Dietary Chitosan Attenuates High-Fat Diet-Induced Oxidative Stress, Apoptosis, and Inflammation in Nile Tilapia (Oreochromis niloticus) through Regulation of Nrf2/Kaep1 and Bcl-2/Bax Pathways

1. Introduction

The aquaculture sector has grown and prospered over the past few decades [1]. Despite its advancement, the aquaculture sector faces significant challenges, such as the limited availability of high-performing fry strains, the elevated cost of aquafeeds, and limited disease resistance [2].

In fact, short-term feeding of fish benefits from boosting dietary lipid levels within specified bounds [3]. However, as the concentration of dietary lipids increases, a series of adverse effects begins to emerge [4].

The primary organ in charge of regulating lipid uptake, synthesis, redistribution, catabolism, and storage is the liver. Steatosis is largely caused by fatty acid and triacylglycerol imbalances [5,6].

A high-fat diet is posited as the instigator of hepatic lipidosis in farmed fish [3,7,8,9]. This can subsequently compromise fish health and reduce production [10]. Fat accumulation has been shown in several studies utilizing animal models to trigger inflammation [11,12,13,14,15] and contribute to liver damage, hypoimmunity, and decreased appetite [8]. Furthermore, fat buildup can accelerate lipid peroxidation, inducing oxidative stress [16], and disrupt lipid metabolism, thereby inhibiting growth [3,8,10,17].

nrf2 serves as a pivotal regulator of the adaptive response to oxidative stress. Upon exposure to oxidative stress, nrf2 is activated through its dissociation from the kelch-like ECH-associated protein-1 (Keap1), subsequently translocating to the nucleus [18], where it activates a battery of antioxidant genes [19]. However, severe oxidative stress suppresses the nrf2 pathway [20,21,22]. To counteract hepatic steatosis, nrf2 activation inhibits lipogenesis and promotes beta-oxidation of fatty acids [23], while nrf2 pathway inactivation could potentially exacerbate liver damage induced by hepatotoxicants [24,25]. Genetically regulated cell death is called apoptosis, and it may be brought on by a stream of physiological events. It is widely accepted in the scientific community that mitochondrial malfunction is a crucial indicator of apoptosis [26]. cyt-c is released by dysfunctional mitochondria, which then activates the downstream effector cas-3, resulting in the execution of apoptotic changes [27].

Occludin, a trans-membrane protein, members of the claudin family, junction adhesion molecules, and linker proteins such as zo-1 are all components of tight junctions [28]. Increased intestinal permeability results from decreased intestinal tight junction protein expression, which can seriously jeopardize intestinal barrier integrity [29]. Impaired intestinal integrity is accompanied by intestinal permeability changes and histological changes [30,31,32]. The intestine predominantly responds to various stressors, including oxidative stress and inflammation [33,34]. Hence, oxidative stress and inflammation in the intestines are crucial for maintaining intestinal function. Consequently, to maintain intestinal function in the face of oxidative stress, an optimal candidate with free radical scavenging activity as well as antioxidant and anti-inflammatory capacity is urgently needed.

Natural materials have recently shown distinctive, affordable, and secure antibacterial properties. Recently, a remarkable number of organic bioactive substances have been investigated for potential use as methods of reducing the detrimental effects of an HFD in farmed fish [35,36,37].

Chitosan (β-(1-4)-N-acetyl-D-glucosamine) has proven to be among the most intelligent and safe natural cationic biopolymers. It is a member of a class of glycosaminoglycan-like polysaccharides having structural characteristics [38]. Following cellulose, chitosan is the second-most abundant polymer [39]. It offers exceptional qualities such as a non-toxic nature, biocompatibility, biodegradability, and increased solubility, along with immunological restoration attributes [40]. Chitosan is derived from the de-acetylation process of chitin and is recognized as a crucial constituent found in the exoskeletons of aquatic crustaceans, such as crab, crayfish, and shrimp, as well as in certain terrestrial organisms and the cell walls of some microorganisms [41]. Chitosan is receiving considerable interest and has already begun to contribute significantly to aquaculture’s sustainability. It satisfies environmental standards since it is an eco-friendly substance that also promotes the effective use of reagents and minimizes potential waste [42]. There are several uses for chitosan in the biomedical and pharmaceutical industries [15]. Chitosan is an active growth promoter that can be considered a crucial element for the growth of aquatic animals. The major effect of chitosan is improving the morphological structure of the small intestine, which may potentially augment nutrient absorption and subsequently improve growth performance [43]. Even at low concentrations, dietary chitosan was found to enhance nitrogen utilization and amino acid digestibility [44].

Nile tilapia (Oreochromis niloticus) is highly favored among cultured fish, ranking as the third most prominent group of cultured fish globally, surpassed by carp and salmonids. It is extensively cultured in about 100 countries in both tropical and subtropical regions [45,46]. The success of Nile tilapia in aquaculture is ascribed to its superior survival rate in poorly oxygenated environments, comparatively high resistance to diseases, and adaptability to consume a diverse range of foods [47]. Moreover, in cultured fish, there is a paucity of evidence supporting the potential pathogenesis of fatty liver injury. Consequently, a prevailing challenge in aquaculture is the development of strategies to mitigate the oxidative stress and inflammation induced by high-fat diets. Chitosan’s antioxidant activity has garnered significant attention. Research indicates that chitosan exhibits hepatoprotective effects due to its antioxidant properties [48]. Similarly, AlKandari et al. [49] stated that supplementing with natural antioxidants like propolis, chitosan, or their combination during ibuprofen use enhanced the reduction in toxic effects and improved the antioxidant system and anti-inflammatory response.

Our study constituted novel insights on hepatotoxicity and intestinal injury induced by an HFD and evaluated the ameliorative effects of dietary chitosan supplementation on intestinal integrity, oxidative status, and apoptosis/anti-apoptosis.

2. Materials and Methods

3. Results

4. Discussion

Lipids are a dietary energy resource that plays a consequential role in fish growth. However, excessive amounts of lipids in the diet will have adverse impacts on fish growth and health, including poor feed intake, slow growth, low immunity, and oxidative stress [67,68]. The formation of a fatty liver is mostly a result of the high content of fat in feed, which renders the body unable to consume too much fat itself. The deposition of fat will consequently cause an increase in the fat content in the fish body, which is principally deposited in liver cells and can result in a fatty liver [69].

In mammals, lipotoxicity is the primary contributor to several diseases associated with excess fat accumulation, such as a fatty liver, obesity, and diabetes [70,71]. Excessive deposition of triglycerides in the liver is considered an important biomarker of a fatty liver. Moreover, excessive dietary fat might also cause adverse effects on fish [72].

The negative impacts caused by an HFD in fish have been investigated in different species. It has been noticed in grass carp (Ctenopharyngodon idella) and tilapia (Oreochromis niloticus) that diets high in fat (15 or 16%) decreased feed intake and growth performance, lowered immunological function, and changed lipid metabolism [67,68,73]. Moreover, it has been reported in turbot (Scophthalmus maximus) and black seabream (Acanthopagrus schlegelii) that a high-fat diet (17–19.5%) induces lipid peroxidation and oxidative stress [9,74]. An HFD (11 or 12% fat) exacerbated intestinal and liver health in tilapia [17] and triggered endoplasmic reticulum (ER) stress to modulate hepatic lipid secretion in blunt snout bream (Megalobrama amblycephala) [3]. Excessive lipid consumption beyond the body’s capacity for utilization is the main risk factor for hepatic steatosis in most cultured fish. Hepatic steatosis is a typical form of abnormal lipid metabolism in the liver that is characterized by excessive deposition of triacylglycerol in hepatic tissue [3,72]. Growth, feed utilization rate, immunity, and stress tolerance are negatively impacted by steatosis [8,69,75]. It was explained by Tanaka et al. [76] that an HFD induces hepatic steatosis by inhibiting hepatic autophagic activity, which triggers fat accumulation. Similarly, in mammals, lipotoxicity is the main contributor to various diseases, such as a fatty liver, obesity, and diabetes [70,71]. The present study showed that Nile tilapia exposed to a high-fat diet without chitosan supplementation (F12Ch0) had the lowest values for FBW, BWG, SGR, PER, and body protein content and the highest values for FCR, HSI, IPF, liver, and body fat content after 8 weeks of a feeding trial. Tilapia groups offered trial diets fortified with 5 and 10 g/kg chitosan (100%) exhibited the opposite trend. Previous research studied the negative impacts of an HFD on tilapia and indicated that an HFD reduces feed intake, impairs growth performance, damages fish’s liver and intestinal tissue, and causes oxidative damage [75,77,78]. Also, Dai et al. [8] stated that an HFD-related inflammation response reduces the appetite of blunt snout bream and is closely related to lipid utilization abnormality and exacerbation of growth performance. The lowered growth performance and poor body indices obtained in Nile tilapia fed with an HFD (F12Ch0) could be due to the reduced feed intake, efficiency, digestion, absorption, and metabolism that resulted from the oxidative damage and inflammatory effect of an HFD on the liver and intestine, which are considered the main organs involved in nutrient digestion, absorption, and metabolism, as explained previously by Ding et al. [79]. This evidence is confirmed by the results of serum antioxidant enzymes and liver and intestine histopathology (Table 6 and Figure 2 and Figure 3). Subsequently, the nutrient deposition in the whole fish body, such as protein, decreased in this group, as seen in Table 4, as did the distorted tight junction proteins (Figure 8). Furthermore, excessive lipid intake causes over-deposition of fat in hepatic tissue and viscera, causing an increase in relative liver size (HSI), viscerosomal index (VSI), metabolic disturbances, and a reduction in liver activity [72,79,80,81]. Similar findings were observed in grass carp (Ctenopharyngodon idella) [13], turbot (Scophthalmus maximus) [74], black seabream (Acanthopagrus schlegelii) [9], largemouth bass (Micropterus salmoides), and orgiant croaker (Nibea japonica) fed with a high-fat diet [72,81].

In this feeding trial, Nile tilapia fed the high-fat diet (F12) supplemented with chitosan demonstrated better growth, feed utilization, body indices, body protein content, and lower fat accumulation in the body and liver. It indicates that adding chitosan mitigated the deleterious impacts induced by the high-fat diet (12% fat) without chitosan. The beneficial effects of chitosan supplementation on fish diets have been studied in different species. Chitosan plays various biological roles, including hypolipidemic, immunomodulatory, and antioxidant properties [82,83,84]. It has been observed that diets containing chitosan enhance the growth of shrimp (Penaeus monodon), loaches (Misgurnus anguillicadatus), and caspian kutum (Rutilus frisii kutum Kamenskii) fingerlings [85,86,87,88]. Similarly, Wu [50] noticed that diets containing 4 g kg⁻¹ chitosan increased tilapia body weight gain, feed conversion rate, specific growth rate, body protein content, decreased lipid in the whole body, and hepatopancreas. It is linked to the modulatory action of chitosan on a variety of receptors, including the calcium-sensing receptor (CaSR), olfactory receptor, epidermal growth factor receptor, Tolllike receptor 4, TLR4/MD-2 receptor, scavenger receptor BI, and CYP7A1 [89,90]. Thus, certain receptors mediated the effects of chitosan, which in turn triggered the production of proteins. Furthermore, a large dose of chitosan probably displayed hypolipidemic action, restricting the tilapia from synthesizing lipids [83].

Hematological response is an essential index of fish health, which varies according to the type of stressor [91]. In the present study, it was notable that dietary chitosan positively impacts the hematological parameters in Nile tilapia by enhancing the RBC, PCV, and hemoglobin concentration. This may be attributed to the ability of chitosan to improve the absorption of macronutrients and micronutrients [92] that lead to good health. The count of WBCs is used to evaluate both fish innate immunity and health [93]. Moreover, chitosan-treated fish might receive a sufficient amount of oxygen for respiration and high metabolic activity [94,95]. Mubarak Ali et al. [96] stated that dietary chitosan and chitosan nanoparticles recorded better non-specific defenses due to the immunostimulatory effect and antimicrobial properties of chitosan nanoparticles. Similarly, Meshkini et al. [97] indicated that dietary chitosan at concentrations of 0.25% showed improving effects on the hematological indices, a result that agreed with the biochemical findings of the present study regarding enhanced total serum proteins in fish fed chitosan, which suggest better innate immunity of the fish. Comparable outcomes have been reported in many fish species when fed chitosan [98] and chitosan nanoparticles [99].

Fish biochemical parameters are regularly used as reliable diagnostic tools in biomonitoring, allowing for detection of the pathophysiological changes attributable to nutrition [100]. In this study, we found that fish fed an HFD, F12Ch0, showed significantly higher serum activities of AST, ALT, and LDH compared to the F6Ch0 group (Table 6). Their abnormal elevations implied the occurrence of both liver injury and hepatotoxicity, which are firmly associated with both hyperlipidemia and hepatic steatosis [101]. This is consistent with the work of Li et al. [102] and Chen et al. [103] in M. amblycephala and in blunt snout bream (Megalobrama amblycephala) fed an HFD. However, in the present study, AST and ALT activities were reduced by increasing dietary chitosan in HFD-fed fish, implying that dietary chitosan incorporation could mitigate the HFD-induced damage in Nile tilapia, which may be attributed to the antioxidative capability of chitosan that protects liver cells from damage. Current results coincided with Mehrpak et al. [104] and El-Naby et al. [92], who reported declining AST and ALT activities in fish fed a chitosan-supplemented diet. Elevated ALT and AST with raised creatinine levels may also affect other organs, such as the gills and kidney [105].

The concentrations of energetic metabolites triglycerides and cholesterol, as well as VLDL-C and LDL-C, were significantly increased in fish fed an HFD (F12Ch0). According to Mensinger et al. [106], cholesterol levels can result in disorders of lipid and lipoprotein metabolism, particularly liver dysfunction. Higher levels of lipid in fish feeds also make hepatocytes work harder, probably stressing them and contributing to liver damage [3]. Moreover, chitosan supplementation in an HFD-fed fish exhibited a beneficial effect on the lipid profile, as seen in a previous study on Nile tilapia (Oreochromis niloticus) [107,108]. Kang et al. [109] stated that chitosan oligosaccharide (COS) can inhibit the activity of pancreatic lipase and reduce the absorption of intestinal fat in combination with bile acids, as well as increase the excretion of fecal fat [110]. Accumulated shreds of evidence have demonstrated that these results were analogous to data in B. bidyanus [111], C. carpio [112], and Ctenopharyngodon Idella [113], and Xu et al. [114] indicated that adding 5% chitosan can promote liver LDL receptor (LDLR) mRNA expression, improve the clearance of LDL into the liver, and diminish the plasma cholesterol level. Furthermore, the current reduction in glucose levels may indicate the ability of chitosan to reduce liver gluconeogenesis and increase glucose consumption in the skeletal muscles [115].

Oxidative stress is a major cause of the progression of liver disease, which is induced by an HFD and leads to mitochondrial dysfunction, apoptosis, ER stress, and an inflammatory response [5,116]. Fish fed an HFD have a notable hepatic lipid accumulation and peroxidation [81,117]. As a result of lipid deposition and peroxidation, excessive production of ROS will deteriorate the integrity of organelles and cause severe damage to cells and tissues [81,118]. In this study, we found that the liver and intestine of HFD-fed Nile tilapia (F12Ch0) signally downregulated the mRNA levels of antioxidant enzymes (SOD and GPX), as well as nrf2, while elevating kaep1, which reflected the occurrence of severe oxidative stress and redox imbalance. SOD, GSH-Px, and CAT were considered the main antioxidant enzymes for free radical scavenging [119]. In this work, dietary chitosan could alleviate intestinal mucosal oxidative stress by upregulating the gene expression of the antioxidative enzyme activities of SOD and GSH-PX. These findings are consistent with Lan et al. [120], who indicated COS could increase SOD, CAT, GSH-Px, and T-AOC activity and decrease the MDA level after H2O2 challenge. Li et al. [121] also indicated COS enhanced SOD activity in the duodenum’s mucosa and decreased the MDA level in the jejunum and ileum’s mucosa in broilers. Furthermore, Li et al. [122] indicated that dietary COS supplementation elevated the inhibition of hydroxy radical capacity while decreasing MDA content in the ileum mucosa of broilers. On the same line, Assar et al. [123] stated that chitosan dietary inclusion in HFD-fed Zaraibi goat bucks caused a notable enhancement in antioxidant enzyme activities and suppressed the elevated MDA levels.

Many studies concluded that the accumulation of fat intensified vulnerability to oxidative stress and debilitated the antioxidant defense system in HFD-induced liver injury [116,124,125]. However, dietary chitosan enhanced nrf2 mRNA expression in the liver and intestine, similar to other similar studies on doxorubicin-challenged rats [126] and mice fed a high-fat diet [127].

Some studies have concluded that adding chitosan induces nuclear factor erythroid-derived2-like2 (nrf2) activation, which plays a vital role in cellular protection in opposition to free radical damage and reduces the incidence of severe oxidative stress as well as redox imbalance and chronic inflammation in the liver [3,8].

In this study, we explore the mechanism by which an HFD promotes hepatic and intestinal injury by focusing on mitochondrial dysfunction, which has been identified as a crucial driver for both cell injury and cell death. Apoptosis is a genetically programmed type of cell death that can be triggered by a variety of physiological changes. It is commonly accepted that mitochondrial dysfunction is a crucial indicator of apoptosis, which is mediated by the bcl-2 family of proteins, including both bcl-2 and bax [26]. Mitochondrial dysfunction causes the release of cyt c, which activates the downstream effector cas-3 and finally executes the apoptotic changes [27]. In the current investigation, apoptosis of hepatocytes was also associated with intrinsic mitochondrial pathways characterized by mitochondrial damage with the release of cytochrome c and activation of caspase-9 and, subsequently, caspase 3 as well as baxa in the HFD-fed group (F12Ch0). This is in line with Lu et al. [128], who detected that cytochrome c was released from mitochondria into the cytosol after feeding fish an HFD, causing damage to mitochondrial permeability. Cytochrome c release from mitochondria is considered the principal event for apoptosis. Therefore, we believe that the anti-apoptotic activity of chitosan stems from its ability to protect mitochondrial integrity. Among proapoptotic members, bax is perhaps the best-studied protein and is essential for mitochondrion-mediated apoptosis [129]. Functional analysis showed that bax could promote the release of cytochrome c from mitochondria [129,130]. Dietary chitosan supplementation significantly downregulated the expressions of cytochrome c, caspase-3, and caspase-9 compared to the HFD-fed group (F12Ch0). Conversely, bcl-2 plays an anti-apoptosis role by ameliorating cytochrome c release, and a decrease in the bax/bcl-2 ratio alleviates the amount of apoptosis [131,132]. In this study, dietary chitosan inclusion downregulated the bax expression level while upregulating the bcl-2 expression level compared with the HFD-fed fish group, F12Ch0. An HFD induces hepatocyte apoptosis in the blunt snout bream [133]. In line with earlier findings, apoptosis was also enhanced through upregulating pro-apoptotic genes cytc, bax, cas-3, cas-8, and p53 and downregulating anti-apoptotic genes bcl-2 and xiap in HFD-fed tilapia [134]. Similarly, Dai et al. [8] observed an increase in hepatocyte gene expression of caspase-3, caspase-9, and CD68. These genes are known for their functions in promoting intrinsic apoptosis, regulating physiological cell death, and regulating pathological tissue degeneration in M. amblycephala fish.

High-fat diets are well recognized to induce metabolic inflammation throughout the tissues. High-fat diets elevate the amounts of endotoxins, circulating free fatty acids, and inflammatory mediators, resulting in low-grade systemic inflammation and disturbed homeostasis in many tissues [135]. Li et al. [122] indicated that long-term HFD feeding induced an increase in inflammatory markers in zebrafish (Danio rerio). Inflammatory cytokines are intimately associated with the development of metabolic disorders [131,132,136]. Many studies demonstrated that an HFD could impair lipid homeostasis and the rate of fat peroxidation and induce oxidative stress, thus inducing inflammatory responses in various marine fish species, notably black seabream (Acanthopagrus schlegelii), blunt snout bream (Megalobrama amblycephala), and large yellow croaker (Larimichthys crocea) [8,9,128,137,138]. We relate these adverse inflammatory responses to oxidative stress induced by an HFD diet (Figure 8). Previous investigations have revealed that oxidative stress precedes metabolic disorders induced by a high-fat diet in mice [139] and rats [140] and mitochondrial damage and apoptosis in rats [141]. The Nile tilapia, when under sustained oxidative stress, modulates the mRNA of nrf2/kaep1 signaling as a signal for cell damage. Our results were consistent with earlier research demonstrating that inflammation was a key factor for liver injury after feeding an HFD to tilapia [142]. Meanwhile, dietary chitosan supplementation could attenuate inflammation by modulating cox2 signaling molecules, increasing expression of anti-inflammatory markers il-10, and decreasing tnf-α and il-1b levels [124,143,144]. Bai et al. [145] reported that chitosan oligosaccharide supplementation resulted in downregulation of mRNA levels of pro-inflammatory cytokines (il-6, tnf-α, and monocyte chemo-attractant protein 1(MCP-1) in the liver of mice fed an HFD.

A healthy intestine morphology improves the capacity to absorb different nutrients and acts as an immune barrier [36]. However, consumption of an HFD causes an increase in intestinal permeability, impairs its mucosal defenses, and promotes intestinal inflammation [146]. A high-fat diet (15%) fed to Nile tilapia for a period of 8 weeks significantly reduced the length of intestinal villi, decreased the number of goblet cells, downregulated the mRNA expressions of tight junction proteins, i.e., occludin and claudin, and prompted the expression of the intestinal inflammatory factor il-1b [146]. The accumulating literature illustrates that inflammation is an important marker in intestinal dysfunction [147,148]. Chen et al. [147] demonstrated that overproduction of inflammatory cytokines could alter the intestinal permeability and tight junction structure by modulating tight junction-related gene expression in weaning piglets. At the same time, the overproduction of il-1β, il-6, and tnf-α directly causes intestinal mucosal injury [149]. Therefore, suppressing the overproduction of intestinal mucosal il-1β, il-6, and tnf-α proved to be effective in maintaining intestinal function. Previous research suggested that stressors could disturb the balance between anti-inflammatory and pro-inflammatory responses by boosting the production of pro-inflammatory cytokines [147,150]. In this study, intestinal il-1b and tnf-α levels were higher, whereas intestinal il-10 levels were lower in the HFD-fed group compared to the F6Ch0 group, indicating that oxidative stress resulted in inflammation in the intestine. Dietary chitosan supplementation decreased the intestinal il-1b and tnf-α levels while enhancing the intestinal il-10 levels, all of which was inconsistent with the results of Hu et al. [151], who reported that COS reduced il-1β and tnf-α mRNA expression levels in the jejunum mucosa of weaning pigs. Also, COS reduced the il-6 and tnf-α mRNA expression levels in the liver of mice after being fed a high-fat diet [123]. These findings suggest that COS may mitigate intestinal inflammation by suppressing the levels of il-1β, il-6, and tnf-α [152,153].

Normal intestinal permeability is maintained by tight junctions, which are multi-protein complexes made up of various proteins, namely Zonula-Occludens, Occludin, and Claudin [154]. Paola et al. [155] suggested that intestinal hyperpermeability is a causative agent of both liver fibrosis and hepatic inflammation. A high-fat diet can significantly decrease the expression of tight junction proteins and thus increase intestinal permeability by weakening the intestinal barrier. Previous studies have demonstrated that mice fed with an HFD showed increased intestinal damage when compared to mice fed a low-fat diet [156].

In this study, HFD-induced oxidative stress downregulated the protein expression of claudin3, claudin7, tjp1, and tjp2, which reflected intestinal barrier dysfunction and was consistent with the results reported by Song et al. [157] and Cao et al. [158]. Moreover, dietary chitosan upregulated the expression of tight junction proteins, similar to other studies on mice fed high-fat diets [159], dexamethasone-challenged broilers [160], and weaning pigs [151,161]. These results indicated that chitosan could modulate oxidative-induced intestinal barrier function relatively well by maintaining the intestinal structure, intestinal permeability, and tight junction functionality.

In this investigation, we observed histological alterations in the liver, kidney, and intestine that are linked to a high-fat diet. These alterations were mainly in the form of renal tubular damage, hepatic necrosis, and shortening of intestinal villi. However, these changes showed less severe patterns emerging in the groups that received chitosan supplements.

Surprisingly, the F12Ch10 group had the best results, providing features of healing in the form of enlarged intestinal villi with a minor loss of apical mucosa, normal renal tubular endothelial lining, and restricted hepatic inflammatory cell infiltration, suggesting that increased chitosan supplementation may have a protective effect against fat droplet accumulation. Similar results were previously reported on common carp [162].

High fat storage in hepatocytes disrupts the normal regulation of glycogen metabolism in the liver. It decreases glycogen synthesis, increases gluconeogenesis, and impairs the response to insulin [163,164]. These changes, in turn, result in reduced glycogen storage in the liver and explain the low glycogen level in hepatocytes in the F12Ch0 group. Also, such results suggest the effect of chitosan treatment in alleviating fat accumulation and maintaining the normal metabolism of glucose, as observed by the higher storage of glycogen in hepatocytes from the F12Ch10 group.

Collectively, the histopathological findings suggest that chitosan supplementation, particularly at higher levels, may have a protective role in mitigating the severe damage induced by a high-fat diet.

In summary, the present study demonstrated that an HFD could promote liver and intestinal injury that might be closely related to oxidative damage, inflammation, and apoptosis. An impaired nrf2 pathway may depress the antioxidant defense system, leading to oxidative damage. Concurrently, apoptosis was induced by activating the mitochondria pathway after feeding an HFD, which triggered cox2, led to the release of pro-inflammatory factors, and exaggerated liver and intestinal injury.

5. Conclusions

This study suggests that dietary chitosan supplementation may be an effective nutritional strategy to mitigate the deleterious effects of HFD-induced oxidative stress via modulating the nrf2/kaep1 signaling pathway and anti-apoptotic action by increasing the expression of bcl-2. Therefore, it attenuates intestinal barrier damage by improving intestinal morphology and tight junction protein expression, which may be involved in suppressing intestinal inflammation and increasing antioxidant capacity in HFD-fed Nile tilapia.

Author Contributions

A.G.R.: Conceptualization, Methodology, Software. D.H.A.: Data curation, Methodology, Writing—Original draft preparation. A.S.S.: Visualization, Investigation, Statistical analysis, Methodology. X.L.: Resources, Writing—Reviewing and editing. I.I.A.-H.: Supervision, reviewing-editing final manuscript. M.H.A.-A.: Resources, Writing—Reviewing and editing. S.M.R.S.: Methodology, Feed formulation, Analysis, Reviewing. K.K., A.A. and N.A.N.H.: Pathological analysis, Validation. L.S.: Supervision, Resources, Writing—Reviewing and editing. Z.I.E.: Methodology, Molecular analysis, Writing—Reviewing and editing. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

The study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board (or Ethics Committee) of the Faculty of Aquatic and Fisheries Sciences Ethics Committee, Kafrelsheikh University, Egypt (IAACUC-KSU-028-2022). All precautions were followed to diminish animal suffering during the experiment.

Informed Consent Statement

Not applicable.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Conflicts of Interest

The authors declare no conflicts of interest.

Funding Statement

This work was funded by the Deanship of Graduate Studies and Scientific Research at the University of Bisha, Saudi Arabia, through the Promising Program under grant number (UB-Promising-34-1445).