The economic cost of and liver and kidney diseases in Sub Saharan Africa remain prohibitive, hence the quest for medicinal foods that can reverse hepato-renal damages. The aim of this study is to investigate the hepato-renal protective effect of wild edible P.tuber-regium on carbon tetrachloride (CCl4) induced oxidative stress in male Sprague–Dawley rats.
Method
Thirty six rats were divided into six groups of six animals each. Group I (negative control) received 10 ml/kg olive oil intraperitoneal weekly in addition to feed and water ad libitum. Group II (positive control) received CCl4 10 ml/Kg (30% in Olive oil) weekly. Group III, IV and V received 100 mg, 200 mg and 500 mg wild edible P.tuber-regium (33.3% in feed) daily in addition to 10 ml/Kg CCl4 weekly. Group VI received 500 mg P.tuber-regium (33.3% in feed) daily. After four weeks, the serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatinine, bilirubin, urea and fasting blood glucose were determined. Also, the histopathologic examinations of the kidney and liver were carried out.
Results
Administration of CCl4 to rats significantly (p < 0.05) decreased the levels of Bilirubin (1.21 ± 0.07), Creatinine (0.91 ± 0.04) and Urea (45.76 ± 3.10 mg/dl) when compared to control (0.66 ± 0.07, 0.76 ± 0.05 and 24.48 ± 4.70 respectively). Liver and kidney MDA from 14.00 ± 2.60 and 14.00 ± 3.50 in CCl4 only treated groups to 8.60 ± 1.5 and 1.70 ± 0.15 μmol/mg in the 500 mg P.tuber-regium treated groups. Photomicrographs also showed that P.tuber-regium prevented the fibrosis of the bile duct, glomeruli and tubules seen in CCl4 group.
Conclusion
P.tuber-regium may be protective against the CCl4 induced oxidative damage of the hepato-renal system.
Background
Despite recent developments in medical sciences, treatment of diseases relating to liver and kidney is very expensive, relatively unavailable with high incidence of adverse effects and failure [1]. This spurs the need for a more efficient, cost effective and easily available regime to combat liver and kidney pathologies especially in the developing nations. Natural products with their secondary metabolites may be efficacious and possess antioxidant potentials that scavenge free radicals and reduce oxidative stress [2]. Edible mushrooms had been used in the treatment of different ailments in folk medicine and possesses secondary metabolites that could treat or slow down the pathogenesis of diseases like liver and kidney pathologies [3, 4].
Pleurotus tuber-regium a fungus that grows on drill dust of the wood Treculia Africana (bread fruit), Elaeis guineensis, or Daniella oliver tree. It belongs to the genus basidiomycete, and is found mainly in the tropics and subtropics. It forms a large spherical to ovoid sclerptia (underground tuber) [5]. Pleurotus tuber-regium is a popular edible mushroom found in decaying vegetation [6] that had been attributed with many medicinal properties like anticancer, anti-inflammatory, antihypertensive, antilipidemic and antidiabetic [6‐8]. Edible mushrooms are known to contain several secondary metabolites like phenolic, tannins, alkaloids, and flavonoids etc. that account for their diverse pharmacological activities and in some instance their antioxidant properties. Antioxidants in mushrooms are known to combat free radical mediated injuries via its unique ability to donate electrons and therefore stabilize highly reactive free radicals [9].
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The aim of this study is to evaluate the hepato-renal protective activity of P.tuber-regium in a free radical model using rat.
Methods
Harvesting of the mushroom
Fresh fruiting bodies of wild P.tuber-regium were collected from a forest at the back of University of Nigeria Nsukka by a taxonomist working with the university. These fresh fruiting bodies were cleaned and air dried away from direct sunlight. The mushroom were ground and stored in a clean dry plastic container until use [10]. Little is known about bacterial contamination of the Nigerian harvested P.tuber-regium. P.tuber-regium were not tested for micriobiological contaminations
Animals husbandry
Thirty six male Sprague–Dawley rats with body weights 180–200 g acclimatized for two weeks were maintained under controlled conditions of temperature (23 ± 2 °C) and humidity (50 ± 5%) and a 12-h light–dark cycle, were used for the experiment. The animals were housed in sanitized polypropylene cages containing sterile paddy husk as bedding. The bedding of the cages was changed daily and the cages were cleaned as well. They had free access to standard rat
pellet diet and water ad libitum. All the experimental procedures were performed according to the committee for the purpose of control and supervision of experiments on animals, norms and approved by the University of Port Harcourt Animal Ethical Committee with an approval number UNIPORT/PHARM/0133.
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Carbon tetrachloride (CCl4)
Thirty percent carbon tetrachloride (Sigma Aldrich) in Olive oil [11] was used to induce renal and hepatic damage at a dose of 10 ml/kg (i.p) [12].
Acute toxicity studies
Different concentrations of P.tuber-regium (50–5000 mg/kg body weight b.w.) were administered orally to male rats. These animals were observed daily for toxicological manifestations like behavioral changes, neural and autonomic toxicities, feeding pattern changes etc. There was no mortality recorded during this period even up to the dose of 5000 mg/kg [13].
Experimental design
The animals were divided into six groups with each group consisting of six animals each. The administration of CCl4 10 ml/kg body weight of 30% CCl4 in olive oil was given on days 0, 7, 14, and 21 concomitantly with the daily feeding of the mushroom.
The animals were treated for 4 weeks as follows
Group I – normal control received olive oil 10 ml/kg i.p. weekly in addition to standard food and water.
Group II – Positive control received CCl4 (30% CCl4 in olive oil) at a dose of 10 ml/kg weekly in addition to standard feed and water.
Group III – rats were treated orally with 100 mg/kg b.w. of P.tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.
Group IV – rats were treated orally with 200 mg/Kg b.w. of P.tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly.
Group V – rats were treated orally with 500 mg/kg b.w. of P.tuber-regium in feed (33.3% w/w) along with 10 ml/kg CCl4 (30% in olive oil) weekly. In all CCl4 was given intraperitoneally.
Group VI – rats were treated orally with 500 mg/kg b.w. of P.tuber-regium in feed (33.3% w/w) only with standard feed and water.
Necropsy
Animals were sacrificed 24 h after the last treatment. Blood was collected by retero orbital sinus puncture and serum was separated by centrifugation at 3000 r.p.m for 10mins at 4 °C for assay of biochemical parameters. Rats were sacrificed under ether anesthesia; liver and kidney were excised, rinsed clean in saline, weighed and preserved in 10% formalin for histopathological study.
Determination of biochemical parameters
Commercial reagent kits for the determination of bilirubin, cholesterol, creatinine and urea concentrations and alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), were assayed using Quimica Clinica Aplicada (QCA) Spain. Fasting blood glucose was determined using commercial glucometer made by accu-chek.
Antioxidant assay
The liver and kidney were minced separately into small pieces and homogenzed with ice cold 0.05 M potassium phosphate buffer (pH 7.4) to make 10% homogenates. The homogenates were centrifuged at 6000 rpm for 15 mins at 4 °C. The supernatant was collected for the estimation superoxide dismutate (SOD) and malondialdehyde (MDA) assays. Superoxide dismutate (SOD) was assayed by the method described by Misra and Fridovich [14]. Lipid peroxidation was quantified as malondialdehyde (MDA) according to the method described by Ohkawa et al. [15]. and the MDA level was calculated according to the method of Todorova et al. [16] and expressed as μmol MDA/mg protein.
Histopathological studies
Portions of the liver and kidney from all the experimental groups were fixed in 10% formaldehyde, dehydrated in graded alcohol, cleared in xylene and then embedded in paraffin. Microtome sections (5 μm thick) were prepared from each liver and kidney sample and stained with heamtoxylin-eosin (H&E) dye. The sections were examined for the pathological findings.
Statistical analysis
Data obtained were analyzed using graph pad prism 5. The values represent means and their standard deviations. Differences between the means were determined using one way analysis of variance (one way ANOVA) followed by Bonferroni’s test. P values of 0.05 or less were considered statistically significant.
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Results
Effect of P.tuber-regium on the liver weight of CCl4 treated animals
Figures 1 and 2, shows the effect of the P.tuber-regium on the weights of the liver and kidney after administration of CCl4. Administration of CCl4 to the rats resulted in a significant (p < 0.05) increase in the absolute (0.77 ± 0.04) and relative weight (0.40 ± 0.04 g) of the kidneys in all the treatment groups when compared to control (absolute 0.63 ± 0.03 and relative 0.34 ± 0.01 g). Carbon tetrachloride significantly (p < 0.05) increased in both the absolute (10.94 ± 0.41) and relative weights (5.70 ± 0.21) of the liver in all the treated groups when compared to the control. There was a significant dose – dependent reduction in the weights of the liver and kidney in the rats that received CCl4 and P.tuber-regium.
Fig. 1
Effect of Pleurotus tuber-regium treatment on the average kidney weight of CCl4 administered rats
Fig. 2
Effect of Pleurotus tuber-regium treatment on the liver weight of CCl4 administered rats
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The effect of P.tuber-regium on the Liver function tests: ALT, AST and ALP are shown on Table 1. Administration of CCl4 significantly (p < 0.05) increased the liver enzymes ALT (42.82 ± 2.70), AST (92.97 ± 6.34) and ALP (421.10 ± 12.89 iu/L) in all the treated rats when compared to control rats 26.85 ± 2.63 . 75.68 ± 5.20 and 293.10 ± 15.15 iu/L respectively. In the CCl4 plus P.tuber-regium treated groups 200 and 500 mg P.tuber-regium tended to significantly reverse the effects of CCl4 on the ALT, AST and ALP in dose dependent fashion. Administration of P.tuber-regium at 500 mg only without CCl4 showed no significant (p < 0.05) difference from the control.
Table 1
Effect of P.tuber-regium on ALT, AST and ALP
Treatment
ALT (iu/L)
AST (iu/L)
ALP (iu/L)
Control
26.85 ± 2.63
75.68 ± 5.20
293.10 ± 15.15
CCl4 Only
42.82 ± 2.70 ***
92.97 ± 6.34 **
421.10 ± 12.89 ***
CCl4 + 100 mg P.t.
34.67 ± 1.34
83.98 ± 5.18
352.90 ± 15.44
CCl4 + 200 mg P.t.
29.82 ± 8.34 ##
78.47 ± 4.53 ##
331.60 ± 12.76 #
CCl4 + 500 mg P.t.
30.74 ± 5.53 ##
78.09 ± 8.23 ##
336.70 ± 16.22 #
500 mg P.t. Only
26.64 ± 2.45 ###
75.74 ± 4.68 ###
263.00 ± 13.92 ###
Each value represents mean ± SD, n = 6. Values marked with an asterisk (*) differ significantly from control value (** p < 0.01, *** p < 0.001) while those marked with # differ significantly from CCl4 only group (#p < 0.05, ##p < 0.01, ###p < 0.001)
Effect of P.tuber-regium on the Kidney function tests
Administration of CCl4 to rats significantly (p < 0.05) decreased the serum levels of Bilirubin (1.21 ± 0.07), Creatinine (0.91 ± 0.04) and Urea (45.76 ± 3.10 mg/dl) when compared to control (0.66 ± 0.07, 0.76 ± 0.05 and 24.48 ± 4.70 respectively) (Table 2). Also, fasting blood glucose was increased significantly (p < 0.05) when compared to control from 75.67 ± 2.94 to 90.60 ± 4.62 mg/dl. Treatment with P.tuber-regium restored the levels of these markers in a dose dependent manner when compared to the control group. However, treatment with P.tuber-regium alone did not cause any significant (p < 0.05) change in the markers studied as compared to the control group.
Table 2
Effect of P.tuber-regium on bilirubin, creatinine urea and fasting blood glucose
Treatment
Bilirubin
Creatinine
Urea
Fasting Blood Glucose
(mg/dL)
(mg/dL)
(mg/dL)
(mg/dL)
Control
0.66 ± 0.07
0.76 ± 0.05
24.48 ± 4.70
75.67 ± 2.94
CCl4 Only
1.21 ± 0.07 **
0.91 ± 0.04 **
45.76 ± 3.10 ***
90.60 ± 4.62 ***
CCl4 + 100 mg P.t.
0.99 ± 0.04
0.82 ± 0.07
35.47 ± 11.68
79.25 ± 8.30 #
CCl4 + 200 mg P.t.
0.78 ± 0.02 #
0.79 ± 0.03 #
27.31 ± 7.13 ##
76.50 ± 3.79 ##
CCl4 + 500 mg P.t.
0.73 ± 0.09 #
0.76 ± 0.05 ##
27.88 ± 6.44 ##
76.25 ± 4.35 ##
500 mg P.t. Only
0.66 ± 0.09 ##
0.75 ± 0.06 ##
24.25 ± 4.73 ###
75.00 ± 3.92 ##
Each value represents mean ± SD, n = 6. Values marked with an asterisk (*) differ significantly from control value (** p < 0.01, *** p < 0.001) while those marked with # differ significantly from CCl4 only group (#p < 0.05, ##p < 0.01, ###p < 0.001)
The effect of P.tuber-regium on the superoxide dismutase (SOD) and malondialdehyde
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(MDA) in liver and kidney is shown on Table 3. There was significant increase in the MDA of kidney 1.40 ± 0.32(control) and 14.00 ± 3.50 (CCl4) and liver 1.90 ± 0.42(control) and 14.00 ± 2.60 (CCl4) treated groups. The superoxide dismutase (SOD) in the liver and kidney were also significantly decreased from 7.58 ± 0.59 and 8.49 ± 0.38 in control to 1.65 ± 0.48 and 3.18 ± 0.31 μg/mg protein after CCl4 administration. P.tuber-regium administration decreased the level of MDA and increased the level of SOD in the CCl4 treated groups significantly in dose dependent manner.
Table 3
Effect of P.tuber-regium on the superoxide dismutate (SOD) and malondialdehyde (MDA) in liver and kidney
Groups
Control
CCL4 Only
CCL4 + 100 mg P.t.
CCL4 + 200 mg P.t.
CCL4 + 500 mg P.t.
MDA (μmol/mg)
Kidney
1.40 ± 0.32
14.00 ± 3.50 ***
3.50 ± 0.61 ###
1.60 ± 0.36 ***
1.70 ± 0.15 ###
Liver
1.90 ± 0.42
14.00 ± 2.60 ***
10.00 ± 1.80 ***
8.90 ± 1.00 ** #
8.60 ± 1.5 ** ##
SOD (μg/mg protein)
Kidney
8.49 ± 0.38
2.78 ± 0.30 ***
3.18 ± 0.31 ***
5.31 ± 0.37 ***###
6.48 ± 0.50 *** ###
Liver
7.58 ± 0.59
1.48 ± 0.33 ***
1.65 ± 0.48 ***
3.60 ± 0.36 *** ###
3.88 ± 0.47 *** ###
Each value represents mean ± SD, n = 6. Values marked with an asterisk (*) differ significantly from control value (** p < 0.01, *** p < 0.001) while those marked with # differ significantly from CCl4 only group (#p < 0.05, ##p < 0.01, ###p < 0.001)
Histological studies of the liver
The histological architecture of the liver in control rats showed normal and well preserved structures with the hepatocytes well arranged around a central vein with alternations of blood sinusoids. The liver cells show a membrane, a nucleus and nucleoli (Fig. 3a). Examination of the hepatocytes of CCl4 treated rats show proliferation of bile duct epithelium with dilated and congested sinusoids (Fig. 3b). Treatment with P.tuber-regium reversed these changes (Fig. 3c, d and e) with the reversal being more pronounced in the higher dose of 500 mg P.tuber-regium. Examination of P.tuber-regium only group shows a well preserved and normal hepatic cells comparable the control (Fig. 3f).
Fig. 3
Histopathology showing the alterations induced with CCl4 and preventive effects of P.tuber-regium in hepatic tissues. a Hepatic section showing the normal architecture of the liver (b) CCl4 induced injuries with severe proliferation of bile ducts and congested sinusoids (c, d and e) CCl4 induced injuries and protective effect of P.tuber-regium in a dose dependent manner (f) A well preserved architecture of the hepatocytes comparable to the control
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Histological studies of the kidney
The histological studies of the kidney showed normal well preserved architecture of the kidney with normal glomeruli and tubules (Fig. 4a). Administration of CCl4 markedly disrupted the normal architecture of the kidney cell by degenerating the tubules and collapsing the glomerulus (Fig. 4b). Treatment with P.tuber-regium improved these pathological changes in a dose dependent manner (Fig. 4c, d and e). Slides of the 500 mg P.tuber-regium only group showed well preserved architecture comparable to the control (Fig. 4f).
Fig. 4
Photomicrograph showing the alterations induced with CCl4 and preventive effects of P.tuber-regium in nephrotic tissues. a Normal structure of nephrotic tissue showing well preserved glomerulus and tubules. b Nephrotic alterations induced with CCl4 with pronounced tubules and glomeruli due to fibrosis (f). c, d and e alterations in the nephrotic tissue following CCl4 intoxication and the protective effect of Pleurotus tuber-regium in a dose dependent manner. f A well preserved nephron comparable to the control
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Discussion
Carbon tetrachloride is a reference toxicant in mammalian systems with established toxicities in the kidney, liver, testis, heart, lungs etc. [17, 18]. Once injected into a mammalian system, it undergoes extensive biotransformation in the livers P-450 system to generate trichloromethyl radical (CCl3°) and chloride radical Cl° which being electron deficient, have very high affinity for electrons in biological tissues. This extortion of electrons from biological systems leads to peroxidation of proteins, distortion of enzymes and DNA [19]. In this study, we tested the hypothesis that wild edible mushrooms like P.tuber-regium could protect against CCl4 induced hepato-renal toxicity.
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Administration of CCl4 resulted in significant (p < 0.05) increases in both the absolute and relative weights of the liver and kidney when compared with the control. This increase in liver and kidney weights may be attributed to lesions and injuries associated with xenobiotics [2] like CCl4 which peroxidizes cell proteins thereby activating the inflammatory pathway. This is evident from the photomicrographs of both the kidneys and liver that showed proliferation of the bile ducts, tubules and the glomeruli. The enlargement of the liver and kidney were significantly reduced in the treatment groups that received P.tuber-regium suggesting that the mushroom contains some protective phytomedicinals. This result is in agreement with other works on mushrooms in a free radical mediated injury where mushroom reduced oxidative stress mediated injury by gentamicin [2].
The treatment of rats with CCl4 caused a significant increase in the serum levels of liver and kidney biomarkers like Bilirubin, Creatinin, Urea, Fasting blood glucose FBG, ALT, AST and ALP. The implication of these observations is possible hepato-renal dysfunction as a result of the free radicals; trichloromethyl radical (CCl3°), trichloromethylperoxy radical (OOCCl3°), chloride radical (Cl°) etc. generated by the CCl4 following biotransformation by CYP 2E1 of the liver [20]. These radicals being electron deficient react with electron rich proteins to cause peroxidation of macromolecules like the membranes with subsequent leakage of the enzymes into the serum [21]. Some of these leaked enzymes found in the serum like ALT, AST and ALP are biomarkers of liver that is elevated during hepatotoxicity. This result is agreement with other works that had recorded elevated serum levels of liver markers following CCl4 intoxication [22]. These elevated biomarkers of liver toxicity were markedly reduced by the administration of P.tuber-regium in a dose dependent manner. P.tuber-regium is known to be rich in antioxidant molecules like phenolics, tannins, flavonoids [22] which are efficient electron donor thereby helping to terminate free radical reaction. This process spares the body’s antioxidant systems and macromolecules which are the primary targets of peroxidation. The protective effect in the liver is also seen in the photomicrographs with well-preserved structures comparable to untreated control group.
The protective activity of P.tuber-regium seen in the liver was also seen in the kidney. The major renal biomarkers suggested significant renoprotection in a dose dependent manner after administration P.tuber-regium, which may be due to anti- oxidative stress mechanism [23]. The significant increase in SOD and decrease in MDA following P.tuber-regium administration in CCl4 treated animals lend credence to the antioxidant potential of this mushroom and its folkloric use. The oxidation of membrane lipid bilayer affected lipid structures of the nephron which resulted in a subcellular structural damage as revealed by the micrographs of the kidney (tubular and glomerular fibrosis). These finding are similar to the protection observed with Digera muricata where the structural changes observed in CCl4 group were attenuated [24]. This hepato-renal protective effectof P.tuber-regium may be due to the presence of antioxidant phytochemicals that may not only attenuate the toxicity of CCl4 but may have restorative properties.
The histopathological findings have provided direct evidence of the possibility of the P.tuber-regium to attenuate the disruption of structure of both the liver and kidney cells. These observations complemented the results of the cytoplasmic enzymes namely (alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and bilirubin, creatinine and urea concentrations. A direct radical-scavenging activity of the P.tuber-regium might be involved in the hepato- and nephron- protective activity against CCl4 exposure.
Conclusion
Taken together it may be concluded that the P.tuber-regium is able to combat the effect of CCl4 exposure in albino rats, which might be attributed to its antioxidant defense mechanism.
Acknowledgements
Authors’ contributions
KOO: Designed study, carried out the bench work and analysed data. IMS: Design. OEO: Designed the study, analysed data and write up. All authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Source of support
Nill.
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