The serum or plasma concentrations of a variety of cytokines and cytokine antagonists are elevated in patients with liver disease. However, the potential pathogenic role of elevated circulating cytokines in the development of hepatic inflammation is not clear. Increased circulating proinflammatory cytokines can contribute to the multiorgan failure seen in some patients with liver disease. Elevated circulating TNFa or ILlp has been observed in patients with alcoholic liver disease, especially those who are malnourished, and has been correlated with survival (Means et al, 1996). Increased circulating TNFol has been reported in patients with the HELLP (haemolysis, elevated liver enzymes and low platelets) syndrome (Haeger et al, 1996) and others with acute liver failure (Keane et al, 1996). Stellate cell proliferation and collagen synthesis can be influenced by Kupffer cell (e.g. TGFP and TNFor), endothelial cell (e.g. PDGF) and hepatocyte (e.g. insulinlike growth factor and IGF-binding protein) derived factors (Gressner et al, 1995).

TNF-alpha is one of the principal mediators of the inflammatory response in mammals, transducing differential signals that regulate cellular activation and proliferation, cytotoxicity and apoptosis (Buetler, 1995; Jacob, 1992). In addition to its role in acute septic shock, TNF alpha has been implicated in the pathogenesis of a wide variety of inflammatory diseases (Jacob, 1992).

Tumor necrosis factor (TNF)- α is a pleiotropic cytokine that has both inflammatory and growth factor properties (Tracey, 1997). Dr. William Cooley first noted that some cancers spontaneously regressed when patients developed infection. Over a century later, TNF was identified as a macrophage-derived factor that caused cytotoxicity in murine tumor (Carswell et al, 1975). At the same time, Buetler and Cerami isolated a 17 kDa protein that they termed cachectin, because it caused severe wasting in infected animals (Buetler et al, 1985). It was subsequently determined that cachectin and TNF were identical. The membrane-bound form of TNF (26 kDa) has biologic activity and can induce cytotoxicity. The secreted (17 kDa) monomer is thought to fold back on itself and associates with other monomers to form a biologically active trimer that can activate either of the two TNF receptors (TNF R1-55 kDa) or (TNF R2-75 kDa) (Tracey, 1997).

Numerous cell types can produce TNF, with macrophages and monocytes being major sources. Fixed macrophages in the liver (Kupffer cells) are thought to be an important contributor to overall TNF production, especially that appearing in the bloodstream during endotoxemia. Multiple stimuli can induce TNF production, including lipopolysaccharide (LPS) or endotoxin and other cytokines such as interleukin (1L)-1, irradiation, oxygen radicals, viruses, and leukotrienes (Tracey, 1997). TNF can cause a host of diverse biologic actions, including a sepsis-like picture, anorexia, muscle wasting, fever, neutrophilia, alterations in intestinal permeability, and cell injury cytotoxicity (Hill et al, 1997). The present study interested in the role of TNF has been shown to play a role in many forms of experimental liver injury (Hill et al, 1997).

MATERIALS AND METHODS:

Animals

Adult male albino rats of Wister strain weighing 170-200 g were used for the study. The rats were housed in polypropylene cages and kept under standard laboratory conditions (temperature 25± 2⁰C; natural light-dark cycle). The rats had free access to drinking water and commercial standard pellet diet (Lipton India Ltd, Mumbai, India). The commercial rat feed contained 5% fat, 21% protein, 55% nitrogen free extract and 4% fiber (w/w) with adequate minerals and vitamin contents.

Experimental:

The toxic dose of the Cd was selected based on the study by Shibasaki et al. (1994). In this experiment, a total of 24 rats were used. The rats were randomly divided into 4 groups of 6 rats in each group.

Group 1 : Control rats subcutaneously treated with isotonic saline

Group 2 : Rats orally administered with PAC (100 mg/kg body weight/day) dissolved in water for 3 weeks using intragastric tube.

Group 3: Rats subcutaneously received Cd as cadmium chloride
(3 mg/kg body weight/day) in isotonic saline for 3 weeks.

Group 4: Rats subcutaneously administered with Cd (3 mg/kg body weight/day) followed by oral administration of PAC (100 mg/kg body weight/day) in water for 3 weeks.

REVERSE TRANSCRIPTASE-POLYMERASE CHAIN REACTION:

All glass wares were rinsed with diethyl-pyrocarbonate (DEPC) treated water to inhibit RNases. Total RNA was isolated using guanidium thiocynate-chloroform-phenol method of Chomczynski and Sacchi (1987).

Procedure:

Total RNA from the various tissue samples were isolated following the method of Chomczynski and Sacchi (1987). The tissue samples were minced and homogenized (100 mg/1 mL) in RNA isolation buffer. The homogenate was transferred to a 15 mL polypropylene tube and added in order: 2.5 mL of 2.5 M sodium acetate (pH 4.6), 0.5 mL of saturated phenol (80%) and 2.5 mL CHCl₃: Isoamyl alcohol (24:1). Mixed thoroughly by inversion, following the addition of each reagent. After incubation on ice for 15 min, the samples were centrifuged at 10,000 rpm for 15 min at 4⁰C. To the aqueous phase equal volume of ice-cold isopropanol was added and kept at -20 ⁰C for 1 h. The RNA was precipitated at 12,000 rpm for 15 min at 4 ºC, discard the supernatant and the pellet washed with 80% ethanol. The resulting pellet was dried briefly in vacuum and dissolved in minimal volume of sterile DEPC treated MQ water. The amount of RNA was quantified spectrophotometrically.

The RNA was quantified by UV-absorbance spectrophotometry. Total RNA (2 µg) was reverse transcribed and 4 µL cDNA obtained was used for polymerase chain reaction (PCR) amplification to estimate the expression of Transforming Growth Factor (TNF-α) and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal standard. Primer sequences and the resultant PCR products (Gene expressed) are listed in table 1.

The PCR thermocycling conditions for TNFα

Cycle	Step	Temperature	Time	Description
1X	1	95 ºC	5 min	Initial denaturation
30X	2	95 ºC	10 s	Template denaturation
	3	55 ºC	10 s	Primer annealing
	4	72 ºC	30 s	Primer extension
1X	5	72 ºC	7 min	Final extension

The PCR thermocycling conditions for GAPDH

Cycle	Step	Temperature	Time	Description
1X	1	94 ºC	4 min	Initial denaturation
30X	2	95 ºC	45 s	Template denaturation
	3	60 ºC	45 s	Primer annealing
	4	72 ºC	45 s	Primer extension
1X	5	72 ºC	10 min	Final extension

Table 1. Primer sequences and the resulting polymerase chain reaction products

PCR products

Primer sequences

TNF-α

Sense: 5′- ATG AGC ACA GAA AGC ATG ATG -3′

Antisense: 5′- TAC AGG CTT GTC ACT CGA ATT

-3′

GAPDH

Sense: 5′-TCGAGTCTACTGGCGTCTT-3′

Antisense: 5′-ATGAGCCCTTCCACGAT-3′

Statistical analysis:

All quantitative measurements were expressed as means ± SD for control and experimental animals. The data were analyzed using one way analysis of variance (ANOVA) on SPSS/PC* (statistical package for social sciences, personal computer) Ver. 10 and the group means were compared by Duncan’s Multiple Range Test (DMRT). The results were considered statistically significant if the p value is less than 0.05.

RESULTS:

Figure 1. showed mRNA expression levels of TNF- α in liver of control and experimental rats. The transcript analysis of 4 different groups revealed notable increase in the mRNA expression of TNF-α in the liver of cadmium chloride group rats (Group 3) when compared to control rats (Group 1). While protocatechuic acid supplementation showed significant down regulation of TNF- α levels when compared with cadmium chloride group rats (group 4). Protocatechuic acid supplementation alone (Group 2) did not produce any significant change in the expression level of TNF- α as compared to control rats.

Figure 1: Effect of drug on TNF- α mRNA level in the liver of hepatotoxicity rats.

a) Photograph a shows agarose gel electrophorotogram of mRNA level.

TNF- α GADPH Lanes

1 2 3 4

TNF- α - Transforming Growth Factor GAPDH - Glyceraldehyde 3-phosphate dehydrogenase

b) Band intensities were scanned by densitometer. The data were expressed as percentage of TNF-α/ GAPDH ratio and given as means ± S.D. for six experiments

P<0.05 compared with control rats; a P<0.05 compared with ethanol rats. Lanes-1: Control; 2: Control + Drug; 3: Cadmium chloride; 4: Cadmium chloride + protocatechuic acid ;GAPDH-glyceraldehyde 3-phosphate dehydrogenase (internal standard).

DISCUSSION:

TNF- α is a central proinflammatory cytokine. Activated Kupffer cells produce various mediators, including cytokines, eicosanoids, proteases, and oxygen radicals, that participate in inflammation, immune responses, and modulation of hepatocyte metabolism (Strieter et al, 1993). In the present study, increased TNF-α expression hepatic cadmium chloride rats. Tumor necrosis factor (TNF)-α levels are greater in hepatitis, and levels correlate with survival (Stahnke et al, 1991). Because TNF-α is produced predominantly by the monocyte-macrophage lineage and the major population of this lineage in the liver is Kupffer cells (Decker et al,1989), increased production of TNF-α by activated Kupffer cells may be responsible for hepatitis.

Increased expression of TNF-α in cadmium chloride rats may be due to inflammation, necrosis and the oxidative stress. Supplementation of protocatechuic acid effectifely decreased TNF-α expression in hepatic cadmium chloride rats. Decreased TNF-α expression may be due to attenuated inflammation, necrosis and reduce the oxidative stress.

Reverse transcriptase PCR analysis of TNF-α expression of hepatic of cadmium chloride treated rats showed increased expression of TNF-α as compared to control rats. Supplementation with protocatechuic acid to cadmium chloride treated rats showed down regulation of TNF-α expression as compared with cadmium chloride alone treated rats.

ACKNOWLEDGEMENTS:

The authors are grateful to the management of STET Women’s College, Mannargudi for their encouragement and support.

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Received on 19.09.2012

Modified on 30.09.2012

Accepted on 09.10.2012

Research Journal of Pharmaceutical Dosage Forms and Technology. 4(6): November–December, 2012, 324-327