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6J mice with high-fat diet-induced type 2 diabetes
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Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso and Trigonella foenum-graecum L. on liver fat accumulation in C57BL/6J mice with high-fat diet-induced type 2 diabetes Nawel Hamza a,b,n, Bénédicte Berke a, Catherine Cheze a, Sébastien Marais d, Simon Lorrain a, Abdelilah Abdouelfath a, Regis Lassalle a, Dominique Carles e, Henri Gin c, Nicholas Moore a,n a
Département de Pharmacologie, Université de Bordeaux, Bordeaux, France Département de Nutrition, Université des frères Mentouri, INATAA, Constantine, Algerie Service de Nutrition Diabétologie et Maladies Métaboliques, CHU de Bordeaux, Haut-Levèque, Bordeaux, France d Bordeaux Imaging Center, UMS 3420 CNRS, Université de Bordeaux, Bordeaux, France e Unité de pathologie fœtoplacentaire, Université de Bordeaux, CHU de Bordeaux, Hôpital Pellegrin, Bordeaux, France b c
art ic l e i nf o
a b s t r a c t
Article history: Received 13 March 2015 Received in revised form 8 May 2015 Accepted 16 May 2015
Ethnopharmacological relevance: Centaurium erythraea Rafn (CE), Artemisia herba-alba Asso (AHA) and Trigonella foenum-graecum L. (TFG) are traditionally used to treat type 2 diabetes in Algeria, previous studies have found that extracts of these plants were effective to treat or prevent experimental diabetes induced by high-fat diet (HFD). Aim of the study: Describe the additional effects of these extracts on lipid tissue deposition in HFD. Materials and methods: Male C57BL/6J mice were fed with HFD to induce type 2 Diabetes. Groups of mice were given plant extracts orally at 2 g/kg/bodyweight daily for 20 weeks during establishment of diabetes, or for 18 weeks after confirmation of diabetes at the 17th week. Liver and other tissue samples were stained with Oil Red O. Results: Liver steatosis was confirmed with HFD. CE, AHA and TFG extracts improved liver steatosis by the end of the preventive (20 weeks) and curative periods (35 weeks). This was most marked for CE extract (po0.05), less so with TFG and AHA. No steatosis was found in other tissues. Conclusion: CE extract had a clear hepatoprotective effect in this mouse model of diet-induced type 2 diabetes. AHA and TFG had a minimal or no significant effect on steatosis. Beyond its effect as an antidiabetic agent, CE may also be promising to prevent or treat non-alcoholic liver steatosis. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Type 2 diabetes/therapy/prevention Non-alcoholic liver steatosis Artemisia herba-alba Centaurium erythraea Trigonella foenum-graecum High fat diet Mice
1. Introduction In Algeria, a wide range of medicinal plants are used in folk medicine for the treatment of different disease and many are used for the treatment of diabetes such as Centaurium erythraea Rafn (CE) or Mraret lahnach, Artemisia herba-alba Asso (AHA) or Chih
Abbreviations: HFD, High fat diet; TFG, Trigonella foenum-graecum; AHA, Artemisia herba-alba Asso; CE, Centaurium erythraea Rafn; HDL-cholesterol, High-density lipoprotein; HOMA, Homeostasis model assessment; STD, Standard diet; NASH, Non alcoholic fatty liver disease; i.p, Intraperitoneal administration; OR, Odds ratio; TG, Triglycerides; ADP, Adenosine 50 -diphosphate n Corresponding author at: Département de Pharmacologie, INSERM U657, Université de Bordeaux, Bât 1A, Carreire Zone Nord, Case 36, 33076 Bordeaux Cedex, France. Tel.: þ33 5 57 57 15 60; fax: þ 33 5 57 57 46 71. E-mail addresses: [email protected] (N. Hamza), [email protected] (N. Moore).
and Trigonella foenum-graecum L. (TFG) or Halba (Helba) as they are commonly named in Algeria (Hamza et al., 2010, 2011, 2012). C. erythraea Rafn (CE) has been used in traditional medicine for its depurative, sedative, antipyretic and anti-inflammatory effects (Kumarasamy et al., 2003; Valentao et al., 2001, 2002). CE is also used in the treatment of diabetes (Hamza et al., 2010, 2011; Stefkov et al., 2014). A. herba-alba Asso (AHA) is also used for the treatment of diabetes, for its antihyperglycemic (Al-Khazraji et al., 1993; Al-Shamaony et al., 1994; Hamza et al., 2010; Mansi et al., 2007; Marrif et al., 1995; Stefkov et al., 2014; Testekin et al., 2007) and hypoglycemic effect (Hamza et al., 2011). T. foenum-graecum L. (TFG) has been traditionally used in the treatment of diabetes but most models on which TFG was tested were experimentally induced by streptozotocin or alloxan, which results in models closer to type 1 diabetes than the type 2 diabetes that is the most prevalent in human adults (Abdel-Barry et al., 1997; Ajabnoor and Tilmisany, 1988; Broca et al., 1999; Hannan et al., 2007; Khosla et
http://dx.doi.org/10.1016/j.jep.2015.05.027 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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al., 1995; Mowla et al., 2009; Vats et al., 2003; Zia et al., 2001). We have tested TFG during development of high fat diet induced diabetes and in mice with established diabetes (Hamza et al., 2010, 2011). TFG has also been reported to have antioxidant properties (Anuradha and Ravikumar, 2001; Belguith-Hadriche et al., 2010; Dixit et al., 2005) and hypocholesterolaemic effect (BelguithHadriche et al., 2010; Hamza et al., 2012; Hannan et al., 2007; Valette et al., 1984). In addition to the development of diabetes mellitus, high-fat diet results in dyslipidemia and tissue steatosis, especially potentially severe non-alcoholic liver steatosis. In addition, there is a link between the presence of type 2 diabetes and the severity of the liver injury (Hickman and Macdonald, 2007). Non alcoholic fatty liver disease or steatohepatitis (NASH) is characterized by fat accumulation in the liver, which can progress to hepatic cirrhosis (Agro et al., 2009). The worldwide prevalence of non-alcoholic fatty liver disease continues to increase and corresponds to the frequency of the systemic complex known as metabolic syndrome (Farrell and Larter, 2006). NASH is increasingly recognized as the hepatic manifestation of insulin resistance and metabolic syndrome (Marchesini et al., 2001, 2003). Dixon et al. (2001) showed that insulin resistance and systemic hypertension, features of the metabolic syndrome, are independently associated with advanced forms of NASH. At present, no efficient remedy for NASH exists. Treatment is still centered on lifestyle changes, weight loss, healthy eating and exercise (Caldwell and Lazo, 2009). There are no reports on the effects of herbal therapies on experimental NASH. In a previous study we found that extracts of C. erythraea Rafn (CE), A. herba-alba Asso (AHA) and T. foenum-graecum L. (TFG) could prevent and oppose the development of high fat dietinduced diabetes in C57BL/6J mice (Hamza et al., 2010, 2011, 2012). We report here the effects of diet and treatment on liver and tissue histology and lipid deposits in these HFD diabetic mice.
2. Materials and methods 2.1. Plant extracts The air-dried parts of plants were botanically authenticated, and voucher specimens (NH-H 21, C. erythraea; NH-H 07, A. herbaalba; NH-H10, T. foenum-graecum) have been deposited in the Nutrition Department (Constantine 1 University, Algeria). Hydroethanolic plant extracts of C. erythraea Rafn (CE), A. herba-alba Asso (AHA) and T. foenum-graecum L. (TFG) were prepared as previously reported (Hamza et al., 2010, 2011, 2012). In brief, the dried aerial parts of CE, AHA and dried seeds of TFG were homogenized to a fine powder. Extracts were prepared as follows: the powdered plants were soaked in water–ethanol (2/8, v-v) solution (1:5, plant weight:solvent volume) for 24 h, then the solution was filtered, solvent replaced and the maceration renewed for another 24 h. After filtration, the filtrate was concentrated under reduced pressure at 50 1C and then freeze-dried. 2.2. Study design Ten groups of 10 male C57BL/6J mice (4 weeks old at the start of the study) were studied, five for the preventive treatment study (weeks 0–20), and five for the curative study (weeks 17–35). In each part, a control groups was fed standard diet (STD); and four groups of animals were fed a high fat diet (HFD): one group was fed HFD alone and the three other groups were fed HFD and treated daily with plant extracts CE, AHA and TFG at the dose of 2 g/kg bodyweight distilled in water via intragastric intubation. Extracts were administered from the beginning of the study to week 20 in the
preventive study and from the 17th week to the 35th week of study in the curative group (Hamza et al., 2010, 2011, 2012). Principles of laboratory animal care (Guide for the Care and Use of Laboratory Animals), published by the US National Institutes of Health (NIH Publication 85-23 revised 1996) were followed. 2.3. Biological sample collection and surgical procedure After 4 h of fasting, the mice were anaesthetized using pentobarbitone (100 ml/100 mg i.p.) repeated if necessary and then sacrificed by cardiac puncture. Immediately after death, the abdomen was opened by a midline incision and the liver, kidney and muscle were quickly removed, harvested and weighed. Livers, kidney and skeletal muscle were then prepared for histological examination. 2.4. Histological examination of mice liver, kidney and skeletal muscle Livers were weighed and dissected. Liver samples from different lobes were assembled, coded and sections embedded in the OCT medium (medium for frozen section), and immediately flash frozen in isopentane cooled in liquid nitrogen. They were then stored at 80 1C until further use. Organs were cut into 5-mm thick sections (Leica CM1850 UV microtome), mounted on glass slides and stained with Oil red O. The sections were counterstained with Mayer's hematoxylin. Steatosis was analyzed by light microscopy. The kidneys were rapidly excised longitudinally. One longitudinal section was prepared as above. One fragment of the right quadriceps muscle, of about 10 5 mm2 in size was immediately placed in liquid nitrogen and then stored at 80 1C until further use. Each stained liver, kidney and muscle section was analyzed for the severity of hepatocytes, kidney and intramyocellular fat accumulation. The extent of hepatocyte lipid accumulation was then scored based on the percentage of hepatocytes that contained macrovesicular fat. Macrovesicular steatosis was graded 0–3 based on percent of hepatocytes in the biopsy involved (grade 0: none or absent; 1: less than 33%; 2: 33–66%; 3: more than 66%) (Kleiner et al., 2005); zonal distribution of steatosis and the presence of macrovesicular or microvesicular steatosis were noted. The pathological changes were evaluated and photographed using an Eclipse 50i Nikon optical microscope controlled by the software Nickon-NiSelements D. Histological evaluation included a semi-quantitative and quantitative analysis of the presence of micro and macrovesicular fat. All sections were coded and analyzed blindly by the pathologist without knowledge of related characteristics or diet (semi-quantitative analysis). The area of Oil red O-stained fatty droplets to the total tissue area was calculated (quantitative analysis) by using the analysis software ImageJ (ImageJ, US National Institutes of Health, Bethesda, Maryland, USA). The results of the histological analysis were confronted to the biochemical results (blood glucose, plasma triglyceride, total cholesterol, HDL-cholesterol, insulin and HOMA) obtained in the same animals in our previous studies (Hamza et al., 2010, 2011, 2012). 2.5. Statistical analysis Data analysis was conducted after database lock using SASs software (SAS Institute, Version 9.4, North Carolina, USA). Descriptive variables are presented as mean and standard deviation (SD). Comparison between mice groups were performed using Kruskal– Wallis test. The significant multiple comparisons tests between each
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Fig. 1. Liver histology in the preventive study. Representative Oil red O staining of C57BL/6J liver tissue from high-fat diet (HFD) induced diabetes. The standard diet STD control group (panel A; 1A) received 20 weeks of standard diet and had normal histology. The HFD control group (panel B; 1B) received high-fat diet during 20 weeks and had steatosis without ballooning, inflammation, necrosis or fibrosis. Treatment with plant extracts during 20 weeks (TFG (panel C; 1C), CE (panel D; 1D), AHA (panel E; 1E)), at a dose of 2 g/kg bodyweight, displayed mild, minor or absent steatosis. Micrometer scale on the left side of the figure (1 mark is 100 mm). All magnification, 20.
pair of groups of semi-quantitative and quantitative analysis of steatosis, blood glucose, triglyceride, liver weight and insulin resistance were performed by Fisher's test with Bonferroni correction. A multivariate ANOVA test was performed to examine association with steatosis and mice groups adjusted for blood glucose, triglyceride, liver weight and insulin resistance. Data in untreated HFD mice was compared to the results in STD control mice; data in treated HFD mice were compared to the results in untreated HFD mice. Statistical significance was set at p o0.05.
which appeared to localize in periportal hepatocytes. There was no liver fibrosis and no inflammation. In the groups treated with TFG, CE and AHA extracts, morphological abnormalities were less marked than in the HFD group. The extent of steatosis was considerably reduced, histology was normal or with minimal periportal lipid accumulation (Figs. 1C–E and 2C–E), seen as microvesicules of fat within periportal hepatocytes. Compared to the HFD group, the lipid drops in liver of treated groups had a lighter color and smaller volume.
3. Results
3.1.1. Semi quantitative assessment of liver fat (visual scoring) 3.1.1.1. Preventive study. Treatment during the establishment of obesity and diabetes. The semi quantitative data of liver fat accumulation is shown in Table 1. HFD group had grade 2 (50%) or 3 (30%) macrovesicular steatosis. The groups treated with TFG, CE and AHA had lower grades of liver fat compared to HFD group Table 1 and Fig. 1. The effect was most marked with CE, with no grade 3 deposition. There was a modest decrease in liver fat in treated groups but the difference were not significant (p4 0.05).
3.1. Histological finding of the liver The STD group had normal hepatic histology (Figs. 1 and 2A). The continuous consumption of high-fat diet for 8 months resulted in macrovesicular hepatic steatosis, with periportal hepatocytes lipid accumulation (Figs. 1 and 2B). There was widespread deposition of fat globules of different size inside parenchymal cells. The whole surface was occupied by macrovesicles of fat,
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Fig. 2. Liver histology in the curative study. Representative Oil red O staining of C57BL/6J liver tissues from high-fat diet (HFD) induced diabetes. The STD control group (panel A; 2A) received 32 weeks of standard diet and showed normal histology. The HFD control group (panel B; 2B) received high-fat diet during 32 weeks and revealed severe steatosis without ballooning, inflammation, necrosis or fibrosis. Treatment with plant extracts for 15 weeks (TFG (panel C; 2C), CE (panel D; 2D), AHA (panel E; 2E)), at a dose of 2 g/kg bodyweight starting at the 17th week of HFD, displayed mild, minor or absent steatosis. Micrometer scale on the left side of the figure (1 mark is 100 mm). All magnification, 20.
Table 1 Liver histology scoring (visual scoring) for macrovesicular steatosis in C57BL/6J mice after 20 weeks. Steatosis
STD control HFD control CE-treated HFD AHA-treated HFD TFG-treated HFD
n
0
1
2
3
10 10 9 9 8
8 2 6 3 5
1 0 1 3 1
1 5 2 0 1
0 3 0 3 1
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum.
3.1.1.2. Curative study. Treatment after establishment of obesity and diabetes. In livers from HFD group the whole surface was occupied by fat macrovesicles. HFD group had two-fold (100% grade of 3) greater
lipid accumulation than the normal control group and CE-treated group (p o0.05). Livers from groups treated with TFG and AHA had a sparse distribution of small microvesicules (Table 2, Fig. 1C and E and Fig. 2Cand E). There was no inflammation except for 2 mice in the HFD group. 3.1.2. Quantitative assessment of liver fat (ImageJ scoring) 3.1.2.1. Preventive study. Liver sections in the STD control group showed normal morphological features. The tissues of HFD group had macrovesicular steatosis of grade 0 (20%) or grade 1 (80%) (Table 3 and Fig. 1A and B). The groups treated with TFG, CE and AHA had lower grades of steatosis compared to HFD group but the differences were not significant (p 40.05). 3.1.2.2. Curative study. In the liver sections from mice in the HFD group, more than one-third of hepatocytes contained macrovesicles of fat, corresponding to grade 1 or 2 steatosis (Table 4 and Fig. 2B). Treatment with CE extract resulted in significant improvement compared with HFD alone (Table 4; po 0.05). There was also decreased liver fat in groups treated
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Table 2 Liver histology scoring (visual scoring) for macrovesicular steatosis in C57BL/6 J mice after 35 weeks.
STD control HFD controlb CE-treated HFDa AHA-treated HFDb TFG-treated HFDb
n
0
1
2
3
10 8 10 10 10
10 0 7 0 1
0 0 0 1 1
0 0 3 2 3
0 8 0 7 5
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum. a b
p o 0.05, vs. HFD control. p o0.05, vs. STD control.
Table 3 Liver histology scoring (ImageJ scoring) for macrovesicular steatosis in C57BL/6J mice after 20 weeks. Steatosis
STD controlb HFD control CE-treated HFD AHA-treated HFD TFG-treated HFD
n
0
1
2
3
10 10 10 9 8
10 2 8 6 5
0 7 2 2 2
0 1 0 1 1
0 0 0 0 0
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum. a p o 0.05, vs. STD control. b
p o0.05, vs. HFD control.
Table 4 Liver histology scoring (ImageJ scoring) for macrovesicular steatosis in C57BL/6J mice after 35 weeks. Steatosis
STD controla HFD control CE-treated HFDa AHA-treated HFDb TFG-treated HFD
n
0
1
2
3
10 8 10 10 10
10 0 7 0 1
0 0 0 1 1
0 0 3 2 3
0 8 0 7 5
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum. a b
was excellent in the preventive and curative studies (kappa coefficient ¼0.8244 and 0.6364 respectively). 3.2. Skeletal muscle and kidney
Steatosis
a
5
p o 0.05, vs. HFD control. p o0.05, vs. STD control.
with TFG and AHA but the differences were not significant (p 40.05). The concordance between semi-quantitative assessment of liver fat (visual scoring) and the quantitative assessment of liver fat (ImageJ scoring) was low in both the preventive and curative studies (kappa coefficient ¼0.3708 and 0.1753 respectively). However, when the steatosis grades were grouped the concordance
Histological analysis of muscle and kidney tissues did not show any deposition of droplets in either HFD or treated groups (Figs. 3 and 4A and B). Kidney and muscle sections showed normal morphological features in the controls and treated groups. 3.3. Biochemical parameters, liver weight and steatosis The specific treatment-related changes in blood chemistry have already been reported (Hamza et al., 2010, 2011, 2012). In brief, all three extracts were associated with decreased blood glucose and plasma lipids, and improved insulin sensitivity, both during the preventive and the curative parts of the study. Here we found no association of steatosis with blood glucose or TG in the preventive study, but there was an association between steatosis and liver weight (OR ¼2.86 in the visual scoring; OR¼9.97 in the ImageJ scoring). There was an association in both ImageJ scoring and visual scoring between steatosis and HOMA. When HOMA increased, the risk of steatosis greater than 0 increased (OR ¼1.06—p ¼0.018 in the visual scoring; OR ¼1.06— p¼ 0.014 in the ImageJ scoring). Liver weights in TFG, AHA and CEtreated groups in the preventive study were also lower than in the HFD group but the differences were not significant (respectively 1.6 70.5; 1.870.6; 1.770.5 vs. 1.970.4 g, p 40.05). In the curative study, blood glucose, TG, liver weight, and HOMA were not associated with the importance of steatosis (p 40.05). The liver weight increased significantly in the HFD groups and AHA-treated group compared with the STD group respectively (2.2 7 0.4; 2.3 70.6 vs. 1.5 70.2 g, p o0.05).
4. Discussion We have previously shown the preventive and anti-diabetic effect of AHA, CE and TFG on high fat diet induced diabetes in C57BL/6J mice, related to a reduction of insulin resistance (Hamza et al., 2010, 2011, 2012). Plant extracts also reduced the increased insulin, triglyceride and total cholesterol concentrations, as well as insulin-resistance seen in untreated HFD mice. In the present study we report on the effect of the plant extracts on liver steatosis, which was decreased by the extracts in both preventive and curative models, but to a greater extend by the CE extract than by AHA or TFG extracts. The effect of CE, AHA and TFG on the histological finding of liver steatosis in high fat diet induced diabetic mice had not yet been reported. It had been shown that a TFG extract protects the renal tissues against functional and morphologic injuries in rats with diabetes induced by both enriched diets and streptozotocin possibly via its antioxidant activity (Xue et al., 2011). We found no change in renal histology in our HFD mouse model. Musso et al. (2003) have shown that the dietary intake of NASH patients was richer in saturated fat and in cholesterol, and was poorer in polyunsaturated fat, fiber and the anti-oxidant vitamins C and E. An association was found between saturated fat intake and insulin sensitivity, suggesting that dietary habits may promote NASH directly by modulating hepatic triglyceride accumulation and inflammatory activity, as well as indirectly by affecting insulin sensitivity. Toshimitsu et al. (2007) have confirmed these data, showing that in NASH patients, in comparison to simple fatty liver, there is an excess intake of carbohydrates, a low intake of protein and zinc, and lower ratio of intake of polyinsaturated fatty acid to
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Fig. 3. Kidney histology. Photomicrographs of kidney sections in the preventive study (20 weeks): STD control group (panel A; 3A); HFD control group (panel B; 3B); There was no measurable steatosis in the HFD group, nor in the treatment groups (not shown). Aspects at 32 weeks (curative) were identical (not shown). Micrometer scale on the left side of the figure (1 mark is equivalent to 100 mm). All magnification, 20.
Fig. 4. Quadriceps muscle histology. Photomicrographs of muscle sections in the preventive study (20 weeks): STD control group (panel A; 4A); HFD control group (panel B; 4B); There was no measurable steatosis in the HFD group, nor in the treatment groups (not shown). Aspects at 32 weeks (curative) were identical (not shown). Micrometer scale on the left side of the figure (1 mark is 100 mm). All magnification, 20.
saturated fatty acid. Experimental models suggest that a diet richer in defined carbohydrates, especially fructose, is able to promote steatosis accumulation and NASH progression (Bergheim et al., 2008; Tetri et al., 2008). Additionally, beside liver, skeletal muscle is another insulinresponsive tissue and a very strong relationship between insulin resistance and muscle lipid content has been reported (Laybutt et al., 1999). Several studies have shown that the most common form of insulin resistance is associated with an accumulation of visceral fat (Slawik and Vidal-Puig, 2006). In this study, there was no accumulation of intramyocellular lipids, maybe because of the disease model used. In morbidly obese patients, insulin resistance in muscle might occur at a late point in the insulin signaling cascade and be associated with intramyocellular lipids and nonalcoholic fatty liver disease severity (Machado et al., 2012). In our high-fat diet mice, liver steatosis developed alongside diabetes, which may resemble NASH, though there were no signs of liver inflammation at this stage. CE treatment reduced the development of liver steatosis by 70% in the preventive study. In addition, after the experimental diabetes and NASH had developed (curative approach), CE extract reversed the steatosis, despite the continued intake of the high fat diet. CE extract reduced the severity of hepatic macrovesicular steatosis and triglyceride accumulation, in addition to the reduction of hypertriglyceridaemia and glycemia and improvement of insulin sensitivity (reduced HOMA-IR). In contrast, TFG and AHA had minimal or no significant effect on steatosis. The mechanisms by which CE reduces lipid accumulation in liver are unclear. One of several possible mechanisms may be related to the reduction of plasma triglyceride and cholesterol concentration. We have shown in previous studies that plant
extracts improved hypercholesterolemia and hypertriglyceridemia and now we show that extracts improve liver steatosis (Hamza et al., 2010, 2011, 2012). Accumulation of fat in the liver can occur because of increased delivery of free fatty acids to the liver, increased synthesis of fatty acids in the liver, or decreased betaoxidation of free fatty acids, which may, in turn, cause fat accumulation in the liver. Fat in hepatocytes results in cellular dysfunction and may damage the liver parenchyma (Shelh et al., 1997). Hypertriglyceridemia is a characteristic lipid abnormality in obesity and type 2 diabetes and has been linked to the development of fatty liver in obesity (Assy et al., 2000; Osno et al., 1995). In obesity, accumulation of triglycerides in the liver has been suggested to occur in response to an increased flux of fatty acids to the liver from dietary sources, from adipose tissue or from increased endogenous synthesis of fatty acids (Fong et al., 2000). Excessive ingested fat and metabolic syndrome conditions, such as obesity, insulin resistance, and type 2 diabetes, can result in increased free fatty acids levels in the blood. In metabolic syndrome, the lipolysis of lipids in adipose tissue increases. As a result, the delivery of free fatty acids to the liver is amplified (Clark and Diehl, 2002). Excess carbohydrates consumption can also result in free fatty acids overload in hepatocytes through de novo synthesis, perhaps magnified by the use of fructose (Neuschwander-Tetri, 2010). There is also quite a strong association of NASH with type 2 diabetes (Angulo, 2002; Clark and Diehl, 2002; Kelley et al., 2003) and insulin resistance (Dixon et al., 2001). Insulin resistance may contribute to the development of hepatocyte steatosis by impairing the ability of insulin to suppress lipolysis, resulting in increased circulating free fatty acid and liver lipid accumulation
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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(Day and Saksena, 2001; Marchesini et al., 2001). The progression from steatosis to NASH is usually a result of a combination of insulin resistance, oxidative stress and inflammation the two latter being likely a consequence of insulin resistance (Das and Kar, 2005; Malhi and Gores, 2008). Uygun et al. (2000) concluded that leptin might contribute to hepatic steatosis by promoting insulin resistance and also by altering insulin signaling in hepatocytes, thereby promoting increased intracellular fatty acids. Plant extracts (CE, AHA and TFG) restored several metabolic parameters associated with steatosis (Hamza et al., 2010, 2011, 2012). This was the case with insulin resistance, as attested by the normalization of HOMA. The decrease in plasma lipid concentration may also have contributed to restoring insulin sensitivity in treated groups. However improvement in HOMA was not associated with reduced steatosis with TFG and AHA, only with CE. It is not clear which components(s) present in CE, AHA or TFG are responsible for the observed effects. The bioactivity of plant extracts may be associated with polyphenols. The benefits of polyphenols in suppressing TG accumulation in hepatocytes have been also reported in previous studies. Wu et al. (2011) proved that rutin can significantly reduce lipid accumulation and had a good antioxidant capacity in an oleic acid-induced in vitro model of fatty liver by decreasing lipogenesis and oxidative stress in hepatocytes. The benefit of phenolic compounds on preventing steatosis has also been observed. Kimura et al. (1985) showed that the extracts from leaves of Artemisia species, as well as caffeic acid and chlorogenic acid inhibit liver injury and lipid metabolism in peroxidized corn oil-fed rats. Previously, the same authors reported that in vitro caffeic acid and its derivatives inhibited the production of lipid peroxide induced by ADP and NADPH in liver microsomes and by ADP and ascorbic acid in mitochondria. Coffee and two of its components (polyphenol and melanoidin) can reduce fat accumulation, improve inflammation in rat model of HFD induced steatohepatitis. This result may be associated with chlorogenic acid (Vitaglione et al., 2010). It has been reported that chlorogenic acid improved glucose tolerance and decreased plasma and hepatic lipids without changing triglyceride concentration in adipose tissue in an animal model for obese type 2 diabetes (Rodriguez de Sotillo and Hadley, 2002). Cho et al. (2010) reported that chlorogenic and caffeic acids increase fatty acid β-oxidation activity and PPAR α expression in the liver of HFD mice. Hsu et al. (2009) indicated that o-coumaric acid in HFDinduced obesity impedes the development of hepatosteatosis by inhibiting the elevation of hepatic triacylglycerol and cholesterol levels. Hsu and Yen (2007) showed that the addition of gallic acid, a phenolic compound, decreased hepatic steatosis, serum parameters (triglyceride, phospholipid, total cholesterol, LDL-cholesterol) insulin, leptin and oxidative stress markers in HFD-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. Recently Stefkov et al. (2014) identified groups of bitter compounds (flavonoids, iridoids and xanthones) in the methanol extract of the aerial parts of the CE that may have an influence on the expressed antihyperglycemic effect in streptozotocin rats.
5. Conclusion Plant extracts improved liver steatosis in HFD mice. C. erythraea Rafn (CE) extract produced a more pronounced effect in reducing fat accumulation in liver. Our study clearly for the first time demonstrates that treatment with CE extract is effective in reversing steatosis in type 2 diabetes induced by a high fat diet in mice. CE may offer an interesting treatment of insulinoresistance and consequent steatosis and diabetes, and may provide a new
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therapeutic strategy to reduce fatty liver. A. herba-alba Asso (AHA) and T. foenum-graecum L. (TFG) have shown minimal or no effect on steatosis. What remains to be done is on one hand to identify the active compounds in CE involved in this effect, and on the other confirm these effects in patients suffering from type 2 diabetes and/or NASH.
Acknowledgments The authors are grateful to Joelle Amédée and Robin Siadous (INSERM Université Bordeaux, Inserm U1026, Bioingénierie Tissulaire (BioTis)) for the use of a microtome. The authors wish to acknowledge the Bordeaux Imaging Center for the use of image analysis system, Université de Bordeaux, for the use of the image analyzing system. The authors wish to acknowledge Paulette Bioulac and Jean Rosenbaum (INSERM 1053) for the use of their facilities. The authors would also like to thank Béatrice Mougenot, for her technical assistance. References Abdel-Barry, J.A., Abdel-Hassan, I.A., Al-Hakiem, M.H., 1997. Hypoglycaemic and antihyperglycaemic effects of Trigonella foenum-graecum leaf in normal and alloxan induced diabetic rats. J. Ethnopharmacol. 58, 149–155. Agro, C.K., Northup, P.G., AL-Osaimi, A.M., Caldwell, S.H., 2009. Systematic review of risk factors for fibrosis progresion in non-alcoholic steatohepatitis. J. Hepatol. 51, 371–379. Ajabnoor, M.A., Tilmisany, A.K., 1988. Effect of Trigonella foenum graceum on blood glucose levels in normal and alloxan-diabetic mice. J. Ethnopharmacol. 22, 45–49. Al-Khazraji, S.M., Al-Shamaony, L.A., Twaij, H.A., 1993. Hypoglycaemic effect of Artemisia herba alba. I. Effect of different parts and influence of the solvent on hypoglycaemic activity. J. Ethnopharmacol. 40, 163–166. Al-Shamaony, L., Al-Khazraji, S.M., Twaij, H.A., 1994. Hypoglycaemic effect of Artemisia herba alba. II. Effect of a valuable extract on some blood parameters in diabetic animals. J. Ethnopharmacol. 43, 167–171. Angulo, P., 2002. Nonalcoholic fatty liver disease. N. Engl. J. Med. 346, 1221–1231. Anuradha, C.V., Ravikumar, P., 2001. Restoration on tissue antioxidants by fenugreek seeds (Trigonella Foenum Graecum) in alloxan-diabetic rats. Indian J. Physiol. Pharmacol. 45, 408–420. Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., Minuk, G., 2000. Fatty infiltration of liver in hyperlipidemic patients. Dig. Dis. Sci. 45, 1929–1934. Belguith-Hadriche, O., Bouaziz, M., Jamoussi, K., El Feki, A., Sayadi, S., Makni-Ayedi, F., 2010. Lipid-lowering and antioxidant effects of an ethyl acetate extract of fenugreek seeds in high-cholesterol-fed rats. J. Agric. Food Chem. 58, 2116–2122. Bergheim, I., Weber, S., Vos, M., Kramer, S., Volynets, V., Kaserouni, S., McClain, C.J., Bischoff, S.C., 2008. Antibiotics protect against fructose-induced hepatic lipid accumulation in mice: role of endotoxin. J. Hepatol. 48, 983–992. Broca, C., Gross, R., Petit, P., Sauvaire, Y., Manteghetti, M., Tournier, M., Masiello, P., Gomis, R., Ribes, G., 1999. 4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties. Am. J. Physiol. 277, E617–623. Caldwell, S., Lazo, M., 2009. Is exercice an effective treatment for NASH? Known and unknown. Ann. Hepatol. 8, S60–S66. Cho, A.S., Jeon, S.M., Kim, M.J., Yeo, J., Seo, K.I., Choi, M.S., Lee, M.K., 2010. Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice. Food Chem. Toxicol. 48, 937–943. Clark, J.M., Diehl, A.M., 2002. Hepatic steatosis and type 2 diabetes mellitus. Curr. Diabetes Rep. 2, 210–215. Das, K., Kar, P., 2005. Non-alcoholic steatohepatitis. J. Assoc. Physicians India 53, 195–199. Day, C.P., Saksena, S., 2001. Non-alcoholic steatohepatitis: definitions and pathogenesis. J. Gastroenterol. Hepatol. 17 (3), S337–S384. Dixit, P., Ghaskadbi, S., Mohan, H., Devasagayam, T.P., 2005. Antioxidant properties of germinated fenugreek seeds. Phytother. Res. 19, 977–983. Dixon, J.B., Bhathal, P.S., O'Brien, P.E., 2001. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology 121, 91–100. Farrell, G.C., Larter, C.Z., 2006. Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 43, S99–S112. Fong, D.G., Nehra, V., Lindor, K.D., Buchman, A.L., 2000. Metabolic and nutritional considerations in nonalcoholic fatty liver. Hepatology 32, 3–10. Hamza, N., Berke, B., Cheze, C., Agli, A.N., Robinson, P., Gin, H., Moore, N., 2010. Prevention of type 2 diabetes induced by high fat diet in the C57BL/6J mouse by two medicinal plants used in traditional treatment of diabetes in the east of Algeria. J. Ethnopharmacol. 128, 513–518.
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jep
Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso and Trigonella foenum-graecum L. on liver fat accumulation in C57BL/6J mice with high-fat diet-induced type 2 diabetes Nawel Hamza a,b,n, Bénédicte Berke a, Catherine Cheze a, Sébastien Marais d, Simon Lorrain a, Abdelilah Abdouelfath a, Regis Lassalle a, Dominique Carles e, Henri Gin c, Nicholas Moore a,n a
Département de Pharmacologie, Université de Bordeaux, Bordeaux, France Département de Nutrition, Université des frères Mentouri, INATAA, Constantine, Algerie Service de Nutrition Diabétologie et Maladies Métaboliques, CHU de Bordeaux, Haut-Levèque, Bordeaux, France d Bordeaux Imaging Center, UMS 3420 CNRS, Université de Bordeaux, Bordeaux, France e Unité de pathologie fœtoplacentaire, Université de Bordeaux, CHU de Bordeaux, Hôpital Pellegrin, Bordeaux, France b c
art ic l e i nf o
a b s t r a c t
Article history: Received 13 March 2015 Received in revised form 8 May 2015 Accepted 16 May 2015
Ethnopharmacological relevance: Centaurium erythraea Rafn (CE), Artemisia herba-alba Asso (AHA) and Trigonella foenum-graecum L. (TFG) are traditionally used to treat type 2 diabetes in Algeria, previous studies have found that extracts of these plants were effective to treat or prevent experimental diabetes induced by high-fat diet (HFD). Aim of the study: Describe the additional effects of these extracts on lipid tissue deposition in HFD. Materials and methods: Male C57BL/6J mice were fed with HFD to induce type 2 Diabetes. Groups of mice were given plant extracts orally at 2 g/kg/bodyweight daily for 20 weeks during establishment of diabetes, or for 18 weeks after confirmation of diabetes at the 17th week. Liver and other tissue samples were stained with Oil Red O. Results: Liver steatosis was confirmed with HFD. CE, AHA and TFG extracts improved liver steatosis by the end of the preventive (20 weeks) and curative periods (35 weeks). This was most marked for CE extract (po0.05), less so with TFG and AHA. No steatosis was found in other tissues. Conclusion: CE extract had a clear hepatoprotective effect in this mouse model of diet-induced type 2 diabetes. AHA and TFG had a minimal or no significant effect on steatosis. Beyond its effect as an antidiabetic agent, CE may also be promising to prevent or treat non-alcoholic liver steatosis. & 2015 Published by Elsevier Ireland Ltd.
Keywords: Type 2 diabetes/therapy/prevention Non-alcoholic liver steatosis Artemisia herba-alba Centaurium erythraea Trigonella foenum-graecum High fat diet Mice
1. Introduction In Algeria, a wide range of medicinal plants are used in folk medicine for the treatment of different disease and many are used for the treatment of diabetes such as Centaurium erythraea Rafn (CE) or Mraret lahnach, Artemisia herba-alba Asso (AHA) or Chih
Abbreviations: HFD, High fat diet; TFG, Trigonella foenum-graecum; AHA, Artemisia herba-alba Asso; CE, Centaurium erythraea Rafn; HDL-cholesterol, High-density lipoprotein; HOMA, Homeostasis model assessment; STD, Standard diet; NASH, Non alcoholic fatty liver disease; i.p, Intraperitoneal administration; OR, Odds ratio; TG, Triglycerides; ADP, Adenosine 50 -diphosphate n Corresponding author at: Département de Pharmacologie, INSERM U657, Université de Bordeaux, Bât 1A, Carreire Zone Nord, Case 36, 33076 Bordeaux Cedex, France. Tel.: þ33 5 57 57 15 60; fax: þ 33 5 57 57 46 71. E-mail addresses: [email protected] (N. Hamza), [email protected] (N. Moore).
and Trigonella foenum-graecum L. (TFG) or Halba (Helba) as they are commonly named in Algeria (Hamza et al., 2010, 2011, 2012). C. erythraea Rafn (CE) has been used in traditional medicine for its depurative, sedative, antipyretic and anti-inflammatory effects (Kumarasamy et al., 2003; Valentao et al., 2001, 2002). CE is also used in the treatment of diabetes (Hamza et al., 2010, 2011; Stefkov et al., 2014). A. herba-alba Asso (AHA) is also used for the treatment of diabetes, for its antihyperglycemic (Al-Khazraji et al., 1993; Al-Shamaony et al., 1994; Hamza et al., 2010; Mansi et al., 2007; Marrif et al., 1995; Stefkov et al., 2014; Testekin et al., 2007) and hypoglycemic effect (Hamza et al., 2011). T. foenum-graecum L. (TFG) has been traditionally used in the treatment of diabetes but most models on which TFG was tested were experimentally induced by streptozotocin or alloxan, which results in models closer to type 1 diabetes than the type 2 diabetes that is the most prevalent in human adults (Abdel-Barry et al., 1997; Ajabnoor and Tilmisany, 1988; Broca et al., 1999; Hannan et al., 2007; Khosla et
http://dx.doi.org/10.1016/j.jep.2015.05.027 0378-8741/& 2015 Published by Elsevier Ireland Ltd.
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al., 1995; Mowla et al., 2009; Vats et al., 2003; Zia et al., 2001). We have tested TFG during development of high fat diet induced diabetes and in mice with established diabetes (Hamza et al., 2010, 2011). TFG has also been reported to have antioxidant properties (Anuradha and Ravikumar, 2001; Belguith-Hadriche et al., 2010; Dixit et al., 2005) and hypocholesterolaemic effect (BelguithHadriche et al., 2010; Hamza et al., 2012; Hannan et al., 2007; Valette et al., 1984). In addition to the development of diabetes mellitus, high-fat diet results in dyslipidemia and tissue steatosis, especially potentially severe non-alcoholic liver steatosis. In addition, there is a link between the presence of type 2 diabetes and the severity of the liver injury (Hickman and Macdonald, 2007). Non alcoholic fatty liver disease or steatohepatitis (NASH) is characterized by fat accumulation in the liver, which can progress to hepatic cirrhosis (Agro et al., 2009). The worldwide prevalence of non-alcoholic fatty liver disease continues to increase and corresponds to the frequency of the systemic complex known as metabolic syndrome (Farrell and Larter, 2006). NASH is increasingly recognized as the hepatic manifestation of insulin resistance and metabolic syndrome (Marchesini et al., 2001, 2003). Dixon et al. (2001) showed that insulin resistance and systemic hypertension, features of the metabolic syndrome, are independently associated with advanced forms of NASH. At present, no efficient remedy for NASH exists. Treatment is still centered on lifestyle changes, weight loss, healthy eating and exercise (Caldwell and Lazo, 2009). There are no reports on the effects of herbal therapies on experimental NASH. In a previous study we found that extracts of C. erythraea Rafn (CE), A. herba-alba Asso (AHA) and T. foenum-graecum L. (TFG) could prevent and oppose the development of high fat dietinduced diabetes in C57BL/6J mice (Hamza et al., 2010, 2011, 2012). We report here the effects of diet and treatment on liver and tissue histology and lipid deposits in these HFD diabetic mice.
2. Materials and methods 2.1. Plant extracts The air-dried parts of plants were botanically authenticated, and voucher specimens (NH-H 21, C. erythraea; NH-H 07, A. herbaalba; NH-H10, T. foenum-graecum) have been deposited in the Nutrition Department (Constantine 1 University, Algeria). Hydroethanolic plant extracts of C. erythraea Rafn (CE), A. herba-alba Asso (AHA) and T. foenum-graecum L. (TFG) were prepared as previously reported (Hamza et al., 2010, 2011, 2012). In brief, the dried aerial parts of CE, AHA and dried seeds of TFG were homogenized to a fine powder. Extracts were prepared as follows: the powdered plants were soaked in water–ethanol (2/8, v-v) solution (1:5, plant weight:solvent volume) for 24 h, then the solution was filtered, solvent replaced and the maceration renewed for another 24 h. After filtration, the filtrate was concentrated under reduced pressure at 50 1C and then freeze-dried. 2.2. Study design Ten groups of 10 male C57BL/6J mice (4 weeks old at the start of the study) were studied, five for the preventive treatment study (weeks 0–20), and five for the curative study (weeks 17–35). In each part, a control groups was fed standard diet (STD); and four groups of animals were fed a high fat diet (HFD): one group was fed HFD alone and the three other groups were fed HFD and treated daily with plant extracts CE, AHA and TFG at the dose of 2 g/kg bodyweight distilled in water via intragastric intubation. Extracts were administered from the beginning of the study to week 20 in the
preventive study and from the 17th week to the 35th week of study in the curative group (Hamza et al., 2010, 2011, 2012). Principles of laboratory animal care (Guide for the Care and Use of Laboratory Animals), published by the US National Institutes of Health (NIH Publication 85-23 revised 1996) were followed. 2.3. Biological sample collection and surgical procedure After 4 h of fasting, the mice were anaesthetized using pentobarbitone (100 ml/100 mg i.p.) repeated if necessary and then sacrificed by cardiac puncture. Immediately after death, the abdomen was opened by a midline incision and the liver, kidney and muscle were quickly removed, harvested and weighed. Livers, kidney and skeletal muscle were then prepared for histological examination. 2.4. Histological examination of mice liver, kidney and skeletal muscle Livers were weighed and dissected. Liver samples from different lobes were assembled, coded and sections embedded in the OCT medium (medium for frozen section), and immediately flash frozen in isopentane cooled in liquid nitrogen. They were then stored at 80 1C until further use. Organs were cut into 5-mm thick sections (Leica CM1850 UV microtome), mounted on glass slides and stained with Oil red O. The sections were counterstained with Mayer's hematoxylin. Steatosis was analyzed by light microscopy. The kidneys were rapidly excised longitudinally. One longitudinal section was prepared as above. One fragment of the right quadriceps muscle, of about 10 5 mm2 in size was immediately placed in liquid nitrogen and then stored at 80 1C until further use. Each stained liver, kidney and muscle section was analyzed for the severity of hepatocytes, kidney and intramyocellular fat accumulation. The extent of hepatocyte lipid accumulation was then scored based on the percentage of hepatocytes that contained macrovesicular fat. Macrovesicular steatosis was graded 0–3 based on percent of hepatocytes in the biopsy involved (grade 0: none or absent; 1: less than 33%; 2: 33–66%; 3: more than 66%) (Kleiner et al., 2005); zonal distribution of steatosis and the presence of macrovesicular or microvesicular steatosis were noted. The pathological changes were evaluated and photographed using an Eclipse 50i Nikon optical microscope controlled by the software Nickon-NiSelements D. Histological evaluation included a semi-quantitative and quantitative analysis of the presence of micro and macrovesicular fat. All sections were coded and analyzed blindly by the pathologist without knowledge of related characteristics or diet (semi-quantitative analysis). The area of Oil red O-stained fatty droplets to the total tissue area was calculated (quantitative analysis) by using the analysis software ImageJ (ImageJ, US National Institutes of Health, Bethesda, Maryland, USA). The results of the histological analysis were confronted to the biochemical results (blood glucose, plasma triglyceride, total cholesterol, HDL-cholesterol, insulin and HOMA) obtained in the same animals in our previous studies (Hamza et al., 2010, 2011, 2012). 2.5. Statistical analysis Data analysis was conducted after database lock using SASs software (SAS Institute, Version 9.4, North Carolina, USA). Descriptive variables are presented as mean and standard deviation (SD). Comparison between mice groups were performed using Kruskal– Wallis test. The significant multiple comparisons tests between each
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Fig. 1. Liver histology in the preventive study. Representative Oil red O staining of C57BL/6J liver tissue from high-fat diet (HFD) induced diabetes. The standard diet STD control group (panel A; 1A) received 20 weeks of standard diet and had normal histology. The HFD control group (panel B; 1B) received high-fat diet during 20 weeks and had steatosis without ballooning, inflammation, necrosis or fibrosis. Treatment with plant extracts during 20 weeks (TFG (panel C; 1C), CE (panel D; 1D), AHA (panel E; 1E)), at a dose of 2 g/kg bodyweight, displayed mild, minor or absent steatosis. Micrometer scale on the left side of the figure (1 mark is 100 mm). All magnification, 20.
pair of groups of semi-quantitative and quantitative analysis of steatosis, blood glucose, triglyceride, liver weight and insulin resistance were performed by Fisher's test with Bonferroni correction. A multivariate ANOVA test was performed to examine association with steatosis and mice groups adjusted for blood glucose, triglyceride, liver weight and insulin resistance. Data in untreated HFD mice was compared to the results in STD control mice; data in treated HFD mice were compared to the results in untreated HFD mice. Statistical significance was set at p o0.05.
which appeared to localize in periportal hepatocytes. There was no liver fibrosis and no inflammation. In the groups treated with TFG, CE and AHA extracts, morphological abnormalities were less marked than in the HFD group. The extent of steatosis was considerably reduced, histology was normal or with minimal periportal lipid accumulation (Figs. 1C–E and 2C–E), seen as microvesicules of fat within periportal hepatocytes. Compared to the HFD group, the lipid drops in liver of treated groups had a lighter color and smaller volume.
3. Results
3.1.1. Semi quantitative assessment of liver fat (visual scoring) 3.1.1.1. Preventive study. Treatment during the establishment of obesity and diabetes. The semi quantitative data of liver fat accumulation is shown in Table 1. HFD group had grade 2 (50%) or 3 (30%) macrovesicular steatosis. The groups treated with TFG, CE and AHA had lower grades of liver fat compared to HFD group Table 1 and Fig. 1. The effect was most marked with CE, with no grade 3 deposition. There was a modest decrease in liver fat in treated groups but the difference were not significant (p4 0.05).
3.1. Histological finding of the liver The STD group had normal hepatic histology (Figs. 1 and 2A). The continuous consumption of high-fat diet for 8 months resulted in macrovesicular hepatic steatosis, with periportal hepatocytes lipid accumulation (Figs. 1 and 2B). There was widespread deposition of fat globules of different size inside parenchymal cells. The whole surface was occupied by macrovesicles of fat,
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Fig. 2. Liver histology in the curative study. Representative Oil red O staining of C57BL/6J liver tissues from high-fat diet (HFD) induced diabetes. The STD control group (panel A; 2A) received 32 weeks of standard diet and showed normal histology. The HFD control group (panel B; 2B) received high-fat diet during 32 weeks and revealed severe steatosis without ballooning, inflammation, necrosis or fibrosis. Treatment with plant extracts for 15 weeks (TFG (panel C; 2C), CE (panel D; 2D), AHA (panel E; 2E)), at a dose of 2 g/kg bodyweight starting at the 17th week of HFD, displayed mild, minor or absent steatosis. Micrometer scale on the left side of the figure (1 mark is 100 mm). All magnification, 20.
Table 1 Liver histology scoring (visual scoring) for macrovesicular steatosis in C57BL/6J mice after 20 weeks. Steatosis
STD control HFD control CE-treated HFD AHA-treated HFD TFG-treated HFD
n
0
1
2
3
10 10 9 9 8
8 2 6 3 5
1 0 1 3 1
1 5 2 0 1
0 3 0 3 1
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum.
3.1.1.2. Curative study. Treatment after establishment of obesity and diabetes. In livers from HFD group the whole surface was occupied by fat macrovesicles. HFD group had two-fold (100% grade of 3) greater
lipid accumulation than the normal control group and CE-treated group (p o0.05). Livers from groups treated with TFG and AHA had a sparse distribution of small microvesicules (Table 2, Fig. 1C and E and Fig. 2Cand E). There was no inflammation except for 2 mice in the HFD group. 3.1.2. Quantitative assessment of liver fat (ImageJ scoring) 3.1.2.1. Preventive study. Liver sections in the STD control group showed normal morphological features. The tissues of HFD group had macrovesicular steatosis of grade 0 (20%) or grade 1 (80%) (Table 3 and Fig. 1A and B). The groups treated with TFG, CE and AHA had lower grades of steatosis compared to HFD group but the differences were not significant (p 40.05). 3.1.2.2. Curative study. In the liver sections from mice in the HFD group, more than one-third of hepatocytes contained macrovesicles of fat, corresponding to grade 1 or 2 steatosis (Table 4 and Fig. 2B). Treatment with CE extract resulted in significant improvement compared with HFD alone (Table 4; po 0.05). There was also decreased liver fat in groups treated
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Table 2 Liver histology scoring (visual scoring) for macrovesicular steatosis in C57BL/6 J mice after 35 weeks.
STD control HFD controlb CE-treated HFDa AHA-treated HFDb TFG-treated HFDb
n
0
1
2
3
10 8 10 10 10
10 0 7 0 1
0 0 0 1 1
0 0 3 2 3
0 8 0 7 5
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum. a b
p o 0.05, vs. HFD control. p o0.05, vs. STD control.
Table 3 Liver histology scoring (ImageJ scoring) for macrovesicular steatosis in C57BL/6J mice after 20 weeks. Steatosis
STD controlb HFD control CE-treated HFD AHA-treated HFD TFG-treated HFD
n
0
1
2
3
10 10 10 9 8
10 2 8 6 5
0 7 2 2 2
0 1 0 1 1
0 0 0 0 0
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum. a p o 0.05, vs. STD control. b
p o0.05, vs. HFD control.
Table 4 Liver histology scoring (ImageJ scoring) for macrovesicular steatosis in C57BL/6J mice after 35 weeks. Steatosis
STD controla HFD control CE-treated HFDa AHA-treated HFDb TFG-treated HFD
n
0
1
2
3
10 8 10 10 10
10 0 7 0 1
0 0 0 1 1
0 0 3 2 3
0 8 0 7 5
STD control: group fed standard diet (STD); HFD control: group fed high fat diet (HFD) alone; CE-treated HFD: group fed high fat diet (HFD) and treated with Centaurium erythraea (CE); AHA-treated HFD: group fed high fat diet (HFD) and treated with Artemisia herba-alba (AHA); TFG-treated HFD: group fed high fat diet (HFD) and treated with Trigonella foenum-graecum. a b
was excellent in the preventive and curative studies (kappa coefficient ¼0.8244 and 0.6364 respectively). 3.2. Skeletal muscle and kidney
Steatosis
a
5
p o 0.05, vs. HFD control. p o0.05, vs. STD control.
with TFG and AHA but the differences were not significant (p 40.05). The concordance between semi-quantitative assessment of liver fat (visual scoring) and the quantitative assessment of liver fat (ImageJ scoring) was low in both the preventive and curative studies (kappa coefficient ¼0.3708 and 0.1753 respectively). However, when the steatosis grades were grouped the concordance
Histological analysis of muscle and kidney tissues did not show any deposition of droplets in either HFD or treated groups (Figs. 3 and 4A and B). Kidney and muscle sections showed normal morphological features in the controls and treated groups. 3.3. Biochemical parameters, liver weight and steatosis The specific treatment-related changes in blood chemistry have already been reported (Hamza et al., 2010, 2011, 2012). In brief, all three extracts were associated with decreased blood glucose and plasma lipids, and improved insulin sensitivity, both during the preventive and the curative parts of the study. Here we found no association of steatosis with blood glucose or TG in the preventive study, but there was an association between steatosis and liver weight (OR ¼2.86 in the visual scoring; OR¼9.97 in the ImageJ scoring). There was an association in both ImageJ scoring and visual scoring between steatosis and HOMA. When HOMA increased, the risk of steatosis greater than 0 increased (OR ¼1.06—p ¼0.018 in the visual scoring; OR ¼1.06— p¼ 0.014 in the ImageJ scoring). Liver weights in TFG, AHA and CEtreated groups in the preventive study were also lower than in the HFD group but the differences were not significant (respectively 1.6 70.5; 1.870.6; 1.770.5 vs. 1.970.4 g, p 40.05). In the curative study, blood glucose, TG, liver weight, and HOMA were not associated with the importance of steatosis (p 40.05). The liver weight increased significantly in the HFD groups and AHA-treated group compared with the STD group respectively (2.2 7 0.4; 2.3 70.6 vs. 1.5 70.2 g, p o0.05).
4. Discussion We have previously shown the preventive and anti-diabetic effect of AHA, CE and TFG on high fat diet induced diabetes in C57BL/6J mice, related to a reduction of insulin resistance (Hamza et al., 2010, 2011, 2012). Plant extracts also reduced the increased insulin, triglyceride and total cholesterol concentrations, as well as insulin-resistance seen in untreated HFD mice. In the present study we report on the effect of the plant extracts on liver steatosis, which was decreased by the extracts in both preventive and curative models, but to a greater extend by the CE extract than by AHA or TFG extracts. The effect of CE, AHA and TFG on the histological finding of liver steatosis in high fat diet induced diabetic mice had not yet been reported. It had been shown that a TFG extract protects the renal tissues against functional and morphologic injuries in rats with diabetes induced by both enriched diets and streptozotocin possibly via its antioxidant activity (Xue et al., 2011). We found no change in renal histology in our HFD mouse model. Musso et al. (2003) have shown that the dietary intake of NASH patients was richer in saturated fat and in cholesterol, and was poorer in polyunsaturated fat, fiber and the anti-oxidant vitamins C and E. An association was found between saturated fat intake and insulin sensitivity, suggesting that dietary habits may promote NASH directly by modulating hepatic triglyceride accumulation and inflammatory activity, as well as indirectly by affecting insulin sensitivity. Toshimitsu et al. (2007) have confirmed these data, showing that in NASH patients, in comparison to simple fatty liver, there is an excess intake of carbohydrates, a low intake of protein and zinc, and lower ratio of intake of polyinsaturated fatty acid to
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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Fig. 3. Kidney histology. Photomicrographs of kidney sections in the preventive study (20 weeks): STD control group (panel A; 3A); HFD control group (panel B; 3B); There was no measurable steatosis in the HFD group, nor in the treatment groups (not shown). Aspects at 32 weeks (curative) were identical (not shown). Micrometer scale on the left side of the figure (1 mark is equivalent to 100 mm). All magnification, 20.
Fig. 4. Quadriceps muscle histology. Photomicrographs of muscle sections in the preventive study (20 weeks): STD control group (panel A; 4A); HFD control group (panel B; 4B); There was no measurable steatosis in the HFD group, nor in the treatment groups (not shown). Aspects at 32 weeks (curative) were identical (not shown). Micrometer scale on the left side of the figure (1 mark is 100 mm). All magnification, 20.
saturated fatty acid. Experimental models suggest that a diet richer in defined carbohydrates, especially fructose, is able to promote steatosis accumulation and NASH progression (Bergheim et al., 2008; Tetri et al., 2008). Additionally, beside liver, skeletal muscle is another insulinresponsive tissue and a very strong relationship between insulin resistance and muscle lipid content has been reported (Laybutt et al., 1999). Several studies have shown that the most common form of insulin resistance is associated with an accumulation of visceral fat (Slawik and Vidal-Puig, 2006). In this study, there was no accumulation of intramyocellular lipids, maybe because of the disease model used. In morbidly obese patients, insulin resistance in muscle might occur at a late point in the insulin signaling cascade and be associated with intramyocellular lipids and nonalcoholic fatty liver disease severity (Machado et al., 2012). In our high-fat diet mice, liver steatosis developed alongside diabetes, which may resemble NASH, though there were no signs of liver inflammation at this stage. CE treatment reduced the development of liver steatosis by 70% in the preventive study. In addition, after the experimental diabetes and NASH had developed (curative approach), CE extract reversed the steatosis, despite the continued intake of the high fat diet. CE extract reduced the severity of hepatic macrovesicular steatosis and triglyceride accumulation, in addition to the reduction of hypertriglyceridaemia and glycemia and improvement of insulin sensitivity (reduced HOMA-IR). In contrast, TFG and AHA had minimal or no significant effect on steatosis. The mechanisms by which CE reduces lipid accumulation in liver are unclear. One of several possible mechanisms may be related to the reduction of plasma triglyceride and cholesterol concentration. We have shown in previous studies that plant
extracts improved hypercholesterolemia and hypertriglyceridemia and now we show that extracts improve liver steatosis (Hamza et al., 2010, 2011, 2012). Accumulation of fat in the liver can occur because of increased delivery of free fatty acids to the liver, increased synthesis of fatty acids in the liver, or decreased betaoxidation of free fatty acids, which may, in turn, cause fat accumulation in the liver. Fat in hepatocytes results in cellular dysfunction and may damage the liver parenchyma (Shelh et al., 1997). Hypertriglyceridemia is a characteristic lipid abnormality in obesity and type 2 diabetes and has been linked to the development of fatty liver in obesity (Assy et al., 2000; Osno et al., 1995). In obesity, accumulation of triglycerides in the liver has been suggested to occur in response to an increased flux of fatty acids to the liver from dietary sources, from adipose tissue or from increased endogenous synthesis of fatty acids (Fong et al., 2000). Excessive ingested fat and metabolic syndrome conditions, such as obesity, insulin resistance, and type 2 diabetes, can result in increased free fatty acids levels in the blood. In metabolic syndrome, the lipolysis of lipids in adipose tissue increases. As a result, the delivery of free fatty acids to the liver is amplified (Clark and Diehl, 2002). Excess carbohydrates consumption can also result in free fatty acids overload in hepatocytes through de novo synthesis, perhaps magnified by the use of fructose (Neuschwander-Tetri, 2010). There is also quite a strong association of NASH with type 2 diabetes (Angulo, 2002; Clark and Diehl, 2002; Kelley et al., 2003) and insulin resistance (Dixon et al., 2001). Insulin resistance may contribute to the development of hepatocyte steatosis by impairing the ability of insulin to suppress lipolysis, resulting in increased circulating free fatty acid and liver lipid accumulation
Please cite this article as: Hamza, N., et al., Effect of Centaurium erythraea Rafn, Artemisia herba-alba Asso.... Journal of Ethnopharmacology (2015), http://dx.doi.org/10.1016/j.jep.2015.05.027i
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(Day and Saksena, 2001; Marchesini et al., 2001). The progression from steatosis to NASH is usually a result of a combination of insulin resistance, oxidative stress and inflammation the two latter being likely a consequence of insulin resistance (Das and Kar, 2005; Malhi and Gores, 2008). Uygun et al. (2000) concluded that leptin might contribute to hepatic steatosis by promoting insulin resistance and also by altering insulin signaling in hepatocytes, thereby promoting increased intracellular fatty acids. Plant extracts (CE, AHA and TFG) restored several metabolic parameters associated with steatosis (Hamza et al., 2010, 2011, 2012). This was the case with insulin resistance, as attested by the normalization of HOMA. The decrease in plasma lipid concentration may also have contributed to restoring insulin sensitivity in treated groups. However improvement in HOMA was not associated with reduced steatosis with TFG and AHA, only with CE. It is not clear which components(s) present in CE, AHA or TFG are responsible for the observed effects. The bioactivity of plant extracts may be associated with polyphenols. The benefits of polyphenols in suppressing TG accumulation in hepatocytes have been also reported in previous studies. Wu et al. (2011) proved that rutin can significantly reduce lipid accumulation and had a good antioxidant capacity in an oleic acid-induced in vitro model of fatty liver by decreasing lipogenesis and oxidative stress in hepatocytes. The benefit of phenolic compounds on preventing steatosis has also been observed. Kimura et al. (1985) showed that the extracts from leaves of Artemisia species, as well as caffeic acid and chlorogenic acid inhibit liver injury and lipid metabolism in peroxidized corn oil-fed rats. Previously, the same authors reported that in vitro caffeic acid and its derivatives inhibited the production of lipid peroxide induced by ADP and NADPH in liver microsomes and by ADP and ascorbic acid in mitochondria. Coffee and two of its components (polyphenol and melanoidin) can reduce fat accumulation, improve inflammation in rat model of HFD induced steatohepatitis. This result may be associated with chlorogenic acid (Vitaglione et al., 2010). It has been reported that chlorogenic acid improved glucose tolerance and decreased plasma and hepatic lipids without changing triglyceride concentration in adipose tissue in an animal model for obese type 2 diabetes (Rodriguez de Sotillo and Hadley, 2002). Cho et al. (2010) reported that chlorogenic and caffeic acids increase fatty acid β-oxidation activity and PPAR α expression in the liver of HFD mice. Hsu et al. (2009) indicated that o-coumaric acid in HFDinduced obesity impedes the development of hepatosteatosis by inhibiting the elevation of hepatic triacylglycerol and cholesterol levels. Hsu and Yen (2007) showed that the addition of gallic acid, a phenolic compound, decreased hepatic steatosis, serum parameters (triglyceride, phospholipid, total cholesterol, LDL-cholesterol) insulin, leptin and oxidative stress markers in HFD-induced dyslipidaemia, hepatosteatosis and oxidative stress in rats. Recently Stefkov et al. (2014) identified groups of bitter compounds (flavonoids, iridoids and xanthones) in the methanol extract of the aerial parts of the CE that may have an influence on the expressed antihyperglycemic effect in streptozotocin rats.
5. Conclusion Plant extracts improved liver steatosis in HFD mice. C. erythraea Rafn (CE) extract produced a more pronounced effect in reducing fat accumulation in liver. Our study clearly for the first time demonstrates that treatment with CE extract is effective in reversing steatosis in type 2 diabetes induced by a high fat diet in mice. CE may offer an interesting treatment of insulinoresistance and consequent steatosis and diabetes, and may provide a new
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therapeutic strategy to reduce fatty liver. A. herba-alba Asso (AHA) and T. foenum-graecum L. (TFG) have shown minimal or no effect on steatosis. What remains to be done is on one hand to identify the active compounds in CE involved in this effect, and on the other confirm these effects in patients suffering from type 2 diabetes and/or NASH.
Acknowledgments The authors are grateful to Joelle Amédée and Robin Siadous (INSERM Université Bordeaux, Inserm U1026, Bioingénierie Tissulaire (BioTis)) for the use of a microtome. The authors wish to acknowledge the Bordeaux Imaging Center for the use of image analysis system, Université de Bordeaux, for the use of the image analyzing system. The authors wish to acknowledge Paulette Bioulac and Jean Rosenbaum (INSERM 1053) for the use of their facilities. The authors would also like to thank Béatrice Mougenot, for her technical assistance. References Abdel-Barry, J.A., Abdel-Hassan, I.A., Al-Hakiem, M.H., 1997. Hypoglycaemic and antihyperglycaemic effects of Trigonella foenum-graecum leaf in normal and alloxan induced diabetic rats. J. Ethnopharmacol. 58, 149–155. Agro, C.K., Northup, P.G., AL-Osaimi, A.M., Caldwell, S.H., 2009. Systematic review of risk factors for fibrosis progresion in non-alcoholic steatohepatitis. J. Hepatol. 51, 371–379. Ajabnoor, M.A., Tilmisany, A.K., 1988. Effect of Trigonella foenum graceum on blood glucose levels in normal and alloxan-diabetic mice. J. Ethnopharmacol. 22, 45–49. Al-Khazraji, S.M., Al-Shamaony, L.A., Twaij, H.A., 1993. Hypoglycaemic effect of Artemisia herba alba. I. Effect of different parts and influence of the solvent on hypoglycaemic activity. J. Ethnopharmacol. 40, 163–166. Al-Shamaony, L., Al-Khazraji, S.M., Twaij, H.A., 1994. Hypoglycaemic effect of Artemisia herba alba. II. Effect of a valuable extract on some blood parameters in diabetic animals. J. Ethnopharmacol. 43, 167–171. Angulo, P., 2002. Nonalcoholic fatty liver disease. N. Engl. J. Med. 346, 1221–1231. Anuradha, C.V., Ravikumar, P., 2001. Restoration on tissue antioxidants by fenugreek seeds (Trigonella Foenum Graecum) in alloxan-diabetic rats. Indian J. Physiol. Pharmacol. 45, 408–420. Assy, N., Kaita, K., Mymin, D., Levy, C., Rosser, B., Minuk, G., 2000. Fatty infiltration of liver in hyperlipidemic patients. Dig. Dis. Sci. 45, 1929–1934. Belguith-Hadriche, O., Bouaziz, M., Jamoussi, K., El Feki, A., Sayadi, S., Makni-Ayedi, F., 2010. Lipid-lowering and antioxidant effects of an ethyl acetate extract of fenugreek seeds in high-cholesterol-fed rats. J. Agric. Food Chem. 58, 2116–2122. Bergheim, I., Weber, S., Vos, M., Kramer, S., Volynets, V., Kaserouni, S., McClain, C.J., Bischoff, S.C., 2008. Antibiotics protect against fructose-induced hepatic lipid accumulation in mice: role of endotoxin. J. Hepatol. 48, 983–992. Broca, C., Gross, R., Petit, P., Sauvaire, Y., Manteghetti, M., Tournier, M., Masiello, P., Gomis, R., Ribes, G., 1999. 4-Hydroxyisoleucine: experimental evidence of its insulinotropic and antidiabetic properties. Am. J. Physiol. 277, E617–623. Caldwell, S., Lazo, M., 2009. Is exercice an effective treatment for NASH? Known and unknown. Ann. Hepatol. 8, S60–S66. Cho, A.S., Jeon, S.M., Kim, M.J., Yeo, J., Seo, K.I., Choi, M.S., Lee, M.K., 2010. Chlorogenic acid exhibits anti-obesity property and improves lipid metabolism in high-fat diet-induced-obese mice. Food Chem. Toxicol. 48, 937–943. Clark, J.M., Diehl, A.M., 2002. Hepatic steatosis and type 2 diabetes mellitus. Curr. Diabetes Rep. 2, 210–215. Das, K., Kar, P., 2005. Non-alcoholic steatohepatitis. J. Assoc. Physicians India 53, 195–199. Day, C.P., Saksena, S., 2001. Non-alcoholic steatohepatitis: definitions and pathogenesis. J. Gastroenterol. Hepatol. 17 (3), S337–S384. Dixit, P., Ghaskadbi, S., Mohan, H., Devasagayam, T.P., 2005. Antioxidant properties of germinated fenugreek seeds. Phytother. Res. 19, 977–983. Dixon, J.B., Bhathal, P.S., O'Brien, P.E., 2001. Nonalcoholic fatty liver disease: predictors of nonalcoholic steatohepatitis and liver fibrosis in the severely obese. Gastroenterology 121, 91–100. Farrell, G.C., Larter, C.Z., 2006. Nonalcoholic fatty liver disease: from steatosis to cirrhosis. Hepatology 43, S99–S112. Fong, D.G., Nehra, V., Lindor, K.D., Buchman, A.L., 2000. Metabolic and nutritional considerations in nonalcoholic fatty liver. Hepatology 32, 3–10. Hamza, N., Berke, B., Cheze, C., Agli, A.N., Robinson, P., Gin, H., Moore, N., 2010. Prevention of type 2 diabetes induced by high fat diet in the C57BL/6J mouse by two medicinal plants used in traditional treatment of diabetes in the east of Algeria. J. Ethnopharmacol. 128, 513–518.
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