Teucrium polium reversed the MCD diet-induced liver injury in rats

Abstract

In the present study, we evaluated the ability of Teucrium polium ethyl acetate fraction, with high antioxidant activity, in the treatment of nonalcoholic steatohepatitis (NASH) in rats and its possible effect on factors involved in pathogenesis of the disease. To induce NASH, a methionine and choline deficient (MCD) diet was given to N-Mary rats for 8 weeks. After NASH development, MCD-fed rats were divided into 2 groups: NASH group that received MCD diet and NASH + T group which was fed MCD diet plus ethyl acetate fraction of T. polium orally for 3 weeks. Histopathological evaluations revealed that treatment with the extract has abated the severity of NASH among the MCD-fed rats. In addition, the fraction reduced the elevated levels of hepatic tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) gene expression and also the elevated level of malondialdehyde (MDA). In addition, the extract increased the activities of superoxide dismutase (SOD), glutathione peroxidase (GPx) and enhanced the level of hepatic glutathione (GSH). Moreover, the fraction treatments lowered caspase-3 level and the phosphorylated form of C-Jun N-terminal kinase (JNK) and augmented the phosphrylated level of extracellular regulated kinase1/2 (ERK1/2). These results indicate that the ethyl acetate fraction of T. poium effectively reversed NASH, mainly due to its strong antioxidant and anti-inflammatory properties.

Introduction

Nonalcoholic fatty liver disease (NAFLD) is a metabolic disorder associated with a wide spectrum of liver abnormalities ranging from simple steatosis (fatty liver) to nonalcoholic steatohepatitis (NASH), hepatocellular carcinoma and cirrhosis. ¹ Liver injury in NASH resembles alcoholic steatohepatitis (ASH), usually characterized by the presence of steatosis, along with ballooning degeneration and lobular inflammation with or without fibrosis, in the absence of alcohol abuse. ²

Although the exact mechanism(s) that mediate the transition from steatosis to steatohepatitis remain unknown, increased oxidative stress, production of inflammatory cytokines and incidence of hepatocyte apoptosis are believed to play key roles in NASH pathogenesis. ^2,3 It has been demonstrated that markers of oxidative stress such as MDA and 4-hydroxynonenal (4-HNE) increase significantly among NASH patients, while the levels of antioxidants including GSH and SOD are suppressed. ⁴ Increased generation of reactive oxygen species (ROS), under oxidative stress conditions, is known to lead to membrane lipid peroxidation, inflammatory responses and stimulation of stellate cells followed by fibrosis. ^2,3 Tumor necrosis factor-alpha (TNF-α) is an inflammatory cytokine that plays major roles in pathogenesis of NASH and is considered as the ‘second hit’ element of the ‘two hit’ hypothesis. ⁵ Crespo and co-workers have observed increased expression of TNF-α mRNA and its receptors in liver of patients with NASH and have also shown that higher serum TNF-α level is correlated with increased severity of NASH as manifested by more inflammation and fibrosis. ⁶ The other cytokine involved in liver injuries is transforming growth factor-beta (TGF-β). This profibrogenic cytokine released by the activated kupffer cells and the hepatocytes play a pivotal role in hepatic fibrogenesis by activation of hepatic stellate cells. ⁷ In addition, several investigators have shown that hepatocyte apoptosis is significantly increased among NASH patients as well as experimental models of NASH. It has been demonstrated that the degree of apoptosis correlates with the severity of steatohepatitis including the degree of inflammation and the stage of fibrosis. ^8,9

Mitogen-activated protein kinases (MAPKs) have been revealed to be an important group of regulators of gene expression that are potently activated by environmental stress and proinflammatory cytokines to regulate many cellular processes, such as cell proliferation, development, differentiation, apoptosis and inflammatory responses. ¹⁰ In humans, three major groups of MAPKs including extracellular regulated kinase (ERK), C-Jun N-terminal kinase (JNK) and p38 MAPK have been identified. Recent studies have pointed to the involvement of some members of MAPKs in NASH incidence. ^11,12

Although, there is yet no effective drug therapy, the implication of oxidative stress in the etiology and progression of NASH has led to the suggestion that antioxidants can have health benefits as prophylactic agents. Among these agents, polyphenolic compounds, mainly flavonoids and isoflavonoids, have been evaluated as the most efficient natural-made free oxygen radical scavengers. ^13,14 Teucrium polium L. (Lamiaceae) is a medicinal plant used for various purposes such as anti-inflammatory, anti-nociceptive, anti-bacterial, anti-hypertensive, anti-hyperlipidemia, anti-rheumatoid and anti-hyperglycemia. ^15,16 The polyphenolic content of T. polium extract along with their antioxidant activity have been evaluated and published by several independent groups. ^17,18 We have previously reported that ethyl acetate fraction of T. polium efficiently prevents the incidence of NASH among the experimental animals. ¹⁹

In this study, we evaluated the ability of T. polium ethyl acetate fraction in treatment of NASH induced by methionine and choline deficient (MCD) diet in rats and its possible effect on factors involved in pathogenesis of the disease including oxidative stress, cytokine levels, apoptosis and MAPKs.

Materials and methods

Material

Reduced glutathione was obtained from Fluka (Buchs, Switzerland). Ethylenediaminetetraacetic acid (EDTA) was obtained from Sigma–Aldrich Chemical Co. Ltd. (England). Trichloroacetic acid (TCA) was obtained from Sigma Chemical Co. (Missouri, USA). Nitroblue tetrazolium (NBT), 5, 5'-dithiobisnitro benzoic acid (DTNB), thiobarbituric acid (TBA), nicotinamide adenine dinucleotide reduced (NADH), nicotinamide adenine dinucleotide phosphate reduced (NADPH) and bovine serum albumin (BSA) were obtained from Merck Co. (Germany). Sulphosalicylic acid was obtained from Carlo Erba (Milan, Italy). All primers and probes were obtained from Bioneer (Chungwon, Korea). All other chemicals used were analytical grade.

Animals and experimental protocols

Male N-Mary rats, weighing 175−220 g and with an average age of 8 weeks, were housed in cages with 12-h/12-h light/dark cycle and were allowed free access to food and water ad libitum. All procedures for animal experiment were in accordance with the animal ethics committee of University of Tehran. Animals were divided into 2 groups: group 1 (control, n = 5) was fed a normal diet for 11 weeks and group 2 (NASH) received a MCD diet for 8 weeks. After confirmation of NASH development, group 2 was divided into two subgroups: NASH group (n = 5) continued to get MCD diet and group 3 (NASH + T, n = 5) was fed an MCD diet with T. polium ethyl acetate extract (equivalent to 0.5 g of plant leaves powder/kg body weight/day) by gavages for 3 weeks. The diet composition has been described previously. ¹³ Finally, all rats were sacrificed under diethyl ether anaesthesia and the blood and the livers were taken for biochemical, histopathological and molecular examinations.

Plant extract preparation

The aerial parts of T. polium L. were collected from Fars province, Iran, during spring. A voucher herbarium specimen (No. 570) was deposited in the herbarium of the school of pharmacy, Shaheed Beheshti University of Medical Sciences, Tehran, Iran. The plant aerial parts were air-dried, protected from direct sunlight, and then powdered. The powdered plant material (300 g) was extracted three times with ethanol (EtOH 80%), at room temperature (RT) overnight. The EtOH extracts were combined and concentrated under reduced pressure on a rotary evaporator and the volume was adjusted to 300 mL. The EtOH extract was then re-extracted four times with ethyl acetate. The extract was evaporated to dryness to give the ethyl acetate residue. This residue (0.75 g) was used for in vivo studies after dissolution in distilled water at a concentration of 0.25 mg/mL.

Biochemical analysis

The plasma alkaline phosphatase (ALP), aspartate aminotransefrase (AST) and alanine aminotransferase (ALT) activities were assayed using the corresponding commercial kits (Pars Azmoon, Iran).

Preparation of liver homogenate

The liver samples were cut into small pieces and homogenized in Tris-HCl buffer (25 mM, pH 7.5) with a homogenizer to give a 10% (w/v) liver homogenate. The homogenates were then centrifuged at 12,000 rpm for 15 min at 4°C (Beckman). The protein concentration of each extract was determined by the method of Lowry ²⁰ using BSA as the standard.

Measurement of hepatic lipid peroxidation

Hepatic MDA level of each sample was determined by the double-heating method. ²¹ The method is based on spectrophotometeric measurement of the purple color generated by the reaction of TBA with MDA. Briefly, 0.5 mL of the liver homogenate was mixed with 2.5 mL of TCA (10%, w/v) solution followed by boiling in a water bath for 15 min. After cooling to room temperature, the sample was centrifuged at 3000 rpm for 10 min and 2 mL of each sample supernatant was transferred to a test tube containing 1 mL of TBA solution (0.67%, w/v). Each tube was then placed in a boiling water bath for 15 min. After cooling to room temperature, the absorbance was measured at 532 nm with respect to the blank solution. The concentration of MDA was calculated based on the absorbance coefficient of the TBA–MDA complex (ϵ = 1.56 × 10⁵ cm⁻¹ M⁻¹) and it was expressed as nmol/mg protein.

Determination of reduced glutathione

Hepatic GSH content was determined by the method of Jollow et al. ²² An aliquot of 0.5 mL of each tissue homogenate was precipitated with 1 mL of sulphosalicylic acid (4% w/v). The precipitate was removed by centrifugation. The filtered sample (0.5 mL) was mixed with 0.1 mL DTNB (4 mg/mL) and 0.9 mL phosphate buffer (0.1 M, pH 7.4). The yellow color developed was read at 412 nm. Reduced glutathione was expressed as µg/mg of protein.

Superoxide dismutase activity assay

Superoxide dismutase activity was measured based on the extent inhibition of amino blue tetrazolium formazan formation in the reaction mixture of nicotinamide adenine dinucleotide, phenazine methosulphate and nitroblue tetrazolium (NADH–PMS–NBT), according to method of Kakkar et al. ²³ The assay mixture contained 0.1 mL of supernatant, 1.2 mL of sodium pyrophosphate buffer (pH 8.3; 0.052 M), 0.1 mL of phenazine methosulphate (186 µM), 0.3 mL of nitroblue tetrazolium (300 µM) and 0.2 mL of NADH (750 µM). The reaction was started by addition of 0.2 mL of NADH solution (750 µM). After incubation at 30°C for 90 s, the reaction was stopped by addition of 0.1 mL of glacial acetic acid. The reaction mixture was stirred vigorously with 4.0 mL of n-butanol. Colour intensity of the chromogen in the butanol was measured spectrophotometrically at 560 nm. One unit of enzyme activity was defined as that amount of enzyme which caused 50% inhibition of NBT reduction/mg protein.

Glutathione peroxidase assay

Liver glutathione peroxidase activity was assayed in a cuvette containing 890 µL of 100 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1 mM NaN₃, 0.2 mM NADPH, 1 U/mL GSH reductase and 1 mM GSH. Each liver homogenate (10 µL) was added to make a total volume of 900 µL. The reaction was initiated by the addition of 100 µL of 2.5 mM H₂O₂, and the conversion of NADPH to NADP⁺ was monitored with a spectrophotometer at 340 nm for 3 min. GPx activity was expressed as nmoles of NADPH oxidized to NADP⁺/ (min. mg protein), using a molar extinction coefficient of 6.22 × 10⁶ (cm⁻¹ M⁻¹) for NADPH. ²⁴

Histopathological examination

Liver tissue samples were kept in 10% formalin, and paraffin blocks were prepared. The sections from blocks were stained with hematoxylin-eosin (HE) and masson trichrom. The histopathological evaluation was performed blindly by an expert pathologist using a scoring system proposed by Kleiner et al.: steatosis (0−3), lobular inflammation (0−3) and ballooning degeneration (0−2). ²⁵ Fibrosis was evaluated as absent/present.

Determination of mRNA level using real-time PCR

Total RNA was isolated from the frozen liver tissues using the TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Carlsbard, California). The RNA concentration and the quality were determined spectrophotometrically at 260 nm and by the A260/A280 ratio, respectively. Total RNA (4 µg) was reversed transcribed into complementary DNA (cDNA) with the use of 200 U of M-MuLV reverse transcriptase (Fermentas, Lithuania) and 0.2 µg of random hexamer (Fermentas, Lithuania) as the primer. The amplification reactions were performed on a Roche light cycler instrument (Roche diagnostics GmbH, Mannheim, GER) applying the following thermal cycling conditions: an initial activation step for 3 min at 95°C followed by 45 cycles including a denaturation step for 10 s at 95°C, annealing step for 15 s at 55°C and extension step for 20 s at 72°C. β-actin was used as a normalizer and the fold change in expression of each target mRNA relative to β-actin was calculated based on 2^-ΔΔct comparative expression method. The primers and probes were synthesized to the published primer sequences. ^{26 –28}

Immunoblot analyses

Each frozen liver tissue (10−20 mg) was homogenized with 0.2 mL lysis buffer containing 10 mM Tris (pH 7.4), 100 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 20 mM sodium pyrophosphate, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulphonyl fluoride (PMSF), 10 μg/mL leupeptin, 1 µg/mL pepstatin and 60 µg/mL aprotinin. After 30 min, liver homogenates were centrifuged at 14,000 rpm for 15 min at 4°C. Protein concentration of each sample was determined using Lowry’s procedure. ²⁰ Equal quantities of protein (50 µg/lane) were resolved on a 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electroblotted to a polyvinylidene difluoride (PVDF) membrane (Amersham, Bioscience, UK). Membranes were blocked in Tris-buffered saline pH 7.6 containing 0.1% Tween-20, 0.05% sodium azide and 4% non-fat dry milk overnight at 4°C. Blocked blots were incubated with primary antibodies for 2 h at room temperature using diluted antibody in Tris-buffered saline pH 7.6, 0.05% Tween-20 and 1% non-fat dry milk as recommended by the manufacturer. After 1 h incubation at room temperature with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (Biosource, Belgium), the proteins were detected by an enhanced chemiluminescence (ECL) detection system (Amersham-Pharmacia, Piscataway, New Jersey, USA) according to the manufacture’s instructions. The specific signal was revealed by autoradiography. In all experiments, equal protein loadings have been confirmed by the β-tubulin content.

Statistical analyses

Data are expressed as mean ± SD of three independent experiments and statistically analyzed using Student’s t-test. Values of p < 0.05 were considered significant.

Results

Effect of ethyl acetate fraction on histopathological lesions and liver injuries

As shown in Figure 1 , in the control group no pathological changes were observed and histologically the livers appeared normal. However, the MCD diet led to grade 1 liver steatosis, lobular inflammation and ballooning degeneration. In the NASH + T group, lobular inflammation and ballooning degeneration abated to grade 0 in 100% of rats, while grade 1 steatosis was found in the remaining 50% of rats. Masson trichrom staining did not confirm the incidence of fibrosis in either group of rats. In order to assess the liver function, the sera levels of ALP, ALT and AST enzymes were determined. As it can be observed in Figure 2 , the plasma level of ALP, ALT and AST increased in the NASH group compared to the control group. However, treatment with the ethyl acetate extract of T. polium decreased the activities of these enzymes by 58%, 37% and 11%, respectively, compared to animals of NASH group.

Figure 1.

Histological examination of liver samples of rats. (A) Control group: normal liver histology (×100). (B) nonalcoholic steatohepatitis (NASH) group: macro and microsteatosis, ballooning degeneration and lobular inflammation (×400). (C) NASH + T group: marked reduction in steatosis, ballooning degeneration and lobular inflammation (×100).

Figure 2.

Effect of T. polium ethyl acetate fraction on activities of hepatic enzymes in methionine and choline deficient (MCD)-induced nonalcoholic steatohepatitis (NASH) in rats. Data represent means ± SD (n = 5) of triplicate measurements of each sample. *Significantly different from control group (p < 0.05). **Significantly different from NASH group (p < 0.05).

Effect of ethyl acetate fraction on liver oxidative status

In order to explore the effect of T. polium ethyl acetate fraction on the liver antioxidant status of rats fed MCD diet, the extent of lipid peroxidation along with antioxidant defense system capabilities were evaluated. Although MCD-fed rats showed enhanced levels of lipid peroxidation, upon ethyl acetate extract therapy, MDA level of the liver was decreased by 48%, compared to NASH group (Figure 3A). As shown in Figure 3A, The MCD diet has caused a severe decrease in hepatic GSH content relative to control group. However, administration of T. polium ethyl acetate fraction increased the GSH level by 80%, compared to NASH group. Figure 3B shows that the activity of SOD has decreased after 11 weeks of MCD diet administration, relative to control group. However, T. polium extract administration caused an increase in activities of hepatic antioxidant enzymes such as SOD and GPx by almost 95% and 26%, respectively, relative to NASH group.

Figure 3.

Effect of T. polium ethyl acetate fraction on hepatic levels of malondialdehyde (MDA) and glutathione (GSH) (A) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzymes (B) in methionine and choline deficient (MCD)-induced nonalcoholic steatohepatitis (NASH) in rats. Data represent means ± SD (n = 5) of triplicate measurements of each sample. *Significantly different from control group (p < 0.05). **Significantly different from NASH group (p < 0.05).

Effect of ethyl acetate fraction on hepatic TNF-α and TGF-β expression

To investigate the effect of T. polium ethyl acetate fraction on the inflammatory and fibrotic cytokines in rats fed MCD diet, TNF-α and TGF-β gene expressions were measured by real time-PCR analysis in the liver tissue. As shown in Figure 4 , in NASH group the TNF-α and TGF-β mRNA levels have increased by 1.9- and 1.5- folds, respectively, compared to normal healthy rats. However, treatment with ethyl acetate fraction of T. polium significantly decreased the TNF-α and TGF-β gene expressions by 54% and 39%, respectively, relative to rats of NASH group.

Figure 4.

Effect of T. polium ethyl acetate fraction on hepatic tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta (TGF-β) expression in methionine and choline deficient (MCD)-induced nonalcoholic steatohepatitis (NASH) in rats. The relative mRNA expressions of TNF-α and TGF-β were measured using real-time PCR after normalizing the cycle thresholds (Ct) of each sample against their corresponding β-actin. Data represent means ± SD (n = 4). *Significantly different from control group (p < 0.05). **Significantly different from NASH group (p < 0.05).

Effect of ethyl acetate fraction on pro-caspase-3 activation

Procaspase-3 is commonly cleaved to active caspase-3 during caspase-dependent apoptosis. To evaluate the effect of T. polium ethyl acetate fraction on caspase-dependent apoptosis induced by MCD diet, activation of pro-caspase–3 was examined by immunoblot analysis. According to Figure 5 , MCD feeding caused significant increase in the level of caspase-3 relative to the control group. However, treatment by ethyl acetate fraction of T. polium markedly decreased MCD-induced activation of pro-caspase–3.

Figure 5.

Effect of T. polium ethyl acetate fraction on hepatic pro-caspase–3 activation (A) and mitogen-activated protein kinases (MAPKs) (C) in methionine and choline deficient (MCD)-induced NASH in rats. Following the process described in the materials and methods section, equal amount of protein of each liver sample was subjected to western blot analyses using specific antibodies for the respective caspase–3, C-Jun N-terminal kinase (JNK)/pJNK and extracellular regulated kinase1/2 (ERK1/2)/p-ERK1/2 and p38/p-p38. Equal protein loading in each gel lane was confirmed by the β–tubulin content. The densitometry analyses of the gel documents are presented in B and D in which the data are the means ± SD (n = 4). *Significantly different from control group (p < 0.05). **Significantly different from NASH group (p < 0.05).

Effect of ethyl acetate fraction on the involvement of MAPKs

The activation of MAPKs by oxidative stress and inflammatory cytokines has been reported by many investigations. ⁹ In that regard, the possibility and pattern of MAPKs involvement were evaluated among the rats in NASH and NASH + T groups. As shown in Figure 5, keeping the rats on an MCD diet has led to significant phosphorylation of JNK in NASH group as compared to control group. However, no considerable alternation was observed in the phosphorylated forms of ERK and p38 among NASH group relative to the control group. However, treatment with ethyl acetate fraction of T. polium resulted in significant decrease in the extent of JNK phosphorylation and increase in level of ERK phosphorylation. In addition, as it is also evident in Figure 5, no alternation in total hepatic JNK, ERK and p38 contents has occurred in either group of rats.

Discussion

Feeding diet devoid of methionine and choline to experimental animals serves as an established approach for induction of steatohepatitis. ²⁹ The suggested biochemical basis of lipid build up under methionine and choline deficiency is attributed to the impaired biosynthesis of phosphatidiylcholoine which is essential for processing, packaging and secretion of VLDL from hepatocytes. Under this circumstance, triglycerides are accumulated in hepatocytes leading to high fatty liver incidence. Rodents on an MCD diet have developed steatohepatitis which is pathologically similar to NASH observed in human. ³⁰ It is known that MCD diet causes oxidative stress, lipid peroxidation and secretion of several inflammatory and fibrotic cytokines such as TNF-α and TGF-β. ^29,30

Our results indicated that the ethyl acetate fraction of T. polium not only effectively improved the grade of steatosis, ballooning degeneration and lobular inflammation but also considerably decreased markers of hepatic injury including the serum aminotransferases levels. In other words, T. polium effectively cured steatohepatitis, if not totally, but to high extent based on histological and biochemical measurements.

Oxidative stress is produced under steatosis condition and it is involved in the development and progression of NASH. ^2,3 High level of ROS may lead to biological damages through direct chemical reactions with cellular macromolecules such as proteins, nucleic acids and lipids. Lipid peroxidation is an important biological consequence of oxidative stress which increases drastically in NASH. Lipid peroxidation end-products such as MDA can assist the formation of Mallory bodies, induce mitochondrial dysfunction by inhibiting mitochondrial respiration and active stellate cells, along with promoting collagen synthesis and fibrogenesis among NASH-affected individuals. ^1,31 It has been shown that antioxidants such as vitamin E, C and some plant flavonoids and isoflavonoids are capable to decrease the extent of liver lipid peroxidation among various NASH experimental models mainly through scavenging the reactive oxygen species. ^13,14,32 Our results also demonstrated that the ethyl acetate fraction of T. polium significantly decreased the MDA content of the liver among the MCD-fed rats, suggesting a reduction in the level of oxidative stress. This antioxidant property of T. polium is certainly due to its chemical constituents. Phytochemical investigation of T. polium has demonstrated the presence of flavonoids. ³³ Flavonoids are a class of secondary plant phenolic compounds with powerful antioxidant property.

Endogenous antioxidants have the capability to prevent the uncontrolled formation of free radicals and reactive oxygen species, and/or to inhibit their reaction with biological molecules. These antioxidants include mainly enzymes such as SOD, GPx and GR and non-enzymatic antioxidants such as GSH. It has been reported that the level of these oxidative stress-related parameters decreases in the livers of patients with NASH. ⁴ The decrease in GSH level in NASH is probably due to its increased utilization by hepatocytes to counteract the increased level of free radicals. In our study, a dramatic rise in the liver GSH level was observed among the extract-treated rats. This probably indicates that the extract can either increase the biosynthesis of GSH or reduce the extent of oxidative stress leading to less GSH degradation, or it may have both effects. On the other hand, the activities of hepatic SOD and GPx were both attenuated (although it was not significant for the latter) in NASH-affected rats relative to that of the control rats. The decreased activity of these enzymes can lead to an excessive availability of superoxide and hydrogen peroxide in biological systems, which in turn will generate hydroxyl radicals involved in initiation and propagation of lipid peroxidation. ³⁴ However, treatment of the NASH-affected rats with ethyl acetate fraction significantly increased the SOD, GPx and GR (data not shown) activities. These results indicate that T. polium can decrease oxidative damages indirectly by activating endogenous defense elements.

Hepatocyte apoptosis is a prominent feature of human NASH and correlates with its severity. ^8,9 Emerging data suggest that a strong correlation exists between the extent of oxidative stress and the rate of apoptosis among NASH-affected hepatocytes. ⁹ Activation of caspases, mainly pro-caspase-3, has been documented among the hepatocytes undergoing apoptosis. Our data also supported the induction of apoptosis, as determined on the basis of caspase-3 level, among the liver cells of the NASH-affected rats. However, treatment of NASH-affected rats with the T. polium extract significantly decreased caspase-3 level.

Tumor necrosis factor-alpha is well known as a proinflammatory cytokine involved in almost all steps of NASH development. It has been reported that TNF-α can promote inflammation, impair mitochondrial respiration and induce apoptosis in hepatocytes. ^1,3,5 Similar to what is observed in human cases, we found a significant increase in hepatic TNF-α gene expression among rats with steatohepatitis. This increase might be due to several factors including oxidative stress. It has been demonstrated that the excess ROS leads to over-expression of TNF-α via nuclear factor-kB (NF-kB) activation. ² Several recent reports have pointed that antioxidants decrease hepatic expression and the level of serum TNF-α among various NASH experimental models. ^14,35 Our results also demonstrated that the ethyl acetate fraction of T. polium significantly decreased TNF-α gene expression among MCD-fed rats.

Liver fibrosis, as a wound-healing response to chronic liver injury, is characterized by excessive extracellular matrix (ECM) protein deposition. ³⁶ TGF-β is the most potent fibrogenic factor for hepatic stellate cells. It stimulates the synthesis and deposition of ECM components, such as type I, II and IV collagens. ³⁶ In our study, although no evidence of fibrosis was found among the NASH group, the TGF-β expression level was elevated. This observation probably demonstrates the initiation step of fibrosis process in rats and it seems that more than 11 weeks of exposure to MCD diet is required for detection of fibrosis. It has been shown that the oxidative stress activates the nuclear factor κB/Jun kinase pathway, generating nuclear C-Jun that is known to have co-activator effect on TGF-β. ⁷ Thus, the reduced oxidative stress caused by T. polium administration might be responsible for lower TGF-β gene expression induced by MCD diet. In addition, T. polium might exert a favorable effect on liver fibrosis through decreasing TNF-α expression. Recently, Tomita and colleagues have demonstrated that enhancement of TNF-α signaling via activation of kupffer cells might be critically involved in the pathogenesis of liver fibrosis in NASH. ³⁷

The C-Jun N-terminal kinase is activated by inflammatory cytokines such as TNF-α and also by exposure to internal and external insults such as oxidative stress. ¹⁰ Recent experimental studies have demonstrated that JNK activation is involved in the various steps of NASH pathogenesis. ^11,12 The JNK/MAPK pathway mediates hepatocyte injuries including steatosis, insulin resistance and apoptosis. ^11,12,38 It is believed that the activated JNK induces cell injuries through both transcriptional and non-transcriptional mechanisms. ³⁹ Our data, in full agreement with the published reports, demonstrated that MCD diet has led to higher phosphorylation of JNK. However, this activation markedly decreased by administration of T. polium ethyl acetate fraction probably via diminishing oxidative stress or TNF-α level. In contrast, the other member of MAPK family, ERK which is known as a cell survival factor in response to a variety of insults including ROS, ¹⁰ remained unaffected by MCD diet. However, treatment of MCD-fed rats with ethyl acetate fraction of T. polium increased the active form of ERK1/2. These results indicated that although ERK MAPK pathway is not involved in MCD-induced NASH, its activation is probably required for the treatment of the disease. In addition, our results showed that activation of p38 was not affected by MCD diet and T. polium administration.

In conclusion, the ethyl acetate fraction of T. poium effectively reversed NASH among the experimental animals. This beneficial effect of T. poium might be attributed to its strong antioxidant and anti-inflammatory properties that leads to suppression of intra-hepatocyte ROS level followed by suppression of TNF-α and TGF-β expressions, also JNK inhibition and increased activation of ERK1/2.