- Email: [email protected]
6 mice
Chinese Journal of Natural Medicines 2014, 12(3): 0194−0198
Chinese Journal of Natural Medicines
Effect of Trifolium pratense extract on methionine-cholinedeficient diet-induced steatohepatitis in C57BL/6 mice CHEN Tong1, 2, ZHONG Fo-Jin1, HONG Ya-Min1, SU Wei-Jiao1, ZHUANG Li-Li 1, QIU Long-Xin1, 2* 1
School of Life Sciences, Longyan University, Longyan 364000, China;
2
Fujian Key Laboratory of Preventive Veterinary Medicine and Biotechnology, Longyan 364000, China Available online 20 Mar. 2014
[ABSTRACT] AIM: The potential of Trifolium pratense (red clover) extract in the prevention of lipid disorder has attracted increasing attention in recent years. In this study, the aim was to determine whether and how red clover extract affected the development of murine diet-induced non-alcoholic steatohepatitis. METHOD: Non-alcoholic steatohepatitis was induced in C57BL/6 mice by feeding mice with a methionine-choline-deficient (MCD) diet. Hematoxylin and eosin staining was used for histological analyses. Real-time PCR was used to analyze the mRNA expression levels. RESULTS: Hepatic steatosis and necroinflammation was observed in MCD diet-fed mice, and this diet-induced steatosis was significantly attenuated, whereas liver inflammation was not significantly attenuated, by red clover extract treatment. Consistent with the results of H&E staining, the MCD diet-induced increase of liver triglycerides and cholesterol levels were significantly reduced by red clover extract treatment. However, with the improvement in hepatic steatosis, mRNA levels of acetyl CoA oxidase, carnitine palmitoyl transferase-1, and liver fatty acid-binding protein, three genes regulated by peroxisome proliferator-activated receptor (PPAR) α, were unaffected. CONCLUSION: Red clover extract alleviated MCD diet-induced hepatic steatosis, but did not ameliorate liver inflammation in C57BL/6 mice, and the improvement in hepatic steatosis was not through activating PPARα. [KEY WORDS] Trifolium pratense; Steatohepatitis; Hepatic steatosis; Liver inflammation; Peroxisome proliferator-activated receptor α
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)03-0194-05
Introduction The term “non-alcoholic steatohepatitis” (NASH) is used to describe the pathological and clinical features of non-alcoholic diseases of the liver associated with pathological features most commonly seen in alcoholic liver disease itself [1]. Features of NASH on liver biopsy include steatosis, inflammation, liver cell injury, and varying degrees of fibrosis. It is a more advanced form of non-alcoholic fatty liver disease [Received on] 14-Nov.-2012 [Research Funding] This project was supported by the Science and Technology Planning Project of Educational Commission of Fujian Province, China (No. JB12198) and Fujian Province, China (No. 2010N0023). [*Corresponding author] QIU Long-Xin: Ph.D., Tel/Fax: 86-5972793889, Email: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
(NAFLD). NASH is becoming a major public health problem. However, current treatments for NASH have side effects and do not prove to be efficient [2]. Thus, the requirement for alternative and natural medicines to prevent NASH has been increasing rapidly and considerably. The potential of dietary isoflavones in the prevention of NASH has attracted increased attention among the public and in the medical community in recent years [3]. Soy isoflavones were reported to be beneficial for preventing NAFLD in Sprague–Dawley rats [4], high-fat-diet fed C57BL/6J mice [5-7], and Wistar rats [8]. Whereas soy isoflavones are the most studied isoflavones used for the studies on NAFLD, very few data are available for the isoflavones of red clover, Trifolium pratense L. (Fabaceae). The isoflavones from red clover differ from those of soy. The principal isoflavones in red clover are biochanin A, formononetin, genistein, and diadzein while those in soy consist solely of genistein and diadzein. In a previous report from this laboratory, red clover extract reduced liver triglycerides in db/db mice [9], however, whether red clover
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extract ameliorates steatohepatitis remained unclear. The four isoflavones from red clover extract (RCE) are reported to be the agonists of peroxisome proliferator-activated receptor (PPAR) α [10-12]. Since PPARα is a major lipid sensor and a transcriptional regulator for lipid metabolic enzymes in the liver [13], and some PPARα agonists are beneficial for alleviating NASH [14-15], it is of interest to study the effect of red clover extract on NASH. Methionine-choline-deficient (MCD) dietinduced steatohepatitis is a well established model for the study of NASH [15-16]. Thus, in this study, we wanted to examine the effect of red clover extract on the development of nutrition- induced murine NASH in C57BL/6 mice fed a MCD diet.
Materials and Methods Materials. Red clover extract was purchased from a common Chinese pharmacy (Rimian, Fuzhou, China) and was standardized to 10% isoflavones (consisting of 5.1% formononetin, 4.8% biochanin A, 0.16% genistein, and 0.04% daidzein). Animal experiments. C57BL/6 mice were obtained from the SLAC Laboratory (Shanghai, China). All animals were maintained under a 12/12 h light/dark schedule. Male mice, 7−8 weeks of age, were randomly divided into four experimental groups (each containing six animals): control diet, MCD diet, MCD diet + 50 mg·kg−1·d−1 red clover extract treated, MCD diet + 200 mg·kg−1·d−1 red clover extract treated. Red clover extract was administered orally in 0.5% sodium carboxymethyl cellulose (CMC) suspension and continued for 35 days. Serum biochemical measurements. At the end of the red clover extract treatment, mice were sacrificed and blood was collected from orbit. Serum alanine aminotransferase (ALT) was measured using a IDEXX analyser (Westbrook, ME, USA). Liver lipid analyses. Liver lipid was extracted by chloroform/methanol. Briefly, pulverized liver was homogenized in PBS, then extracted with chloroform/methanol (2:1), dried overnight and re-suspended in a solution of 60% butanol:40% Triton X-114/methanol (2:1). Liver triglycerides (TG) and cholesterol levels were measured using colorimetric assays (Jianchen, Nanjing, China). Quantitative analyses of mRNA expression by real-time PCR. Total RNA was isolated from tissues using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from hepatic mRNA using RevertAid First Strand cDNA Synthesis kits (Fermentas, Vilnius, Lithuania). Hepatic PPARα, acetyl CoA oxidase (ACO), carnitine palmitoyl transferase-1 (CPT-1), liver fatty acid- binding protein (L-FABP) mRNA were analyzed with the specific primers listed in Table 1. Real-time polymerase chain reactions were assayed using the FastStart Universal SYBR Green Master (Rox) (Roche Applied Science, Mannheim, Germany). Each Ct value was normalized to β-actin. Histological examination. Formalin-fixed liver tissue was processed and 5 μm-thick paraffin sections were stained with hematoxylin and eosin (H&E) for histological analyses. Steatosis and inflammation on all sections was blindly as-
Table 1 Primer sequences used for amplification of mRNA by real-time PCR Gene PPARα ACO CPT-1 L-FABP β-actin
F R F R F R F R F R
Sequences 5’-AAGAGGGCTGAGCGTAGGT-3’ 5’-GGCCGGTTAAGACCAGACT-3’ 5’-CCACATATGACCCCAAGACC-3’ 5’-AGGCATGTAACCCGTAGCAC-3’ 5’-GTCAAGCCAGACGAAGAACA-3’ 5’-CGAGAAGACCTTGACCATAG-3’ 5’-GTGGTCCGCAATGAGTTCAC-3’ 5’-GTATTGGTGATTGTGTCTCC-3’ 5’-CTATTGGCAACGAGCGGTTCC-3’ 5’-GCACTGTGTTGGCATAGAGGTC-3’
sessed by a hepatopathologist as previously described [17]. Hepatic steatosis was graded according to the percentage of lipid-laden hepatocytes as 0: 0%, 1: 0%–33%, 2: 33%–67%, 3: 67%–100%. Hepatic necroinflammation was given a score from 0 to 3, as follows: 0: no inflammatory foci, 1: mild, 2: moderate, and 3: severe. Statistical Analysis. Quantitative data are expressed as mean ± SEM. Students’ t test was used for pairwise comparisons and One-way ANOVA with Newman-Keuls Multiple Comparison Test for multigroup analyses. Differences were considered significant when probability values were less than 0.05.
Results Red clover extract attenuated diet-induced hepatic steatosis significantly, but did not ameliorate liver inflammation. Mice fed the MCD diet are a useful small animal model of progressive NAFLD [15-16]. Feeding C57BL/6 mice a MCD diet induces NASH and liver fibrosis within 4-8 weeks. To investigate the effect of red clover extract treatment on the development of MCD diet-induced steatohepatitis, mice fed the MCD diet were treated with red clover extract for 5 weeks. As shown in Fig. 1 and Table 2, examination of
Fig. 1 Effect of red clover extract on MCD diet-induced hepatic steatosis and necroinflammation. Hematoxylin and eosin–stained liver sections from mice fed: (a) Control diet; (b) MCD diet; (c) MCD diet + 50 mg·kg−1·d−1 red clover extract treated; and (d) MCD diet + 200 mg·kg−1·d−1 red clover extract treated. Liver shows foci of necroinflammation (arrows) and macrovesicular fat droplets. Slides are representative of four separate experiments
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CHEN Tong, et al. / Chin J Nat Med, 2014, 12(3): 194−198 Table 2 Effect of red clover extract on scores for hepatic steatosis and necroinflammation (mean ± SEM, n = 4) Measurements Controls Steatosis 0.00 ± 0.00 Necroinflammation 0.00 ± 0.00 * P < 0.05 vs MCD diet fed mice
MCD + 50 mg·kg−1 RCE 1.53 ± 0.29 1.13 ± 024
MCD 1.90 ± 0.25 1.25 ± 0.35
H&E-stained liver sections demonstrated marked steatosis and lobular inflammation in mice fed the MCD diet, while mice fed the control diet did not exhibit significant histological steatosis and inflammation. Co-administered with 200 mg/kg/day red clover extract for five weeks, steatosis in mice fed the MCD diet was attenuated significantly (P < 0.05). However, red clover extract treatment did not improve the lobular inflammation in mice fed the MCD diet. Consistent with the histological findings, red clover extract treatment did not result in a significant decrease in serum ALT levels in mice fed the MCD diet (Fig. 2), while it significantly decreased the level of total liver triglycerides (Fig. 3A; 103.70 ± 3.16 mg·g−1 tissue for mice fed the MCD diet versus 78.01 ± 8.13 mg·g−1 tissue for mice fed the MCD diet + 200 mg·kg−1·d−1 RCE, P < 0.05). Moreover, red clover extract treatment resulted in a significant decrease in liver cholesterol levels in mice fed the MCD diet (Fig. 3B; 16.34 ± 1.02 mg·g−1 tissue for mice fed MCD diet versus 9.39 ± 1.30 mg·g−1
MCD + 200 mg·kg−1 RCE 0.85 ± 0.13* 1.00 ± 0.22
tissue for mice fed the MCD diet + 200 mg·kg−1·d−1 RCE, P < 0.01). Together, these data indicate that red clover extract treatment is beneficial for the improvement of MCD dietinduced hepatic steatosis, but not for the improvement of MCD diet-induced liver inflammation.
Fig. 2 Effect of MCD diet and red clover extract (RCE) treatment on serum ALT levels in C57BL/6 mice (means ± SEM, n = 6). **P < 0.01
Fig. 3 Effect of red clover extract treatment on serum and liver lipid profiles in C57BL/6 mice (means ± SEM, n = 6). Red clover extract (RCE) treatment significantly decreased the liver TG level (A) and cholesterol level (B) in MCD diet-fed C57BL/6 mice. *P < 0.05; **P < 0.01; ***P < 0.001
Red clover extract did not affect the hepatic expression of some PPARα target genes involved in the development of steatosis. To clarify the mechanism whereby red clover extract exerts a beneficial effect on the development of dietinduced steatosis, the effects of red clover extract on hepatic mRNA expression of PPARα and its target genes, ACO, L-FABP, and CPT-1 were investigated. As shown in Fig. 4, intake of the MCD diet resulted in a marked decrease in ACO and L-FABP mRNA expression, compared with that of the control diet, whereas the mRNA expression of PPARα and CPT-1 were not affected significantly. However, the decrease in ACO and L-FABP mRNA expression was not recovered significantly by treating the MCD diet-fed mice with red clover extract. These data suggest that red clover extract does not activate hepatic PPARα, the effect of red clover extract on MCD diet-induced steatosis is therefore not through the activation of PPARα.
Discussion Red clover extract has been the focus of many studies during the last decade due to the protective effects against menopausal symptoms and a variety of disorders, including cardiovascular disease, cancer, and osteoporosis, etc [18]. Additionally, the potential of red clover extract in the prevention of lipid disorders has attracted increased attention. However, there are some debates about the hypolipidemic effects of red clover isoflavones under different disease conditions. Several studies reported the effect of red clover extract on improving lipid profile [19-22], whereas other studies did not show such an effect [23-24]. A previous report demonstrated that red clover extract reduced liver triglycerides and cholesterol in type 2 diabetic db/db mice [9]. In this report, it was demonstrated that red clover extract reduced liver triglycerides and cholesterol
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liver inflammation in animals with NASH. These data suggest the uncertainness of red clover extract as an anti-inflammatory drug.
References [1]
[2]
[3]
Fig. 4 Effect of red clover extract (RCE) treatment on hepatic mRNA levels of genes involved in hepatic steatosis (means ± SEM, n = 4). Hepatic mRNA levels were assessed using reverse transcription-real time PCR, standardized against an internal control (β-actin) and are expressed as fold differences compared with values obtained in mice fed the control diet. **P < 0.01
in mice with NASH. This present report further confirms the lipid-disorder-correcting effect of red clover extract. PPARα is an important metabolic nuclear receptor that regulates lipid metabolism through direct transcriptional control of the genes involved in peroxisomal and mitochondrial β-oxidation pathways, fatty acid uptake, and TG catabolism, etc. [13]. Hepatic activation of PPARα by its agonists, such as WY 14,643, decreases hepatic steatosis in MCD diet fed C57BL/6 mice [14-15]. In this report, however, the mRNA expression of ACO, CPT-1, and L-FABP, three PPARα regulated genes, was not altered by red clover extract treatment. This in vivo result is incompatible with some other previous in vitro reports. In those reports, red clover extract and its isoflavones were demonstrated to be agonists of PPARα [10-11]. This current study failed to demonstrate the PPARα agonist activity of red clover extract in mice with NASH. The mechanism whereby red clover extract ameliorates hepatic steatosis remains to be further studied. Red clover extract has been demonstrated to have anti-inflammatory properties in vitro [10-11]. It suppressed the lipopolysaccharide-induced secretion of proinflammatory cytokines, TNF-α and IL-6, in macrophage RAW264.7 cells. Moreover, isoflavones were reported to possess antioxidant and radical scavenging activities [25-26]. The anti-inflammatory, antioxidant and radical scavenging activities are beneficial for improving steatohepatitis. Surprisingly, in this report, it was demonstrated that red clover extract could not prevent liver inflammation. Thus, further in vivo studies are needed to investigate the anti-inflammatory effects of red clover extract under different inflammation conditions. In conclusion, the present study demonstrated the hepatic lipid lowering effect of red clover extract in animals with NASH, and that the effect was not achieved by activating PPARα. Moreover, red clover extract could not ameliorate the
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Ludwig J, Viggiano TR, McGill DB, et al. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease [J]. Mayo Clin Proc, 1980, 55 (7): 434-438. Dowman JK, Armstrong MJ, Tomlinson JW, et al. Current therapeutic strategies in non-alcoholic fatty liver disease [J]. Diabetes Obes Metab, 2011, 13 (8): 692-702. Kim MH, Kang KS. Isoflavones as a smart curer for non-alcoholic fatty liver disease and pathological adiposity via ChREBP and Wnt signaling [J]. Prev Med, 2012, 54 (Suppl): S57-63. Demonty I, Lamarche B, Deshaies Y, et al. Role of soy isoflavones in the hypotriglyceridemic effect of soy protein in the rat [J]. J Nutr Biochem, 2002, 13 (11): 671-677. Kim MH, Kang KS, Lee YS. The inhibitory effect of genistein on hepatic steatosis is linked to visceral adipocyte metabolism in mice with diet-induced non-alcoholic fatty liver disease [J]. Br J Nutr, 2010, 104 (9): 1333-1342. Kim MH, Park JS, Jung JW, et al. Daidzein supplementation prevents non-alcoholic fatty liver disease through alternation of hepatic gene expression profiles and adipocyte metabolism [J]. Int J Obes (Lond), 2011, 35 (8): 1019-1030. Lee YM, Choi JS, Kim MH, et al. Effects of dietary genistein on hepatic lipid metabolism and mitochondrial function in mice fed high-fat diets [J]. Nutrition, 2006, 22 (9): 956-964. Mohamed Salih S, Nallasamy P, Muniyandi P, et al. Genistein improves liver function and attenuates non-alcoholic fatty liver disease in a rat model of insulin resistance [J]. J Diabetes, 2009, 1 (4): 278-287. Qiu L, Chen T, Zhong F, et al. Red clover extract exerts antidiabetic and hypolipidemic effects in db/db mice [J]. Exp Ther Med, 2012, 4 (4): 699-704. Mueller M, Hobiger S, Jungbauer A. Red clover extract: a source for substances that activate peroxisome proliferator- activated receptor alpha and ameliorate the cytokine secretion profile of lipopolysaccharide-stimulated macrophages [J]. Menopause, 2010, 17 (2): 379-387. Qiu L, Lin B, Lin Z, et al. Biochanin A ameliorates the cytokine secretion profile of lipopolysaccharide-stimulated macrophages by a PPARgamma-dependent pathway [J]. Mol Med Report, 2012, 5 (1): 217-222. Qiu L, Ye H, Chen L, et al. Red clover extract ameliorates dyslipidemia in streptozotocin-induced diabetic C57BL/6 mice by activating hepatic PPARalpha [J]. Phytother Res, 2012, 26 (6): 860-864. Kota BP, Huang TH, Roufogalis BD. An overview on biological mechanisms of PPARs [J]. Pharmacol Res, 2005, 51 (2): 85-94. Ip E, Farrell G, Hall P, et al. Administration of the potent PPARalpha agonist, Wy-14,643, reverses nutritional fibrosis and steatohepatitis in mice [J]. Hepatology, 2004, 39 (5): 1286-1296. Ip E, Farrell GC, Robertson G, et al. Central role of PPARalpha-dependent hepatic lipid turnover in dietary steatohepatitis in mice [J]. Hepatology 2003, 38 (1): 123-132.
CHEN Tong, et al. / Chin J Nat Med, 2014, 12(3): 194−198 [16] Leclercq IA, Farrell GC, Field J, et al. CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis [J]. J Clin Invest, 2000, 105 (8): 1067-1075. [17] Brunt EM. Nonalcoholic steatohepatitis: definition and pathology [J]. Semin Liver Dis, 2001, 21 (1): 3-16. [18] Beck V, Rohr U, Jungbauer A. Phytoestrogens derived from red clover: an alternative to estrogen replacement therapy? [J]. J Steroid Biochem Mol Biol, 2005, 94 (5): 499-518. [19] Asgary S, Moshtaghian J, Naderi G, et al. Effects of dietary red clover on blood factors and cardiovascular fatty streak formation in hypercholesterolemic rabbits [J]. Phytother Res, 2007, 21 8): 768-770. [20] Geller SE, Studee L. Soy and red clover for mid-life and aging [J]. Climacteric, 2006, 9 (4): 245-263. [21] Lukaczer D, Darland G, Tripp M, et al. Clinical effects of a proprietary combination isoflavone nutritional supplement in menopausal women: a pilot trial [J]. Altern Ther Health Med, 2005, 11 (5): 60-65.
[22] Schult TM, Ensrud KE, Blackwell T, et al. Effect of isoflavones on lipids and bone turnover markers in menopausal women [J]. Maturitas, 2004, 48 (3): 209-218. [23] Haines C, James A, Sahota D, et al. Comparison between phytoestrogens and estradiol in the prevention of atheroma in ovariectomized cholesterol-fed rabbits [J]. Climacteric, 2006, 9 (6): 430-436. [24] Howes JB, Sullivan D, Lai N, et al. The effects of dietary supplementation with isoflavones from red clover on the lipoprotein profiles of post menopausal women with mild to moderate hypercholesterolaemia [J]. Atherosclerosis, 2000, 152 (1): 143-147. [25] Khan N, Afaq F, Mukhtar H. Cancer chemoprevention through dietary antioxidants: progress and promise [J]. Antioxid Redox Signal, 2008, 10 (3): 475-510. [26] Win W, Cao Z, Peng X, et al. Different effects of genistein and resveratrol on oxidative DNA damage in vitro [J]. Mutat Res, 2002, 513 (1-2): 113-120.
Cite this articls as: CHEN Tong, ZHONG Fo-Jin, HONG Ya-Min, SU Wei-Jiao, ZHUANG Li-Li, QIU Long-Xin. Effect of Trifolium pratense extract on methionine-choline-deficient diet-induced steatohepatitis in C57BL/6 mice [J]. Chinese Journal of Natural Medicines, 2014, 12(3): 194-198
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Chinese Journal of Natural Medicines
Effect of Trifolium pratense extract on methionine-cholinedeficient diet-induced steatohepatitis in C57BL/6 mice CHEN Tong1, 2, ZHONG Fo-Jin1, HONG Ya-Min1, SU Wei-Jiao1, ZHUANG Li-Li 1, QIU Long-Xin1, 2* 1
School of Life Sciences, Longyan University, Longyan 364000, China;
2
Fujian Key Laboratory of Preventive Veterinary Medicine and Biotechnology, Longyan 364000, China Available online 20 Mar. 2014
[ABSTRACT] AIM: The potential of Trifolium pratense (red clover) extract in the prevention of lipid disorder has attracted increasing attention in recent years. In this study, the aim was to determine whether and how red clover extract affected the development of murine diet-induced non-alcoholic steatohepatitis. METHOD: Non-alcoholic steatohepatitis was induced in C57BL/6 mice by feeding mice with a methionine-choline-deficient (MCD) diet. Hematoxylin and eosin staining was used for histological analyses. Real-time PCR was used to analyze the mRNA expression levels. RESULTS: Hepatic steatosis and necroinflammation was observed in MCD diet-fed mice, and this diet-induced steatosis was significantly attenuated, whereas liver inflammation was not significantly attenuated, by red clover extract treatment. Consistent with the results of H&E staining, the MCD diet-induced increase of liver triglycerides and cholesterol levels were significantly reduced by red clover extract treatment. However, with the improvement in hepatic steatosis, mRNA levels of acetyl CoA oxidase, carnitine palmitoyl transferase-1, and liver fatty acid-binding protein, three genes regulated by peroxisome proliferator-activated receptor (PPAR) α, were unaffected. CONCLUSION: Red clover extract alleviated MCD diet-induced hepatic steatosis, but did not ameliorate liver inflammation in C57BL/6 mice, and the improvement in hepatic steatosis was not through activating PPARα. [KEY WORDS] Trifolium pratense; Steatohepatitis; Hepatic steatosis; Liver inflammation; Peroxisome proliferator-activated receptor α
[CLC Number] R965
[Document code] A
[Article ID] 2095-6975(2014)03-0194-05
Introduction The term “non-alcoholic steatohepatitis” (NASH) is used to describe the pathological and clinical features of non-alcoholic diseases of the liver associated with pathological features most commonly seen in alcoholic liver disease itself [1]. Features of NASH on liver biopsy include steatosis, inflammation, liver cell injury, and varying degrees of fibrosis. It is a more advanced form of non-alcoholic fatty liver disease [Received on] 14-Nov.-2012 [Research Funding] This project was supported by the Science and Technology Planning Project of Educational Commission of Fujian Province, China (No. JB12198) and Fujian Province, China (No. 2010N0023). [*Corresponding author] QIU Long-Xin: Ph.D., Tel/Fax: 86-5972793889, Email: [email protected] These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
(NAFLD). NASH is becoming a major public health problem. However, current treatments for NASH have side effects and do not prove to be efficient [2]. Thus, the requirement for alternative and natural medicines to prevent NASH has been increasing rapidly and considerably. The potential of dietary isoflavones in the prevention of NASH has attracted increased attention among the public and in the medical community in recent years [3]. Soy isoflavones were reported to be beneficial for preventing NAFLD in Sprague–Dawley rats [4], high-fat-diet fed C57BL/6J mice [5-7], and Wistar rats [8]. Whereas soy isoflavones are the most studied isoflavones used for the studies on NAFLD, very few data are available for the isoflavones of red clover, Trifolium pratense L. (Fabaceae). The isoflavones from red clover differ from those of soy. The principal isoflavones in red clover are biochanin A, formononetin, genistein, and diadzein while those in soy consist solely of genistein and diadzein. In a previous report from this laboratory, red clover extract reduced liver triglycerides in db/db mice [9], however, whether red clover
– 194 –
CHEN Tong, et al. / Chin J Nat Med, 2014, 12(3): 194−198
extract ameliorates steatohepatitis remained unclear. The four isoflavones from red clover extract (RCE) are reported to be the agonists of peroxisome proliferator-activated receptor (PPAR) α [10-12]. Since PPARα is a major lipid sensor and a transcriptional regulator for lipid metabolic enzymes in the liver [13], and some PPARα agonists are beneficial for alleviating NASH [14-15], it is of interest to study the effect of red clover extract on NASH. Methionine-choline-deficient (MCD) dietinduced steatohepatitis is a well established model for the study of NASH [15-16]. Thus, in this study, we wanted to examine the effect of red clover extract on the development of nutrition- induced murine NASH in C57BL/6 mice fed a MCD diet.
Materials and Methods Materials. Red clover extract was purchased from a common Chinese pharmacy (Rimian, Fuzhou, China) and was standardized to 10% isoflavones (consisting of 5.1% formononetin, 4.8% biochanin A, 0.16% genistein, and 0.04% daidzein). Animal experiments. C57BL/6 mice were obtained from the SLAC Laboratory (Shanghai, China). All animals were maintained under a 12/12 h light/dark schedule. Male mice, 7−8 weeks of age, were randomly divided into four experimental groups (each containing six animals): control diet, MCD diet, MCD diet + 50 mg·kg−1·d−1 red clover extract treated, MCD diet + 200 mg·kg−1·d−1 red clover extract treated. Red clover extract was administered orally in 0.5% sodium carboxymethyl cellulose (CMC) suspension and continued for 35 days. Serum biochemical measurements. At the end of the red clover extract treatment, mice were sacrificed and blood was collected from orbit. Serum alanine aminotransferase (ALT) was measured using a IDEXX analyser (Westbrook, ME, USA). Liver lipid analyses. Liver lipid was extracted by chloroform/methanol. Briefly, pulverized liver was homogenized in PBS, then extracted with chloroform/methanol (2:1), dried overnight and re-suspended in a solution of 60% butanol:40% Triton X-114/methanol (2:1). Liver triglycerides (TG) and cholesterol levels were measured using colorimetric assays (Jianchen, Nanjing, China). Quantitative analyses of mRNA expression by real-time PCR. Total RNA was isolated from tissues using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. Complementary DNA (cDNA) was synthesized from hepatic mRNA using RevertAid First Strand cDNA Synthesis kits (Fermentas, Vilnius, Lithuania). Hepatic PPARα, acetyl CoA oxidase (ACO), carnitine palmitoyl transferase-1 (CPT-1), liver fatty acid- binding protein (L-FABP) mRNA were analyzed with the specific primers listed in Table 1. Real-time polymerase chain reactions were assayed using the FastStart Universal SYBR Green Master (Rox) (Roche Applied Science, Mannheim, Germany). Each Ct value was normalized to β-actin. Histological examination. Formalin-fixed liver tissue was processed and 5 μm-thick paraffin sections were stained with hematoxylin and eosin (H&E) for histological analyses. Steatosis and inflammation on all sections was blindly as-
Table 1 Primer sequences used for amplification of mRNA by real-time PCR Gene PPARα ACO CPT-1 L-FABP β-actin
F R F R F R F R F R
Sequences 5’-AAGAGGGCTGAGCGTAGGT-3’ 5’-GGCCGGTTAAGACCAGACT-3’ 5’-CCACATATGACCCCAAGACC-3’ 5’-AGGCATGTAACCCGTAGCAC-3’ 5’-GTCAAGCCAGACGAAGAACA-3’ 5’-CGAGAAGACCTTGACCATAG-3’ 5’-GTGGTCCGCAATGAGTTCAC-3’ 5’-GTATTGGTGATTGTGTCTCC-3’ 5’-CTATTGGCAACGAGCGGTTCC-3’ 5’-GCACTGTGTTGGCATAGAGGTC-3’
sessed by a hepatopathologist as previously described [17]. Hepatic steatosis was graded according to the percentage of lipid-laden hepatocytes as 0: 0%, 1: 0%–33%, 2: 33%–67%, 3: 67%–100%. Hepatic necroinflammation was given a score from 0 to 3, as follows: 0: no inflammatory foci, 1: mild, 2: moderate, and 3: severe. Statistical Analysis. Quantitative data are expressed as mean ± SEM. Students’ t test was used for pairwise comparisons and One-way ANOVA with Newman-Keuls Multiple Comparison Test for multigroup analyses. Differences were considered significant when probability values were less than 0.05.
Results Red clover extract attenuated diet-induced hepatic steatosis significantly, but did not ameliorate liver inflammation. Mice fed the MCD diet are a useful small animal model of progressive NAFLD [15-16]. Feeding C57BL/6 mice a MCD diet induces NASH and liver fibrosis within 4-8 weeks. To investigate the effect of red clover extract treatment on the development of MCD diet-induced steatohepatitis, mice fed the MCD diet were treated with red clover extract for 5 weeks. As shown in Fig. 1 and Table 2, examination of
Fig. 1 Effect of red clover extract on MCD diet-induced hepatic steatosis and necroinflammation. Hematoxylin and eosin–stained liver sections from mice fed: (a) Control diet; (b) MCD diet; (c) MCD diet + 50 mg·kg−1·d−1 red clover extract treated; and (d) MCD diet + 200 mg·kg−1·d−1 red clover extract treated. Liver shows foci of necroinflammation (arrows) and macrovesicular fat droplets. Slides are representative of four separate experiments
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CHEN Tong, et al. / Chin J Nat Med, 2014, 12(3): 194−198 Table 2 Effect of red clover extract on scores for hepatic steatosis and necroinflammation (mean ± SEM, n = 4) Measurements Controls Steatosis 0.00 ± 0.00 Necroinflammation 0.00 ± 0.00 * P < 0.05 vs MCD diet fed mice
MCD + 50 mg·kg−1 RCE 1.53 ± 0.29 1.13 ± 024
MCD 1.90 ± 0.25 1.25 ± 0.35
H&E-stained liver sections demonstrated marked steatosis and lobular inflammation in mice fed the MCD diet, while mice fed the control diet did not exhibit significant histological steatosis and inflammation. Co-administered with 200 mg/kg/day red clover extract for five weeks, steatosis in mice fed the MCD diet was attenuated significantly (P < 0.05). However, red clover extract treatment did not improve the lobular inflammation in mice fed the MCD diet. Consistent with the histological findings, red clover extract treatment did not result in a significant decrease in serum ALT levels in mice fed the MCD diet (Fig. 2), while it significantly decreased the level of total liver triglycerides (Fig. 3A; 103.70 ± 3.16 mg·g−1 tissue for mice fed the MCD diet versus 78.01 ± 8.13 mg·g−1 tissue for mice fed the MCD diet + 200 mg·kg−1·d−1 RCE, P < 0.05). Moreover, red clover extract treatment resulted in a significant decrease in liver cholesterol levels in mice fed the MCD diet (Fig. 3B; 16.34 ± 1.02 mg·g−1 tissue for mice fed MCD diet versus 9.39 ± 1.30 mg·g−1
MCD + 200 mg·kg−1 RCE 0.85 ± 0.13* 1.00 ± 0.22
tissue for mice fed the MCD diet + 200 mg·kg−1·d−1 RCE, P < 0.01). Together, these data indicate that red clover extract treatment is beneficial for the improvement of MCD dietinduced hepatic steatosis, but not for the improvement of MCD diet-induced liver inflammation.
Fig. 2 Effect of MCD diet and red clover extract (RCE) treatment on serum ALT levels in C57BL/6 mice (means ± SEM, n = 6). **P < 0.01
Fig. 3 Effect of red clover extract treatment on serum and liver lipid profiles in C57BL/6 mice (means ± SEM, n = 6). Red clover extract (RCE) treatment significantly decreased the liver TG level (A) and cholesterol level (B) in MCD diet-fed C57BL/6 mice. *P < 0.05; **P < 0.01; ***P < 0.001
Red clover extract did not affect the hepatic expression of some PPARα target genes involved in the development of steatosis. To clarify the mechanism whereby red clover extract exerts a beneficial effect on the development of dietinduced steatosis, the effects of red clover extract on hepatic mRNA expression of PPARα and its target genes, ACO, L-FABP, and CPT-1 were investigated. As shown in Fig. 4, intake of the MCD diet resulted in a marked decrease in ACO and L-FABP mRNA expression, compared with that of the control diet, whereas the mRNA expression of PPARα and CPT-1 were not affected significantly. However, the decrease in ACO and L-FABP mRNA expression was not recovered significantly by treating the MCD diet-fed mice with red clover extract. These data suggest that red clover extract does not activate hepatic PPARα, the effect of red clover extract on MCD diet-induced steatosis is therefore not through the activation of PPARα.
Discussion Red clover extract has been the focus of many studies during the last decade due to the protective effects against menopausal symptoms and a variety of disorders, including cardiovascular disease, cancer, and osteoporosis, etc [18]. Additionally, the potential of red clover extract in the prevention of lipid disorders has attracted increased attention. However, there are some debates about the hypolipidemic effects of red clover isoflavones under different disease conditions. Several studies reported the effect of red clover extract on improving lipid profile [19-22], whereas other studies did not show such an effect [23-24]. A previous report demonstrated that red clover extract reduced liver triglycerides and cholesterol in type 2 diabetic db/db mice [9]. In this report, it was demonstrated that red clover extract reduced liver triglycerides and cholesterol
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liver inflammation in animals with NASH. These data suggest the uncertainness of red clover extract as an anti-inflammatory drug.
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Fig. 4 Effect of red clover extract (RCE) treatment on hepatic mRNA levels of genes involved in hepatic steatosis (means ± SEM, n = 4). Hepatic mRNA levels were assessed using reverse transcription-real time PCR, standardized against an internal control (β-actin) and are expressed as fold differences compared with values obtained in mice fed the control diet. **P < 0.01
in mice with NASH. This present report further confirms the lipid-disorder-correcting effect of red clover extract. PPARα is an important metabolic nuclear receptor that regulates lipid metabolism through direct transcriptional control of the genes involved in peroxisomal and mitochondrial β-oxidation pathways, fatty acid uptake, and TG catabolism, etc. [13]. Hepatic activation of PPARα by its agonists, such as WY 14,643, decreases hepatic steatosis in MCD diet fed C57BL/6 mice [14-15]. In this report, however, the mRNA expression of ACO, CPT-1, and L-FABP, three PPARα regulated genes, was not altered by red clover extract treatment. This in vivo result is incompatible with some other previous in vitro reports. In those reports, red clover extract and its isoflavones were demonstrated to be agonists of PPARα [10-11]. This current study failed to demonstrate the PPARα agonist activity of red clover extract in mice with NASH. The mechanism whereby red clover extract ameliorates hepatic steatosis remains to be further studied. Red clover extract has been demonstrated to have anti-inflammatory properties in vitro [10-11]. It suppressed the lipopolysaccharide-induced secretion of proinflammatory cytokines, TNF-α and IL-6, in macrophage RAW264.7 cells. Moreover, isoflavones were reported to possess antioxidant and radical scavenging activities [25-26]. The anti-inflammatory, antioxidant and radical scavenging activities are beneficial for improving steatohepatitis. Surprisingly, in this report, it was demonstrated that red clover extract could not prevent liver inflammation. Thus, further in vivo studies are needed to investigate the anti-inflammatory effects of red clover extract under different inflammation conditions. In conclusion, the present study demonstrated the hepatic lipid lowering effect of red clover extract in animals with NASH, and that the effect was not achieved by activating PPARα. Moreover, red clover extract could not ameliorate the
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Cite this articls as: CHEN Tong, ZHONG Fo-Jin, HONG Ya-Min, SU Wei-Jiao, ZHUANG Li-Li, QIU Long-Xin. Effect of Trifolium pratense extract on methionine-choline-deficient diet-induced steatohepatitis in C57BL/6 mice [J]. Chinese Journal of Natural Medicines, 2014, 12(3): 194-198
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