A review of the hepatoprotective effects of hesperidin, a flavanon glycoside in citrus fruits, against natural and chemical toxicities

Abstract

Objectives

Liver is the most important and functional organ in the body to metabolize and detoxify endogenous compounds and xenobiotics. The major goal of the present narrative review is to assess the hepatoprotective properties of hesperidin against a variety of natural and chemical hepatotoxins via different mechanisms.

Evidence acquisition

Scientific databases such as Scopus, Medline, Web of Science and Google scholar were thoroughly searched, based on different keywords.

Results

A variety of natural hepatotoxins such as lipopolysaccharide, concanavalin A and microcystins, and chemical hepatotoxins such as ethanol, acrylamide and carbon tetrachloride have been shown to damage hepatocytes as well as other liver cells. In addition to hepatocytes, ethanol can also damage liver hepatic stellate cells, Kupffer cells and sinusoidal endothelial cells. In this regard, the flavanone hesperidin, occur in the rind of citrus fruits, had been demonstrated to possess widespread pharmacological properties. Hesperidin exerts its hepatoprotective properties via different mechanisms including elevation in the activities of nuclear factor-like 2/antioxidant response element and heme oxygenase 1 as well as the levels of enzymatic and non-enzymatic antioxidants. Furthermore, reduction in the levels of high-mobility group box 1 protein, inhibitor of kappa B protein-alpha, matrix metalloproteinase-9 and C-reactive protein are some other important hesperidin-derived hepatoprotective mechanisms.

Conclusion

Based on several research papers, it could be concluded that hesperidin is able to protect against liver damage from inflammation and/or oxidative stress-mediated natural and chemical toxins.

Introduction

Liver, as the most important digestive organ in the body, has the duty of metabolizing xenobiotics via a variety of pathways including conjugation, hydrolysis, hydration, reduction and oxidation [1]. Any interruption in these processes results in liver damage or hepatotoxicity which can be elicited by different mechanisms such as a disruption in mitochondria, hepatocyte DNA damage and apoptosis, and an increase in the serum levels of liver enzymes [2]. According to the reports, more than 50 million people are affected by different xenobiotics-induced hepatotoxicity worldwide [2].

A large variety of medicinal plants contain compounds with hepatoprotective activities including flavonoids, phenols, alkaloids, carotenoids, coumarins, xanthines, essential oils and monoterpenes [3]. Herein, flavonoids are a class of secondary plant metabolites. These phenolic compounds are abundant in plants, particularly in fruits and vegetables [4]. The abundance in the diet, potential health effects and the moderately low toxicity of flavonoids in comparison with other phytochemicals, have led researchers to consider them as a research interest [5].

Hesperidin (HSP), abundantly found in citrus species (blood orange, orange, lemon and lime), is a pharmacologically active flavonoid aglycone and subclass of flavonoids [6]. It has been demonstrated that this flavanone exhibits a variety of pharmacological properties such as antioxidant, anti-inflammatory, analgesic, anticarcinogenic, antiviral [7], anti-coagulant, hypolipidemic and hypoglycemic activities [8]. According to Zanwar et al., HSP in herbal formulations is safe, with no side effects and/or toxicities because the LD₅₀ of this flavonoid in acute oral toxicity testing is more than 2000 mg/kg [9]. Good safety profile of HSP in animal experiments has also been confirmed by Li et al. [10].

Considerable evidence has implicated the effects of different medicinal plants and/or naturally occurring compounds on natural and chemical toxins-provoked organ toxicities [11–14]. In addition, liver plays a central role in detoxifying the xenobiotics and its proper function is vital for the well-being of humans. Hence, in this review, we discussed, for the first time, the hepatoprotective effects of HSP against natural and chemical toxicities.

Literature search strategy

The databases Scopus, Medline, Web of Science and Google scholar were thoroughly searched on the hepatoprotective effects of HSP against natural and chemical toxicities. The search terms included: “hepatoprotective”, “liver protection”, “liver protective effects”, “liver toxicity”, hesperidin”, “and citrus fruits”. The reference list of related articles was searched for more relevant articles. Duplicated, non-relevant and non-English language articles were excluded. Besides, the search in the databases was carried out from inception until February 2020. Following this search criteria, we found 131 articles in online database, among which 41 were excluded, while the rest of 90 articles were included in the current study (Fig. 1).

Fig. 1.

Flow diagram of narrative literature search representing the included and excluded studies

Hepatoprotective effects of hesperidin against natural toxicants

Lipopolysaccharide

Lipopolysaccharide (LPS) is a key glycolipid component of the cell wall of gram-negative bacteria [15]. In a study, LPS was injected intraperitoneally (i.p.) in rats (1 mg/kg, single administration) to induce liver toxicity which was evidenced by an elevation in the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bilirubin. Thiobarbituric acid reactive substances (TBARS) levels and tissue and serum nitrite levels also augmented whereas the content of glutathione (GSH) and superoxide dismutase (SOD) activity reduced. However, administration of HSP reversed all these anomalies and the authors concluded that HSP could attenuate the nitric oxide and reactive oxygen species (ROS) generation and prevent the LPS-induced hepatotoxicity. HSP also improved the histopathological changes induced by LPS such as Kupffer cell hyperplasia, necrosis and pyknotic nuclei [16] (Fig. 2). In another investigation, LPS (0.1 μg, intravenously (i.v.), single administration) was injected to mice, and the levels of tumor necrosis factor alpha (TNF-α) as well as high-mobility group box 1 protein (HMGB1) increased. LPS also elevated the number of apoptotic cells in the liver. On the other hand, HSP (0.3, 1 and 3 mg, i.p.), in a dose-dependent fashion, was able to normalize the imbalances provoked by LPS and exhibited hepatoprotective effects via its anti-inflammatory activity [17].

Fig. 2.

Schematic of possible mechanisms underlying hepatoprotective effects of hesperidin

Concanavalin A

Concanavalin A, a plant lectin, is used as a model of hepatotoxicity in animals. This lectin triggers Kupffer cells to generate TNF-α and interferon gamma (IFNγ), as markers of hepatocytes damage [18]. In an investigation, concanavalin A (15 mg/kg, i.v., single administration) was administered to mice and the induced hepatotoxicity was evidenced by an increase in the hepatic malondialdehyde (MDA) content and a decrease in SOD activity, an increase in the generation of TNF-α and IFNγ as well as expression of HMGB1, and activation of T cells. An elevation in AST and ALT activities was also observed. Interestingly, HSP (1000 mg/kg, i.v., for 10 days) was capable of counteracting these imbalances. Inhibiting hepatic oxidative stress, cytokines and HMGB1 secretion, and activating T cells, were proposed as underlying mechanisms. In addition, HSP alleviated the liver inflammatory infiltration and necrosis induced by concanavalin A [19].

Microcystin

As a group of cyclic heptapeptide hepatotoxic peptides, microcystins are produced by cyanobacteria. Via preventing the activities of protein phosphatases, microcystin-LR overproduces ROS [20]. In a research, microcystin-LR (200 μg/kg, i.p., single administration) was injected to mice to produce hepatotoxicity. The serum levels of ALT, lactate dehydrogemse (LDH) and gamma glutamyl transferase (GGT) were increased. It also inhibited protein phosphatase, and increased MDA and methylglyoxal contents. On the other hand, HSP (300 mg/kg/d, orally (p.o.), for 10 days) reversed all these alterations. As a potent antioxidant, HSP protected the liver from microcystin-LR-induced toxicity [21].

Hepatoprotective effects of hesperidin against chemical toxicants

Ethanol

It has been demonstrated that consuming ethanol (EtOH) leads to hepatocyte injury and death, via apoptotic and necroptotic pathways. Injury to hepatocytes happens, partly, due to oxidative stress induced by EtOH along with the pro-inflammatory factors in the liver [22]. In a study, EtOH was administered to mice, and a substantial elevation in the levels of inhibitor of kappa B protein-alpha (IκBα), TNF-α, interleukin 1 beta (IL-1β) and interleukin 6 (IL-6) were observed in hepatocytes. In addition, EtOH increased the levels of serum AST and ALT while decreased catalase (CAT), glutathione reductase (GR), glutathione-S-transferase (GST) and glutathione peroxidase (GPx) activities. This alcohol increased the liver content of MDA and lowered the levels of hepatic GSH. Promisingly, HSP reversed these alterations and showed its hepatoprotective effects by increasing the antioxidant capacity, preventing inflammation and excessive lipid formation in hepatocytes [23]. In an alcoholic liver disease model, HSP (6.25, 12.5 and 25 μg/ml, for 24 h) could attenuate the morphological changes induced by EtOH (350 mM, for 32 h). Additionally, HSP diminished the expressions of cyp2y3, cyp3a65, hmgcra, hmgcrb, fasn and fads2 (the genes related to alcohol and lipid metabolism) as well as chop, gadd45훼a, and edem1 (the genes related to endoplasmic reticulum stress and DNA damage) provoked by EtOH. Collectively, HSP via preventing endoplasmic reticulum stress and DNA damage and modulating alcohol and lipid metabolism, exhibited its protective effects against alcoholic liver damage [24]. In a combination treatment model of study, HSP (200 mg/kg, p.o., for 7 weeks) and diethylcarbamazine (50 mg/kg, p.o., for 7 weeks) were administered to rats to evaluate their possible hepatoprotective effects against EtOH (1 ml/100 g/d, p.o., twice a week, for seven weeks). EtOH substantially elevated the serum activities of AST, ALT, ALP and GGT as well as MDA content. It also augmented the content of hepatic nitric oxide and 4-hydroxyproline while decreased GSH content. Hepatic interleukin (IL)-6 content and serum levels of transforming growth factor-β (TGF-β) as well as the expression of alpha smooth muscle actin (α-SMA) were increased by EtOH. HSP and diethylcarbamazine combination treatment was able to normalize these imbalances. The underlying mechanisms for their hepatoprotective effects were via increasing the antioxidants content in the liver, preventing the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) as evidenced by decreasing hepatic IL6, and inhibiting the activation of hepatic stellate cells [25].

Organic compounds

Acetaldehyde

A very reactive intermediate metabolite of EtOH, acetaldehyde has been shown to play an important role in alcoholic liver disease, and its production in hepatocytes leads to cell injury [26].

In an in vitro model of study, HepG2 cells were exposed to acetaldehyde (100 μM). The results indicated cell invasion, expression and secretion of matrix metalloproteinase-9 (MMP-9), and an increase in NF-κB and activator protein 1 (AP-1) activities provoked by acetaldehyde. The translocation of NF-κB into the nucleus was elevated via IκBα signaling pathway. The activity of AP-1 increased via phosphorylation of c-Jun N-terminal kinase (JNK) and P38 kinase signaling pathways. After treatment of the cells with HSP (50, 100 and 200 μM), NF-κB and AP-1 activities were inhibited via IκBα, JNK and P38 signaling pathways. Besides, HSP decreased the expression and secretion of MMP-9 and cellular invasion, and in this way, showed its hepatoprotective effects [27].

Acrylamide

As one of the most important contaminants in the environment, acrylamide is broadly used to produce polyacrylamides []. This chemical leads to toxic effects in many organs such as liver, kidney and central nervous system [28]. After administration of rats with acrylamide (15 mg/kg/d, p.o., for 4 months), a considerable elevation in the serum levels of AST, ALT and LDH, creatinine and urea, protein carbonyls, MDA content and carcinoembryonic antigen was observed. In addition, acrylamide resulted in a remarkable decrease in SOD, CAT and GPx activities, and GSH levels. Histopathologically, acrylamide resulted in degenerative alterations in several hepatocytes, pyknotic nuclei, and lymphocyte infiltration between hepatocytes and around the central vein. From these results, the authors concluded that via counteracting all these anomalies and attenuating histopathological changes, HSP (50 mg/kg/d, p.o., for 2 weeks) showed its antioxidant properties and protected the liver from acrylamide toxicity [29].

Carbon tetrachloride

Carbon tetrachloride (CCl₄) is known as a hepatotoxin which produces apoptosis and inflammation in experimental animals [30]. CCl₄ (2 ml/kg, single administration) was administered to rats, leading to a noticeable increase in the activities of AST and ALT, along with a substantial decrease in the liver GSH content, and the activities of SOD and GPx. However, administration of HSP (10 and 100 mg/kg, p.o., for 28 days) protected the liver via antioxidant activity which was proposed by the authors as a novel drug candidate against hepatotoxicity [31]. In another animal model of CCl₄-induced hepatotoxicity (2 ml/kg, subcutaneously (s.c.), single administration), rats received HSP (100 and 200 mg/kg, p.o., for 10 days), and exhibited a substantial reduction in the serum levels of AST and ALT, TNF-α, and MDA content. In contrast, the activity of SOD and GSH content, and total antioxidant capacity were remarkably elevated which was approved by histopathology findings. Taken together, HSP exerted hepatoprotective effects in a dose-dependent manner [32]. Injection of CCl₄ (0.4 g/kg, i.p., 3 times per week, for 8 weeks) to rats led to an elevation in the serum levels of ALT, GGT, NF-κB, TGF-β, connective tissue growth factor (CTGF), IL-1β and IL-10, and hydroxyproline content; but, glycogen and GSH content decreased. On the other hand, HSP (200 mg/kg/d, p.o., for 8 weeks) not only was able to counteract all these changes, but also elevated the levels of IL-10. Consequently, HSP via its antioxidant, anti-inflammatory, anti-necrotic and anti-fibrotic properties protected the liver from CCl₄-induced damage [33]. A study accomplished by Tirkey et al. showed that treatment of rats with CCl₄ (2 ml/kg in olive oil, s.c.), substantially augmented the serum levels of AST and ALT, and TBARS while diminished the activities of SOD and CAT, and the content of GSH in the liver. Though, pretreatment of rats with HSP (100 and 200 mg/kg, p.o., for 1 week) could counteract these alterations. The antioxidant activity was suggested to be the mechanism behind the hepatoprotective effect of this flavanone [34].

In a combination treatment study, rats were exposed to CCl₄ (1 ml/kg in olive oil, i.p., for 2 days) to induce hepatotoxicity as marked by elevated serum levels of AST and ALT, and lowered activities of GGT and GPx and GSH content. It also increased the inflammatory factors such as C-reactive protein and IL-6, along with alpha-fetoprotein (AFP) and extracellular matrix proteins. The diosmin-HSP complex (known as daflon) (100 mg/kg/d, p.o., for 16 days) was able to prevent liver damage via its antioxidant and anti-inflammatory activities [35]. In another combination therapy, HSP and quercetin exhibited a substantial antioxidant activity in rats via inhibiting the formation of nitric oxide radicals, produced by CCl₄ [36]. Cetin et al. indicated that exposure of rats to CCl₄ (2 ml/kg, i.p., single administration) resulted in liver oxidative stress as reflected by elevated levels of TBARS along with diminished content of GSH, and SOD and CAT activities. CCl₄ also increased the levels of caspase 3, leading to hepatocyte apoptosis. Promisingly, HSP (50 mg/kg/d, p.o., for 10 days) reversed all these anomalies of which the underlying mechanisms include antioxidant and anti-apoptotic actions [37]. Additionally, CCl₄ (2 ml/kg in olive oil, s.c., single administration) augmented the serum levels of AST and ALT, and liver MDA content whereas reduced GSH content and SOD activity, when administered to rats. HSP (100 and 200 mg/kg, p.o., for 8 days), in a dose-dependent fashion, presented protection against CCl₄-induced hepatotoxicity via normalizing these changes [38]. Exposure of rats to CCl₄ (2 ml/kg, i.p., single dose) led to a considerable increase in the levels of myeloperoxidase, nitric oxide, low-density lipoprotein cholesterol (LDL), very low-density lipoprotein cholesterol (VLDL), high density lipoprotein cholesterol (HDL), total cholesterol, triglycerides (TG), albumin/globulin ratio, globulins (α1, α2, β, and γ), MDA, indirect bilirubin, direct bilirubin, total bilirubin, AST, ALT, ALP and GGT. A significant reduction in the levels of SOD, CAT, GSH, and albumin concentrations were also observed after CCl₄ treatment. However, HSP (100 mg/kg/twice a week, p.o., for 4 weeks) could counteract all these abnormalities and protect the liver from the toxicity induced by CCl₄ [39]. In another investigation, liver fibrosis was produced by CCl₄ (2 ml/kg, i.p., for 5 weeks) exposure to rats. Administration of HSP (200 mg/kg, p.o., for 5 weeks) resulted in a pronounced reduction in the levels of TGF-β1, hydroxyproline, AST, ALT, total bilirubin, MDA, myeloperoxidase, TNF-α, LDL and TG along with an increase in the levels of albumin, GSH and CAT. For these results, the authors concluded that HSP could be considered as a promising candidate against liver fibrosis via antioxidant and anti-inflammatory properties [40].

A derivative of HSP known as HSP derivative-14 (100 mg/kg, p.o., for 7 days) was shown to possess hepatoprotective and anti-inflammatory effects in mice against CCl₄-elicited liver injury (10 ml/kg in oil, single administration). It also led to upregulation of peroxisome proliferator-activated receptor gamma (PPARγ) expression resulting in reduced levels of TNF-α, IL-6, and IL-1β. Additionally, this derivative substantially suppressed the expression of phosphorylated Janus kinase (P-JAK)1 and phosphorylated signal transducer and activator of transcription (P-STAT)1. JAK1/STAT1 signaling pathway was suggested as anti-inflammatory activity of HSP derivative-14 [41].

Diethylnitrosamine

A potential chemical carcinogen, diethylnitrosamine is used to induce tumors in different organs such as liver [42]. In a model of hepatocarcinogenesis, rats received diethylnitrosamine (200 mg/kg, i.p., single administration) and after 2 weeks, CCl₄ (3 ml/kg/week in mineral oil, s.c., for 16 weeks). The results were as follows: a substantial increase in the serum levels of AST, ALT, ALP, LDH and bilirubin as well as liver MDA content; a diminution in the GSH content, SOD, GPx and GST activities; an elevation in AFP and carcinoembryonic antigen as circulating tumor markers, and the number of nuclear positive cells containing proliferating cell nuclear antigen (PCNA); an augmentation in TNF-α, NF-κB, and TGF-β1 protein expressions and Smad3 phosphorylation levels; a decrease in nuclear factor (erythroid-derived 2)-like 2/antioxidant response element (Nrf2/ARE) mRNA expression and heme oxygenase 1 (HO-1) protein expression; and a downregulation of PPARγ expression. Promisingly, HSP (50 and 100 mg/kg/d, p.o., for 18 weeks) was capable of normalizing all these changes. The authors concluded that HSP prevented hepatocarcinogenesis via anti-inflammatory and antioxidant activities as well as inhibiting TGF-β1/Smad3 signaling and cell proliferation, which were attributed to Nrf2/ARE/HO-1 and PPARγ pathways activation [43]. Similarly, other researchers approved these results and showed that diethylnitrosamine-induced hepatocarcinogenesis (0.01% in drinking water, p.o., for 12 weeks) was abolished by HSP (250, 500 and 1000 ppm, p.o., for 12 weeks). They suggested HSP as a therapeutic candidate in chemoprevention [44].

Dimethylnitrosamine

As a conservative, utilized in processed meats and industrial products, dimethylnitrosamine (DMN) is a potent hepatotoxin, carcinogen and mutagen which leads to hepatic fibrosis [45].

DMN (10 mg/kg/d, i.p., three times per week, for 4 weeks) was given to rats to induce liver fibrosis as indicated from a remarkable increase in the serum levels of AST, ALT, ALP, and total and direct bilirubin, MDA content, gene expression of inducible nitric oxide synthase (iNOS), α-SMA and caspase 3 activity. Moreover, DMN led to a marked reduction in the serum total protein, albumin and liver GSH content. Treatment of rats with HSP (100 or 200 mg/kg, p.o., for 4 weeks) counteracted these alterations. Overall, the antioxidant, anti-inflammatory, anti-apoptotic and anti-fibrotic activities of HSP were demonstrated in this study [46].

Tert-butyl hydroperoxide

As a toxic agent, tert-butyl hydroperoxide is used to assess the antioxidant capacity in liver cells, because it produces free radicals via cytochrome P450 enzymes, leading to lipid peroxidation [47]. Chen et al. demonstrated that HSP (20, 40 and 80 μM) could substantially prevent hepatotoxicity provoked by tert-butyl hydroperoxide (150 μM) in human liver L02 cells. In fact, HSP normalized mitochondrial membrane potential and LDH activity. Furthermore, it reduced the formation of ROS and elevated the levels of MDA in a dose-dependent way. This flavonon increased the mRNA and protein expression levels of HO-1 as well as enzyme activity. HSP led to a marked elevation in the Nrf2 expression in the cell nucleus as well as ERK phosphorylation. Collectively, HSP could be considered as a potential candidate in the prevention and treatment of liver toxicity [48, 49].

Metals

Iron

A very important trace element within the body, iron is found in functional form in hemoglobin, myoglobin and cytochrome enzymes. Iron and other trace metals are of vital importance [50]. Trace metals are important in the environmental pathology because they have a wide range of toxic reactions in organ systems [51]. In an investigation, iron (30 mg/kg, i.p., for 10 days) was administered to rats to induce toxicity in the liver. The serum levels of AST, ALT, ALP, LDH, GGT, and bilirubin were drastically elevated. In addition, serum and tissue TG, free fatty acids, total cholesterol and phospholipids were increased. Iron resulted in an elevation in the liver lipid peroxidation markers such as TBARS and lipid hydroperoxides while lowered the activities of enzymatic antioxidants (SOD, CAT, GPx and GST) and non-enzymatic antioxidants (GSH, vitamin C and vitamin E). As pathological changes, inflammatory cell infiltration, focal necrosis and giant cell formation were observed in iron-treated rats. Promisingly, HSP (20, 40 and 80 mg/kg, p.o., for 10 days) was able to reverse all these biological markers and alleviate liver pathology [52].

Mercury

As a heavy metal, mercuric chloride is used in numerous products and agriculture medicines and intoxication with mercury occurs accidentally, occupationally and environmentally [53]. In a study, treatment of rats with mercuric chloride (1.23 mg/kg, p.o., for 7 days) resulted in a considerable increase in the levels of free radicals while decreased the activities of SOD, CAT, GPx as well as GSH content in the liver. Concomitant treatment of rats with HSP (5 mg/kg, p.o., for 7 days) and mercuric chloride ameliorated these alterations. Taken together, the hepatoprotective activity of HSP and ellagic acid was proved in this study [54].

Arsenic

With high toxicity, arsenic is known as a heavy metal and exposure to it, may occur by drink of potable water. This creates a high level of concern because arsenic affects human beings resulting in the formation of cancer [55]. In a study, mice received 10 mg/kg arsenite (i.p.), showing hepatocyte vacuolization, inflammatory cells infiltration, centrilobular swelling, parenchyma disorganization, dilatation of the interhepatocyte space, and hemorrhagic clots. On the other hand, HSP (25 mg/kg, p.o.) protected the liver against arsenite-induced hepatotoxicity via counteracting these changes [56]. In another study, exposure of rats to sodium arsenite (10 mg/kg, p.o., for 15 days) led to oxidative stress via a reduction in the CAT, SOD, GPx activities as well as GSH content, and an elevation in the liver content of MDA. The activities of serum AST and ALT, and urea and creatinine were also increased. Moreover, sodium arsenite resulted in oxidative DNA damage via an elevation in the 8-hydroxy-2′-deoxyguanosine (8-OHdG) levels, apoptosis via an increase in the caspase 3 levels, and inflammation via a raise in the IL-1β, NF-κB and TNF-α levels in rats liver. On the other hand, HSP (100 and 200 mg/kg, p.o., for 15 days) prevented oxidative stress, apoptosis and inflammation via reversing aforementioned alterations [57].

Zinc

An essential trace element in the body, zinc is used as a nutritional supplement, but in the form of nanoparticles, it can lead to toxicity [58]. Due to their highly reactivity, zinc oxide nanoparticles can result in oxidative stress both in experimental animals and humans [59]. In an investigation, treatment of rats with zinc oxide nanoparticles (600 mg/kg, i.p., single administration) showed a substantial elevation in the serum activities of AST and ALT, and MDA content accompanied by a significant reduction in the liver content of GSH, and the activities of CAT, SOD and GPx. Though, HSP (100 mg/kg, p.o., for 7 days) alleviated liver toxicity via counteracting these changes [60].

Anticancers

Cisplatin

The results of a study demonstrated that cisplatin (7.5 mg/kg, i.p., single administration), as an anti-cancer drug, increased the serum activities of AST and ALT, and TG and total cholesterol levels when administered to rats. Oxidative stress was evidenced by a considerable increase in the hepatic MDA and nitric oxide content, and a decrease in the GSH content. Additionally, the expression of NF-κB elevated while the expression of P-Akt decreased. After examining the pathology of the liver, it was determined that HSP (100 and 200 mg/kg/d, p.o., for 7 days) had protective effects against cisplatin-provoked damage. Consequently, cisplatin-provoked liver injury was inhibited by HSP in a dose-dependent way [61]. In another study, hepatotoxicity was induced by injecting a single dose of cisplatin (7 mg/kg) to rats, as reflected by a substantial increase in the levels of MDA and nitric oxide, along with a marked reduction in the activity of CAT, and levels of GSH. Interestingly, administration of HSP was able to counteract all these anomalies and showed its hepatoprotective effects via antioxidant activities [62].

Cyclophosphamide

An anti-cancer drug and alkylating agent, cyclophosphamide has application in organ transplantation to suppress the immune system [63]. One of the most known adverse effects of this drug is hepatotoxicity [64]. It has been shown that administration of cyclophosphamide (200 mg/kg, i.p., single administration) to rats led to several consequences including a substantial elevation in the serum levels of AST, ALT and GGT, total bilirubin and albumin concentrations, TNF-α, IL-1β and IL-6 levels, liver MDA and nitric oxide contents; a significant reduction in the liver GSH content, and the activities of CAT, SOD and GPx; a downregulation of liver PPARγ mRNA expression; and an upregulation of NF-κB and iNOS expressions. HSP, dose-dependently, was able to normalize the measured parameters. The proposed hepatoprotective mechanisms of HSP (25 and 50 mg/kg, p.o., for 11 days) were antioxidant and anti-inflammatory activities accompanies by upregulation of liver PPARγ expression [65].

Doxorubicin

Doxorubicin is a widely-used anti-cancer drug with hepatotoxicity as a common adverse effect [66]. The results of an investigation indicated that exposure of rats to doxorubicin (25 mg/kg, i.p., for 2 weeks) significantly increased the serum activities of AST, ALT, ALP and GGT as well as the serum levels of AFP, total bilirubin and sialic acid. A pronounced reduction in the liver content of GSH, and activities of GST, GPx and peroxidase were also observed. Overall, the authors concluded that HSP (50 mg/kg, i.p., for 3 weeks) via restoring aforementioned changes, could alleviate doxorubicin-induced hepatotoxicity [67].

Streptozotocin

In a diabetic model, streptozotocin (STZ) (50 mg/kg, i.p., single administration) was administered to rats, which led to a diminution in the serum and liver levels of alpha-klotho, however, administration of HSP (100 mg/kg, p.o., for 2 weeks) substantially increased alpha-klotho levels in the serum and liver. The serum and liver levels of fibroblast growth factor-23 (FGF-23) elevated in the STZ-exposed rats, but concomitant treatment of animals with HSP reduced FGF-23 levels. Collectively, the alpha-klotho/FGF-23 pathway was demonstrated to possess a key role in the systemic toxicity including hepatotoxicity, and the positive effect of HSP in ameliorating STZ-liver injury was revealed. HSP could be considered as a novel therapeutic agent in this regard [68]. In another diabetic model, rats received STZ (60 mg/kg, i.p., single administration), and showed diminished activities of the liver sirtuin 1, SOD and CAT while the levels of NF-κB and MDA content increased. HSP (100 mg/kg, p.o., for 15 days) was demonstrated to reverse these alterations. Based on the obtained results from this study, the suggested hepatoprotective mechanisms of HSP were antioxidant and anti-inflammatory properties [69]. STZ (45 mg/kg, i.p.) was administered to rats to induce diabetes. STZ elevated the serum and liver levels of TBARS and hydroperoxides while declined the liver activities of SOD, CAT, GPx, GST, and the plasma and liver levels of GSH, vitamin C and vitamin E. However, administration of HSP (25 and 50 mg/kg, p.o., for 35 days) reversed all these alterations, demonstrating its hepatoprotective activity [70]. In a combination treatment of curcumin, HSP and rutin (CHR), rats were exposed to 100 mg/kg/d STZ (i.p., for 2 days). STZ increased the levels of total nitrate/nitrite while decreased the liver content of GSH and SOD activity. A reduction in B cell lymphoma 2 (Bcl-2) and B cell lymphoma-extra large (Bcl-XL), an elevation in Bcl-2 agonist of cell death (Bad) and cytosolic/mitochondrial cytochrome C was also observed. Hopefully, CHR (200 mg/kg/d p.o., for 14 days) counteracted all these changes. Taken these results together, STZ-induced liver oxidative stress was alleviated by CHR [71].

Other drugs

Acetaminophen

Also known as APAP in the United States and paracetamol in Europe and other countries, acetaminophen is a widely-used drug in the world as an analgesic or anti-pyretic drug with a potential liver toxicity [72]. In an in vivo study, administration of APAP (100 and 200 mg/kg, p.o., for 14 days) to rats caused a significant reduction in the liver content of GSH, and the activities of GST, GR, GPx and CAT, accompanied by a substantial elevation in the serum levels of AST, ALT, LDH, blood urea nitrogen and creatinine. On the other hand, HSP (750 mg/kg, p.o., for 14 days) normalized these changes. From these results, the authors deduced that HSP could prevent oxidative stress and toxicity provoked by APAP [73]. In another experiment, mice were given APAP, leading to an elevation in the serum activities of AST and ALT, and the liver content of MDA, paralleled with a decrease in the activities of SOD and GPx and hepatic GSH content. Hepatocyte apoptosis was observed as evidenced by a substantial decrease in the ratio of Bax/Bcl-2. All these imbalances were reversed by HSP. Preventing lipid peroxidation and modulating the gene expressions related to apoptosis, were concluded to be the underlying mechanisms of this flavonon [74].

Isoproterenol

In a model of myocardial injury, rats were given an injection of isoproterenol hydrochloride (85 mg/kg, s.c., for 2 days) and the following results were observed: an elevation in the serum levels of cholesterol, VLDL, TG, free fatty acids and phospholipids as wells as a reduction in the serum levels of HDL. In the liver tissue, increased levels of cholesterol, TG and free fatty acids alongside with decreased levels of phospholipids were also evident. Interestingly, HSP (200 mg/kg, p.o. for 7 days) was able to normalize these changes. Overall, these results showed that HSP had hypolipidemic effects in the liver of rats [75].

Nicotine

In a study, hepatotoxicity was produced by injection of nicotine (2.5 mg/kg, s.c., 5 days a week, for 22 weeks) to rats as evidenced by a marked augmentation in the serum levels of AST, ALT and LDH as well as phospholipids, cholesterol, free fatty acids and TG levels. HSP at the dose of 25 mg/kg (p.o., 5 days a week, for 22 weeks) was able to protect the liver from nicotine-induced toxicity by normalizing the mentioned markers which was supported by histopathological evaluations in liver tissue [76]. Similarly, the same investigators injected nicotine (2.5 mg/kg, s.c., 5 days a week, for 22 weeks) to rats to induce hepatotoxicity as reflected by a remarkable rise in the TBARS and hydroperoxides levels, concomitant with AST, ALT and ALP activities. Nicotine also elevated the liver levels of phospholipids, cholesterol, free fatty acids and TG. However, HSP at different doses of 25, 50, 75, 100 and 150 mg/kg (p.o., 5 days a week, for 22 weeks) could counteract these imbalances. Regulating the extent of lipid peroxidation was suggested to be the underlying mechanism of HSP against nicotine-induced hepatotoxicity [77].

Isoniazid/rifampin

As first line medications in the treatment of tuberculosis, isoniazid and rifampicin have been reported to cause liver toxicity [78]. The findings of an investigation indicated that co-administration of isoniazid (75 mg/kg) and rifampin (150 mg/kg) p.o., for 7 days, significantly increased the serum levels of AST, ALT, total bile acid, bilirubin and GPx while substantially reduced the liver GSH content and multidrug resistance proteins 2 (Mrp2) expression. HSP (50, 100, and 200 mg/kg, p.o., for 7 days) was capable of reversing these anomalies. The authors concluded that an upregulation in Mrp2 and a decrease in oxidative damage could be the possible mechanisms behind the hepatoprotective effects of HSP [79]. In another experiment, liver toxicity was induced in rats by a single-dose oral administration of isoniazid (27 mg/kg), rifampicin (54 mg/kg) and pyrazinamide (135 mg/kg) for 50 days. A noticeable raise in the activities of AST, ALT, ALP and LDH as well as lipid peroxides were observed while the levels of SOD, CAT, GSH, vitamin C and vitamin E were reduced by antitubercular drugs. Inflammation and apoptosis were evident by a remarkable elevation in the levels of NF-κB, IL- 10, TNF- α, Bax, caspase 3, caspase 9, and a reduction in the Bcl-2 expression. However, HSP (200 mg/kg, p.o., single administration) was capable of restoring these biochemical parameters near to normal levels, indicating its anti-oxidative stress, anti-inflammatory and anti-apoptotic properties [80].

Valproate

As one of the most used antiepileptic medications, valproate sodium has been reported to possess several adverse effects such as hepatotoxicity. Oxidative stress is one of the most known mechanisms of valproate-mediated hepatotoxicity [81]. In a model of valproate-induced liver toxicity, rats received 300 mg/kg/d (p.o., for 60 days) of valproate resulting in a pronounced escalation in the serum activities of AST, ALT, ALP, GGT and LDH as well as the levels of hydrogen peroxide, superoxide and hydroxyl radicals, and the content of protein carbonyl. Conversely, the activities of liver SOD, CAT, GPx and GR, along with the contents of vitamin C, vitamin E and GSH were declined by valproate. The intrinsic pathway of apoptosis was also affected by this drug as marked by increased gene expression of Bad, Bax and caspase 3, and decreased gene expression of Bcl-2. HSP (100 mg/kg/d, p.o., for 60 days) reversed all these alterations. Collectively, antioxidant and anti-apoptotic properties of HSP were suggested in this study [82].

Pesticides

2,3,7,8-tetrachlorodibenzo-p-dioxin

As an environmental pollutant released from plastics manufacturing and paper processing, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been demonstrated to have some toxic consequences such as hepatotoxicity, both in humans and experimental animals [83]. In an experimental study, rats were given TCDD (2 μg/kg/week in corn oil, p.o., for 60 days) to induce liver toxicity. TCDD resulted in a drastic elevation in the serum levels of TNF-α and IL-1β while liver SOD, CAT, GSH and TBARS levels were reduced. HSP (60 mg/kg/week in corn oil, p.o., for 60 days) normalized these values. From these observations, it was concluded that TCDD caused liver toxicity via modifying cytokine balances, changing liver histology and exerting oxidative stress in the experimental animals. Nevertheless, exposure of rats to HSP attenuated the aforementioned imbalances and in this way, protected the liver from TCDD-induced hepatotoxicity [84].

Paraquat

As a well-known herbicide, paraquat is able to induce toxicity against internal organs such as liver [85]. Paraquat at the dose of 75 mg/kg (i.p., single administration) noticeably elevated the serum activities of AST and ALT as well as the levels of ROS and TBARS while diminished the activities of GST, GPx and CAT along with the liver content of GSH and total antioxidant capacity. The expressions of cyclooxygenase-2, iNOS and NF-κB were upregulated by this herbicide. In addition, paraquat reduced IκBα and elevated P-IκBα, IL-1β and IL-6 levels. The expressions of pro-apoptotic factors Bax, caspase 3 and caspase 9 were increased while the expression of anti-apoptotic factor Bcl-2 was declined. Paraquat-elicited hepatotoxicy was ameliorated by HSP (200 mg/kg, p.o., for 6 days) which counteracted these biofactors. Anti-apoptotic, anti-inflammatory and antioxidant activities of HSP were demonstrated in this experimental study [86].

Miscellaneous

Hydrogen peroxide

In an in vitro model of study, L02 cells were concomitantly treated with hydrogen peroxide (200 μM) and HSP (20, 40 and 80 μM). The results indicated that HSP was able to upregulate the expression of HO-1 and in this way, protected liver cells from oxidative stress produced by hydrogen peroxide. HSP also led to an increase in the Nrf2 nuclear translocation as well as ERK activation. From these data, the authors concluded that HSP improved hepatocyte total antioxidant capacity via enhancing HO-1 through ERK/Nrf2 signaling pathway. Consequently, HSP could be considered as a novel drug candidate in the therapy of hepatic dysfunction as a result of oxidative stress [87].

Thioacetamide

In a model of hepatocellular carcinoma, rats received 200 mg/kg (i.p., twice a week, for 14 weeks) thioacetamide which resulted in a substantial elevation in the gene and protein expressions of Wnt3a, β-catenin, Cyclin D1 and Wnt5a. Oxidative stress was marked by a considerable rise in the content of liver MDA accompanied by a noticeable decline in the liver total antioxidant capacity and SOD activity. In addition, liver inflammation and hepatocyte apoptosis were reflected by a considerable elevation in the myeloperoxidase and caspase 3 activities, respectively. Overall, the authors proposed that Wnt3a/β-catenin and Wnt5a pathways were involved in the hepatocarcinogenocity of thioacetamide. HSP (150 mg/kg/d, p.o., for 14 weeks) via blocking these pathways had the potential to inhibit hepatic carcinoma [88].

X- and γ-radiation

In an X-radiation-induced hepatocyte damage model, mice were exposed to four Gy radiations, which resulted in decreased serum activities of SOD, CAT and GR, and liver GSH content followed by a striking elevation in the TBARS levels. It also increased tail length, tail moment, olive tail moment and percentage DNA in the tail. HSP (25 mg/kg, p.o., for 7 days) counteracted all these imbalances. On the basis of these results, the radiation-induced hepatotoxicity was ameliorated by HSP [89]. In another study, when rats were exposed to γ-irradiation (1, 3 and 5 Gy), liver injury was produced as demonstrated by drastically-increased levels of AST, ALT, ALP, LDH and GGT, accompanied with diminished body and liver weights, in a dose-dependent fashion. Lipid peroxidation assay showed increased concentration of TBARS in hepatic tissue while activities of SOD, CAT and GR as well as the levels of liver GSH, vitamin C and vitamin E decreased. Though, HSP (50 and 100 mg/kg, p.o., for 7 days), remarkably and dose-dependently reversed all these anomalies. Collectively, γ-irradiation-induced oxidative stress and liver damage were alleviated by HSP, which was supported by histopathological findings. The proposed underlying mechanisms behind the hepatoprotective action of HSP were membrane stabilizing and free radical quenching abilities [90]. Hepatotoxicity was induced by exposing rats to five Gy of γ-radiation as evidenced by elevated serum levels of AST, ALT, ALP, GGT and LDH, along with reduced levels of hepatic SOD, CAT, GPx, GSH, vitamin C and vitamin E. The authors found that administration of HSP (100 mg/kg, p.o., for 7 days) to γ-radiated rats could restore the changed levels of these biomarkers. Antioxidant and free radicals hunting activities were attributed to the hepatoprotective effects of HSP [91].

Conclusion and future perspective

In the recent years, there are plenty of studies already published on the subject of hepatoprotective mechanisms of HSP against a variety of natural and chemical toxins. Antioxidant, anti-inflammatory and anti-apoptotic properties of HSP have been demonstrated in such studies. The current review represents several in vivo and in vitro models of experiments related to natural and chemical toxicants-induced hepatotoxicity, showing the underlying mechanisms behind HSP hepatoprotective activities. HSP produces its antioxidant activity through an elevation in the levels of GSH, SOD, CAT, GST and GPx concomitant with a reduction in the ROS and MDA levels. Anti-apoptotic activity of HSP is reflected by a decrease in the Bax/Bcl-2 ratio, and the levels of caspase 3 and Bad. While its anti-inflammatory property is evidenced by a diminution in the TNF-α, Il-1β, Il-6 and NF-κB levels.

Insufficient clinical trials on the therapeutic activity of HSP are a significant limitation, which has to be further considered. The bioavailability, proper dose, tolerability, and efficacy of HSP and its metabolites on liver diseases, are less known clinical aspects of HSP. HSP can be considered, in near future, as a promising candidate for the treatment of liver-related diseases, although well-designed clinical trials in patients with different types of liver diseases are needed to be conducted. Taken together, we can say that HSP is a protective agent against inflammation and/or oxidative stress-mediated hepatotoxicity.

Abbreviations

HSP

Hesperidin

TNF-α

Tumor necrosis factor alpha

HMGB1

High-mobility group box 1 protein

i.v.

Intravenously

i.p.

Intraperitoneally

AST

Aspartate aminotransferase

ALT

Alanine aminotransferase

ALP

Alkaline phosphatase

TBARS

Thiobarbituric acid reactive substances

GSH

Glutathione

SOD

Superoxide dismutase

ROS

Reactive oxygen species

LPS

Lipopolysaccharides

IFNγ

Interferon gamma

MDA

Malondialdehyde

LDH

Lactate dehydrogenase

GGT

Gamma-glutamyl transpeptidase

p.o.

Orally

EtOH

ETHANOL

IL-6

Interleukin 6

TGF-β

Transforming growth factor-β

α-SMA

Alpha smooth muscle actin

NF-κB

Nuclear factor kappa-light-chain-enhancer of activated B cells

IκBα

inhibitor of kappa B protein-alpha

IL-1β

Interleukin 1 beta

GP_X

Glutathione peroxidase

CAT

Catalase

GR

Glutathione reductase

GST

Glutathione-S-transferase

JNK

C-Jun N-terminal kinase

AP-1

Activator protein 1

CCl₄

Carbon tetrachloride

PPARγ

Peroxisome proliferator-activated receptor gamma

P-JAK

Phosphorylated Janus kinase

P-STAT

Phosphorylated signal transducer and activator of transcription

s.c.

Subcutaneously

CTGF

Connective tissue growth factor

AFP

Alpha-fetoprotein

Nrf2/ARE

Nuclear factor (erythroid-derived 2)-like 2/antioxidant response element

HO-1

Heme oxygenase 1

iNOS

Inducible nitric oxide synthase

8-OHdG

8-hydroxy-2′-deoxyguanosine

FGF-23

Fibroblast growth factor-23

STZ

Streptozotocin

Bcl-2

B cell lymphoma 2

Bcl-XL

B cell lymphoma-extra large

Bad

Bcl-2 agonist of cell death

APAP

N-acetyl-para-aminophenol

Mrp2

Multidrug resistance proteins 2

LDL

Low-density lipoprotein cholesterol

VLDL

Very low-density lipoprotein cholesterol

HDL

High density lipoprotein cholesterol

TG

Triglycerides.

Compliance with ethical standards

Conflict of interest

No potential conflict of interest was reported by the authors.