the hepatoprotective effects of ganoderma lucidum peptides against carbon tetrachloride-induced...

THE HEPATOPROTECTIVE EFFECTS OF GANODERMALUCIDUM PEPTIDES AGAINST CARBON

TETRACHLORIDE-INDUCED LIVER INJURY IN MICE

HUI HE1,3, JU-PING HE1, YU-JIE SUI1, SHI-QI ZHOU2 and JIN WANG1

1College of Food Science and TechnologyHuazhong Agricultural University

Wuhan, Hubei 430070, China

2College of Animal Science and TechnologyHuazhong Agricultural University

Wuhan, Hubei 430070, China

Accepted for Publication June 23, 2007

ABSTRACT

Ganoderma lucidum, a traditional Chinese medicinal mushroom, hasbeen widely used for the treatment of chronic hepatopathy of various etiolo-gies. Our previous study showed that peptides isolated from G. lucidum (GLP)possessed potent free radical-scavenging capacities. Here, we evaluated thehepatoprotective effects of GLP, using mice with CCl4-induced liver injuries.Results showed that the serum aminotransferase (ALT/AST) activities andnumbers of necrotic and pathological hepatocytes were reduced in a dose-dependent manner in GLP-treated groups. At the dose of 260 mg/kg·bw GLP,the serum malondialdehyde, ALT and AST were statistically significant differ-ent to that of positive control groups (P < 0.05). CCl4-induced liver damagewas largely prevented as suggested by histopathological examination. In con-clusion, our study for the first time demonstrated that GLP showed preventiveeffects against CCl4-induced liver damage in mice, suggesting that GLP alsoplays a role for the hepatoprotective effects of G. lucidum extracts.

PRACTICAL APPLICATION

Liver is one of the major organs actively involved in metabolic functionsand frequently targeted by toxicants. Hepatitis is a severe disease with thehighest incidence rate around the world. Thus, interest in liver protection drugs

3 Corresponding author. TEL:+011-86-27-87280346; FAX: +011-86-27-87282966; EMAIL: [email protected]

Journal of Food Biochemistry 32 (2008) 628–641. All Rights Reserved.© 2008, The Author(s)Journal compilation © 2008, Wiley Periodicals, Inc.

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has never been higher. Ganoderma lucidum, a traditional Chinese medicinalmushroom, has been widely used for the treatment of chronic hepatopathy ofvarious etiologies with little or no side effects. A group of peptides from G.lucidum (GLP) was isolated with potent free radical-scavenging capacities andimmune system regulation ability. In general, GLP could be used as a tonic forhealth, energy and longevity. GLP could be manufactured into various forms,such as powder, tinctures, tablet, capsule and syrup. The future applications ofGLP would be mostly as a medicine, supplement or functional foods.

INTRODUCTION

Hepatitis, a high incidence disease around the world, is induced byviruses, alcohol, lipid peroxidative products and various drugs (Hwang et al.2005). The liver has great capacity to detoxicate toxic substances and tosynthesize useful principles. Therefore, damage to the liver inflicted by hepa-totoxic agents is of grave consequences (Girish et al. 2004). Unfortunately,both the environment and food are becoming increasingly polluted by hepa-totoxic agents such as pesticide and there are not many drugs available for thetreatment of liver disorders despite tremendous strides in modern medicine(Chaterrjee 2000). Carbon tetrachloride-induced liver injury in mice is one ofseveral commonly used models for the screening of hepatoprotective drugs(Janbaz and Gilani 1995). The hepatotoxicity mechanism of CCl4 had beenstudied extensively. Free radical formation, the subsequent peroxidative chainreaction, and oxidative stress have been considered as among the main hepa-totoxicity mechanisms of CCl4 (Shah et al. 1979). Potent hepatoprotectiveactivities have been shown among many antioxidative Chinese traditionalherbs, including Scutellaria radix (Hwang et al. 2005), Ginkgo biloba (Oze-nirler et al. 1997) and Ganoderma lucidum (Lin et al. 1995). Some of them arealso good functional foods.

G. lucidum, a Chinese medicinal mushroom, has been widely used for thetreatment of chronic hepatopathy of various etiologies with little or no sideeffects (Gao et al. 2003). Polysaccharide and triterpenoid components in G.lucidum have been proposed as the bioactive constituents responsible for theprotective activities against toxin-induced liver injury (Gao et al. 2003). Previ-ous studies indicated that polysaccharides of G. lucidum could effectivelyprevent ethanol-induced hepatic damage in mice (Zhou et al. 2002). Thetriterpenoids isolated from G. lucidum also showed protective effects againstliver injury induced by CCl4, D-galactosamine (D-Gal) and Bacillus Calmette-Guerin plus lipopolysaccharide in vivo (Wang et al. 2000). Ganoderenic acidA,one of the triterpenoids, was proven to be a potent inhibitor of b-glucuronidaseactivity, a good indicator of hepatic damage (Kim et al. 1999).

629HEPATOPROTECTIVE EFFECTS OF GANODERMA LUCIDUM PEPTIDES

Some bioactive peptides, defined as peptides with molecular masses lessthan 6000 Da, have been found to possess anti-hepatotoxic activities that wereinduced by toxins such as CCl4 (Guo et al. 2000; Sun et al. 2002), thioaceta-mide (Sun et al. 2002), D-Gal (Guo et al. 2000) and cyclophosphamid (Huangand Wu 2005). Bioactive peptides with anti-hepatotoxicity activities wereisolated from both plant and animal origins, such as corn and shark. Cai et al.(2001) reported that three kinds of peptides isolated from animal stomachs andintestines could prevent CCl4-induced liver injury in rats. Previously, we havereported that peptides isolated from G. lucidum (GLP) are strong antioxidantsand possess potent free radical-scavenging activities (Sun et al. 2004). Thus,we hypothesized that GLP may possess hepatoprotective effects through itsantioxidant and free radical-scavenging abilities. This hypothesis was testedhere by evaluating the hepatoprotective activities of GLP against CCl4-inducedliver injury in vivo.

MATERIALS AND METHODS

Chemicals and Reagents

Malondialdehyde (MDA) detection kits were purchased from JianchenBiological Engineering Institute (Nanjin, China). Alanine aminotransferase(ALT) and aspartate aminotransferase (AST) detection kits were purchasedfrom Huili Biological Technique Company (Changchun, China). SephadexG-25 was purchased from Sigma Chemical Co. (St. Louis, MO). D201 chelat-ing resin was purchased from Guangfu Fine Chemical Research Institute(Tianjin, China). All other chemicals used were of analytical grade.

GLP Preparation (Sun et al. 2004)

Fermented G. lucidum was purchased from Guoyao Co. (Wuhan, China).The peptides were separated from fermented G. lucidum. Briefly, G. lucidumwater extraction was separated into 70% filtrate and 30% retentate throughultrafiltration with a 10,000 Da membrane (He et al. 2001). The peptides werefurther purified from the filtrate using a gel-based ligand chromatogrammethod specifically designed for low molecular weight peptides purification(Rothenbühler et al. 1979). The peptides fraction was analyzed by HitachiL-8800 Amino Acid Analyzer (Japan). The amino acid contents of originalpeptide fractions were determined first. Then the peptides fraction was hydro-lyzed 24 h at 110C by 6 mol/L hydrochloric acid for amino acid analysis.Comparing the amino acid contents of this peptide fraction before and afterhydrolysis, the final purity of peptides fraction was calculated as 91.5%.

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Animals and Treatment (Dang and Xiao 2002)

Male mice (Qunming, 18–20 g) were purchased from Hubei LaboratoryAnimal Center (Hubei, China). The mice were housed in wire-bottomed stain-less steel cages and acclimated under laboratory conditions (25–28C, relativehumidity 65%, 12 h light/dark cycle) for 1 week. Mice were then randomlydivided into groups with 10 animals per group. Five groups were used, namelythree GLP-treated groups (Groups III, IV and V) and two control groupsincluding a negative control group (Group I) and a positive control group(Group II, CCl4-treated). Mice among Groups III, IV and V were daily giventhe doses at 65, 130 and 260 mg/kg·bw GLP, respectively, as aqueous solutionadministered via gavage tube for 2 weeks prior to the CCl4 treatment. In themeantime, control groups were given the same amount of physiological saline(0.85% NaCl) for 2 weeks. On the 15th day, mice from positive control group(Group II) were treated with a 0.1% CCl4 mineral oil solution at 5 ml/kg·bw(Xu et al. 1999), while the negative control group (Group I) was given thesame amount mineral oil. Mice from experimental groups (III, IV, V) receivedthe 0.1% CCl4 mineral oil solution at 5 ml/kg·bw 1 h after being given the GLPtreatment. Animals were then fasted for 24 h before they were sacrificed.Blood samples were collected and serum was isolated for further tests. Thelivers were removed and fixed in 10% buffered formalin saline, subsequentlydehydrated and embedded in wax. The tissue wax was cut into 3 mm sections.The fixed sections were then stained with both hematoxylin and eosin. Thehistopathological characters were observed under light microscopy.

Biochemical Determinations

AST and ALT activities and MDA level in serum were measured by thedetection kits according to the manufacturer’s protocols.

Statistical Analysis

All results are reported as mean � SD for at least 10 replicates. The datawere analyzed using one-way analysis of variance followed by Tukey’s test formultiple comparisons. P < 0.05 is regarded as statistically significant.

RESULTS

The Effect of GLP on MDA Level, ALT and AST Activities

CCl4 is activated by enzymes in liver microsomes and forms trichlorom-ethyl free radicals (CCl3) and peroxy trichloromethyl free radicals (CCl3OO).They covalently combine with biomolecules such as protein, nucleic acid and


lipid, resulting in cellular membrane degeneration, permeation increase andaminotransferase (ALT and AST) leakage. Free radicals also attack unsatur-ated lipids, which leads to lipid peroxidation. MDA is an important lipidperoxidative product that can reveal the degree of lipid peroxidation in organ-isms and, indirectly, the degree of cell degeneration. Tang et al. (2002)reported that ALT/AST activities and MDA levels were commonly usedindices for hepatotoxicity in the CCl4-induced liver damage model.

MDA level and AST and ALT activities in serum are shown in Table 1.Compared with the negative control group, the three indices in serumsamples from the positive control group significantly increased (P < 0.05),implicating the model was successful. Among GLP-treated groups, the serumAST and ALT activities were reduced in a dose-dependent manner comparedwith that of the positive control group. At the dosage of 260 mg/kg·bw GLP,MDA, AST and ALT were all statistically significantly different from thepositive control group (P < 0.05). The levels of these three indices were notstatistically significantly different when compared with the negative group,suggesting a potent protective effect of GLP against CCl4-induced liverdamage in vivo.

The Effect of GLP on Liver Histopathology

In the liver histological samples from the negative control group, thestructures of lobular, hepatic cell cords and hepatic sinusoid were intact andclear. The cell cords were arranged in neat radiant order (Fig. 1) with identicalcell sizes. The cellular nuclei and plasma could be clearly observed (Fig. 2). Incontrast, the cells focal necrosis in the center of the hepatic lobule and theturbulence of hepatic cell cords were clearly observed in positive control group(Fig. 3). Swollen hepatocytes, condensed or disappeared cellular nuclei and a

TABLE 1.THE LEVEL OF MDA AND ACTIVITIEST OF ALT/AST IN THE SERUM OF MICE

Index Group I(negativecontrol)

Group II(positivecontrol)

Group III(65 mg/kg·bwGLP-treated)

Group IV(130 mg/kg·bwGLP-treated)

Group V(260 mg/kg·bwGLP-treated)

ALT (U/L) 102.4 � 4.4d 494.1 � 20.8a 427.9 � 62.6b 159.2 � 15.2c 132.9 � 26.4cdAST (U/L) 233.7 � 19.6c 338.2 � 37.6a 330.5 � 28.7a 289.8 � 20.4b 256.8 � 35.7bcMDA (mol/L) 5.54 � 0.44c 7.69 � 0.63a 6.58 � 0.82b 6.33 � 0.60bc 5.53 � 0.76c

All values are mean � SD (n = 10).Means that do not share same subscripts in the same row differ at P < 0.05 in the Tukey honestlysignificant difference comparison.ALT, alanine aminotransferase; AST, aspartate aminotransferase; GLP, Ganoderma lucidum peptide;MDA, malondialdehyde.

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significant decrease of nuclei numbers were also observed (Fig. 4). Together,this histological evidence suggests that the model was working well, and CCl4

did induce liver injuries in the positive groups. In the center of some hepaticlobules of the sample from 65 mg/kg·bw GLP-treated group, the outlinesamong hepatocytes were obscured (Fig. 5). Hepatocyte hydropic degeneration,nuclei condensation and a large number of necrotic cells were observed(Fig. 6). At the dose of 130 mg/kg·bw GLP, numbers of the necrotic cells

FIG. 1. HISTOLOGY OF THE LIVER OF NEGATIVE CONTROL MOUSE SHOWINGHEPATIC CELLS ARCHITECTURE (hematoxylin and eosin, 25¥)

FIG. 2. HISTOLOGY OF THE LIVER OF NEGATIVE CONTROL MOUSE SHOWINGHEPATIC CELLS ARCHITECTURE (hematoxylin and eosin, 100¥)


decreased, the intensity of plasma dye decreased and hydropic degeneratedhepatocytes were seen in various shapes and sizes (Fig. 7). The cell cords werestill not clearly organized (Fig. 8). In the 260 mg/kg·bw GLP-treated group,the structures of hepatic lobules were mostly intact and cell cords were wellorganized (Fig. 9). A significant decrease of necrotic cells and ballooned cellswas evident (Fig. 10).

FIG. 3. HISTOLOGY OF THE LIVER OF POSITIVE CONTROL MOUSE SHOWING HEPATICCELLS ARCHITECTURE (hematoxylin and eosin, 25¥)

FIG. 4. HISTOLOGY OF THE LIVER OF POSITIVE CONTROL MOUSE SHOWING HEPATICCELLS ARCHITECTURE (hematoxylin and eosin, 100¥)

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DISCUSSION AND CONCLUSIONS

It is generally accepted that the hepatotoxicity of CCl4 is the result ofhighly unstable CCl3·catalyzed by the cytochrome P450 system in livermicrosome. The cleavage of CCl4 leads to the formation of CCl3·and

FIG. 5. HISTOLOGY OF THE LIVER AFTER GANODERMA LUCIDUM PEPTIDETREATMENT (65 mg/kg·bw) MOUSE SHOWING HEPATIC CELLS ARCHITECTURE

(hematoxylin and eosin, 25¥)




CCl3OO·free radicals (William and Lee 1995). These free radicals may causecellular damage by initiating lipid peroxidation through covalent bindingcombined with macro-cellular molecules (protein, nucleic acid, lipid), andthus ultimately lead to cell death (Basu 2003; Weber et al. 2003). Enzymaticactivity of serum ALT has been widely used as a sensitive index for liver





636 H. HE ET AL.

damage (Tang et al. 2002). Under healthy conditions, ALT is mostly locatedin the cellular plasma, while AST is mostly located in the mitochondria.When liver injury happened, hepatocytes membrane integrity was damagedand ALT started to permeate from the cell plasma into the blood. Only underserious liver damage conditions is AST released from mitochondria into






blood (Chen et al. 2003). Our experimental results confirmed that serumALT activity is a more sensitive index for liver damage than serum ASTactivity (Table 1). Compared with the negative control group, the serum ALTactivity has a greater amplitude than the AST activity level in the positivecontrol group. In the GLP-treated groups, serum ALT levels also had agreater decline compared with serum AST levels. At the dose of 65 mg/kg·bw GLP, the ALT level was significantly reduced (P < 0.05), while theAST level showed no significant difference compared with serum AST levelof the positive control group. Our results clearly indicated that GLP signifi-cantly decreased serum aminotransferase activities in a dose-dependentmanner.

Sun et al. (2002) reported that corn peptides reduced CCl4-inducedhepatocyte degeneration and necrosis by inhibiting hepatocyte lipid peroxi-dation and stabilizing cell membranes, thus causing a rapid recovery of liverfunctions. Shahepatide, a bioactive peptide isolated from shark liver, wasshown to exert its hepatoprotective effects through the regulation of immunesystem function (Huang and Wu 2005). Huang et al. (2004) suggested thatShahepatide could attenuate liver damage by stabilizing hepatocyte mem-branes, repairing tissue and scavenging free radicals. From our previousstudy, we proposed that the capability of GLP to reduce serum aminotrans-ferase activities is due to its potent radical-scavenging ability (Sun et al.2004), as well as its ability to prevent lecithin liposomes oxidation (He et al.2004), which protects membrane tissue against oxidation and reduces mem-brane permeability.

Our results demonstrated that 260 mg/kg·bw GLP significantly reducedserum MDA levels compared with concentrations in the positive controlgroup (P < 0.05), which is consistent with results from our previouslyin vitro experiments (Sun et al. 2004). Our previous studies showed thatGLP could inhibit MDA formation in rat liver tissue and mitochondriain vitro, and that GLP could also inhibit the swelling of rat liver mito-chondria in vitro. Our current study further demonstrated that GLP hadthe ability to reduce the MDA level in vivo. Bringing these results together,it is reasonable to suggest that GLP’s ability to prevent MDA formationis due to GLP’s potent radical-scavenging ability and low redox potential(Sun et al. 2004). Another possibility is that GLP could reduce the consump-tion of glutathione (GSH), which in turn could help inhibit oxidation of livercellular membrane.

In conclusion, GLP possesses potent protective abilities against CCl4-induced liver injury in vivo. GLP is found to be another hepatoprotectivecomponent in G. lucidum besides polysaccharide and triterpenoid compo-nents. Further studies are planned to reveal the mechanism of action of thehepatoprotective effects of GLP.

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ACKNOWLEDGMENTS

We are thankful to Dr. Heiko Schoenfuss (St. Cloud State University,MN) for his enormous help with proofreading. We gratefully thank theNational Natural Science Foundation of China for the financial support (GrantNo. 30570190).

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