Effects of carvacrol upon the liver of rats undergoing partial hepatectomy.

Introduction

When liver lobs are surgically excised or damaged due to various reasons, a quick regeneration occurs in the liver. The regeneration will last until the liver reaches its own original mass. Researches concerning regular liver growth are about partial hepatectomy and chemical damages incurred. Experimental partial hepatectomy is applied by excising 67-70% of the liver in rats (Borowiak et al., 2004). The existence of cytokines and growth factors, autocrine, paracrine and endocrine interactions are crucial as well during trigger and stoppage of liver regeneration (Tang et al., 1997; Ankoma-Sey, 1999; Andiran et al., 2000). Cytokines are important mitogen and antiapoptotic factors stimulating hepatic regeneration following interleukin-6 (IL-6) partial hepatectomy (Fausto, 2000; Galun and Axelrod, 2002). IL-6 is activated by the activation of tumour necrosis factor-alpha (TNF-[alpha]). TNF-[alpha] is a proinflammatory cytokine generated by Kuppfer and endothelium cells during liver regeneration. TNF-[alpha] stimulates the proliferation of hepatocytes, and affects the activation of bcl-2, bax, bcl-x genes (Galun and Axelrod, 2002). The determination of IL-6 and TNF-[alpha] amounts gives information relating to the state of regeneration occurring after partial hepatectomy.

Volatile oils are being used by people for some respiratory, digestive and excretory system disorders. Carvacrol obtained from Origanum onites L. (Lamiaceae) plant is a monoterpenoid having the chemical name 2-methyl-5-(1-methylethyl)phenol. Volatile oils also have antiaging, antibacterial, antifungal and antiseptic properties (Baser, 2002). Therefore, our study presents the effects of carvacrol on liver regeneration of rats which have undergone partial hepatectomy by performing mitotic index, IL-6 and TNF-[alpha] measures.

Materials and methods

Plant extract

The plant material examined in this study, carvacrol (2-methyl-5- (1-methyl ethyl) phenol), was isolated from steam-distilled essential oil of Origanum onites L. collected from West Anatolia. A fractional distillation was performed using a lab-size glass fractional distillation unit containing column packed with S/S Knit Mesh packing material (2.8cm-1.35m). Reflux ratio was adjusted at 10/1 to 20/1 and the medium pressure was 8-10 mmHg. Carvacrol-rich fractions were bulked to obtain carvacrol with 99% purity (GCMS).

Animals

In this study male Wistar Albino rats, weighing 230 [+ or -] 30 g, were used after 2 weeks of adaptation. They were housed in polycarbonate cages in an air-conditioned room (12L/12D, 22 [+ or -] 2 [degrees]C, 50 [+ or -] 5% humidity). Water and food supplied ad libitum.

The experimental design and procedures were approved by the Institutional Ethical Committee for Animal Care and Use at the Eskisehir Osmangazi University, Eskisehir, Turkey.

Experimental protocols

The rats were randomly divided into three groups, each containing eight animals.

Group I: Sham operation.

Group II: Hepatectomized (the animals were subjected to 68% partial hepatectomy and received 0.5 mL saline solution intraperitonally).

Group III: Hepatectomized and 73 mg/kg carvacrol treated (MSDS; NIOSH).

One dose of the test material was injected to Group III 1 h before (about 68% removal of the liver) partial hepatectomy. Carvacrol in aqueous suspension was administered intraperitonally to rats.

After 72 h, partial hepatectomy of rats of the all groups were done under thiopental sodium (50mg/kg) anaesthesia (Tang et al., 1997) and the blood was collected intracardiacally. Then livers were immediately removed for the assessment of liver regeneration. The liver regeneration rate of rats was calculated measuring the weights of the liver (Tang et al., 1997). Aspartate transaminase (AST), alanine transaminase (ALT), TNF-[alpha] (ELISA) and IL-6 levels (ELISA) were also determined in serum samples. Livers were stained for mitotic index with haematoxylin and eosin (H & E), proliferating cell nuclear antigen (PCNA) and IL-6.

Immunohistochemical staining

Liver cross-sections were deparaffinized, treated with gradual alcohol series and the dehydration of cross-sections was provided for IL-6 monoclonal antibody (IL-6 M-19 sc-1265, Santa Cruz Biotechnology) and PCNA mouse monoclonal antibody (NeoMarkers, Ab-1 Clone PC10) immunohistochemical staining. PCNA labeling index was issued for determining the proliferative activity. Hepatocytes with particles of brown pigment over their nuclei were considered labeled. A ratio of the number of PCNA-positive hepatocytes to the total number of hepatocytes was counted. The mitosis rate was determined by random evaluation of at least 1000 hepatocytes and was expressed as a percentage (number of cells with mitosis/total hepatocytes x 100) (Hou et al., 2003).

Statistical analysis

The results were expressed as the mean [+ or -] SD of eight animals per group. One-way analysis of variance (ANOVA) and Tukey test were used for the analysis and comparison of data within and between groups. Mann--Whitney U tests were used only for nonparametric IL-6 results. Differences were considered significant at p<0.05.

Results

Liver histology

In H & E-stained sections, in Group I fewer mitosis and normal histologic structure were seen in the liver. In Group II, mitosis and vacuolization were prevalent in hepatocytes. Sinusoidal congestion was evident, but neutrophils were fewer. Similarly in Group III mitosis was prevalent in hepatocytes, but vacuolization reduced.

Hepatocyte proliferative activity

Fig. 1 and Table 1 summarizes the liver regeneration rate, mitotic index and PCNA index of the hepatocytes of various groups of animals.

Biochemistry

In AST, ALT, TNF-[alpha]. IL-6 levels there were no differences among the groups.

Discussion

The complex mediator network such as cytokines and growth factors plays a role in liver regeneration in the case of substantial tissue losses (Higgins and Anderson, 1931; Hou et al., 2003). It is required to measure AST, ALT, alkaline phosphatase (ALP) enzymes (El-Ashmawy et al., 2005) and TNF-[alpha] and IL-6 cytokines (Scotte et al., 1997; Iwai et al., 2001) in the serum and to derive the mitotic index of hepatocytes (Furuta et al., 2000; Palmes and Spiegel, 2004) in order to evaluate hepatocyte functions, and the results give information about the state of regeneration. Sixty-eight percent of the right and left liver lobs of animals were removed during the partial hepatectomy administered in our research. AST. ALT, ALP enzyme values were periodically examined during the partial hepatectomy. While AST and ALT values were initially high in Groups II and III, which were applied hepatectomy, they reached a normal level at the 72nd hour and livers improved. Some other researches using various hepatectomy models also indicated that, similar to our results, AST and ALT enzyme values at the 72nd hour following partial hepatectomy were similar to the control groups (Furuta et al., 2000; Aoki et al., 2001). These results were considered significant in terms of carvacrol having no adverse effect on liver functions.

[FIGURE 1 OMITTED]

The histological examination of regenerated liver sections revealed that vacuolization was only prevalent in hepatocytes of animals, which were applied only hepatectomy in Group II. Parallel to our findings, prevalent vacuolization was observed in hepatocytes following hepatectomy in a research. Researchers also indicated that vacuolization had lipid accumulation and that they prevented vacuolization by means of the organic compound they used (Hou et al., 2003). Our findings indicated that vacuolization was significantly decreased in groups that were treated with carvacrol before the partial hepatectomy. Thus, we think that the protective effects of carvacrol on livers are important following hepatectomy.

TNF-[alpha] and IL-6 cytokines play an important role in liver regeneration, It was indicated in some researches that while serum levels of TNF-[alpha] and IL-6 cytokines were at their highest values at the 24th or 48th hour following hepatectomy, they reached normal values at the 72nd hour (Scotte et al., 1997; Iwai et al., 2001). DNA synthesis in hepatocytes of both normal and regenerated livers increased in animals administered exogenous TNF-[alpha] (Hou et al., 2003). However, such an application in aged animals caused formation of hepatic adenoma and carcinomas (Kay and Fausto, 1997). Our findings indicated that serum values of TNF-[alpha] and IL-6 cytokines in Groups II and III are similar to the sham group at the 72nd hour following hepatectomy. Mitotic and PCNA indexes are very important and they are frequently used in researches about liver regeneration (Assy et al., 1998). Mitotic and PCNA indexes were found to be high in Groups II and III in our research. We think that carvacrol stimulates the mitosis of hepatocytes following hepatectomy.

It is important to note that carvacrol lacks genotoxic and mutagenic effects, it has antibiotic features and it protects the liver and increases the regeneration rate. Thus, we are in the opinion that carvacrol may be used as an alternative to drugs following new molecular studies to be carried out with this substance.

Acknowledgments

The study was supported by Eskisehir Osmangazi University Scientific Research Projects Committee (Project number: 200419025).

References

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Mustafa Uyanoglu (a,*), Mediha Canbek (a), Erinc Aral (b), K. Husnu Can Baser (c)

(a) Faculty of Science, Department of Biology, Eskisehir Osmangazi University, 26480, Eskisehir, Turkey

(b) Medicine Faculty, Department of Histology, Eskisehir Osmangazi University, 26480, Eskisehir, Turkey

(c) Faculty of Pharmacy, Department of Pharmacognosy, Anadolu University, 26470, Eskisehir, Turkey

Received 2 March 2007; accepted 18 June 2007

Abbreviations: H & E, hematoxylin and eosin; DNA, deoxyribonucleic acid; TNF-[alpha], tumor necrosis factor-alpha; PCNA, proliferating cell nuclear antigen; AST, aspartate transaminase; ALT, alanine transaminase; ALP, alkaline phosphatase; ELISA, enzyme-linked immunoassay; IL-6, interleukin-6; GCMS, gas chromatography and mass spectrometry analyses

*Corresponding author. Tel.: + 90 222 239 37 509/2433; fax: [phone].

E-mail address: [e-mail] (M. Uyanoglu).

Table 1. The regeneration rate, mitotic index, PCNA, IL-6 of the
hepatocytes of various groups of animals

Group  Regeneration rate (%)  Mitosis (%)

I      --                      1.65 [+ or -] 0.85
II     40.96 [+ or -] 7.40    70.17 [+ or -] 10.80
III    57.85 [+ or -] 12.26*  81.96 [+ or -] 4.39**
P      II-III: p<0.05 *       II-III: p<0.01**

Group  PCNA (%)                IL-6

I       0.78 [+ or -] 0.46     0.38
II     46.53 [+ or -]10.23     0.88
III    65.01 [+ or -]15.60***  0.88
P      II-III: p<0.001***      p>0.05

The liver regeneration rate in Group III (carvacrol treatment) was
significantly different than that of Group II. The mitotic index and
PCNA index increased significantly in regenerating liver of rats of
Group III over the Group II at the 72nd hour of post partial
hepatectomy. Group III further raised the mitotic and PCNA index. At
IL-6 levels there were no significant differences among these groups.
Each value was the mean [+ or -] SD of eight animals per group.
Differences at p values of <0.05 were considered significant.