NAFLD and Atherosclerosis Are Prevented by a Natural Dietary Supplement Containing Curcumin, Silymarin, Guggul, Chlorogenic Acid and Inulin in Mice Fed a High-Fat Diet

Abstract

Non-alcoholic fatty liver disease (NAFLD) confers an increased risk of cardiovascular diseases. NAFDL is associated with atherogenic dyslipidemia, inflammation and renin-angiotensin system (RAS) imbalance, which in turn lead to atherosclerotic lesions. In the present study, the impact of a natural dietary supplement (NDS) containing Curcuma longa, silymarin, guggul, chlorogenic acid and inulin on NAFLD and atherosclerosis was evaluated, and the mechanism of action was examined. C57BL/6 mice were fed an HFD for 16 weeks; half of the mice were simultaneously treated with a daily oral administration (os) of the NDS. NAFLD and atherogenic lesions in aorta and carotid artery (histological analysis), hepatic expression of genes involved in the NAFLD (PCR array), hepatic angiotensinogen (AGT) and AT₁R mRNA expression (real-time PCR) and plasma angiotensin (ANG)-II levels (ELISA) were evaluated. In the NDS group, steatosis, aortic lesions or carotid artery thickening was not observed. PCR array showed upregulation of some genes involved in lipid metabolism and anti-inflammatory activity (Cpt2, Ifng) and downregulation of some genes involved in pro-inflammatory response and in free fatty acid up-take (Fabp5, Socs3). Hepatic AGT, AT₁R mRNA and ANG II plasma levels were significantly lower with respect to the untreated-group. Furthermore, NDS inhibited the dyslipidemia observed in the untreated animals. Altogether, these results suggest that NDS prevents NAFLD and atherogenesis by modulating the expression of different genes involved in NAFLD and avoiding RAS imbalance.

Keywords: Profiler PCR array; atherogenic lesions; diet-induced obesity; natural dietary supplement; non-alcoholic fatty liver disease; renin-angiotensin system imbalance.

Figure 1

NDS reduces food intake, body and liver weight in HFD mice. Effects of the NDS treatment (0.9 mg/mouse) on body weight (A), food intake (B), liver weight (C) and the ratio of liver weight/body weight (D), in mice fed a high-fat diet (HFD). Data are the means ± S.E.M. (n = 6/group). * p ≤ 0.05.

Figure 2

NDS prevents NAFLD development in diet-induced obesity (DIO) animals. Representative images of gross anatomy and histological cross-sections of liver from NDS-treated and untreated high-fat diet (HFD) mice. Liver morphologies of untreated HFD mice (A) and NDS-treated HFD mice (B). Cross-sections from untreated HFD mice (C,E) show micro- and macro-vesicular steatosis (black arrowhead) with polymorphonuclear cell infiltration (black arrow). Histological cross-sections from NDS-treated HFD mice (D,F) show normal hepatocytes and maintaining of the lobular architecture. Hematoxylin and eosin stain. Original magnification: (C,D) = ×200, (E,F) = ×400. (G) Plasma levels of AST and ALT. Data are the mean values ± S.E.M. (n = 6/group). * p ≤ 0.05.

Figure 3

NDS prevents altered the plasma lipid profile. Effects of NDS treatment (0.9 mg/mouse) on plasma lipid concentrations. Data are the mean values ± S.E.M. (n = 6/group).* p ≤ 0.05.

Figure 4

NDS reduces the expression of genes involved in NAFLD pathogenesis. Livers of untreated and treated high-fat diet (HFD) mice were used to perform the RNA for the PCR array analysis. (A) Scatter plot of relative expression levels for each gene in mice samples (treated vs. untreated). The figure depicts a log transformation plot of the relative expression level of each gene (2^−ΔCt) between untreated (x-axis) and treated mice (y-axis). The grey lines indicate a two-fold change in gene expression threshold. Red rings indicate upregulated genes; green rings indicate downregulated genes. (B) Histogram of some up- and down-regulated genes with a greater than two-fold expression change. (C) Table of the names and functions of the quantitative real-time PCR of the chosen genes.

Figure 5

NDS prevents the development of atherosclerotic lesions. Representative images of histological cross-sections of aortic arch and carotid artery in high-fat diet (HFD) mice. The black arrow (A,B) denotes the prominent fatty streak between elastic laminae (arrowhead) in the aortic root wall of untreated vs. treated (C,D) HFD mice. Carotid artery shows a hyperplasia of the intima (i) in untreated HFD mice (E,F). On the contrary, no alterations of carotid artery are detected in treated HFD mice (G,H). a = tunica adventitia. s = smooth muscle cells. Hematoxylin and eosin stain. Original magnification: (A,C) = ×400; (B,D,F) and (H) = ×600; (E,G) = ×200.

Figure 6

Atherogenic index serum (AIS) in HFD mice is reduced by NDS. Effects of NDS treatment (0.9 mg/mouse) on AIS of high-fat diet (HFD)-fed mice. Data are the mean values ± S.E.M. (n = 6/group). * p ≤ 0.05.

Figure 7

NDS reduces RAS component expression. (A) Expression levels of AGT and AT1R mRNAs in liver of treated high-fat diet (HFD) mice and the untreated HFD group, by real-time RT-PCR analysis. Data are the mean values ± S.E.M. (n = 3/group). * p ≤ 0.05. (B) Effects of NDS treatment (0.9 mg/mouse) on circulating levels of angiotensin II in HFD mice, by ELISA. Data are the mean values ± S.E.M. (n = 6/group). * p ≤ 0.05.