DOI: 10.5897/SRE11.1070 ISSN 1992-2248 ©2011 Academic Journals

Previous investigators have suggested that granulocyte colony-stimulating factor (G-CSF) may accelerate the healing process of liver lesions. This hypothesis has been discussed by some researchers. The action mechanisms remains unclear and further studies are needed to clarify this mechanism. To test this, we establish a rat model of liver injury using dimethylnitrosamine (DMN), transplanted the G-CSF into the rats. To determine whether G-CSF could prevent liver injury in DMN- intoxicated rats, we employed the model of acute and chronic liver injury induced by DMN: our study was limited to the characteristic changes in biochemical and histological parameters seen in liver damage. The DMN injections were given twice weekly for four weeks to establish liver damage. Rat was treated with DMN by intraperitoneal (IP) injection. On the 1st, 7th or 28th days, rats were sacrificed and liver histology was examined. Serial 5 µm sections of the liver were stained with hematoxylin and eosin (H&E). The serial measurement of the end products of hepatic synthetic activity also was used to assess liver damage. Liver injury was determined by biochemical and histological examination. As it is evident from results after administration of DMN, in addition to histologically changes of liver tissue, central vein, liver cord, sinusoids and bile duct were not normal, the activities of the serum liver enzymes also rose. Administration of G-CSF caused a statistically significantly decrease in the liver enzymes activity and sever of biochemical synthesis disruption in the liver. The protective role of G-CSF was showed in this rat model of acute and chronic liver damage induced by DMN. Our results showed beneficial effects of G-CSF treatment after liver injury on liver function. Histological and biochemical assessment of liver function revealed that liver tissue have a unique regenerative capability. 
 
   
 
 Key words: Dimethylnitrosamine, damage, granulocyte colony stimulating factor, liver.


Abbreviations:
DMN, dimethylnitrosamine; G-CSF, granulocyte-colony stimulating factor, IP, intraperitoneal, H&E, hematoxylin and eosin.demonstrated that it is an N-nitroso compound present in processed meats and industrial products (George, 2001).Several experimental studies suggest that G-CSF is a cytokine able to regulate a number of functions in neutrophils (Bergley, 1986;Clark, 1987;Ichinose, 1990;Avalos, 1990).According to the recent work, G-CSF also causes a marked increase in the mobilization of hematopoietic stem cells into the peripheral circulation (Avalos, 1996;Link, 2000).There are some reports in the literature showing the effect of treatment with G-CSF consisted of a slight effect on the inflammatory reaction \ (Ghanem, 2010).According to the recent work G-CSF used as a skeletal muscle regeneration therapy (Hara, 2011).It is established that G-CSF administration could significantly increase the survival rate in the rats (Zhang, 2011).As reported by many investigators, granulocytecolony stimulating factor are shown to reduce liver injury (Takayama, 2011).It is now clear that G-CSF treatment activates serial events of inflammation-thrombosis circuitry (Wei, 2011;Lian et al., 2011).The purpose of the present study was to utilize an established animal model of liver injury, and then to utilize G-CSF administrated to rats in order to observe the effects on liver function, and their kinetics using biochemical and histological criteria.

Experimental animal model
Wistar rats were used at ages from 8 to 12 weeks weighing of 200 to 250 g.Rats were prepared and used at the Animal Center of Babol University of Medical Sciences.Babol, Iran.All the protocols involving animals were approved by the Babol University Animal Care and Use Committee.Effort was made to minimize the number of animals.Age-matched rats were used as control animals.The animals were housed in suspended bracket cages in a climate controlled room with temperature of 23 ± 3°C in a cycle of 12 h light and 12 h darkness with free access to food and water.All animals were carefully maintained under standard animal housing conditions.The animals were divided into four groups of six each as follows: Group I: control group, non-treated animals receiving only PBS buffer, Group II: administration of DMN followed administration of PBS 2 h later.Group III: G-CSF administered and Group IV: administration of DMN followed by administration of G-CSF 2 h later.

Experimental model of liver damage
The experiments had started between 8.00 and 10.00 am and the animals were fasted for 12 h before any manipulation.The DMN, C2H6N2O was purchased from (Sigma, USA), and administered at a dose of 100 µl/kg body weight and G-CSF at 100 µg/kg body weight.Human recombinant G-CSF expressed in E. coli, (Sigma, USA) or PBS was administered by i.p. (intraperitoneal) injection in the respective groups of rats under light ether anesthesia (diethyl ether), (Sigma, USA).

Experimental model of acute liver damage
Acute liver damage on rats were induced by i.p. injection of a DMN 50% (v/v) solution in PBS (Sigma) at a dose of 100 µl/kg for two consecutive days.Control rats received i.p. PBS alone.

Experimental model of chronic liver damage
Chronic liver damage on rats were induced by intraperitoneal injection of a DMN 50% (v/v) solution in PBS at a dose of 100 µl/kg of DMN.The injection was given twice a week for 4 weeks to establish chronic liver damage.After 2 weeks of injury, all rats were submitted to blood tests for the evaluation of liver injury and function.Samples obtained from 6 normal rats referred to as control Qujeq et al. 4647 were used to define the normal range for each blood parameter analyzed.The specific values were: alanine aminotransferase (ALT), aspartate aminotransferase (AST).Only those animals presenting blood values different from the predefined range in all two parameters were used in the study.

Effect of G-CSF administration in healthy rats
determine the effects of G-CSF in healthy animals, we selected six rats that each received daily i.p. injection of G-CSF at the dose of 100 µg/kg in PBS for seven consecutive days.

Effect of G-CSF administration after DMN damage
DMN injections, the animals received i.p. injections of G-CSF.The four groups of six rats were sacrificed under general anesthesia after 1, 7 and 28 days to collect blood and liver tissue samples.Immediately after exsanguinations, the livers were removed, cleaned and weighed.One portion of the livers was separated and immersed in buffered formalin solution and then paraffin-embedded for histological examination.

Histological grading and staining
Following sacrifice of animals by cervical dislocation, the liver was removed, rinsed with phosphate-buffered saline and weighed.Histological features were analyzed by a single histologist who was unaware of animal characteristics.All liver sections were fixed in 10% (volume/volume) phosphate-buffered formalin and embedded in paraffin wax.Sections were cut at 5 µm and stained with hematoxylin and eosin.All specimens were randomized and were given code number however the examiner was unaware of the group to which the specimen belonged.The morphologic changes produced in the liver by the i.p administration of DMN as well as those produced by G-CSF in DMN-administered animals were assessed.The histopathologic index was assayed as the number of cases observed per 10 high power fields (HPF) chosen at random.

Statistics
All data are expressed as mean ± standard error.Data were analyzed by unpaired student's t-test for comparison between two means.A statistical analysis was performed by running the SPSS Version 16.0 for Windows package.Probability values <0.05 were considered as statistically significant.

RESULTS
All animals survived the study without signs of illness .None of the rats receiving the G-CSF and DMN doses died.Representative pictures of hematoxylin and eosin stained liver sections are shown in Figures 1 and 2. There were no abnormalities or histological changes in the livers of control (Group I) rats (Figure 1).Central vein, liver cords and sinusoids, bile duct, parenchymal cells and kupffer cells were normal.Results from histological studies correlated with the level of biochemical parameters.No deaths were observed due to DMN and/or G-CSF treatment during the experiment.We have administered the G-CSF in healthy rats up to 7  consecutive days without finding any evidence of tissue injury.There were no biochemical abnormalities, (data not shown) or histological changes in the livers of (Groups I and III) rats. Figure 2 shows degree of hepatic change in liver sections of rat treated with G-CSF.Our results showed that treated with G-CSF in DMN administered rat caused a significant rise in albumin and total protein compared with untreated group.Accompanied by a drop in the hydroxyproline level compared with untreated group, DMN administration caused slight to moderate increased activities of serum hepatic enzymes compared to those observed treated group; aminotransferases are enzymes widely used to assess liver damage.Elevated aminotransferase activities suggest liver damage.Any injury to the liver that results in necrosis causes the liberation of transaminases enzymes.The measurement of these hepatic enzymes in the serum is used to assess the extent of liver damage.

DISCUSSION
In the present study, the DMN treatment caused liver damage in rats as evidenced by increased serum aminotransferase activities (data not shown) levels.A marked rise in the levels of aminotransferases activities caused by liver injury.Any injury to the liver that results in damaging liver causes the liberation of various biochemical parameters.The measurement of these liver parameters in the serum is used to assess the extent of liver injury.Low serum albumin levels due to liver damage were caused by destruction of liver tissue.The diminution in albumin content was paralled by fall in total serum protein.A decreased serum albumin maybe due to decreased liver protein synthesis.This phenomenon was confirmed by the histological changes.Histological and biochemical evidence from this study supports the effectiveness of G-CSF for treating liver damage by DMN.The histological sections of rat livers following treatment with G-CSF indicated reduced incidence of liver injury, hepatocyte swelling, leukocyte infiltrations and necrosis induced by DMN.Controversial experimental observations ascribe different roles to G-CSF.In this regard many investigators demonstrate that G-CSF decreases aminotransferases activity and subsequently enhance liver regeneration in chemicalinduced liver injury in animal models (Theocharis et al., 2003).Other investigators report that G-CSF administration significantly improves survival of animals in the liver injury model induced by chemical liver injury (Yannaki et al., 2005).Similar results were obtained in the present study.This role of G-CSF in reducing the damaging effect of chemical toxin was challenged by other studies.G-CSF administration did not shown liver repair in chemical induce liver injury in such studies (Ono, 2004;Ogiso, 2007).These results differ from the data in the present study.It is difficult to explain the discrepancies between these studies.The mechanism by which G-CSF induce protective effects on liver injury are not well understood.The precise mechanism will require further basic and clinical studies.To explain the protective action of G-CSF in rat, we hypothesized that the reduction in transaminses activities and sever liver injury following the administration of G-CSF might be attributed to several mechanism: first, it facilitate improving of liver function with regard to hepatocyte function, a unique feature of liver tissue, ability to Qujeq et al. 4649 regenerate.Secondly, with regard to kupffer cells function, G-CSF may aid in the immune mechanism by removing chemical toxin or drug detoxification via reduction reactions.Thirdly, recruitment of stem cells from the bone marrow by the G-CSF is an attractive hypothesis.G-CSF may induce liver homing of bone marrow cells to regenerate liver function.Hematopoiesis may occur in the rat liver in damage state.However, relatively little is known about the mechanism of it.Several, possible mechanisms for the association between G-CSF and liver ability to regeneration must be considered in understanding of its details.In future studies, it will be important to examine the possible roles of G-CSF and tissue injury.Further basic and clinical experimental investigations are needed to explore the role of G-CSF in acute and chronic liver injury.

Conclusions
Our present findings taken together with previous results demonstrated that G-CSF administration facilitate improvement of liver function in the experimental model of acute and chronic induced by DMN.Our findings suggest that administration of G-CSF could be an effective approach to the treatment of liver damage.This observation is in agreement with the results obtained by other investigators.

Figure 1 .
Figure 1.Histological analysis of liver sections from control rat (hematoxylin and eosin staining, original magnification 100×x).

Figure 2 .
Figure 2. Histological analysis of liver sections after administration of G-CSF alonetreated group (hematoxylin and eosin staining, original magnification 100×x).