HEPATOPROTECTIVE EFFECT OF FORSKOLIN IN CARBON TETRACHLORIDE-INDUCED MODEL OF ACUTE LIVER INJURY BY

Liver disease is one of the most common causes of death in the world. Nowadays, research studies have been focused on the development of new drugs for treatment of liver damage. Some natural products from medicinal plants have been found as potent agents for protection against liver injury induced by chemicals. Thus, it is interesting to find more effective natural products for protection against liver injury. Accordingly, the present study was designed to assess the hepatoprotective potential of a diterpenoid forskolin, isolated from the Indian plant Coleus forskohlii, at different doses in rat model with acute liver injury induced by carbon tetrachloride (CCl4) at a dose of 1ml/kg intraperitoneally as a mixture with corn oil. Forkolin was administered in doses of 5,10,20,40 mg/kg intraperitoneally for 7 days. Its protective effect was assessed via liver function tests and histopathological liver sections. Significant reduction in the hepatic enzymes levels was found in animals treated with forskolin at a dose of 10mg/kg as well as restoration of hepatocellular architecture. Therefore, treatment with forskolin showed preventive effect against CCl4-induced liver damage.


INTRODUCTION
Liver disease is a serious health problem throughout the world.A special interest in lowering liver damage arises since liver is a vital organ that plays a pivotal role in metabolism and detoxification of various endogenous and exogenous harmful substances (Yang et al., 2010).Many hepatotoxicants are known to cause liver injury in humans, such as viruses, fungal products, bacterial metabolites, minerals, environmental pollutants and chemotherapeutic agents (Wang et al., 2008).Despite new advances in hepatology, there is a lack of effective therapeutic strategies or specific medicines for the protection against hepatic disorders (Wu et al., 2007).Therefore, herbal medicines that possess hepatoprotective effects have attracted the attention of many researchers in recent years ( Forskolin, a labdane diterpene is the main active ingredient in the ayurvedic herb Coleus forskohlii (Labatiae) that has been used in India since ancient times.The root portion of the plant has been traditionally used for medicinal purposes and contains the active constituent, forskolin.Historically, it has been used to treat hypertension, congestive heart failure, eczema, colic, respiratory disorders, painful urination, insomnia, and convulsions.Clinical studies have justified these traditional uses and indicate its therapeutic potential in asthma, angina, glaucoma, psoriasis, and prevention of cancer metastases (Patel, 2010;Wagh et al., 2012).These properties encouraged us to investigate its hepatoprotective effect in a CCl 4 -induced acute hepatotoxicity model.

Materials
Forskolin was purchased from LC Laboratories (Woburn, MA, USA).It was dissolved in a mixture of dimethyl sulfoxide (DMSO) and saline.All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Animals and experimental protocol
The studies were conducted in accordance with ethical guidelines of Ain Shams University, Egypt.Male albino rats weighing 150-250 g rats were used in this study.They were supplied by Nile Co. for Pharmaceutical and Chemical industries, Egypt and housed in an air-conditioned atmosphere at 22±2°C, under a 12 h light-dark cycle and provided with rodent chow and water ad libitum.
Forty-eight animals were randomly divided into six groups of eight rats each.Group I served as normal control, receiving vehicles only for 7 days.Groups II-V were given forskolin (5, 10, 20 and 40 mg/kg IP, respectively) dissolved in a DMSO/saline solution for 7 days and on the 7th day; after one hour of forskolin injection, a single dose of CCl 4 (1ml/kg,IP,1:1 mixture with corn oil) was given.Group VI served as disease group, receiving vehicles for 7 days and on the 7th day; a single dose of CCl 4 (1ml/kg, IP, 1:1 mixture with corn oil) was given.24 h after CCl 4 injection, Blood samples were collected from the retro-orbital plexus and allowed to clot.Serum was separated by centrifugation at 3000 rpm for 10 min, then stored at -20°C.Rats were sacrificed and liver tissues were dissected, washed and placed in 10% formalin for histopathological examination.

Assessment of liver function tests
The levels of ALT and AST were measured following the commercial kit's instructions (Spectrum diagnostics, Egypt).

Histopathological examination
For light microscopy, liver specimens were fixed in 10% formalin and embedded in paraffin.Sections of 4 um thickness were cut, stained with hematoxylin and eosin and subjected to photomicroscopic examination.

Statistical analysis
Data are presented as mean ± standard deviation (SD).Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey-Kramer as a post hoc test.The 0.05 level of probability was used as the criterion for significance.All statistical analyses were performed using Instat software package (version 3.06).

Effect of Forskolin on ALT and AST in serum
Compared to the control group, CCl 4 induced a significant increase in serum ALT and AST levels reaching 160 and 21.9 fold respectively.Concurrent administration of 5, 20 and 40 mg/kg Forskolin with CCl 4 did not show any significantly decrease in ALT and AST levels as compared to CCl 4 group.However, the 10 mg/kg dose of forskolin showed a significant decrease in ALT and AST reaching 18.6 % and 25.2 % respectively as compared to the CCl 4 group (Table 1).

Histopathological findings
Liver sections from control group stained with H&E showed normal hepatic architecture (Figure 1A).CCl 4 group showed centrilobular necrosis with ballooning degeneration in the hepatocytes associated with dilatation in the central vein (Figure 1B).Pretreatment with 5mg/kg, 20mg/kg and 40mg/kg Forskolin failed to reduce the extensive hepatocellular damage caused by CCl 4 .Liver sections from those 3 groups showed centrilobular necrosis with ballooning degeneration in diffuse manner all over the hepatocytes in association with congestion in the portal vein (Figure 1 C,E,F).However, pretreatment with 10mg/kg Forskolin dose significantly reduced these alterations as liver sections only showed ballooning degeneration in some of the hepatocytes with dilatation in the central vein (Figure 1 D).A histopathologist graded these histopathological findings (Table 2).

DISCUSSION
Acute and chronic liver diseases constitute a global concern, and it is mostly induced by viral hepatitis, alcoholism, iron overload or drug toxicity.Among these types of liver injuries, there is consistent evidence of enhanced production of free radicals and/or a significant decrease in antioxidant defense mechanisms (Hoek and Pastorino, 2002).Many drugs have been used in the treatment of liver damage.However, the medical treatments for acute and chronic liver diseases are often difficult to handle and have limited efficacy.Thus, there is a need to explore new medicines with high efficacy for the protection against liver diseases.
CCl 4 has been widely used for experimental induction of hepatic fibrosis in rats (Hernandez-Munoz et al., 1990; Pierce et al., 1987).CCl 4 undergo metabolism by cytochrome P450 2E1 leading to production of free radicals which cause lipid peroxidation of the hepatocellular membrane, followed by inflammatory cytokines release and, eventually, hepatocellular damage (Recknagel et al., 1989;Weber et al., 2003).The later was evidenced by the significant elevation in the serum levels of the aminotransferases enzymes ALT and AST, because these enzymes are cytoplasmic and are released into the blood after cellular damage (Recknagel et al., 1989).
Results showed that a significant increase in the level of ALT and AST in CCl 4 -treated rats.The level of ALT and AST decreased significantly in administration of 10 mg/kg dose of forskolin only.However, other doses failed to protect the liver against CCl 4 -induced toxicity as evidenced by the high serum levels of ALT and AST.These biochemical results were confirmed by the histopathological findings.Our results were in accordance with a previous study which showed that forskolin reduced serum ALT levels and preserved hepatic architecture in a mouse model of liver ischemia/reperfusion injury (Ji et al., 2012).
Based on the previous findings, this study confirms that forskolin, indeed, has a hepatoprotective effect against CCl 4 -induced acute hepatotoxicity.The anti-oxidant and anti-inflammatory effects reported in previous studies on forskolin might be the reason for its hepatoprotective effect (Irie et al., 2001;Kamata et al., 1996;Niaz and Singh, 1999).
the mean ± SD (n=6).a or b: Significantly different from control or CCl 4 group, respectively at P < 0.001 using ANOVA followed by Tukey-Kramer as a post-hoc test.

McCay et al., 1984).
These free radicals are believed to cause lipid peroxidation, and the breakdown of cellular membranes (