Non-alcoholic fatty liver disease (NAFLD) is the most frequent hepatic disorder in developed countries and may lead to steatohepatitis, cirrhosis and liver cancer. NAFLD is also considered the hepatic component of metabolic syndrome (MetS) because it is associated with atherogenic dyslipidemia, obesity and type 2 diabetes (T2DM) [1
]. The precise mechanism of the onset and progression of NAFLD remains unclear although increased fatty acid syntheses, oxidative stress and inflammation may play a fundamental role [2
Emerging evidence suggests that angiotensin (ANG) II, a pro-oxidant cytokine, synthesized mainly from the hepatic precursor angiotensinogen (AGT), may have a relevant importance in the pathogenesis of NAFLD by generating reactive oxygen species and regulating the production of pro-inflammatory mediators [3
]. Patients with NAFLD present elevated ANG II levels [4
], and animals with liver steatosis show increased hepatic expression of AGT, AGT II and ANG II type 1 receptor (AT1
]. The renin-angiotensin system (RAS) and its primary mediator ANG II have also a direct influence on the progression of the atherosclerotic process via effects on endothelial function, inflammation, fibrinolytic balance and plaque stability [8
]. Increasing clinical evidence supports a strong association between NAFLD and cardiovascular diseases (CVD), which represents the principal cause of death in NAFLD patients, more so than liver-related complications [9
]. Patients with NAFLD have an altered flow-mediated vasodilatation and increased carotid-artery intimal medial thickness, two reliable markers of subclinical atherosclerosis [11
]. Therefore, the involvement of AGT II in NAFLD pathogenesis and in atherosclerotic plaque formation may provide one of the possible links between NAFLD and accelerated atherogenesis. Accordingly, the use of RAS blockers seems to be potentially useful as a therapeutic approach against NAFLD [12
] and atherosclerosis [13
]. To date, there is no single approved pharmacologic therapy to treat metabolic dysfunctions occurring in obesity, such as NAFLD and related atherosclerosis. The backbone of therapy currently includes lifestyle management to induce weight loss and therapeutic treatment to reduce cardiovascular risk or hyperglycemia [14
]. Recently, natural herbs have been the focus of many researches both because of their safety and efficacy and because their potential bio-active ingredients could help to prevent or treat obesity and the related metabolic disorders [15
]. The natural dietary supplement (NDS) used in this study contains extracts from Cynara scolymus
(chlorogenic acid), Silybum marianum
(silymarin), Taraxacum officinale
(inulin), Curcuma Longa
(curcuma) and Commiphora mukul
(guggul), plant extracts that exert protective actions mainly towards the liver. Recent evidence has shown that the treatment with the NDS exerts beneficial effects in patients with MetS, reducing anthropometric parameters and total cholesterol levels, but the mechanism of action is still unknown [16
This study aims to investigate whether the treatment with this NDS is able to prevent the development of NAFLD and related atherosclerotic lesions in aorta and carotid artery in a mouse model of diet-induced obesity (DIO). In order to investigate the mechanism of action of the natural supplement, its ability to modulate the expression of some RAS components (AGT and AT1R mRNA in liver and circulating concentration of AGT II) or of genes involved in NAFLD was examined. In addition, the impact of the NDS treatment in the plasma lipid profile was also analyzed.
2. Materials and Methods
The procedures were performed in accordance with the conventional guidelines for animal experimentation (Italian D.L. (Legislative Decree) No. 116 of 27 January 1992 and subsequent variations) and the recommendations of the European Economic Community (86/609/ECC).
Male C57BL/6J (B6) mice, purchased from Harlan Laboratories (San Pietro al Natisone Udine, Italy) at 4 weeks of age, were housed in a room with controlled temperature and dark-light cycles, with free access to water and food. After acclimatization (1 week), the animals were weighed and divided into two groups, both fed a high-fat diet (HFD) (PF4051/D, Mucedola, Milan, Italy) composed of 60% of energy as fat, 20% protein and 20% carbohydrates, for 16 weeks.
It has been shown that these animals, consequent to an HFD, develop obesity, hyperglycemia [17
], hepatic steatosis [18
], atherosclerosis [19
] and neurodegeneration [20
One group served as a control of obesity-related dysfunctions (n = 6, untreated group), and the other one (n = 6, treated group) received, simultaneously to the HFD, a daily administration of NDS (0.9 mg/mouse) for 16 weeks. The dose given to the DIO mice was extrapolated from the human dosage (1.6 g/day) and calculated on the basis of the average body weight (40 mg).
The NDS used in this study is, in Italy, commercialized under the name Kèpar®
and was provided by Rikrea®
S.r.l. (Modica-RG, Italy). The main constituents of the NDS are plant-derived polyphenolic compounds that are well known for their antioxidant and anti-inflammatory properties. In particular, NDS consists of extract from five plant sources, and each extract was obtained from a different part of the plant (Table 1
The tablets of the NDS (Kèpar, Batch No. SL0010) were ground by pestle and 9 mg of powder dissolved in 200 µL of water and used as stock solution. The daily dose was freshly made up, by diluting 1:10 the stock solution (0.9 mg/mouse in 20 µL), and was administered by oral gavaging. The administrated dose contained: 0.09 mg of Curcuma
, 0.057 mg of silymarin, 0.0135 mg of guggul lipids, 0.008 of chlorogenic acid and 0.002 mg of inulin. During the 16 weeks of the treatment, changes in body weight and food-intake, determined by measuring the difference between the pre-weighed chow and the weight of chow at intervals of 24 h [21
], were periodically monitored and compared between the two groups of animals. After 16 weeks of treatment, mice were sacrificed, and blood, liver, aorta and carotid artery were immediately collected for subsequent analysis.
2.2. Histological Analysis
Aorta, carotid arteries and liver specimens excised from each hepatic lobe were fixed in 4% formalin for 24 h. After this treatment, the tissues were dehydrated in alcohol and embedded in paraffin wax. Paraffin histological sections (5 μm thick) were stained with hematoxylin and eosin and observed using an automated Leica DM5000 B microscope (Leica, Milan, Italy) connected to a high-resolution camera, Leica DC300 F (Leica, Milan, Italy). According to the Non-alcoholic Steatohepatitis Clinical Research Network (NASH CRN) scoring system, steatosis was determined by analyzing the morphology and percentage of lipid vesicles in hepatocytes [22
2.3. Atherogenic Index (AIS)
The atherogenic index serum (AIS), which is the measure of the atherosclerotic lesion extent based on serum lipids [23
], was determined in all groups. The atherogenic index serum is calculated using the formula AIS = total cholesterol (TC)/HDL [24
2.4. Biochemical Analysis
Lipid profile was measured in mice fasted for 6 h with free access to water. After this time, the mice were euthanized, and the blood was drawn by cardiac puncture and immediately transferred into chilled tubes containing a final concentration of 1 mg/mL EDTA. Then, the samples were centrifuged at 825 g for 10 min, and the obtained plasma was stored at −80 °C until analysis. Plasma triglyceride, cholesterol, low density lipoprotein (LDL), high density lipoprotein (HDL) levels and AST- and ALT-serum concentrations were measured using the ILAB 600 Analyzer (Instrumentation Laboratory, Bedford, Massachusetts).
2.5. Quantitative Real-Time qPCR
Total RNA from livers of treated and untreated obese mice were extracted using the RNEasy Mini Kit (Qiagen, Milan, Italy). Two nanograms of RNA were used to synthesize the first strand cDNA using the RT First-Strand kit (Qiagen, Milan, Italy). Synthesized cDNAs were amplified using RT2 SYBR Green/ROX qPCR Mastermix (Qiagen, Milan, Italy) and StepOne Real-Time instrument (Applied Biosystem, Foster City, CA, USA).
Gene expression analysis was performed using sequence primers for mice AGT, AT1R and β-actin (SigmaLife Sciences, Milan, Italy). The primers were as follows: AGT forward 5’-GTA CAG ACA GCA CCC TAC TT-3’, reverse 5’-TTG TTG AAG AGG CAC TGC AC-3’; AT1R forward 5’-GAC CAA CTC AAC CCA GAA AAGC-3’, reverse 5’-CCT TTG TCG AAC CAC CACTA-3’; β-actin forward 5’-CGG GAT CCC CGC CCT AGG CAC CAG GGT-3’, reverse 5’-GGA ATT CGG CTG GGG TGT TGA AGG TCT CAAA-3’. Each PCR reaction was amplified in triplicate, and levels of expression were calculated after normalization to β-actin. On the basis of the Ct value (threshold cycle; the number of reaction cycles after which fluorescence exceeds the defined threshold) of the examined gene and of the internal control gene, the relative expression level of RNA was calculated according to the 2−ΔΔCt approximation method.
2.6. RT2 Profiler PCR Array
Synthesized cDNAs from NDS-treated and NSD-untreated livers were added to 96-well reaction plates of the Mouse Fatty Liver PCR Array (PAMM-157Z, SABiosciences, Qiagen, Milan, Italy) according to the manufacturer's instructions. The array profiles the expression of 84 key genes involved in the mechanisms of non-alcoholic fatty liver disease (NAFLD) and hepatic insulin resistance. The reaction was performed by using a StepOne Real-Time instrument (Applied Biosystem, Foster City, CA, USA). Analysis was performed using the spreadsheet provided by Qiagen Company, Milan, Italy.
2.7. Measurement of Circulating Levels of Angiotensin II
Quantification of plasma AGT II was carried out by the ELISA kit for mice (Enzo Life Sciences, Inc. Farmingdale NY, USA) according to the manufacturer’s instructions. The experimental detection limit of the analysis was 3.9 pg/mL.
2.8. Statistical Analyses
Results are shown as means ± the standard error of the mean (S.E.M.). The letter n indicates the number of animals. Statistical analyses were performed using Prism Version 6.0 Software (Graph Pad Software, Inc., San Diego, CA, USA). The comparison between the groups was performed by ANOVA followed by Bonferroni’s post-test. A p-value ≤ 0.05 was considered statistically significant.
The present study shows that the natural supplement, here utilized, is able to prevent the development of NAFLD and atherogenic lesions in HFD obese mice. Such a preventive role is determined by its ability to reduce the expression of the RAS components (AGT, AT1R and AGT II) and modulate positively the expression of genes involved in NAFLD. The NDS also improves the lipid profile, a typical obesity-related dysfunction.
In recent years, there has been an increasing interest in the use of plant extracts as potential therapeutic agents. A mixture of natural products is used in various therapeutic areas obtaining a number of interesting outcomes due to their synergistic effects [25
]. The natural dietary supplement used in this work, known as Képar, is used in the Italian market to treat liver discomfort, caused by gallstones, cirrhosis and toxic agents. It is composed of several plant extracts (Curcuma
, silymarin, guggul, chlorogenic acid and inulin) that exert, at least individually, beneficial effects on different components of MetS. Curcumin improves insulin resistance and dyslipidemia [26
]; silymarin exerts anti-inflammatory effects in animal models of NAFLD [27
]; guggul lipids have been successfully used in obesity and hypercholesterolemia [28
]; and chlorogenic acid, as well as inulin, may improve lipidic and glycidic metabolism [29
]. In the present study, we have demonstrated that the diet natural supplement is able to prevent body weight gain, hepatic fat accumulation, atherosclerotic lesions development and dysregulation of lipidic metabolism. In fact, NDS-treated HFD mice showed a body weight significantly lower than the untreated HFD animals, without any differences in the food intake. This suggests that NDS prevents body mass gain by an independent mechanism from central control of the feeding behavior.
It is interesting to note that our study represents the first experimental evidence for a preventive role of the natural supplement against obesity-associated steatosis development. In fact, the liver of treated obese mice did not show hepatomegaly or other histomorphological alterations; on the contrary, the untreated group liver showed the presence of moderate micro- and macro-vesicular steatosis in Zone 3, a zonal distribution highly associated with the severity of steatosis [31
]. The positive impact of NDS treatment, besides liver morphology, was also observed on the hepatic function, as suggested by the reduced ALT and AST plasma levels in treated-obese animals compared to the untreated group. In addition, the results showed that plasma triglycerides and LDL were lower and HDL higher in treated HFD animals in comparison to the untreated mice, demonstrating that NDS exerts beneficial effects on lipid metabolism and, consequently, on cardiovascular functions [32
In order to examine the mechanism by which NDS prevents NAFLD, we also analyzed and compared the hepatic expression of genes involved in NAFLD pathogenesis in treated vs. untreated animals. For the first time, it was demonstrated that NDS is able to modulate different signaling pathways involved in de novo hepatic lipogenesis, lipid oxidation and inflammatory responses. In fact, in NDS-treated liver, genes involved in fatty acid turnover (such as Fabp1 and Cpt2) and anti-inflammatory activity (Ifng) were upregulated, while genes involved in FFA uptake (Fabp5), lipogenesis (Scd1) and inflammation (Socs3) were downregulated. The increased level of Cpt-2 and Fabp-1 provides an explanation of the reduced hepatic lipid depots observed in the NDS-treated liver. Cpt-2 is involved in beta-oxidation and Fabp-1 in the rapid removal of fatty acid in the oxidative organelles [33
]. Downregulation of Fabp5 and Scd1 also contributes to preventing hepatic lipid accumulation, as Fabp5 leads to the fatty acid uptake [34
] and Scd1 converts saturated FA to monounsaturated FA, the major substrates necessary for the synthesis of other lipids [35
]. On the other hand, Scd1 knockout mice result in being resistant to the development of obesity and hepatic steatosis [36
], and the fatty livers of ob/ob mice show increased Scd1 expression [37
Microarray analysis also showed that the treatment with the natural supplement is able to modulate the expression of factors involved in the inflammatory process. In particular, the liver of treated HFD mice showed upregulation of Ifng, a protective mediator against the liver inflammatory process [38
], and a very strong downregulation of Socs3, usually overexpressed in inflamed steatotic liver [39
], providing a molecular basis for the protective role of the NDS in the HFD liver.
It is well known that NAFLD shares many risk factors with cardiovascular diseases, implying a close relationship between NAFLD and adverse cardiovascular events, such as hypertension and atherosclerosis [9
]. Accordingly, we analyzed the impact of NDS treatment on the development of atherogenic lesions in our animal models. Our results showed that no atherosclerotic lesions were present in the vessels of NDS-treated obese mice. On the contrary, early hallmarks of atherosclerosis were highlighted in the untreated-HFD group. In fact, in agreement with Whitman’s report [40
], we observed “fatty streak-type” lesions in the aortic root, representative of foam cells, which are lipid-laden macrophages. We also observed carotid artery intimal-medial wall thickening. Additionally, AIS, a measure of the atherosclerotic lesion extent based on serum lipid concentration [23
], was significantly lower in NDS-treated animals compared with untreated mice, suggesting a preventive action of the natural supplement on atherogenesis development. Therefore, the improvement of the lipid profile could explain the absence of lesions in the NDS-treated animals. Different mechanisms explain the increased risk of cardiovascular events in patients with NAFLD. In fact, besides the proatherogenic lipid profile, the disease is associated with an increased production of pro-inflammatory cytokines [41
] and RAS imbalance. Patients with NAFLD present elevated circulating levels of ANG II and over-activation of intrahepatic RAS [4
]. Our results showed that in the liver of NDS-treated obese mice, angiotensinogen and AT1
R mRNA were significantly decreased, as well as the circulating levels of ANG II, suggesting that the treatment with the natural supplement protects from NAFLD and atherogenesis by preventing RAS imbalance.