1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is identified as a variety of liver disorders ranging from hepatic steatosis (presence of macro-vesicular steatosis only) to non-alcoholic steatohepatitis (NASH) (presence of macro-vesicular steatosis with hepatocyte ballooning, lobular and/or portal inflammation and with/without fibrosis), progressing to advanced fibrosis, cirrhosis, and rarely, may develop into hepatocellular carcinoma [
1]. It has been reported that the estimated worldwide prevalence of NAFLD is approximately 25% and this trend is predicted to increase yearly [
2]. NAFLD is caused by an excessive intake of dietary fat. Conversely, alcoholic liver disease, which also demonstrates similar hepatic disorders as NAFLD, is caused by an excessive intake of alcohol [
3].
The liver plays a vital part in glucose and lipid metabolisms. NAFLD is frequently linked with the main features of metabolic syndromes including obesity [
4,
5], hyperglycemia [
6], insulin resistance [
7,
8], hyperlipidemia [
9] and hypertension [
10]. Hence, a new disease named metabolic dysfunction-associated fatty liver disease (MAFLD) has been proposed to replace the old term NAFLD [
11,
12], and the new term MAFLD is used throughout this study. Enhanced hepatic lipid accumulation, oxidative stress and abnormal inflammatory response remain the foremost pillars in defining the pathogenesis of MAFLD [
13,
14]. Many studies have linked increased hepatic markers of oxidative stress and a reduction in antioxidant enzymes’ activity with the pathogenesis of MAFLD and NASH [
15,
16]. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a key transcription factor regulating the antioxidant signaling to defend against oxidative stress in cells [
17]. Increased lipid levels in the hepatic tissues result in the increases in lipid peroxidation and Nrf2 expression in the liver [
18]. Under the stressful conditions, Nrf2 detaches from Kelch-like ECH-associated protein 1 (Keap1), a Nrf2 repressor protein, and translocates to the nucleus to stimulate the antioxidant response [
19]. In the nucleus, Nrf2 forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins and up-regulates the expression of antioxidant response element (ARE)-containing genes including glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), superoxide dismutase (SOD), glutamate cysteine ligase (GCL), NAD(P)H/quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1) [
20].
Even though inflammation is crucial for an effective immune response, its dysregulation exerts a fundamental role in the propagation of MAFLD-related metabolic dysregulations [
21]. In MAFLD, an impaired inflammatory response is commonly depicted by aberrant levels of cytokines as well as chemokines that are tenacious with enhanced hepatic damage [
22]. Under such circumstances, persistent activation of innate immune responses, mostly driven by excessive hepatic lipid accumulation, can stimulate the classic features of pathological liver inflammation [
23]. For instance, continuously increased leptin and pro-inflammatory mediators (cytokines and chemokines), for example, tumor necrosis factor alpha (TNF-α), nuclear factor kappa β (NF-κβ) and chemoattractant protein-1 (MCP-1), and reduced adiponectin and anti-inflammatory markers such as interleukin-10 are likely to promote NASH progression and initiate fibrosis formation [
23,
24,
25,
26].
To date, there is no specific therapy for MAFLD, although lifestyle interventions such as appropriate food consumption, regular exercise and weight loss have been reported to be effective in managing MAFLD [
27]. Still, these treatment options are challenging and difficult to follow due to adherence issues, particularly for long-term management. A variety of pharmacological therapies are available to treat MAFLD such as pioglitazone and vitamin E, nevertheless, these treatments have been reported to have limited efficacy towards MAFLD [
28]. Scientists have, therefore, become more captivated in researching antioxidant and/or anti-inflammatory interventions as a useful approach to mitigate this disease progression. The use of bee products as complementary medicines and dietary supplements to heal numerous illnesses has been practiced for many decades. Today, there is a growing interest in exploring their benefits and pharmacological properties for the potential development of nutraceutical and functional food of bee products including honey, bee pollen, royal jelly, propolis and bee bread. Bee bread or ambrosia refers to the fermented pollen mixed with honey and secretions of the bee’s salivary glands and stored in the hive [
29,
30]. The foraging bees deliver the collected pollen loads to the hive and pack them directly into the empty cells of the honeycomb, where it is later secured and mixed with wax and honey [
31]. The lactic fermentation process of pollen in the honeycomb performed by
Lactobacillus bacteria under anaerobic conditions converts pollen into a more preserved state called “bee bread” [
32], which gives it a high nutritive value [
33]. Bee bread has been reported to have excellent digestibility and abundant chemical compositions [
34]. It contains proteins, vitamins, lipids, microelements, phenolic and flavonoid compounds that are recognized as the added values to its nutritional and therapeutic properties. Bee bread exhibits antibacterial [
35], antioxidant [
36] and anticancer [
37] properties. Previous studies have demonstrated that bee bread possesses hepatoprotective properties as shown by decreases in the severity of liver damage in alcoholic fatty liver disease patients [
38], and in aluminum [
39] and carbon tetrachloride (CCl
4)-induced liver injury [
40] in rats. Bee bread has also shown its medicinal effects in other varieties of ailments including cardiovascular diseases [
41], male fertility [
42], renal dysfunction [
43] and cancer [
44] in in vitro and in vivo studies. Nevertheless, there is only one set of data demonstrating the effects of bee bread on the high-fat diet (HFD)-induced fatty liver disease rat model, which reported that the consumption of bee bread (80, 400 and 800 mg/kg/day) downregulated the expressions of hepatic lipogenic protein and genes such as fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) levels, hence inhibiting hepatic lipid synthesis [
45]. Therefore, the purpose of this study was to assess the potential alleviative effects of bee bread in protecting against MAFLD or the complications implicated in its aggravation including oxidative stress and inflammation. This study measured the changes in the anthropometrical parameters (body weight gain, BMI and abdomino-thoracic circumferences), serum levels of lipids (TC, TG, LDL and HDL), liver enzymes (ALT, AST and ALP), adiponectin, glucose and insulin resistance, liver lipid contents (TC and TG), liver oxidative stress status, immunoexpressions of redox homeostasis regulators (Nrf2 and Keap1) and inflammation, as well as liver histopathological and fibrosis status.
4. Discussion
MAFLD is a metabolic disorder associated with obesity, hyperlipidemia, hyperglycemia, insulin resistance, oxidative stress and inflammation [
61]. There are now growing evidences that have claimed the health benefits of bee bread in reducing the risk of metabolic syndrome [
38,
41,
42,
43,
44,
45]. Consequently, this study was performed to investigate, for the first time, the possible antioxidative, anti-inflammatory, anti-steatotic and anti-fibrotic effects of bee bread in the HFD-induced MAFLD rat model. This study highlights that bee bread could serve as a potential nutraceutical and functional food that can be used as a treatment for MAFLD. Compared with the NC group, the HFD group showed a number of MAFLD-related metabolic disorders, including obesity, hyperlipidemia, liver dysfunction, redox state imbalance and inflammation. The daily administration of bee bread (0.5 g/kg/day) for 12 weeks effectively ameliorated obesity and improved insulin resistance and hyperlipidemia in the rats. Furthermore, bee bread reduced hepatic steatosis and protected the rats from progression into NASH.
Obesity has become the major contributor to the prevalence of MAFLD worldwide. Hence, obesity is one of the predisposing factors for the progression of MAFLD, which is in agreement with our findings [
62]. Our study showed that an excessive ingestion of HFD for 12 weeks significantly increased the obesity parameters such as body weight gain, BMI and AC/TC ratio in the HFD group compared with those in the NC group. This could be attributed to the greater component of fat in the HFD administration, which comprised of 31% fat in comparison with only 12% fat in the standard diet [
41]. Moreover, excess dietary fat ingested by an individual corresponds to more accumulation of fat mass in the abdominal and visceral areas [
63]. The present study demonstrated that bee bread could repress HFD-induced obesity, which was presented by a marked reduction in the obesity parameters after 12 weeks of administration. This is in agreement with a previously reported study [
41]. The beneficial effects of bee bread presented in this study could be attributed to its rich source of phenolic compounds including hydroxycinnamic acid derivatives, caffeic acid, gallic acid, ferulic acid, quercetin, apigenin, kaempferol and mangiferin [
64], which have lipid-lowering and anti-obesity effects, as reported in previous studies [
41,
65,
66].
Parallel to the change in body weight, we also observed a substantial increase in adipose tissue weight in the HFD group compared with those in the NC group. Numerous data from previous studies reported the role of adipose tissue in regulating metabolic activities of the brain, muscle and cardiovascular system [
67]. The adipocytokines secreted by the adipocytes including adiponectin, leptin, TNF-α, resistin and plasminogen activator 1 (PAI-1) control appetite, insulin sensitivity and inflammation; hence, they play their role in the MAFLD’s pathogenesis and its development to NASH [
25]. An elevation in adiponectin level has been connected with the inhibitions of hepatic lipid accumulation and insulin resistance as well as exerting its hepatoprotective actions by reducing the production of pro-inflammatory cytokines and increasing the expressions of anti-inflammatory IL-10 and IL-1 receptor antagonists [
68,
69]. This study also demonstrated a marked decrease in the level of serum adiponectin in the HFD group compared with the NC group. Thus, HFD might interrupt the function of adipose tissue, which in turn results in insulin resistance and obesity-related metabolic diseases [
67]. In contrast, the administration of bee bread for 12 weeks markedly elevated the level of adiponectin compared with the HFD group, indicating that bee bread might have the potential to improve the adipose tissue dysfunction.
An increase in serum liver enzymes is identified as the first manifestation of liver disease [
70]. In order to confirm the hepatoprotective effects of bee bread against liver injury, liver function tests were evaluated in this study. The present study demonstrated the elevation of liver enzymes, notably ALT, AST and ALP, in the HFD group, as compared with the NC group, which is in line with a previous study [
71]. The intake of bee bread significantly reduced the levels of liver enzymes, which is in line with previously reported studies in different models; streptozotocin-induced diabetic rats [
72], aluminum-induced hepatotoxicity rats [
39] and alcohol-induced liver disease patients [
38].
These positive findings were further confirmed by histopathological examination of liver sections obtained from all animal groups. The liver from the HFD group showed degenerative hepatocytes, steatosis, hepatocyte hypertrophy, inflammatory cell infiltration and the presence of active NASH, together with marked increases in collagen fiber as well as periportal fibrosis and bridging fibrosis (portal-central). Hepatic fibrosis is initiated by hepatic lipotoxicity due to excessive hepatic lipid accumulation and results in chronic liver injury, inflammation and activation of hepatic stellate cells leading to excessive buildup of extracellular matrix proteins, primarily collagen in the liver tissue [
73]. It is recognized as one of the main features of MAFLD and if left untreated, MAFLD can further progress into cirrhosis and hepatocellular carcinoma [
1]. The administration of bee bread markedly ameliorated these negative changes. Notably, bee bread has the ability to improve and prevent most of the features of MAFLD, from histological changes to fibrosis, hence, providing some hepatoprotection against MAFLD.
Indeed, type 2 diabetes has been reported to share a link with hyperlipidemia and is recognized as a co-morbidity commonly established in obese patients [
74]. The Increased consumption of an HFD resulted in an increased peripheral insulin resistance, which was demonstrated by the increased serum fasting glucose, hyperinsulinemia and increased HOMA-IR in the HFD group. Bee bread administration significantly alleviated the levels of serum fasting glucose, insulin and HOMA-IR, thus suggesting that it has an insulin-sensitizing effect. This might be ascribed to the up-regulated levels of glucose transporters GLUT1 and GLUT3 as demonstrated in the testes of obese male rats after intervention with bee bread [
42]. Chronic HFD feeding is also responsible for the increases in blood lipid levels [
75]. This study showed increased concentrations of serum TG, TC and LDL and reduced concentration of serum HDL in the HFD group than those in the NC group. Bee bread improved hyperlipidemia by lowering TG, TC and LDL levels, and, in contrast, increased the HDL level. Our results coincide with previous studies that reported the hypolipidemic effects of bee bread [
41,
42,
76]. This beneficial property of bee bread could be ascribed to the presence of flavonoids, which have been reported to exert their antioxidative property towards LDL by inhibiting its susceptibility to oxidation [
77]. Moreover, the interaction between HDL-associated enzyme paraoxonase-1 (PON1) with the flavonoids has been reported to be responsible for many of HDL’s antioxidative properties, which might elucidate the reduced levels of oxidative stress makers in the present study [
78]. Hence, evaluating the level of PON1 in the serum and liver of the HFD-induced MAFLD model is warranted in future studies. In addition, a previous study reported the ability of saponin in bee bread to reduce non-HDL lipids concentration in the blood by its interaction with dietary fat constituents, and, subsequently, increase the excretion of lipid in feces [
79].
Hepatic lipid buildup is recognized as the “first hit” theory to describe the pathogenesis of MAFLD and, subsequently, the “second hit” theory, which consists of inflammatory cytokines, adipokines, mitochondrial dysfunction and oxidative stress [
80]. Imbalance in hepatic lipid metabolism including a disturbance in fatty acid uptake, de novo lipogenesis (DNL), lipolysis and fatty acid oxidation results in abnormal lipid deposition in the hepatocytes [
81]. The present study demonstrated increased hepatic levels of TG and TC and, concomitantly, increases in the absolute liver weight and liver index of the HFD group. In contrast, the intake of bee bread reduced these hepatic lipid contents, liver weights and hepatic steatosis in the bee bread group. In addition, insulin resistance is strongly related to hepatic lipid accumulation [
82] in which peripheral insulin resistance enhances lipolysis and free fatty acid uptake into the liver tissue, hence, leading to hepatic lipid accumulation [
83]. Furthermore, insulin resistance regulates hepatic DNL via its action on sterol regulatory element binding protein-1c (SREBP-1c) and carbohydrate response element binding protein (ChREBP), the main transcription factors, which are essential to regulate the expression of genes involved in DNL and lipid synthesis in the liver [
81]. Hence, reduced hepatic lipid accumulation and insulin resistance after bee bread administration in the present study might be ascribed to its inhibition effect on hepatic genes-related to DNL as reported by a previous study [
45].
The liver is known as a powerhouse organ that performs various crucial tasks, ranging from the production of proteins, cholesterol and bile to storing vitamins, minerals and even carbohydrates, as well as breaking down toxins such as alcohol, medications and natural by-products of metabolism to maintain metabolic homeostasis in an organism. Hence, it is mostly susceptible to oxidative stress [
84,
85], which, in turn, is incriminated in the pathogenesis of MAFLD [
17]. Our present study proved that an HFD stimulated oxidative stress with a marked reduction in the activities of antioxidant enzymes. This is in line with a few previous findings that reported that there were elevations in the levels of free radicals in the rat model of MAFLD, meanwhile, the activities of antioxidant enzymes were reduced [
86,
87]. The present study reported the hepatoprotective role of bee bread on oxidative stress in HFD-induced MAFLD rats in which the intake of bee bread (0.5 g/kg/day) for 12 weeks significantly reduced the levels of oxidative stress markers (TBARS and NO), meanwhile, it increased the activities of enzymatic antioxidants (GPx, GST, GR, SOD and CAT) and GSH level. It is well-known that the transcription factor Nrf2 plays a critical role in the cellular defense mechanism against MAFLD-induced liver oxidative stress by down-regulating the genes responsible for hepatic lipid accumulation in rats, thereby preventing further damage exerted by lipids in the hepatocytes [
88]. It is reported that Keap1 deficiency prevents ethanol-induced ROS overproduction, while Nrf2 knockout triggers ROS up-regulation in mouse primary hepatocytes [
89]. In the present study, we reported the increased translocation of cytoplasmic Nrf2 into the nucleus of the HFD group compared to the NC group. However, the analyses of antioxidant enzymes activities showed a significant reduction in these enzymes GPx, GST, GR, SOD and CAT activities in the HFD group compared with those in the NC group. Although there was an elevation in Nrf2 expression in the nucleus of the HFD group, the decreased antioxidant enzymes’ activity might be due to deactivation or a reduction in enzyme synthesis caused by the overproduction of ROS, as demonstrated by high levels of TBARS and NO in this group [
90]. Apart from that, bee bread enhanced the translocation of Nrf2 from the cytoplasm into the nucleus, as demonstrated by the higher expression of nuclear Nrf2 in comparison with cytoplasmic Nrf2 following bee bread administration, compared to the HFD group. These results might also support our findings on decreased oxidative stress levels and increased antioxidant enzymes’ activity in the bee bread group as compared to the HFD group, probably by enhancing the synthesis of these enzymes. Hence, evaluating their mRNA levels in future study is warranted to further validate the action of bee bread on these enzymes’ syntheses. Similarly, the significantly decreased Keap1 expression following bee bread administration indicated that bee bread might suppress Keap1 to promote Nrf2 translocation and, in turn, might up-regulate the activities of antioxidant enzymes in the HFD-fed rat livers, thus decreasing oxidative stress [
88]. In addition to immunochemistry analysis, it is also recommended to further corroborate the levels of cytoplasmic Nrf2 and Keap1, and nuclear Nrf2 using the Western blot technique and mRNA analysis in future studies.
As mentioned above, defective lipid metabolism leads to hepatic lipid overload, which results in lipid peroxidation, leading to oxidative stress, inflammation and fibrosis. Excessive production of ROS stimulates the hepatocytes to secrete more cytokine, subsequently leading to liver damage [
91]. Elevated hepatic lipid has been linked with activated NF-κβ and increased productions of TNF-α, IL-1 and Il-6, while suppression of NF-κβ in the liver down-regulates the expression of genes encoding these pro-inflammatory mediators [
92]. TNF-α is responsible for a range of intracellular signaling systems, including the activation of NF-κβ, and has been linked with the increased activity of Jun N-terminal kinase, which is essential in promoting hepatic insulin resistance [
93]. It is also reported that TNF-α is involved in hepatic fatty acid synthesis, increases serum triglyceride level, activates the production of VLDL from the liver and stimulates both hepatocytes’ cell death and proliferation, thus, is crucially involved in the pathogenesis of liver fibrosis [
94]. This is confirmed by our present study in which significantly increased levels of pro-inflammatory mediators TNF-α and NF-κβ, and a significantly decreased level of anti-inflammatory cytokine IL-10 were observed in the liver of the HFD group, as well as elevation in serum lipids and the deposition of collagen fibers, which indicated the presence of liver fibrosis in this group. Bee bread (0.5 g/kg/day for 12 weeks) has been previously reported to suppress TNF-α and NF-κβ, and increase IL-10 levels in obesity-induced aortic vascular damage [
76] and HFD-induced renal damage [
43] rats. Our present findings are in agreement with previous reported studies, and further support the beneficial properties of bee bread against inflammation and fibrosis. MCP-1, also known as CCL2, is a prototypical inflammatory chemokine secreted by hepatic stellate cells upon tissue injury [
95]. Previous studies have linked increased hepatic expression of MCP-1 with hepatic steatosis and insulin resistance in HFD-fed rats [
96,
97], as well as in patients with advanced liver fibrosis [
98]. Similarly, the present study demonstrated a significantly increased hepatic MCP-1 expression in the HFD group compared with those in the NC group, further indicating the progression of hepatic steatosis towards NASH and fibrosis, as well as the increased inability of the liver to manage fat infiltration. Furthermore, an increased level of MCP-1 is reported to be stimulated by an increased level of TNF-α in hepatocytes [
69], which was also demonstrated in the HFD group in the present study. Conversely, the intake of bee bread markedly reduced the expression of MCP-1 in the liver tissue of HFD-fed rats, suggesting the anti-inflammatory property of bee bread.