| | Effects of Teucrium polium on Insulin Resistance in Nonalcoholic SteatohepatitisReceived 25 January 2010; accepted 11 March 2010. Abstract Nonalcoholic fatty liver disease, the most common chronic liver disorder, is frequently associated with the clinical features of metabolic syndrome such as insulin resistance. We aimed to determine the effect of the crude and the ethyl acetate extracts of Teucrium polium on insulin resistance in rats with nonalcoholic steatohepatitis. Rats were divided into four groups. Group A was fed a normal diet for 11 weeks. Nonalcoholic steatohepatitis was induced in the remaining groups using a methionine/choline deficient (MCD) diet for 8 weeks. After nonalcoholic steatohepatitis development, group B continued with receiving the MCD diet alone; group C rats were given the MCD diet along with crude extract of T. polium (equivalent to 1 g leaves powder/kg body weight/day); group D rats were given the ethyl acetate fraction of T. polium by intragastric administration for 3 weeks. MCD diet led to grade 1 liver steatosis. In group C and D, these factors abated to grade 0 in 80% of the rats. In the groups receiving the extract, lipoprotein profiles were significantly improved relative to those not receiving the extract. Also, a dramatic reduction was observed in sera alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase activities. In addition, in groups C and D, an increase in the activities of liver superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes were also associated with a decrease in the malondialdehyde level relative to group B. Moreover, both extracts significantly decreased plasma glucose and insulin levels along with insulin resistance. In conclusion, both extracts of T. polium could reverse the adverse effects of an MCD diet.
1. Introduction  The liver is the controller of carbohydrate homeostasis that stores or releases glucose according to metabolic demands. Liver defects may therefore lead to abnormal carbohydrate metabolism. Nonalcoholic fatty liver disease (NAFLD) defines a spectrum of liver diseases comprising simple fatty liver to nonalcoholic steatohepatitis (NASH) which is associated with fibrosis and progression to cirrhosis. Diet abnormalities, such as protein calorie malnutrition and a diet deficient in methionine/choline (MCD), have been considered as the causative factors of NASH [1]. An MCD diet, occurring mainly through inhibition of phosphatidylcholine biosynthesis, tends to increase lipid content in hepatocytes by preventing very low density lipoprotein (VLDL) synthesis and export. Increased intrahepatic levels of fatty acids provide the appropriate environment for lipid peroxidation particularly if the system is under the condition of oxidative stress [2, 3]. This observation might account for disease progression from steatosis to steatohepatitis and cirrhosis due to enhanced levels of lipid peroxidation, cytokines and Fas ligand induction. By contrast, there is some evidence showing that increased fatty acid content and metabolites in NASH is accompanied by erroneous phosphorylation of the insulin receptor substrates, IRS-1 and IRS-2. Thus, in spite of higher than normal levels of insulin, the cascade of insulin dependent reactions are usually attenuated [4]. In light of this, it is anticipated that antioxidants, capable of scavenging free radicals, can attenuate disease onset and progression and thus these agents could constitute the basis for hepatotropic drug development. Teucrium polium (Lamiaceae) is known in some parts of Iran as an antidiabetic herb [5]. The high insulinotropic and antihyperglycemic activity of T. polium crude extract in both animal and/or isolated rat pancreatic islets has already been published [6, 7]. Other pharmacological properties attributed to T. polium include its antipyretic, antibacterial, anti-inflammatory and (more recently) high antioxidative potential [8, 9, 10]. Despite the noticeable pharmacological effects of the T. polium crude extract, there is limited experimental and clinical documentation outlining the adverse effects of the crude extract on liver and kidney functions [11, 12]. The effects of the crude and the ethyl acetate (EtOAc) extract of T. polium on insulin resistance in NASH are investigated in the present study. 2. Materials and Methods 2.1. Chemicals Reduced glutathione and oxidized glutathione were obtained from Fluka (Buchs, Switzerland). Glutathione reductase (GR) was obtained from Sigma-Aldrich Chemical Co. Ltd. (Gillingham, England). Nitroblue tetrazolium (NBT), 5,5′-dithiobisnitro benzoic acid, thiobarbituric acid, nicotinamide adenine dinucleotide reduced (NADH) and nicotinamide adenine dinucleotide phosphate reduced (NADPH) were obtained from Merck & Co. (Dermstadt, Germany), rat insulin enzyme-linked immunosorbent assay kit was obtained from Crystal Chem Inc. (Downers Grove, IL, USA). All other chemicals used were analytical grade. 2.2. Animals and experimental protocols Male N-Mary rats, obtained from the Pasteur Institute (Tehran, Iran), weighing 176–217 g, were housed in groups of five at 60±5% relative humidity and 22±2°C, with a 12-hour light/dark cycle. All rats had free access to water and food. NASH was induced by administration of an MCD diet as described by Ustundag et al (Table 1) [13]. Five rats in group A received a normal diet for 11 weeks. Twenty rats were fed the MCD diet for 8 weeks to induce NASH. At the end of the 8-week period, five rats were sacrificed to assay the NASH status and the remaining fifteen rats were randomly divided into three groups. In group B, rats continued to receive the MCD diet, in group C rats were given the MCD diet along with crude extract of T. polium (equivalent to 1g leaves powder/kg body weight/day) and in group D rats were given the EtOAc fraction of T. polium by intragastric administration. After 3 weeks, rats of all four groups were fasted overnight and then sacrificed under diethyl ether anesthesia. All experiments were performed according to the guiding principles of care and use of experimental animals approved by the state veterinary administration of the University of Tehran. Blood and liver tissue samples from all groups were obtained, each liver was immediately washed with normal saline, blotted on a filter paper, weighed, cut into small pieces and homogenized in Tris-HCl buffer (0.025 M, pH 7.5) using a homogenizer to give a 10% (w/v) liver homogenate. The homogenate was then centrifuged at 12,000 rpm for 15 minutes and the supernatant was aliquoted and frozen until use. The serum was separated from each blood sample and then stored at −70°C pending biochemical analyses. |
*
Values expressed as percentages
†
vitamin B12 and choline deficient. MCD=methionine/choline deficient.
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2.3. Measurement of body weight and diet intake Throughout the study period, diet intake was measured daily and body weights were recorded from before the extract administration to the end of the experiment. 2.4. Plant material Aerial parts of T. polium were collected from the Yasouj province (Iran) during spring. A voucher herbarium specimen (No. 570) was deposited in the herbarium of the School of Pharmacy, Shaeed Beheshti University of Medical Sciences, Tehran, Iran. Aerial parts of the plant were air-dried, protected from direct sunlight and finely ground. The powder was kept in a closed container at 4°C. 2.5. Preparation of T. polium extract The powdered plant material (300 g) was extracted three times with a mixture of ethanol and H2O (70:30) at room temperature overnight. The accumulated, crude extract was concentrated under reduced pressure on a rotary evaporator to a volume of 300 mL. The concentrated extract was then subjected to in sequence re-extraction with diethyl ether and EtOAc, each for four times. Each extract was then concentrated under reduced pressure [14]. EtOAc and the crude extracts were used in this study. 2.6. Biochemical analyses The sera levels of albumin, glucose, triglycerides, high density lipoprotein cholesterol and low density lipoprotein cholesterol were determined using enzymatic kits (Pars Azmoon, Tehran, Iran). Alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities were assessed as a measure of hepatic cell damage using the corresponding commercial kits (Pars Azmoon). Total protein content of each liver homogenate was estimated using the Lowry method [14]. Liver anti-oxidant status was measured in terms of the activities of glutathione reductase [15], and glutathione peroxidase [16]. The amount of reduced glutathione and malondialdehyde were determined according to Jollow et al [15], respectively. 2.7. Assay for superoxide dismutase (SOD) activity SOD activity was measured based on the extent of inhibition of amino blue tetrazolium formazan formation in the reaction mixture of NADH, phenazine methosulfate and nitroblue tetrazolium (NADH-PMS-NBT), according to the method of Kakkar et al [17]. The assay mixture contained 0.1 mL of liver homogenate, 1.2 mL of sodium pyrophosphate buffer (pH 8.3; 52 mM), 0.1 mL of PMS (186 μM) and 0.3 mL of nitroblue tetrazolium (300 μM). The reaction was initiated by addition of 0.2 mL of NADH solution (750 μM). After incubation at 30°C for 90 seconds, the reaction was stopped by addition of 0.1 mL of glacial acetic acid. The reaction mixture was vigorously stirred with 4.0 mL of n-butanol. Color intensity of the chromogen in the butanol was measured spectrophoto-metrically at 560 nm. One unit of enzyme activity was defined as that amount of enzyme which caused 50% inhibition of nitroblue tetrazolium reduction/mg protein. 2.8. Glutathione peroxidase (GPx) assay Liver GPx activity was assayed in a cuvette containing 0.89 mL of 100 mM potassium phosphate buffer (pH 7.0), 1 mM EDTA, 1mM NaN3, 0.2 mM NADPH, 1 U/mL reduced glutathione reductase and 1 mM reduced glutathione. Liver homogenate (10 μL) was added to make a total volume of 0.90 mL. 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 minutes. The GPx activity was expressed as nmol of NADPH oxidized to NADP+/(min · mg protein), using a molar extinction coefficient of 6.22×106/(cm · M) for NADPH [16]. 2.9. Glutathione reductase (GR) assay Liver GR was assayed in a reaction mixture containing 0.99 mL of 100 mM potassium phosphate buffer (pH 7.0), 1.1 mM MgCl2, 5 mM oxidized glutathione and 0.1 mM NADPH. Liver homogenate (10 μL) was added to trigger the NADPH conversion reaction. Changes in absorbance were monitored at 340 nm for 5 minutes at 25°C. The specific enzyme activity was expressed as nmol NADPH oxidized to NADP+/(min · mg protein) with 6.22×106/(cm · M) as the molar extinction coefficient of NADPH [16]. 2.10. Histopathological examination Liver tissue samples were fixed in 10% formalin, whereupon paraffin blocks were prepared. The sections from blocks were stained with hematoxylin and eosin, and Masson trichrome. The histopathological evaluations were performed in a blind manner by an expert pathologist by means of a scoring system proposed by Kleiner et al [18]: steatosis (0–3), lobular inflammatory changes (0–3) and hepatocyte ballooning (0–2). Fibrosis was evaluated as either absent or present. 2.11. Insulin resistance status Serum insulin was assayed using an enzyme-linked immunosorbent assay kit specific for rat insulin (Crystal Chem Inc.). The physiological index of insulin resistance was the homeostasis model assessment of insulin resistance (HOMA-IR) [19, 20], assessed from fasting glucose and fasting insulin concentrations using the following formula: HOMAIR=[fasting insulin (ng/mL)×fasting glucose (mg/dL)]/22.5. 2.12. Statistical analyses All values are expressed as mean±SD. The significance of differences between the means of the tests and controls were calculated by unpaired Student's t test, and p values less than 0.05 were considered significant. 3. Results 3.1. Histopathological findings Normal histology was observed in group A. Pathological damage was observed in group B. MCD diet led to grade 1 liver steatosis, inflammation and ballooning degeneration. In group C, these factors declined to grade 0 in 100% of the rats (Table 2). Grade 1 liver steatosis was observed among 50% of group D rats, although no inflammation or ballooning degeneration remained. Based on data shown in Table 1, there were no rats with fibrosis in any of the four groups (Figure 1). |
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Group A (normal diet), group B (MCD diet), group C (MCD diet + T. polium crude extract) and group D (MCD diet + T. polium ethyl acetate fraction). The histopathological evaluations were performed blindly by an expert pathologist using the following scoring system: steatosis (0−3), lobular inflammatory changes (0−3) and hepatocyte ballooning (0−2). Fibrosis was evaluated as absent or present. Data presented as n (%).
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3.2. Effect of T. polium extracts on serum lipoprotein profile and hepatic enzymes The MCD diet caused a 67% elevation in serum LDL levels among group B rats. However, the levels of VLDL and HDL were reduced by 2% and 33%, respectively. In group C, where rats received the crude extract of T. polium, LDL concentration was dramatically reduced (18%, p < 0.05) relative to group B. The sera levels of HDL and VLDL increased in group C compared with group B by approximately 28% and 9%, respectively. The ALP, AST, ALT and γ-glutamyl transferase (GGT) sera levels were determined to assess liver function. In group B the levels of ALT, ALP, AST and GGT all increased by 128%, 119%, 27% and 76%, respectively, relative to rats in group A. Treatment with crude extract of T. polium (group C) significantly reduced the sera levels of the aforementioned enzymes by 32%, 39%, 16% and 19% (p < 0.05), respectively relative to group B. This finding implies that crude extract of T. polium has a protective effect against MCD diet-induced liver damage. Administration of the EtOAc fraction of T. polium led to a 15% reduction in LDL concentration, but sera levels of HDL and VLDL increased in group D compared with group B by around 15% and 44%, respectively. The levels of ALT, ALP, AST and GGT in group D all decreased by 37%, 58%, 11% and 40%, respectively, relative to group B rats (Figure 2). 3.3. Effect of T. polium extracts on liver oxidative status To determine the effect of T. polium extracts on liver antioxidant status the activity of the main antioxidant enzymes of the system, SOD, GPx and GR were evaluated. The crude extract of T. polium increased the activity of SOD, GPx and GR by 47%, 55% and 48%, respectively, in group C compared with group B. However, treatment with the EtOAc fraction of T. polium increased the above mentioned items by 95%, 26% and 55%, respectively in group D relative to group B. Meanwhile, the extent of malondialdehyde production, as an index of the extent of lipid peroxidation, was also evaluated. The results indicated a 104% elevation in the malondialdehyde level in group B relative to group A rats. Treatment with the crude extract of T. polium caused a 25% reduction in the level of malondialdehyde in group C compared with group B rats. Nonetheless, treatment with the EtOAc fraction of T. polium revealed a 48% reduction in malondialdehyde level in group D relative to group B, as shown in Figure 3. 3.4. Effect of T. polium extracts on insulin resistance The physiological index of insulin resistance was assessed from fasting glucose and fasting insulin concentrations using the HOMA-IR approach. Although the MCD diet caused a 100% elevation in insulin resistance in group B compared with group A rats, treatment with the crude extract of T. polium caused a 50% improvement in insulin resistance among the rats of group C relative to rats of group B. The EtOAc fraction of T. polium caused a 41% reduction in insulin resistance among the rats of group D relative to rats in group B (Figure 4). 4. Discussion NAFLD is associated with variable degrees of lipid accumulation in the liver. An MCD diet, as a causative factor of NAFLD, inhibits VLDL synthesis and depletes the antioxidant capacity of the liver. Accumulated lipids tend to be oxidized by elevated levels of reactive oxygen species, which leads to oxidative damage. There is also evidence suggesting that the MCD diet also causes a decline in insulin receptors and insulin receptor substrate phosphorylation, which diminishes the cascade of insulin induced reactions and leads to hepatic insulin resistance. As a result, when insulin resistance occurs, a higher than normal level of insulin leads to further lipid accumulation and lipid peroxidase stimulation. Thus, antioxidants appear to play a critical role in the field of insulin resistance in NAFLD. In an effort to reduce the unpleasant outcomes of insulin resistance, researchers have studied different natural antioxidants believed to attenuate oxidative damage in several diseases [21, 22]. Flavonoids are a group of secondary plant metabolites found in fruits and vegetables. T. polium is well known for its antihyperglycemic activity in different parts of Iran, with several studies [5, 6, 9] demonstrating its antidiabetic and antioxidative activity in animal and isolated rat pancreatic islets. Phytochemical investigation into the crude extract of T. polium has led to the isolation and structural elucidation of several flavonoids [12], but the pharmacological activities of these compounds have not been established. Crude and EtOAc extracts of T. polium were chosen to be evaluated in this study. In our study, an MCD diet in rats caused NASH which was also associated with an almost 100% elevation in insulin resistance. Applying the crude extract of T. polium to rats with NASH caused noticeable improvements in liver function evident through sera levels of aminotransferases. In a 4-week treatment period, crude extract of T. polium caused a 32% decrease in serum ALT levels (p < 0.001). Serum AST level was also decreased by 16% (p < 0.001). Crude extract, however, enhanced hepatic levels of antioxidant enzymes, such as SOD, GPx and GR, confirming the high antioxidant activity of the plant extract. Histological changes also revealed a dramatic improvement among the crude extract treated rats. Considerable improvements in the level of oxidative defense enzymes and sera aminotransferases were also seen following the administration of EtOAc fraction of T. polium. Reactive oxygen species are considered major mediators in the etiology of NASH, and we suggested that both crude and EtOAc extracts of T. polium probably act by neutralizing free radicals leading to decreased lipid peroxidation. Some concerns over the adverse effects of the crude extract of T. polium on human and animal liver have been raised [23, 24]. Our histopathological findings, along with biochemical measurements, did not reveal hepatotoxic effects for the crude and EtOAc extracts of T. polium during a 4-week study period. Further cytotoxic evaluation is to follow as final purification of the active constituents in T. polium is conducted in our laboratories. Our data suggest that both extracts have improved liver status and also insulin resistance in rats with NASH. Acknowledgments The authors are grateful to the financial support given for this investigation by the Research Council of the University of Tehran. References 1.
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Institute of Biochemistry and Biophysics, University of Tehran, Tehran, Iran  Corresponding author. Institute of Biochemistry and Biophysics, P.O. Box 13145-1384, University of Tehran, Tehran, Iran
PII: S2005-2901(10)60019-2 doi:10.1016/S2005-2901(10)60019-2 © 2010 Korean Pharmacopuncture Institute. Published by Elsevier Inc. All rights reserved. | |
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