to their role in attenuating HFD-induced obesity, primarily through their protective effect on islets by reducing macrophage infiltration and inflammation (Chen W. et al., 2016). The reduction in hyperglycemia observed in HFD-fed rats treated with exosomes could be due to increased insulin sensitivity, potentially through the modulation of AKT phosphorylation levels and enhanced glucose uptake in AML12 cells (Lei et al., 2021). MSC-Exo treatment mitigates the glucose and lipid metabolism dysfunctions via depressing fat mass and obesity-associated gene expression, contributing to non-alcoholic fatty liver disease management (Tamimi et al., 2024). Accordingly, our data indicated an increase in serum leptin levels in HFD-fed rats that confirmed obesity in these groups.

Leptin, a hormone involved in regulating food intake and body weight homeostasis, is typically elevated in obesity, leading to a condition known as leptin resistance (Labban et al., 2020). Hyperleptinemia has been shown to influence pointedly the obesity development and its correlated neurological disorders (Hristov et al., 2019). Obese rats were reported to typically develop leptin resistance (Scarpace et al., 2000). This finding can also be corroborated by the most recent research of Mzhelskaya et al. (2020) and Palacios-Rodriguez et al. (2024). In the context of HFD-induced obesity, leptin’s anorexigenic impact on hypothalamic neurons is diminished, resulting in an increase in appetite and increased fat mass. A diet enriched in polyphenol-based olive byproducts reduced serum levels of leptin (Carpi et al., 2019). Herein, reduced levels of leptin following a combined therapy of Lipo-OLE-Hydrogel + MSC-Exo indicated their modulatory role on serum leptin levels. OLE can be suggested to improve lipid profile, glycemic status, and free fatty acids levels in serum by body weight reduction, increasing serum adiponectin, and decreasing serum levels of leptin (Haidari et al., 2021).

In the current study, HFD-induced hyperlipidemia was detected by a significant increase in LDL, triglyceride, and cholesterol levels. Application of Lipo-OLE-Hydrogel and MSC-Exo not only helped to maintain a healthy body weight but also improved the blood lipid profile. The observed effects of MSC-Exo on reducing hypercholesterolemia and increased triglyceride levels in rats with HFD-induced obesity could be attributed to their reparative effect on hepatic tissue and the enhancement of liver function in promoting lipid oxidation, which aligns with previous findings by Rashidi et al. (2023). Our data were in line with Mikłosz et al. (2022), who stated that MSC-Exo-based therapy was sufficient to reduce body fat mass in diet-induced obesity animals and suppress the increase in body weight, which could be related to the promotion of insulin production, improvement of insulin resistance, and regulation of hepatic metabolism. In the same line, consumption of Lipo-OLE-Hydrogel has been shown to lower weight gain over time (Razquin et al., 2009; Shen et al., 2014) and LDL-C (Solá et al., 2011), increase HDL-P (Flynn et al., 2017), and improve insulin sensitivity, which is attributed to its higher phenolic content. These boosted outcomes related to Lipo-OLE-Hydrogel in the current study are mainly attributed to its encapsulation in nano materials that could enhance its stability and functionality (González-Ortega et al., 2021).

In our study, animals fed a high-fat diet (HFD) and treated with MSC-Exo or the combined Lipo-OLE-Hydrogel + MSC-Exo therapy exhibited improved glucose clearance and reduced hyperlipidemia compared to those treated with Lipo-OLE-Hydrogel alone. These findings suggest that Lipo-OLE-Hydrogel may not be sufficient on its own to fully mitigate obesity-related metabolic disorders, but its effectiveness is significantly enhanced when combined with MSC-Exo therapy.

Growing evidence demonstrates that the time-course effects of HFD-induced obesity significantly impact learning, memory processes, and mood-related behaviors (Pratchayasakul et al., 2011). In this study, a shift to a high-fat diet (HFD) significantly impacted brain function, mood-related behaviors, and performance in memory-related tasks, as evident in altered open-field test outcomes. Notably, impaired glucose regulation, reflected by increased blood glucose levels—particularly after 14 weeks of HFD intake—was associated with deficits in learning, memory, and behavior. We observed pronounced behavioral changes linked to anxiety, consistent with prior studies in rats and mice, which have shown that high-energy diets can lead to anxiety-like behaviors through increased activity of the hypothalamic–pituitary–adrenal (HPA) axis (Pratchayasakul et al., 2011). Notably, the behavioral deficits induced by HFD were rapidly reversed by the combined therapy of Lipo-OLE-Hydrogel and MSC-Exo, as evidenced by improvements in the novelty-suppressed feeding test (NSFT) and home cage feeding (HCF) assessments. This can be explained by the effective properties, stability, and release of OLE active principles from OLE in liposomal hydrogel (Istenič et al., 2016).

It is well understood that dopamine and serotonin play an essential role in homeostatic signaling as neurotransmitters (Tuominen et al., 2015). Based on previous studies in humans and rodents, modulation (either inhibition or stimulation) of these neurotransmitters has been linked to changes in feeding behavior, appetite regulation, energy expenditure, and reward-based learning, as demonstrated in studies such as Halford et al. (2005) and Palmiter (2008). In the case of food-related processes, increased 5-HT is generally thought to suppress feeding behavior and serve as a satiety signal in mammals (Yabut et al., 2019). The notable increase in amounts of brain dopamine in obese rats noted in this study can be clarified by the fundamental contribution of the dopamine system in controlling hyperphagia, response to high-energy diets, and the pathophysiology of obesity (Geiger et al., 2009). We hypothesized that the alterations in the feeding behavior after eating an HFD associated with obesity are a consequence of variations in the pivotal serotonin and dopamine systems, which align with Halford, Harrold, Lawton, and Blundell (2005). The unusual decrease in brain serotonin (5-HT) in HFD-triggered obese rats, as reported here, is reinforced by recent work of Haleem and Mahmood (2021), in which a significant reduction in serotonin was observed in obese subjects. Their findings further support the neurochemical theories underlying disrupted feeding behavior in obesity, highlighting the role of neurotransmitters such as serotonin in the regulation of appetite and energy balance. Bautista et al. (2019) illustrated the association between glutamate and obesity-linked markers of inflammation, dyslipidemia, and oxidative stress. The significant increase in brain glutamate reported in the present study was supported by the work of Valladolid-Acebes et al. (2012). This supports the hypothesis that elevated levels of nutritional glutamate play an etiological role in the development of obesity. This concept underscores the importance of developing weight management medications that target brain neurotransmitters responsible for regulating food intake.

These research findings provide new insights into how high-fat consumption influences neural mechanisms and their potential role in exacerbating non-homeostatic eating behaviors. The synergistic effect of combined Lipo-OLE-Hydrogel + MSC-Exo on the amelioration of brain neurotransmitter impairment suggests their effective role in the treatment of neurocognitive disorders, aligning with a previous study (Harrell et al., 2021). In addition, inflammation is a central feature of obesity and altered glucose tolerance, impacting not only the blood and peripheral systems but also the central nervous system. Pro-inflammatory cytokines interact with multiple signaling pathways, playing a key role in mood regulation and contributing to the development of depression (Haroon et al., 2012). It has also been found that high-fat diet consumption (HFD) can evoke an inflammatory response (Cavaliere et al., 2018). Additionally, significant inflammatory damage following ingestion of HFD was detected in brain tissues of the non-treated group, as displayed by scattered degenerations, a decrease in the viable pyramidal cells in areas of the hippocampus, a decrease in the thickness of granular cells in the dentate gyrus, and observed signs of apoptosis (Hazzaa et al., 2020; Mengr et al., 2023). Accordingly, cognitive impairment, such as impaired glucoregulation (Mielke et al., 2006), increased brain inflammation, and alteration in blood–brain barrier permeability (Cordner and Tamashiro, 2015), has been attributed to consumption of an HFD (Xia et al., 2015).

We observed increased expression levels of inflammatory cytokines, which are associated with higher levels of oxidative stress biomarkers in the brain tissues of HFD rats. This confirms that the neuroinflammation observed in the diet-induced obesity animal model may be linked to the increased expression of inflammatory markers. This could be attributed to the reduced ability of HFD-fed animals to utilize fat as a metabolic fuel, leading to the accumulation of toxic lipid species that trigger ROS overproduction and inflammation (Mollica et al., 2017). NF-κB is a transcription factor implicated in the regulation of a wide range of genes related to apoptosis, inflammation, and immune response, and the expression of many chemokines, cytokines, and other inflammatory mediators, including Nos2, Cox2, and TNF-α, is under the control of NF-κB activation (Duval et al., 2015). Toll-like receptors (TLRs) are danger-associated molecular pattern receptors that also interact with insulin, inflammation, and mTORC1 signaling pathways (Ghasemi et al., 2013). These receptors serve a variety of functions involving the inflammasome, protein synthesis regulation, inflammatory cytokine expression, apoptosis, and cell survival (Hanke and Kielian, 2011). Data from another animal study further demonstrated that HFD triggers TLR4 and endoplasmic reticulum stress signaling pathways (Zhang et al., 2008; Wang X. et al., 2012).

In the current study, inflammatory responses in brain tissues were increased in obese rats, including the upregulation of TLR4 and NF-κB, as well as the production of pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α). This finding is also linked to the significant alterations in serotonin, dopamine, and glutamate, which were discussed earlier in the study as markers of neuronal damage, and is consistent with previous research of Labban et al. (2020). In the current study, progressive inflammatory responses subsided more prominently in the group that received Lipo-OLE-Hydrogel + MSC-Exo. This was attributed to the health-promoting and anti-inflammatory effects of Lipo-OLE-Hydrogel, resulting from its increased absorption rate, higher bioavailability, and increased permeation through the intestine after encapsulation in an appropriate nano-delivery system (Munin and Edwards-Lévy, 2011). Additionally, mesenchymal stem cell-derived extracellular vesicle (MSC-EV) treatment suppressed neuroinflammation by inhibiting NF-κB activation and expression (Xin et al., 2021).

Oxidative stress, resulting from an imbalance between reactive oxygen species (ROS) and the endogenous antioxidant defense system, plays a pivotal role in neurodegeneration. This is particularly significant in the brain, which is characterized by high oxygen consumption, increased ROS production, a high content of polyunsaturated fatty acids, and relatively low antioxidant defenses (Angeloni et al., 2019). Throughout the high-fat feeding, the mismatch between uptake and utilization of fatty acids can lead to toxic lipid accumulation, triggering ROS overproduction and inflammation. Additionally, oxidative stress contributes to brain damage and induces cell injury with impaired learning and memory (Hazzaa et al., 2020). Importantly, this central inflammation in brain tissues can actually contribute to leptin and insulin resistance, favoring weight gain and maintaining an increased body weight, which agrees with Posey et al. (2009). The current study indicated that Lipo-OLE-Hydrogel + MSC-Exo improved brain function by suppressing the toxic effect of lipids via downregulating inflammatory markers and oxidative stress-related genes and biomarkers in the brain (as shown by a reduction in MDA, H₂O₂, and ROS content with significant increases in TAC and GSH-PX content in brain tissues). MDA is one of the final products in the peroxidation of polyunsaturated fatty acids by the cell, and its higher concentration in brain tissues is an indicator of oxidative stress (Labban et al., 2020).

In this context, when mesenchymal stem cells (MSCs) are engrafted into tissues with elevated levels of inflammatory cytokines, they adopt an immunosuppressive phenotype and produce a variety of immunosuppressive factors, leading to the inhibition of inflammatory immune cells (Gazdic et al., 2015). The beneficial effects of MSCs are attributed to their migration toward inflamed areas, driven by the expression of chemokine receptors. These receptors help inhibit or limit inflammatory responses and promote anti-inflammatory pathways (Wang S. et al., 2012). In addition to differentiation and integration into the damaged tissue, the mode of action of MSCs when implanted in the injured region could involve several mechanisms: immunomodulatory and anti-inflammatory properties, induction of neurogenesis, secretion of neurotrophic factors, promotion of axon growth, enhancement of synaptic and cortical connections, and reduction of apoptosis and oxidative stress (Angeloni et al., 2020). MSCs are able to reduce H₂O₂-induced inflammatory factor, downregulate intracellular oxidant factor (MDA), upregulate intracellular antioxidant factor (SOD), and increase neurotrophin expression (Cui et al., 2017), in addition to increasing GSH and SOD activity and decreasing MDA activity (Chang et al., 2021; Yang et al., 2013).

Cells respond to oxidative stress by inducing antioxidant enzymes that can neutralize ROS, counteracting cell damage (Otsuki and Yamamoto, 2020). In particular, nuclear factor erythroid 2-related factor 2 (Nrf2) increases the expression of these antioxidant enzymes, thanks to its binding to a specific sequence in the promoter region of these genes called antioxidant response element (ARE) (Nam and Keum, 2019). Nrf2 is a key transcription factor involved in protecting against ROS generation and oxidative damage (Gao et al., 2020). Once Nrf2 is translocated to the nucleus, it induces the expression of genes including HO-1, SOD, and GSH, to counteract oxidative stress and maintain redox status balance (Kopacz et al., 2022). In this study, we consistently observed that upregulation of Nrf2 significantly reduced the inflammatory response and ROS production, while increasing the expression of antioxidant enzyme-related genes in brain tissues following treatment with Lipo-OLE-Hydrogel + MSC-Exo, highlighting their antioxidant effect.

In the current study, the optimal functionality of the novel nanocomposite formulated with Lipo-OLE-Hydrogel is linked to its higher biodegradability and controlled release of active biomolecules in the targeted area, which accelerates its curing properties. The benefits of combining natural antioxidants, such as Lipo-OLE-Hydrogel, with exosome injection are to reduce oxidative stress and create a balance between oxidants and antioxidants in the brain environment. This can be achieved via multiple pathways, such as regulating the formation of free radicals, the scavenging of excess radicals, and by interfering in the free radical chain reaction cascade, thereby eliminating the oxidative damage that may contribute to better media for survival, proliferation, and therapeutic effectiveness of MSC-Exo (Zheng et al., 2023).

Recent studies have shown that MSC-derived exosomes promote functional recovery, support neurogenesis, and reduce neuroinflammation in rats after traumatic brain injury, due to their protein composition, lipids, miRNAs, and mRNAs, which play a crucial role in cell-to-cell communication (Yang et al., 2017). The prominent impact of MSC-Exo on the inflammatory response in HFD-fed rats was attributed to their role in inhibiting macrophage inflammatory response (Zhao et al., 2018), indicating their role in the treatment of obesity-associated inflammation. Additional effects of Lipo-OLE-Hydrogel are attributed to its polyphenols, such as oleuropein, hydroxytyrosol, tyrosol, and caffeic acid, which play a crucial role in providing antioxidant and anti-inflammatory effects (Covas et al., 2006). The principal mechanisms of action of polyphenol-based olive byproducts in inflammatory response are a decrease in NF-κB activation, a decrease in LDL oxidation, and an improvement in insulin resistance (Assy et al., 2009). In brain tissues, the biological actions of phenols in Lipo-OLE-Hydrogel have been attributed to their ability to scavenge reactive species or through their possible influences on intracellular redox status (Pollard et al., 2006). In addition, olive leaf phenols have been shown to have some neuroprotective effects against Alzheimer’s disease, Parkinson’s disease, and peripheral neuropathy (Monti et al., 2011). Some findings suggested that Lipo-OLE-Hydrogel had a beneficial effect on learning and memory deficits. It was also observed that an HFD activated apoptosis in brain tissues, as indicated by the upregulation of pro-apoptotic genes caspase-3 and cytochrome c, alongside the downregulation of the anti-apoptotic gene Bcl-2, which is consistent with findings of Crespo et al. (2012), Liu and Wang (2011), and Palacios-Rodriguez et al. (2024). These favorable effects of OLE were attributed to its incorporation in a nano formulation comprised of liposomes that can prolong the release of these active substances, thus optimizing its therapeutic delivery (González-Ortega et al., 2021).

Oxidative stress stimulates the expressions of several genes involved in cell death through apoptosis (Crespo et al., 2012). The Bcl-2 family, which incorporates Bcl-2 and Bax, has a significant role in managing apoptosis. Bcl-2 is considered a pro-survival signal, and Bax is a pro-apoptotic member as it binds and antagonizes Bcl-2 actions (Becker et al., 2013). Our data indicate that Lipo-OLE-Hydrogel + MSC-Exo significantly upregulated Bcl-2 expression, as supported by immunostaining, while downregulating the pro-apoptotic genes Bax, cytochrome c, and caspase-3. These findings highlight their key role in attenuating brain apoptosis. These results are consistent with previous studies suggesting that the modulation of brain apoptosis is associated with the mitigation of oxidative stress (Kruidenier et al., 2003).

Endoplasmic reticulum stress appears in brain regions during obesity and has been implicated in the development of obesity (Çakir et al., 2013). Excessive ER stress can lead to apoptosis and neurodegeneration (Tabas and Ron, 2011) and eventually brain atrophy. ER stress can also exacerbate cellular inflammatory pathways and cause ROS production, which in turn can exaggerate ER stress (Hotamisligil, 2010). Thus, inflammation-related ER stress may also contribute to neuronal dysfunction either directly or by modulating oxidative stress and inflammation. The current study proposed a tight correlation between expression of ER stress and inflammation-related genes, as evidenced by the overexpression of eIF2α, ATF6, PERK, JNK, XBP1, and CHOP mRNA levels in brain tissues in HFD-fed rats. These findings are in agreement with higher expression of caspase-3 and cytochrome C in non-treated HFD rats. Moreover, prolonged activation of ER stress in rats fed an HFD consecutively leads to higher expression of NLRP3 inflammasomes and an increase in pro-inflammatory cytokine levels (Oslowski et al., 2012). HFD-induced prolonged unfolded protein response (UPR) activation can induce apoptosis and PERK-mediated phosphorylation of eIF2α, which may contribute to cell death by inhibiting the synthesis of pro-survival proteins. One of the downstream targets of PERK, the transcription factor CHOP, can repress Bcl-2 expression (McCullough et al., 2001). The pro-apoptotic action occurs mainly through activation of the PERK-eIF2α-ATF6-CHOP-Bcl-2 pathway (Lin et al., 2007), which suppresses the synthesis of anti-apoptotic Bcl-2 proteins.^.Bcl-2 is an anti-apoptotic protein that prevents the release of cytochrome c from mitochondria by inhibiting apoptosis-induced mitochondrial pore formation. Translocation of cytochrome c from the mitochondria to the cytoplasm is a key event in the activation of caspase-3 and the induction of apoptosis (Lin et al., 2007). The downregulation of ER-related genes was more prominent in the group treated with MSC-Exo or the combination therapy of Lipo-OLE-Hydrogel + MSC-Exo. This effect could be attributed to the role of exosomes in upregulating Bcl-2 expression and inhibiting caspase-3 activation. Under ER stress, PRP-derived exosomes inhibit CHOP-mediated suppression of Bcl-2 protein expression through the Akt/Bad/Bcl-2/Caspase-3 signaling pathway, thereby protecting cells from apoptosis (Tao et al., 2017). MSC-exosomes can exert anti-inflammatory and neuroprotective effects by inhibiting the expression of NF-kB and endoplasmic reticulum-related genes (Ren et al., 2020).

The above-mentioned findings were also supported by the synergistic healing effect of Lipo-OLE- with MSC-Exo on brain tissue regeneration in the histological picture that is evinced by regeneration and normal architecture of brain tissues in such groups. The enhanced therapeutic effect of combining Lipo-OLE-Hydrogel with MSC-Exo injection can be attributed to Lipo-OLE-Hydrogel’s ability to alleviate neuroinflammation by reducing oxidative stress. This creates a more favorable microenvironment by suppressing cellular senescence, thereby supporting the proliferation and migration of MSC-Exo and enhancing their overall therapeutic efficacy (Zheng et al., 2023).

The neuroprotective effect of Lipo-OLE-Hydrogel, when combined with the regenerative effect of MSC-Exo, exerted a promising curative role against HFD-induced neuroinflammation progression. Taken together, our findings indicate that the Lipo-OLE-Hydrogel + MSC-Exo therapy provides effective neuroprotection and reduces the deleterious impact of an HFD by ameliorating the accumulated oxidative and endoplasmic stress mediators in brain tissues, thereby reducing the excessive inflammatory response and associated damage. More precisely, MSC-Exo, together with Lipo-OLE-Hydrogel, resulted in considerable improvement in memory and behavioral deficits caused by an HFD. Our findings, therefore, open new avenues for potential treatments of brain dysfunction associated with the adverse effects of HFD consumption by combining natural protective and regenerative agents.