Hepatoprotective activity of raspberry ketone is mediated via inhibition of the NF-κB/TNF-α/caspase axis and mitochondrial apoptosis in chemically induced acute liver injury

Possible proposed mechanisms of hepatoprotective activity of raspberry ketone. Raspberry ketone ameliorated hepatic oxidative stress and suppressed inflammation, apoptosis, and DNA fragmentation in CCl₄-injured hepatocytes.

Abstract

Raspberry Ketone (RK) is a natural phenolic compound which is marketed nowadays as a popular weight-reducing remedy, with reported antioxidant and anti-inflammatory activities. However, its biological activity is not fully elucidated. Hepatotoxicity is the leading cause of acute liver failure in Europe and North America, and its management is still challenging. Therefore, this study aimed to assess the therapeutic detoxification activity of RK against liver injury in vivo and to explore the underlying mechanisms using carbon tetrachloride (CCl₄)-induced hepatotoxicity as a model. First, a dose–response study using 4 different doses, 25, 50, 100, and 200 mg kg^–1 day^–1, of RK was conducted. RK was administered for 5 days as a pretreatment, followed by a single dose of CCl₄ (1 ml kg^–1, 1 : 1 v/v CCl₄ : olive oil)_. The RK dose of 200 mg kg^–1 showed the greatest protective effect and was selected for further investigations. CCl₄ hepatotoxicity was confirmed by elevation of liver enzymes, and histopathological examination. CCl₄-induced oxidative stress was evident from increased lipid peroxidation measured as thiobarbituric acid reactive substances (TBARS) along with depleted superoxide dismutase (SOD), reduced glutathione (GSH), and total antioxidant capacity (TAC). Increased oxidative stress was associated with increased cytochrome c expression with subsequent activation of caspase-9 and caspase-3, in addition to DNA fragmentation reflecting apoptosis. CCl₄ also induced the expression of inflammatory cytokines (NF-κB and TNF-α). Interestingly, RK hepatoprotective activity was evident from the reduction of liver enzymes, and maintenance of hepatocyte integrity and microstructures as evaluated by histopathological examination using H and E, and transmission electron microscopy. The antioxidant activity of RK was demonstrated by the increase of TAC, SOD, and GSH, with a concomitant decrease of the TBARS level. Moreover, RK pretreatment inhibited CCl₄-induced upregulation of inflammatory mediators. RK antiapoptotic activity was indicated by the reduction of the expression of cytoplasmic cytochrome-C, a decrease of caspases, and inhibition of DNA fragmentation. In conclusion, this study demonstrates that RK is a promising hepatoprotective agent. The underlying mechanisms include antioxidant, anti-inflammatory, and anti-apoptotic activities. This is the first study reporting RK hepatoprotective activity in acute hepatic injury and approves its antiapoptotic effect in the liver.

1. Introduction

Liver disease is one of the major causes of morbidity and mortality across the world. According to WHO estimates in 2015, approximately 325 million people are living with chronic hepatitis infections, and 1.34 million people die each year.1 There is no common conventional, effective drug therapy which prevents or reverses liver damage.2 Therefore, it is necessary to search for complementary and alternative medicines, for the treatment of liver diseases.3 Medicinal plants serve as a vital source of potentially useful new compounds. Several studies have reported the therapeutic efficacy of phytomedicines, such as silymarin and curcumin, in the management of liver dysfunction.4

Much attention has been directed toward the potential health-promoting properties of phenolic phytochemicals.5–7 Plant-derived phenolic compounds are well known for their antioxidant properties. Recent evidence indicates that these compounds may confer anti-inflammatory and antiapoptotic activities, which would have important implications in health maintenance and disease risk reduction.8

Rheosmin, or raspberry ketone (RK), is a natural phenolic compound. RK, 4-(4-hydroxyphenyl) butan-2-one, was discovered in blackberries by Japanese researchers. It occurs in many other fruits, including raspberries and cranberries. It is biosynthesized from coumaroyl-CoA.9 It is well absorbed orally; approximately 90% of the dose is excreted as metabolites via the urine within 24 h.10 Many products containing this compound are marketed for weight loss; however, the biological activities of RK are not fully elucidated. The effect of RK on the liver was studied by Wang et al.11 who found that RK could protect the liver from high-fat induced steatohepatitis. The mechanism of action included anti-inflammatory and antioxidant activities and a change of lipid metabolism.12–14 The Flavor & Extract Manufacturers Association (FEMA) provided rheosmin a “GRAS” (generally regarded as safe) status.15

CCl₄/trichloromethane (CCl₃˙)-induced liver injury is one of the most widely used experimental models for exogenous toxin-induced hepatotoxicity and is a commonly used model for the screening of anti-hepatotoxic and/or hepatoprotective activities of drugs. CCl₃˙ is produced by metabolic activation by hepatic microsomal cytochrome P450. CCl₃˙ causes a wide array of dysfunction in the liver, including triglyceride accumulation, centrilobular necrosis, polyribosomal desegregation, and depression of protein synthesis.16 CCl₃˙ is an active free radical which first attacks cellular lipids and causes lipid peroxidation (LPO) of unsaturated fatty acids in plasma and organelle membranes, thus leading to serious hepatocyte damage, with oxidative stress being the primary player.17 Increased lipid peroxidation is usually associated with the depletion of protective no-protein sulfhydryl groups such as reduced glutathione (GSH), and increased leakage of liver enzymes.18

Oxidative stress usually results in macrophage activation and release of many pro-inflammatory cytokines, such as nuclear factor-kappa B (NF-κB). NF-κB regulates the expression of various inflammatory mediators, including interleukin (IL)-1β and tumor necrosis factor (TNF)-α.18,19 Increased TNF-α, an essential apoptotic cytokine, induces apoptotic cell death, and thus, the inflammatory response occurs in a vicious cycle which aggravates liver diseases.20,21 Moreover, accumulation of reactive oxygen species (ROS) can also induce apoptosis via stimulating the release of mitochondrial cytochrome c.22,23

The aims of the present study were to assess whether RK exhibits in vivo protective effects against CCl₄/CCl₃˙-induced liver injury in rats and to explore the probable mechanisms of action involved.

2. Materials and methods

2.1. Chemicals

CCl₄ was obtained from Sigma-Aldrich (St Louis, MO, USA). Raspberry ketone was obtained from Extrasynthese (Genay, France). Ether, n-butanol, 1,1,3,3-tetramethoxypropane, potassium dihydrogen phosphate, reduced glutathione (GSH), Ellman's reagent (5,5′-dithio-bis-(2-nitrobenzoic acid)), trichloroacetic acid (TCA), and thiobarbituric acid (TBA) were purchased from Sigma-Aldrich (St Louis, MO). Alanine transaminase (ALT) and aspartate transaminase (AST) kits were obtained from Sigma-Aldrich (St Louis, MO). Rat TNF-α, caspase-3 and caspase-9 enzyme-linked immunosorbent assay (ELISA) kits were purchased from Raybiotech, Inc. (Norcross, USA) and Cloud-Clone Corp. (Houston, USA), respectively. Immunohistochemistry (IHC) antibodies for NF-κB and cytochrome C were obtained from Thermo Fisher Scientific (USA). A DNA extraction kit was obtained from Qiagen (Hilden, Germany). Other chemicals were of high analytical reagent grade.

2.2. Experimental design

Adult healthy male Wistar rats (150–200 g) were obtained from the animal farm at King Saud University, Riyadh, Saudi Arabia. Animals were housed at a maximum of 4 rats per standard polypropylene cage. The rats were acclimatized for 1 week before beginning the experiments. The animals were provided water and standard chow ad libitum and were maintained under controlled conditions of temperature, humidity, and light. Handling of animals was in compliance with the ARRIVE guidelines for the care and use of animals for scientific purposes, and with ethical approval no. KSU-SE_191-023 for the animal protocol, according to King Saud University instructions.

Dose–response study: Forty-eight adult male rats were randomly divided into six groups, with eight rats per group. The experiment was performed over 6 days. Group I (Control), serving as an untreated control, received a vehicle daily via gavage, and was injected intraperitoneally with a single dose of olive oil on the fifth day of the experiment. Group II (CCl₄) rats received the vehicle daily via gavage and a single CCl₄ dose was injected intraperitoneally (1 ml kg^–1 of 1 : 1 v/v CCl₄ : olive oil) on the fifth day.24 Groups III, IV, V, and VI (RK + CCl₄) received an oral administration of 25, 50, 100, and 200 mg kg^–1 RK daily for 5 days via an oral tube,12 respectively, and were injected intraperitoneally with a single dose of CCl₄ (1 ml kg^–1 of 1 : 1 v/v CCl₄ : olive oil) on the fifth day of the experiment, 1 h after the administration of the last RK dose.

The overnight fasting animals of all groups were euthanized by fast decapitation on the sixth day. Animals were anesthetized with ether and euthanized 24 h after CCl₄ administration. Blood samples were collected from the heart, allowed to stand for 30 min, centrifuged at 3000 rpm for 15 min at 4 °C to separate serum, and then stored at –80 °C for the different biochemical assays. The liver was immediately removed, dried, and weighed. Specimens of the liver from different lobes were transferred immediately into 10% phosphate-buffered formaldehyde for histological examination. Another part was weighed and homogenized immediately in ice-cold 50 mM potassium phosphate buffered saline (pH 7.4) to yield a 50% (w/v) homogenate to be used for the assay of GSH, SOD, TAC, and LPO. Histopathological examination of liver tissue using H and E, tissue homogenate levels of GSH, SOD, TAC, and LPO, and assay of serum liver enzymes, ALT and AST, were used to compare different doses. Based on these tests, a dose of RK 200 mg kg^–1 was selected for further investigations.

Hepatoprotective effects of RK (200 mg kg^–1): Twenty-four adult male rats were divided into four groups: Group I rats (Control) received a vehicle daily via gavage and were injected intraperitoneally with a single dose of olive oil on the fifth day of the experiment. Group II (CCl₄) rats received the vehicle daily via gavage, and a single CCl₄ dose was injected intraperitoneally (1 mL kg^–1 of 1 : 1 v/v CCl₄ : olive oil) on the fifth day (Slama et al., 2018).24 Group III rats (RK: 200 mg kg^–1 + CCl₄) received an oral administration of 200 mg kg^–1 RK daily for 5 days via an oral tube and were injected intraperitoneally with a single dose of CCl₄ (1 mL kg^–1 of 1 : 1 v/v CCl₄ : olive oil) on the fifth day of the experiment, 1 h after RK administration. Group IV (RK control: received an oral administration of 200 mg kg^–1 RK daily for 5 days via oral tube and were injected intraperitoneally with a single dose of olive oil on the fifth day of the experiment; 1 h after the RK administration.

Blood samples were collected as described previously. The liver was immediately removed, dried, weighed, and divided into four parts. The first part was transferred immediately into 10% phosphate-buffered formaldehyde for histopathological and immunohistochemical studies. The second part was transferred immediately into 2.5% glutaraldehyde for electron microscopic examination. The third part was weighed and homogenized immediately in ice-cold 50 mM potassium phosphate buffered saline (pH 7.4) to yield a 50% (w/v) homogenate to be used for various biochemical investigations. The fourth part was stored at –80 °C until used.

2.2.1. Liver function tests

To assess liver cell damage, the serum levels of ALT and AST were assayed using diagnostic spectrophotometric kits, according to the manufacturer's instructions.

2.2.2. Determination of oxidative stress markers

Twenty percent liver homogenate was used for determination of lipid peroxidation (LPO), reduced glutathione (GSH), Total Antioxidant Capacity (TAC), and superoxide dismutase (SOD) as markers of oxidative stress. LPO was determined spectrophotometrically as thiobarbituric acid-reactive substances (TBARs), according to the method of Mihara and Uchiyama.25 The colorimetric determination of TBARs was based on the reaction of malondialdehyde (MDA) with thiobarbituric acid at low pH and high temperature. The resulting pink product was extracted with n-butanol, and the absorbance was determined spectrophotometrically at 535 nm.

The estimation of GSH was performed using a spectrophotometer, according to Ellman's method.26 An aliquot of 0.5 mL of the tissue homogenate was used. Proteins were precipitated using TCA, and samples were centrifuged at 3000 rpm for 10 min. The resulting supernatant was used for determination of GSH using Ellman's reagent. The absorbance was measured at 412 nm.

TAC and SOD were analyzed using commercial kits which were purchased from Spectrum Diagnostics, Cairo, Egypt according to the manufacturer's instructions.

2.2.3. Detection of TNF-α, caspase-9 and caspase 3 using ELISA kits

TNF-α, caspase-9, and caspase-3 (Raybiotech® and Cloud-Clone®) were detected in hepatic tissue homogenates using standard ELISA kits, according to the manufacturer's instructions.

2.2.4. Total DNA extraction and fragmentation analysis

Thirty milligrams of each tissue sample were homogenized in RTL lysis buffer (Qiagen) containing 1% 2-mercaptoethanol. Total DNA was extracted using an All Prep DNA/RNA Mini kit (Qiagen, Cat# 80204), following the manufacturer's manual, and the DNA was eluted with 50 μL of the elution buffer provided. The extracted DNA was quantified using a NanoDrop-8000, and the DNA fragmentation was assessed via agarose gel (1.5%) electrophoresis. Gels were transilluminated with 300 nm UV light, and a photographic record was made.27

2.2.5. Histopathological examination

The liver samples were fixed with 10% phosphate-buffered formaldehyde for 24 h and then washed with tap water. Serial dilutions of alcohols (methyl, ethyl, and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56 °C in a hot air oven for 24 h. Paraffin beeswax tissue blocks were prepared, and 5–6 μm-thick sections were cut using a sledge microtome. The tissue sections were mounted on glass slides, deparaffinized, and stained with hematoxylin and eosin for histopathological examinations with a light microscope.28

2.2.6. Observation of changes of the liver tissue ultra-microstructure by transmission electron microscopy

Glutaraldehyde, 2.5% (prepared with dipotassium sodium arsenate), was used to fix liver samples for 2 h. The samples were then washed with phosphate-buffered saline three times. They were then post-fixed in 1% osmium tetroxide, left overnight, and then washed three times with phosphate-buffered saline. The samples were sequentially dehydrated with 50 and 80% alcohol, followed by 80, 90, and 100% acetone. After overnight immersion, the samples were polymerized at 35, 45, and 60 °C for 24 h each. The samples were sectioned (50–80 μm) with an ultramicrotome, treated with a uranyl acetate–lead citrate dye, and then observed with a transmission electron microscope (model H-7650).29

2.2.7. Immunohistochemical detection of NF-κB and cytochrome C

Formalin-fixed tissues were embedded in paraffin and sectioned (5 μm thickness). The liver sections were mounted overnight, deparaffinized in xylene, rehydrated with a graded series of reducing ethanol concentrations, and boiled in antigen unmasking solution (Vector Laboratories, Burlingame, CA, USA) for 5 min. Slides were immersed in a peroxidase-blocking reagent (Dako, Botany Bay, NSW, Australia) for 10 min and incubated in a humidified chamber with blocking goat serum (Dako) for 30 min. Sections were incubated with anti-NF-κB antibody (rabbit polyclonal, 1 : 500) in blocking solution for 12 h at 4 °C for NF-κB detection, or cytochrome c antibody (mouse monoclonal, Clone 7H8.2C1) for cytochrome C detection. They were then re-equilibrated at 25 °C, washed with PBS, and incubated with horseradish peroxidase (HRP) antibody conjugates (1 : 2500) in blocking solution without Tween®20 for 2 h at 25 °C.

Specimens were washed with PBS and incubated at room temperature with 0.2% 3,3′-diaminobenzidine (DAB) until the desired stain intensity developed, followed by washing with distilled water. Sections were counterstained with hematoxylin, dehydrated in a graded series of increasing ethanol concentrations, and mounted with di-n-butylphthalate-polystyrene-xylene (DPX).30 Immunoreactivity was assessed in a blinded manner by three independent observers.

2.2.8. Statistical analysis

Results are reported as the mean ± standard error of the mean (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA). If the overall p-value was found to be statistically significant (p ≤ 0.05), further comparisons among groups were made according to post-hoc Tukey's test. Statistical analyses were performed and graphs were drawn using GraphPad Prism version 5 (GraphPad Software).

3. Results

3.1. Dose–response study

3.1.1. Liver function tests: ALT and AST

The levels of serum ALT and AST were significantly higher in the CCl₄-treated animals compared with those of the control group. Treatment with RK caused a dose-dependent decrease of liver enzymes, with only the 200 mg kg^–1 dose maintaining the levels of both ALT and AST comparable to the control group (Fig. 1A and B).

Fig. 1. Effects of different doses of RK on liver enzymes in the CCl₄-induced hepatotoxicity model. (A) Serum ALT (U L^–1) and (B) AST (U L^–1) in different animal groups. Each bar represents the mean of 6 rats + SEM. ^ap ≤ 0.05 compared to the control group; ^bp ≤ 0.05 compared to the CCl₄ group; ^cp ≤ 0.05 compared to 200 mg kg^–1 RK + CCl₄ group. ALT, alanine transaminase; AST, aspartate transaminase; CCl₄, carbon tetrachloride; and RK, raspberry ketone.

3.1.2. Effects of CCl₄ and RK treatment on oxidative stress markers

CCl₄ treatment significantly reduced the GSH level, TAC, and SOD, and increased the extent of LPO (as evidenced by the increased TBARS level) in the liver tissues, as compared to those of the control group. Pretreatment with different doses of RK of 25, 50, 100, and 200 mg kg^–1 significantly and dose-dependently ameliorated the effects of CCl₄ on GSH, TAC, SOD, and LPO. Pretreatment with RK at doses of 100 and 200 mg kg^–1 maintained the levels of GSH, TAC, and TBARS comparable to those of the control group, and was significantly different from that at lower doses (25 and 50 mg kg^–1). Regarding SOD, a RK dose of 200 mg kg^–1 maintained a significantly higher SOD level as compared to a RK dose of 100 mg kg^–1, and was not statistically significant compared to that of the control group (Fig. 2 and 3).

Fig. 2. Effects of different doses of RK on hepatic oxidative stress-related parameters in the CCl₄-induced hepatotoxicity model. A: Liver GSH (μmole per g tissue protein) and B: liver TBARS (nmol per g tissue protein) in different animal groups. Each value represents the mean of 6 rats + SEM. ^ap ≤ 0.05 compared to the control group; ^bp ≤ 0.05 compared to the CCl₄ group; ^cp ≤ 0.05 compared to the 200 mg kg^–1 RK + CCl₄ group. CCl₄, carbon tetrachloride; GSH, reduced glutathione; RK, raspberry ketone; and TBARS, thiobarbituric acid-reactive substances.

Fig. 3. Effects of different doses of RK on hepatic oxidative stress-related parameters in the CCl₄-induced hepatotoxicity model. A: Liver SOD (U per g tissue) and B: liver TAC (mM L^–1) in different animal groups. Each value represents the mean of 6 rats + SEM. ^ap ≤ 0.05 compared to the control group; ^bp ≤ 0.05 compared to the CCl₄ group; ^cp ≤ 0.05 compared to the 200 mg kg^–1 RK + CCl₄ group. CCl₄, carbon tetrachloride; RK, raspberry ketone; SOD: superoxide dismutase; and TAC: total antioxidant capacity.

3.1.3. Liver histopathology using H and E

Rats in the control group (Fig. 4, image A) showed normal histological characteristics of the liver, intact hepatocytes with large vesicular nuclei with clear nucleoli and intact vasculature without abnormal alterations. CCl₄ administration caused evident histopathological changes of the liver, including vacuolation, lymphocytic infiltration, congestion of the central veins, obliteration of the blood sinusoids, pyknotic nuclei, and massive necrosis in the centrilobular area (Fig. 4, image B). Pretreatment with RK, dose-dependently, reduced the pathological changes in the liver (Fig. 4, images C–F), with a dose of 200 mg kg^–1 closely resembling the control liver.

Fig. 4. Histological examination of liver tissue sections stained with hematoxylin and eosin dye demonstrating the effect of RK on the CCl₄-induced hepatotoxicity model. Light micrographs showing groups treated with the vehicle (image A), a sample with almost intact hepatocytes with large vesicular nuclei and clear nucleoli (arrowhead) as well as intact vasculature without abnormal alterations. CCl₄ (image B) caused severe vacuolar degenerative changes in hepatocytes with karyopyknosis alternating with necrotic hepatocytes in the centrilobular zone of the hepatic lobule (arrows) with a congested central vein and nearby sinusoids (star) accompanied by a focal aggregation of mononuclear inflammatory cells (red arrow). CCl₄ groups pre-treated with either 25 mg kg^–1 RK (C) or 50 mg kg^–1 RK (D) showed the same lesion records as CCl₄ with minimal protective efficacy with few apparent intact cell records. CCl₄ group pre-treated with 100 mg kg^–1 RK (E) showed better protective efficacy with minimal inflammatory cell infiltration and sinusoidal congestion. However, persistence of vacuolar degeneration of hepatocytes was observed in a less extensive manner in most of the samples (arrows). CCl₄ group pre-treated with 200 mg kg^–1 RK (F) showed morphologically the best protective efficacy records with many apparent intact hepatocytes (arrowhead) and fewer sporadic records of necrobiotic changes (arrow); minimal records of inflammatory cell infiltration with mild dilation of hepatic sinusoids were observed. CCl₄, carbon tetrachloride; and RK, raspberry ketone.

3.2. Effect of 200 mg kg^–1 RK on CCl₄-induced liver damage

3.2.1. Effect on liver enzymes and oxidative stress markers

RK either alone or with CCl₄ maintained liver enzymes ALT and AST, GSH, LPO, TAC, and SOD at levels comparable to those of the control group.

3.2.2. Ultra-microstructure determined by transmission electron microscopy

In the control and RK-treated groups (Fig. 5, images A and B), the ultra-microstructure of rat liver cells was normal. The liver cell nuclei were round or nearly round. The rough endoplasmic reticulum, the mitochondria, and the euchromatin were normal and clear. In the CCl₄ group (Fig. 5, image C), the hepatocytes were obviously abnormal, and the glycogen content was reduced. The liver cell matrix was filled with large numbers of lipid droplets, and the nuclei were no longer round, with a pyknotic and eccentric nucleus, and chromatin margination. In the RK + CCl₄ group (Fig. 5, image D), the nuclei of the liver cells were normal but the used dosage of RK was not sufficient to restore the glycogen content to normal. This could indicate that the metabolic state of the hepatocytes was not completely preserved by RK. The rough endoplasmic reticulum was abundant, and only small vacuoles were found in the matrix.

Fig. 5. Effects of RK on the appearance of liver cells determined by transmission electron microscopy in the CCl₄-induced hepatotoxicity model. A: control group, B: RK (200 mg kg^–1) group, C: CCl₄ group, and D: CCl₄ group pre-treated with 200 mg kg^–1 RK. In the control and RK-treated groups (A and B, respectively), the ultra-microstructure of rat liver cells was normal, and the liver cell nuclei were round or nearly round. In the CCl₄ group (C), the hepatocytes were obviously abnormal, and the glycogen content was reduced. The liver cell matrix was filled with large numbers of lipid droplets, and the nuclei were no longer round, with a pyknotic and eccentric nucleus, and chromatin margination. In the RK + CCl₄ group (D), the nuclei of the liver cells were normal, with no lipid droplets, but the used dosage of RK was not sufficient to restore the glycogen content to the normal level. Glyc: glycogen; LD: lipid droplets; N: nucleus; CCl₄, carbon tetrachloride; and RK, raspberry ketone.

3.2.3. Effect on inflammatory response markers, NF-κB and TNF-α

Immunohistochemical analysis of NF-κB expression in the livers of the control group (Fig. 6A) and rats administered RK alone (200 mg kg^–1) (Fig. 6B) showed low NF-κB immunoreactivity. In contrast, strong NF-κB immunoreactivity was found in the hepatocytes of CCl₄-treated rats, with numerous positive nuclei (Fig. 6C). Pretreatment of CCl₄-injured rats with RK (200 mg kg^–1) produced a similar result to that of the control group, lacking nuclear immunoreactivity (Fig. 6D).

Fig. 6. Effect of RK on NF-κB expression in the CCl₄-induced hepatotoxicity model. Immunohistochemical analysis was performed for NF-κB expression in rat livers in different animal groups. Rats treated with the vehicle (A), RK (200 mg kg^–1) (B), and CCl₄ (C), and the CCl₄ group pre-treated with 200 mg kg^–1 RK (D). CCl₄, carbon tetrachloride; and RK, raspberry ketone.

As shown in Fig. 7, the level of TNF-α, quantified via ELISA, markedly increased in the livers of rats treated with CCl₄. Rats treated with RK (200 mg kg^–1) prior to CCl₄ exhibited a significant suppression in the level of TNF-α compared to rats treated with CCl₄ alone. The values of the RK alone and RK + CCl₄ groups were comparable to those of the control group.

Fig. 7. Effect of RK on the TNF-α protein level in the CCl₄-induced hepatotoxicity model. Liver TNF-α (pg per g tissue protein) was detected by the ELISA technique in different animal groups. Each value represents the mean of 6 rats + S.D. ^ap ≤ 0.05 compared to the control group; ^bp ≤ 0.01 compared to the CCl₄ group. CCl₄, carbon tetrachloride; ELISA, enzyme-linked immunosorbent assay; RK, raspberry ketone; and TNF-α, tumor necrosis factor-α.

3.2.4. Effect on apoptosis-related parameters

3.2.4.1. Cytochrome c protein expression

Cytochrome c protein expression was assessed by immunohistochemistry in the livers of the control group (Fig. 8A) and rats which received RK alone (200 mg kg^–1) (Fig. 8B); both groups showed low cytochrome c immunoreactivity. In contrast, strong cytochrome c expression was found in the hepatocytes of CCl₄-treated rats (Fig. 8C). Interestingly, pretreatment of CCl₄-injured rats with RK (200 mg kg^–1) reduced cytochrome c protein expression (Fig. 8D).

Fig. 8. Effect of RK on the cytochrome c expression level in the CCl₄-induced hepatotoxicity model. Immunohistochemical analysis was performed for the expression of cytochrome c in rat livers of different animal groups. Rats treated with the vehicle (A), RK (200 mg kg^–1) (B), and CCl₄ (C), and the CCl₄ group pre-treated with 200 mg kg^–1 RK (D). CCl₄, carbon tetrachloride; and RK, raspberry ketone.

3.2.4.2. Caspase-9 and 3 activity

The apoptosis which was induced in the CCl₄ group was evident from elevated caspase-9 and 3 activity, which is a well-known marker of apoptosis. However, the group treated with RK prior to CCl₄ had significantly reduced caspase-9 and 3 activity (Fig. 9A and B, respectively).1

Fig. 9. Effect of RK on some apoptosis-related parameters in the CCl₄-induced hepatotoxicity model. Liver caspase-9 (pg per g tissue protein) (A) and caspase-3 (ng per g tissue protein) (B) protein levels were detected by ELISA in different animal groups. Each value represents the mean of 6 rats + S.D. ^ap ≤ 0.05 compared to the control group; ^bp ≤ 0.05 compared to the CCl₄ group. CCl₄, carbon tetrachloride; ELISA, enzyme-linked immunosorbent assay; and RK, raspberry ketone.

3.2.4.3. Liver genomic DNA

Fig. 10 shows the qualitative changes in the integrity of the liver genomic DNA, which is indicative of apoptosis. Agarose gel electrophoresis shows that CCl₄ treatment resulted in the fragmentation of DNA into oligonucleosome-length fragments (lane 4). However, DNA isolated from control rats (lane 2) and RK (200 mg kg^–1) treated rats (lane 3) showed no DNA fragmentation. DNA of rats treated with RK (200 mg kg^–1) prior to CCl₄ (lane 5) showed marked improvement in the integrity of the liver genomic DNA.

Fig. 10. Effect of RK on liver DNA fragmentation in different animal group rats. Lane 1: DNA ladder, 2: control group, 3: group treated with RK (200 mg kg^–1), 4: group treated with CCl₄, and 5: group pretreated with RK (200 mg kg^–1) and CCl₄. CCl₄, carbon tetrachloride; ELISA, enzyme-linked immunosorbent assay; and RK, raspberry ketone.

Results of the current study and proposed mechanisms of RK are summarized in Fig. 11.

Fig. 11. Possible proposed mechanisms of hepatoprotective activity of raspberry ketone. Raspberry ketone ameliorated hepatic oxidative stress and suppressed inflammation, apoptosis, and DNA fragmentation in CCl₄-injured hepatocytes.

4. Discussion

In addition to its crucial role in the metabolism of nutrients, the liver is also responsible for the biotransformation of drugs and chemicals.31 In this context, the liver is exposed to high concentrations of toxic chemicals and their metabolites, which may cause liver injury which is considered as one of the major worldwide health problems and its prevention and treatment are still limited.32 The implication of oxidative stress and inflammation in the pathogenesis of hepatic damage is well established and, accordingly, drugs with documented antioxidant and anti-inflammatory activities could be a promising therapeutic strategy for the prevention and treatment of liver injury.33

In recent years many researchers have approved the effects of many plant extracts as hepatoprotective agents. Raspberry fruits contain various components, such as vitamins, organic acids, flavonoids, ellagic acid, and anthocyanins. RK is a major aromatic compound of raspberries and is being used in cosmetics and as a food-flavoring agent.34,35 RK has been reported previously to exhibit antioxidant and anti-inflammatory activity in the liver in high-fat models,8,28 while its activity in acute hepatotoxicity has not been previously elucidated. CCl₄-induced acute liver injury in mice and rats is being widely used as an experimental animal model for screening of the hepatoprotective potential of new drugs.36 Therefore, this study focused on examining the hepatoprotective effect of RK using CCl₄-induced hepatotoxicity in male rats as a model.

Oxidative stress has been considered as the primary hit affecting hepatocytes exposed to CCl₄, which is mediated by the production of free radical metabolites of CCl₄. These radicals lead to the peroxidation of cellular membrane lipids, with subsequent membrane damage and release of intracellular enzymes including ALT and AST, which are considered as markers of hepatotoxicity.37 In the present study, CCl₄-induced toxicity was associated with increased lipid peroxidation and cell membrane damage as evidenced by the increased leakage of ALT and AST. In agreement with these data, our study revealed a significant elevation of TBARS, a marker of lipid peroxidation, which is associated with the high activity of ALT and AST, the cytosolic enzymes of the hepatocyte, reflecting loss of integrity and increased permeability of the cell membrane. CCl₄-induced hepatic damage was further confirmed by histopathological examination which showed degenerative changes and massive necrosis. Our results are consistent with those of Chen et al.35 and Abdelhafez et al.38,39

Biological systems aim to protect themselves against oxidative injury with several antioxidants which include free radical scavenging molecules, e.g. GSH, and antioxidant enzymes, e.g. SOD.38 GSH is one of the key protectors of the hepatocytes and its homeostasis is associated with various toxin-induced liver injuries. GSH plays an important role against CCl₄-induced injury by covalently binding to CCl₃˙ radicals.40 In addition, SOD plays an important role in the exclusion of ROS derived from the metabolism of xenobiotics in the liver.41 In the present study, CCl₄-induced oxidative stress was accompanied by reduced levels of GSH, SOD and TAC suggesting that CCl₄-derived free radicals lead to an impaired antioxidant defense system in the liver. The depletion of GSH could be related to the reduced synthesis by the injured liver. In addition, GSH stores may be rapidly consumed for scavenging free radicals induced by CCl₄, especially when liver necrosis initiates.42 The lipid peroxides and ROS result in reduced levels of TAC and SOD enzyme activity.

In the present study, pretreatment with RK showed hepatoprotective activity against CCl₄-induced cell membrane damage, illustrated by a significant reduction of serum liver enzymes, ALT and AST, suggesting the stabilization of the cell membrane. This effect exerted by RK could be related to its antioxidant activity, indicated in our study by the increased levels of GSH, SOD, and TAC. Such increased cellular antioxidant defense leads to the reduction of lipid peroxidation, and subsequent maintenance of cell membrane integrity. The hepatoprotective effect of RK was further confirmed by histopathological examination which showed less necrotic changes, and minimized inflammation and vacuolation. The effect of RK was dose-dependent, with the prophylactic benefits being significantly more powerful with a higher dose (200 mg kg^–1 day^–1) than for the lower doses. These results coincide with previous reports in which the dose 200 mg kg^–1 of RK was the most protective dose against isoprenaline-induced myocardial injury.12 Therefore, this dose was selected for further investigation of the hepatoprotective mechanisms of RK in the present study. For this purpose, we assessed the effect of the selected dose on the inflammatory mediators, NF-κB and TNF-α, and apoptotic markers including cytochrome C and caspases, in addition to the extent of DNA fragmentation.

Oxidative stress can further lead to the induction of the inflammatory response. Thus, inflammation is another important pathological mechanism through which CCl₄-induced liver injury propagates.43,44 CCl₄-toxicity can stimulate inflammatory cells by being exposed to free radicals and tissue debris. NF-κB and TNF-α are the most representative pro-inflammatory cytokines, and many studies have shown that NF-κB and TNF-α are key players in the development and progression of inflammation.18 NF-κB acts as a key transcriptional factor which can induce the expression of many other inflammatory genes, and thus modulates several steps in the inflammatory pathway.24,40 It has been demonstrated that CCl₄-intoxication results in the activation of liver macrophages44 with the subsequent production of proinflammatory mediators including TNF-α which in turn exaggerate the CCl₄-induced hepatic injury.45,46 Furthermore, CCl₄-induced free radicals lead to the activation of NF-κB and the release of inflammatory mediators into injured livers.47 In parallel with these studies, our results revealed positive immunostaining of NF-κB and significant elevation of TNF-α in the hepatic tissue of the CCl₄ group compared to those of the control group. These results are in agreement with those reported by Al-Rasheed et al., Liu et al. and Han et al.48–50 Moreover, AlSaid et al.51 and Ebaid et al.52 reported a significant up-regulation of TNF-α mRNA expression in the liver of CCl₄-intoxicated rats. The inflammatory response involved in CCl₄-induced acute liver damage was confirmed in the current study by the histological examination which showed focal aggregation of infiltrated inflammatory cells.

Pretreatment with RK (200 mg kg^–1) caused lowered NF-κB expression and reduced hepatic TNF-α levels comparable to those of the control. These results suggest that RK protects against CCl₄-induced liver damage through anti-inflammatory activity. These results are in agreement with those reported by Jeong and Jeong9 who mentioned an inhibitory effect of RK on NF-κB activation by suppressing IκB-α (inhibitor of NF-κB) phosphorylation and subsequent degradation, which could regulate inflammation. The effect of RK on inflammatory mediators may be a direct consequence of its antioxidant activity.

Apoptosis of the hepatocytes is a critical mechanism of liver injury. CCl₄ induces hepatocyte injury by triggering both necrosis and apoptosis.49,53 Apoptosis is a highly organized and controlled type of cell death which may be induced by different physiological and pathological conditions, e.g. accumulation of ROS and the activation of pro-inflammatory cytokines. Two main apoptotic pathways exist: a mitochondrion-initiated intrinsic pathway and a death receptor-triggered extrinsic one.29,53 Both pathways activate a set of caspases. The mitochondrion plays a central role in apoptosis, by releasing specific mediators which trigger apoptosis. One of the most important mediators is cytochrome C, which is a soluble protein located in the mitochondrial intermembrane space. Once in the cytoplasm, it binds to apoptotic protease activation factor-1 (Apaf-1) to complex with caspase-9; the major initiator caspase implicated in the two pathways.22 Another important activator of the apoptotic pathway is TNF-α, which induces the extrinsic pathway of apoptosis by binding to death receptors and caspase activation.20,21,44,54 Both intrinsic and extrinsic pathways finally activate caspase-3 which initiates the apoptotic response and is known as the executioner caspase.

In the present study, CCl₄ treatment induced apoptotic markers as is evident from the increased expression of cytochrome C, and enhanced hepatic levels of activated caspase-9 and 3, in addition to increased DNA fragmentation, which is a characteristic feature of apoptosis. Our results are in accordance with those reported previously.23,55 Apoptotic pathway activation can be partially explained in terms of ROS cellular effects. High levels of ROS are important for apoptosis as they disrupt intracellular Ca²⁺ homeostasis, and thus lead to ATP (adenosine triphosphate) depletion.56 Moreover, ROS-induced lipid peroxidation can alter the mitochondrial permeability and transition potential. These changes induce the release of pro-apoptotic factors (e.g., cytochrome C), and activation of the mitochondrial (intrinsic) apoptotic pathway.57,58 CCl₄-induced TNF-α activation may also contribute to apoptosis via activation of the extrinsic pathway.23,59,60 Therefore, inhibition of TNF-α synthesis or activity could attenuate liver injury induced by various insults.20,50

Cellular DNA degradation represents an important marker of cell death induced by CCl₄.23 This degradation could be attributed to the oxidative damage induced by ROS and free radical metabolites of CCl₄.61 These radicals bind to DNA covalently, resulting in the oxidation of DNA. DNA oxidation causes the formation of DNA adducts, mutations, chromosomal alterations and DNA fragmentation.60 DNA fragmentation may trigger the tumor suppressor gene p53 expression which blocks the cell cycle for DNA repair. When the damage is severe, it triggers apoptosis. The present study showed that CCl₄ induced high levels of DNA fragmentation, confirming its apoptotic effect, which is due to excessive free radicals.61 Our results are in agreement with those of previous reports.55,62

Interestingly, RK treatment counteracted CCl₄-induced apoptotic cell death, by reducing the expression of cytochrome C, and decreasing the protein levels of both caspase-9 and caspase-3. RK pretreatment maintained DNA integrity, which may be explained in terms of its antioxidant activity. These results show the antiapoptotic activity of RK, which may be explained in terms of reduced indices of apoptosis including ROS, TNF-α and DNA fragmentation.30,53,63 Our study is the first investigation reporting the antiapoptotic activity of RK.

Further confirmation of the RK protective effect was obtained by ultrastructural examination using an electron microscope. The hepatocytes were obviously abnormal with a reduced glycogen content, eccentric nucleus, and chromatin margination in CCl₄-treated rats. Chromatin margination is considered as one of the apoptotic signs. In the classical model of apoptosis, different degrees of chromatin margination are observed before the budding of the cell into small apoptotic bodies.64 In accordance with other findings of the current study, pretreatment with RK showed obvious preservation of cellular microstructures, with no chromatin margination.

Collectively, the present study reports the first investigation of the hepatoprotective activity of RK against acute liver damage caused by CCl_4. This study also demonstrated for the first time the anti-apoptotic activity of RK. RK ameliorated hepatic oxidative stress and suppressed inflammation, apoptosis, and DNA fragmentation in CCl₄-injured hepatocytes in a dose dependent manner. RK administered alone at a dose of 200 mg kg^–1 did not show any significant difference from the control group and thus gives an indication that RK is probably not hepatotoxic at this relatively high dose. Our findings suggest that RK could be used safely and effectively in protection against acute liver injury.

5. Conclusion

In conclusion, to the best of our knowledge, this is the first study to demonstrate the hepatoprotective effect of RK against liver injury induced by CCl₄. RK showed dose-dependent cytoprotective effects against liver injury induced by CCl₄. Our data suggested that RK protects the rat's liver from CCl₄-induced injury and can be considered as a potential prophylactic antioxidant agent against hepatotoxicity.

Abbreviations

ALT

Alanine transaminase

AST

Aspartate transaminase

CCl₃˙

Trichloro methane

CCl₄

Carbon tetrachloride

GSH

Reduced glutathione

LPO

Lipid peroxides

NF-κB

Nuclear factor-κB

RK

Raspberry ketone

ROS

Reactive oxygen species

SOD

Superoxide dismutase

TAC

Total antioxidant capacity

TBARS

Thiobarbituric acid reactive substance

TNF-α

Tumor necrosis factor-α

Conflicts of interest

To the best of our knowledge, no conflict of interest, financial or others, exists. The authors are fully aware of this submission. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.