The Role of Pomegranate Peel Extract in Improving Hepatotoxicity, and hMSH2 Expression in CCI4 -Treated Rats
Dalia Mostafa Mohammed Domiaty1*
1Department of Biology, College of Science, University of Jeddah, Jeddah, Saudi Arabia.
*Email: [email protected]
ABSTRACT
Recently, pomegranate fruit and its leaves have found wide usage as a natural treatment for several ailments. In this study, we investigated the restorative potentials of pomegranate (Punica granatum) peel extract against hepatic damage brought on by calcium tetrachloride (CCl4) in rats and its effects on the human mutS homolog 2 (hMSH2) gene expression. Four groups of twenty male Wistar rats (n =5) were created at random. Group I was the untreated control group. Group II was given a dose of 0.4 ml CCl4 intraperitoneally (IP) for two consecutive days/week for 3 weeks. Group III was given a daily dose (500 mg/ kg b.w) of pomegranate peel extract (PPE) for 3 weeks. Group IV received an IP of CCl4 for two consecutive days/week for 3weeks followed by a daily oral dose of (500 mg/ kg b.w) of PPE for 3 weeks. Bodyweight, relative liver weight, serum AST, ALT, bilirubin, tissue GSH, and MDA, the expression of the hMSH2 gene, liver histology, and immunohistochemistry were assessed. Our results showed that the administration of CCl4 to rats did not affect their body weight and relative liver weight. However, CCl4 administration resulted in a decrease in tissue GSH, an increase in serum AST, ALT, bilirubin, lipid peroxidation, modification of liver histology, and immunohistochemistry, and the downregulation of the expression of the hMSH2 gene. Intriguingly, PPE treatment following CCl4 administration to rats attenuated these changes. Taken together, our study reveals the potential of PPE for its use in the treatment of liver damage.
Key words: Hepatotoxicity, Antioxidant, Oxidative stress, Free radicals, Toxicant
INTRODUCTION
The liver is both the largest gland and the largest organ in the mononuclear phagocyte system of the human body [1]. It is composed of kupffer cells, sinusoidal endothelial cells, and hepatic stellate cells and is situated in the upper right region of the abdomen beneath the diaphragm [2]. The liver is essential for the body's detoxification processes, digestion, metabolic control, and maintaining the flow, and safety of compounds that are taken from the digestive system before they enter the circulatory system [1, 3].
Hepatotoxicity can be caused by agents called hepatotoxins or hepatotoxicants [2]. These agents include drugs, (anti-cancer and anti-tubercular drugs, paracetamol, and anti-cancer drugs), chemicals like galactosamine and chloroform, and oxidative species. Anti-tubercular drugs usually result in toxic hepatitis and liver necrosis which could cause encephalopathy and death [4]. Hepatotoxicity can be caused by both direct toxicities of primary compounds and by reactive metabolite/immunologically mediated response [3, 4]. Vaccines, steroids, and antiviral drugs used in liver disease therapy sometimes produce adverse effects [5]. Hepatocytes are damaged by hepatotoxicants by inducing lipid peroxidation and causing oxidative stress to the liver due to chronic treatment or toxic doses [6]. Age, gender, obesity, drug-drug interactions, kidney damage, genetics, and alcohol consumption, are risk factors for hepatotoxicity [7].
Carbon tetrachloride (CCl4) is a clear, colorless, volatile, and fireproof liquid made up of carbon and four chlorine atoms. The chemical compound occurs both naturally and artificially and has been used in the production of cleaning agents and solvents and in the synthesis of chlorofluorocarbons [8]. Due to its strong hepatotoxic, nephrotoxic, and prooxidant nature, CCl4 is used to induce hepatotoxicity in animal research studies and is used to create hepatocellular carcinoma, hepatic cirrhosis, and liver injury [9]. Inhalation, ingestion, and dermal absorption are the different routes through which CCl4 can easily enter the body [9]. Furthermore, CCl4 is a hepatotoxin that is believed to form free radicals and caused peroxidation after entering the hepatocytes which can lead to the disruption in the liver structure and function [10, 11].
One of the oldest fruits still in existence in the world is pomegranate (Punica granatum L.). It belongs to the family Punicaceae and the genus Punica. The fruit is native to the Middle East and can be found in countries like Iran, India, Afghanistan, and some other Mediterranean countries [12]. The pomegranate is a pharmacologically precious fruit and has been used numerously for cooking and medical purposes. Over the years, research studies have discovered the many health benefits of this plant (fruit, seed, and peel) [13]. The pomegranate peel extract (PPE) is a part of the fruit known to contain high levels of phytochemicals, phenolic acids, flavonoids, and tannins. These phytochemicals and compounds are responsible for the antioxidant, antimicrobial, anticancer, antiulcer, and anti-inflammatory properties of the pomegranate peel extract [14, 15]. The flavonoids, phenolic acids, and tannins present in the PPE are very important in preventing damage caused by free radicals in the human body [12].
The DNA mismatch repair pathway includes the mismatch repair gene, the human mutS homolog 2 (hMSH2) gene [16]. This gene encodes an important nuclear protein that plays an essential role in nucleotide mismatch recognition in the DNA repair pathway. However, studies have shown that mutations in the hMSH2 gene are associated with the progression in the development of liver disease including hepatocellular carcinoma [17]. Consequently, in this research, we looked at the potential advantages of PPE extract on liver injury induced by CCl4 and determined its effects on the expression levels of the hMSH2 gene.
MATERIALS AND METHODS
Plant material and preparation of extract
The dried peels of pomegranates were obtained from an herbal and folk medicine market in Jeddah, Saudi Arabia. These peels were washed, air-dried, and powdered followed by the dissolution of 10 g from this powder in 500 ml distilled water. The extract was filtered, concentrated to 8.5 mg/ml of pomegranate peel extract, and kept till usage at 4 °C.
Animals
Male Wistar rats weighing 150-250 g were purchased from the King Fahd Medical Research Center, King Abdulaziz University, Jeddah, Saudi Arabia. Animals were left to acclimatize to the lab ambiance for one week (12hr/12hr light off/on) and fed on a lab animal diet with freely available water. Declaration by Bioethics Committee of Scientific and Medical Research approved this animal experiment with No. HAP-02-J-094.
Chemicals
Carbon tetrachloride (CCl4) was purchased from Sigma-Aldrich, (Missouri, United States) and diluted in olive oil (1:1 v/v). All other chemicals were of analytical grade.
Experimental design
Rats were randomly placed into four groups (n = 5) after acclimatization and given the following treatment:
Group I (Control): the control group, received no treatment. Group II (CCl4): This group received CCl4 in olive oil (1:1) intraperitoneally (IP) at a dose of 0.4 ml for two consecutive days/week for 3weeks. Group III (PPE): Animals in this group received pomegranate peel extract (PPE) orally at a dose level of (500 mg/ kg b.w) daily/for 3 weeks. Group IV (CCl4 + PPE): This group received IP of CCl4 for two consecutive days/week for 3weeks followed by a daily oral dose of (500 mg/ kg b.w) of PPE for 3 weeks.
After the three weeks experimental period, food was withdrawn from the animals overnight and they were later euthanized under diethyl ether anesthesia. Following this, blood was drawn from the animals and the liver tissue was removed, rinsed in normal saline, and weighed. Part of the liver was either stored in 10% buffered formalin for histological analysis and immunohistochemical analysis or kept at -80˚C for extraction of RNA. The leftover liver tissue was blended in a 100 mM phosphate buffer with a pH of 7.4 at 14,000 rpm for 30 min.
Assessment of liver damage biomarkers
Using a commercial kit (Diagnostic System Laboratories Inc., USA), the serum concentrations of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and bilirubin were measured.
Assessment of oxidative stress biomarkers
For this, the supplier’s instructions were followed when measuring the amounts of glutathione (GSH) and malondialdehyde (MDA) in the supernatant collected after centrifugation at 14,000 rpm using a commercial kit (MyBioSource, California, USA).
RNA الدراسة الجزيئية:Extraction and Real-time quantitative PCR (RT-qPCR)الحامض النووي الرنا:
According to the supplier's recommendations, total RNA was extracted from the liver tissues using a (QIAgen RNeasy mini kit, cat # 74104). Next, 200ng of the extracted RNA was used in cDNA synthesis by the use of the M-MLV Reverse Transcriptase System (Promega, USA), and the qPCR reaction was made up of the following components: cDNA, 3 mL; right and left primers (Table 1), 0.5 mL (500 nM); purified water, 1 mL; SYBR Green Master Mix (Applied Biosystems, USA). To assess the relative mRNA expression of the hMSH2 gene, the 2−ΔΔCT method was applied and normalized to the expression of GAPDH.
Table 1. Primer sequences
Primers sequence (5`-3`) Primers sequence (5`-3`) |
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CYP1A1 F hMSH2 - left |
GGG AGG TTA CTG GTT CTG G 5'-TGCTCAAGGACAAAGGCTG-3' |
CYP1A1 R hMSH2 right |
5'-GGAAATCGGCGAAGTAAATC-3' 5'-GGAAATCGGCGAAGTAAATC-3' |
GAPDH F GAPDH - left |
GAT GGT GAA GGT CGG TGT G 5'-GAT GGT GAA GGT CGG TGT G-3' |
GAPDH R GAPDH -right |
ATG AAG GGG TCG TTG ATG G 5'-ATG AAG GGG TCG TTG ATG G-3' |
F – Forward, R – Reverse, GAPDH – glyceraldehydes phosphate 3-dehydrogenase
Histopathological studies
The fixation of liver tissue was carried out in 10% buffered formalin, then embedded in paraffin wax after being dried out graded ethanol for a day at room temperature. To evaluate histopathological alterations, hematoxylin, and eosin (H&E) were used to stain the sections of tissue blocks that were cut into thin sections. Light microscope images of stained liver sections were taken at 400x magnification.
Immunohistochemistry analysis
Immunohistochemical analysis was done for anti- CK7 and CK19 antibodies using streptavidin-biotin. The liver sections of a thickness of 5 μm and at room temperature were deparaffinized followed by incubation in hydrogen peroxide (0.3%) prepared in methanol for half an hour. The liver sections were incubated with anti- CK7 and CK19 antibodies at a dilution of 1:100 respectively followed by counterstaining with hematoxylin and eosin.
Statistical analysis
The statistical analyses for this study were conducted using one-way ANOVA, and the data are presented as mean SEM. Dunnett's multiple comparisons test was used to compare means, and a significance level of p less than 0.05 was chosen.
RESULTS AND DISCUSSION
Body weight and relative liver weight alterations induced by CCl4 administration
The administration of CCl4 to rats has no significant effect on the body weight of rats compared to animals in the control group (Figure 1a). On the other hand, when compared to rats given CCl4, PPE treatment to the rats resulted in a significant (p < 0.05) drop in body weight. In addition, rats treated with PPE after CCl4 administration saw a significant (p < 0.05) reduction in body weight when compared to animals just given only CCl4 (Figure 1a). Furthermore, CCl4 administration to rats resulted in liver weight increased by 9% and 7% in comparison with the PPE-treated group and the control group respectively. Moreover, rats treated with PPE after receiving CCl4 showed a significant reduction in relative liver weight compared to those who received only CCl4 (Figure 1b).
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Figure 1. Body weight and relative liver weight alterations induced by CCl4 administration. a) final body weight. b) relative liver weight |
Protective effects of PPE against liver damage induced by CCl4
To assess the effect of CCl4 on the health status of the liver, we quantified the serum AST and ALT levels, two biomarkers of liver damage. As shown in Figure 2, CCl4 administration to rats, led to a significant increase in the serum AST and ALT levels in comparison to the control (p < 0.0001 for AST and p < 0.01 for ALT) and PPE treated groups (p < 0.001 for AST and p < 0.05 for ALT) respectively. However, PPE administration to rats following CCl4 led to a significant decrease (p < 0.001) in serum AST levels when compared with rats administered only with CCl4. Similarly, rats treated with PPE after receiving CCl4 revealed a significant reduction in the serum levels of ALT in comparison with CCl4-only administered rats (Figure 2b).
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Figure 2. Effects of CCl4 on serum levels of AST and ALT. a) Serum AST level. b) Serum ALT level. |
Antioxidant effects of PPE against CCl4-induced oxidative stress
When compared to the animals in the control group, treatment with CCl4 caused a significant (p < 0.001) drop in the amount of GSH present in the liver tissue. However, when compared to animals given CCl4, rats given PPE saw a considerable rise (p < 0.01) in the GSH content (Figure 3a). In addition, the GSH contents showed no discernible difference from the rats treated with PPE following CCl4 administration and CCl4-only administered rats. Furthermore, CCl4 administration to rats resulted in a significant elevation (p < 0.0001) in the liver MDA content in comparison to the rats in the control group, and the PPE-only administered to rats (Figure 3b). However, PPE treatment to rats after CCl4 administration resulted in non-significant changes in the MDA contents in comparison with rats administered with only CCl4 (Figure 3b).
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Figure 3. Effects of CCl4 and PPE on GSH and MDA levels. a) Serum glutathione (GSH) level. b) Serum malondialdehyde (MDA) level. |
Effects of CCl4 and PPE on serum bilirubin levels and hMSH2 gene expression
Rats administered with CCl4 saw a substantial rise (p < 0.05) in blood bilirubin levels when compared to the control group and rats given PPE, respectively. Interestingly, rats given PPE following CCl4 administration showed a significant reduction in the bilirubin level when compared to the rats in the CCl4-only group. This decrease in serum bilirubin by PPE treatment to the CCl4 administered rats was near normal (Figure 4a). Furthermore, the hMSH2 gene expression levels between the control group and the CCl4-treated group did not differ significantly. Although PPE administration to rats, upregulated the expression of the hMSH2 gene in comparison to the CCl4-only treated group, this does not reach a significant level (Figure 4b). Interestingly, rats receiving both CCl4 and PPE treatment demonstrated significantly higher levels of hMSH2 gene expression (p < 0.01) as compared to rats receiving CCl4 only.
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Figure 4. Effects of CCl4 and PPE on serum bilirubin levels and hMSH2 gene expression. a) serum testosterone level. b) Relative hMSH2 gene expression. |
PPE administration improved liver histology in CCl4-induced liver-injured rats
Hematoxylin and eosin staining revealed that the liver sections from the animals in the control group presented a normal liver histological architecture with normal hepatic lobules, central vein, visible hepatic sinusoids, and hepatic cells (Figure 5a). However, rats administered with CCl4 presented a visible liver injury with an altered liver architecture, and an increased necrotic infiltration (Figure 5b). In addition, rats administered with PPE only showed similar liver architecture when compared to the liver sections from the animals in the control group (Figure 5c). Furthermore, the liver sections from the animals who received treated CCl4 treatment followed by the administration of PPE revealed an improved liver architecture with visibly radiating hepatic cells as compared with the liver sections from CCl4-only administered rats (Figure 5d).
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