Introduction

Metformin is among the first-line treatments for management of the type 2 diabetes mellitus and avoiding its vascular problems (6). The beneficial properties of metformin have been stated to be mediated via stimulation of AMP-activated protein kinase (AMPK) (7), a protein kinase that is stimulated in response to variations in cellular energy contents (8). AMPK is vital for metabolic homeostasis, which is shared to all eukaryotic cells (9), and can be stimulated by metabolic stresses, such as hypoxia, glucose deficiency and so on (10-12). AMPK activation effect of metformin is not restricted to hepatocytes, but is also detected in other organs such as skeletal muscles, endothelial cells and neurons. AMPK is expressed in the neurons of the developing and adult brain, and more functional brain areas have upper AMPK levels (9). It was revealed that the stimulation of AMPK in the hippocampus of the rats involves in the antidepressant effect of some drug such as ketamine (13). It has been shown that AMPK induces astrocyte stellation, the term used for increasing the outgrowth of neuronal processes, the key feature in depressive disorder pathology (14, 15). Also neuroprotective effects of this antidiabetic drug in central nervous system have been proven (16). On the other hand, interaction between AMPK and nitric oxide pathway has been mentioned in several studies (17, 18). AMPK phosphorylates endothelial isoform of nitric oxide synthase (eNOS), resulting in eNOS activation in a calcium-independent way (6). Neuronal isoform of NOS is also phosphorylated by AMPK (19). NO is a gaseous signaling molecule which has been shown to play the role in some mental functions such as depression (20, 21). 

Cholestasis is a kind of liver disease due to structural impairment and dysfunction of hepatobiliary system which at first, results in accumulation of bile acids and other toxins in plasma and liver. At worst, it can leads to hepatic failure and cirrhosis and if untreated ends in death.  It was  previously showed that in BDL mice immobility time decrease compared to sham-operated animals (22). In other word, AMPK activation in CNS is potently increases in response to bile duct ligation (23). Thus, pharmacological activation of AMPK might provide a new strategy for the management of depression.

Putting all these data together, in this study, we designed an experiment, in which for the first time, we tested the anti-depressive effect of metformin on cholestatic mice. Besides, we examined the mechanism through which metformin exerts it effect. We assumed nitric oxide pathway must, at least in part, be involved in it.

Materials and Methods

1. Animals and Housing Conditions

Male adult NMRI (Naval Medical Research Institute) mice having weight of 20-30 grams obtained from Pasteur Institute were used throughout this study. Two weeks before the behavior experiments, all experimental animals in a group of four to five were placed in plastic cages and were housed under standard laboratory condition of temperature (21-23°C), humidity (55%) and light dark cycle (12-hour light/dark). All animals were permitted to have access to water and food except for the short interval of experiment time. All studies involving animals were done in accordance with the standards and guidelines of animal care and Council Directive for Care and Use of Laboratory Animals of Experimental Medicine Research Center, Tehran University of Medical Sciences, Tehran, Iran.

2. Drugs and reagents

The following drugs were utilized during the study: Metformin hydrochloride (Osveh company, Iran), NG-L-arginine methyl ester (L-NAME) (Sigma, Bristol, UK), Aminoguanidine (AG), a specific iNOS inhibitor (Sigma Chemical Co, USA). Usually the drugs were dissolved in water. Drugs were administered through intraperitoneal (i.p.) route in a constant volume of 10 ml/kg body weight.

3. Induction of cholestasis

 Mice were divided in three experimental groups. Each group was comprised of 8 to 10 animals: (I) un-operated controls; (II) sham-operated-operated controls; and (III) bile duct ligated (BDL) animals. Briefly, mice were laparotomized under general anesthesia induced by ketamine (50 mg/kg, i.p.) and xylazine (10 mg/kg, i.p.). The bile duct was left in place after handling with forceps in sham-operated animals. In BDL animals, the bile duct was double ligated and then the abdominal slit was closed in two layers (24).  We evaluated the scratching activity 5 and 7 days after the procedure in BDL and sham-operated animals.

4. Behavioral Tests

4.1. General conditions

All behavioral tests were carried out by an investigator between 08:30 a.m. and 16:00 p.m. Mice were tested every day using one test, which was recorded through video recorder and later it was extrapolated. Also, animals were accustomed to the room for at least one and half an hour before conducting any procedure.

4.2. Open field test

   Before performing the forced swimming test (FST), the locomotors behavior was accessed through an open field test (OFT) as described previously (21,25,26). In short, the apparatus consists of a wooden box (40×60×50 cm³), the floor arena of which was divided into 12 equal rectangles. Each mouse after placing within the center of the arena, the number of rectangles was counted covered with all paws while moving within the box for at least 6 minutes.

4.3. Forced swimming test

FST is considered as a valid test for the interpretation, assessing of the negative mood like depression. Hence it provides an effective tool for evaluating the efficacy of antidepressants (27). Each mouse was challenged by placing it in a cylindrical tank (30 cm height × 20 cm diameter) filled with tap water having temperature 24±1^°C, up to 19 cm of height. Mice were permitted to swim freely for 6 minutes. Mice were considered immobile when it became static in the water except those motions which were necessary to hold their head above the water surface. In FST the mice were subjected to swim for 6 minutes, however, the last four minutes of the test were analyzed.

5. Biochemical measurements

Immediately after euthanasia, blood samples were obtained via cardiac puncture (about 0.5 ml/mouse). These experiments were performed by a lab technician blinded to the kind of treatment. Total bilirubin and hepatic enzymes including alanine aminotransferase (ALT) and aspartate transaminase (AST) were assayed biochemically. A commercially available kit was used to assay these parameters (Zist-Shimi, Tehran, Iran).

6. Experimental design

To distinguish the anti-depressant effects of metformin, distinct doses (25, 50, 200 and 400 mg/kg), were administrated 4 hours before FST to the un-operated animals and 7-days BDL and sham-operated animals. Each group consists of 8 mice. In order to establish the involvement of NO in the antidepressant like effect of metformin, we coinjected the subeffective doses of  L-NAME (3 mg/kg) (28) or aminoguanidin (50 mg/kg, i.p.) (29) both with effective dose of metformin (25 mg/kg, i.p.). The L-NAME and amino-guanidin were administered 45 minutes before FST in metformin or saline-treated groups, seven days after bile-duct ligation.

7. Statistical analysis

Alterations in immobility time and locomotor activity in experimental and control groups were done and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. A value of p<0.05 was respected to be significant in all groups. All data were stated as the Mean ± SEM. All analyses were performed in SPSS software version 20.

Results

Induction of cholestasis

In current experiment, the mortality rate after the BDL operation was 20% (2 of 10 animals were early sacrificed because poor animal situations obtained). The values of both ALT (555.20 ± 247.47 in BDL vs. 63.33 ± 10.03 in sham) and AST (1000.60 ± 560.16 in BDL vs. 182.66 ± 45.50 in sham) that denote well-recognized serum indicators of hepatic injury quickly increased 7 days after BDL. Characteristically, the livers of sham-operated animals still look smooth at the end of the test, while the livers of mice that experienced BDL display architectural changes that are principally characterized by the development of edema and fibrotic nodules on the surface of corresponding livers and hydrops of the gall bladder that is filled with large amounts of bile.

Effect of metformin on forced swimming test

In saline injected groups there was no significant alteration in immobility times of mice before procedure, and also between control and sham-operated groups; but as exhibited in figure 1A, the bile duct ligation decreased immobility time compared with sham-operated animals in forced swimming test in seven days after surgical process. Also, there was no significant difference in locomotor activity (p>0.05) between above mentioned groups (Figure 1B).

Figure 1: The duration of immobility time in forced-swimming test (A) and the locomotor activity of mice in open field test (B) in unoperated (UNOP), sham-operated (SHOP) and bile-duct ligated (BDL) mice. The groups were tested seven days after bile duct ligation or sham operation. Values are expressed as the Mean±S.E.M. from 8 animals and were analyzed using one-way ANOVA followed by Tukey's post hoc test. **p<0.01 compared to the sham-operated and control groups in 7 days studies.