Naringin ameliorates high-fat diet-induced hepatotoxicity and dyslipidemia in experimental rat model via modulation of anti-oxidant enzymes, AMPK and SERBP-1c signaling pathways

Abstract

High-fat diet causes elevation of steatosis, dyslipidemia and oxidative stress which eventually leads to hepatic injury in the form of non-alcoholic fatty liver disease (NAFLD). Naringin, a natural flavonoid, having tremendous potentiality including antioxidant, anti-inflammatory, hypolipidemic role. Based on this proposition, we investigated the role of naringin in hepatotoxicity and its possible underlying mechanism caused by high-fat diet for prolonged time. Fifteen Wistar rats were divided into three groups: Group A (CON) received normal diet; Group B (HFD) was administered with high-fat diet for 16 weeks; and Group C (THN) was treated with naringin (100 mg/kg B.W.) for last 6 weeks after induction of obesity. After autopsy, various parameters were studied like gravimetry, serum biochemistry, ROS activity, anti-oxidant enzymes, genes expression (AMPK and SREBP-1C), histochemistry, histopathology and ultrastructure of hepatic tissue. In HFD group, Masson's trichome stain intensity increased 6.8-folds, indicating the onset of liver fibrosis; ROS generation and lipid peroxidation (TBARS) were significantly (p < 0.01) increased, whereas SOD and CAT were decreased by 36.7 % and 49.7 %, respectively. Furthermore, these parameters were remained normal in THN group. Besides, HFD group displayed extreme elevation in hepatic SREBP-1C expression (147 %) and downregulation of AMPK gene (77 %) compared to control. The ultrastructural study revealed most important and new insight of this study where HFD induced extreme reticule stress in hepatic tissue which was significantly improved by the treatment of naringin. These findings demonstrate that the naringin may be used as a potential therapeutic agent to combat obesity related hyperlipidemia and NAFLD.

Keywords: AMPK signaling; HFD; Hepatotoxicity; Lipid metabolism; NAFLD; Naringin; Oxidative stress.

Graphical abstract

Fig. 1

Graphical representation of serum lipid profile in control and treatment groups. The results are expressed as Mean ± SEM, significance levels were analysed by using one-way ANOVA followed by Tukey test. N = 5; Comparison was done between CON with HFD and HFD with THN. Statistical significance *^ap < 0.05, * *^ap < 0.01 CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01 level.

Fig. 2

Alteration of liver biomarker in different groups. Values are expressed as Mean ± SEM, N = 5; the mean difference is significant at: *^ap < 0.05, * *^ap < 0.01; CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01. Significance levels were analysed by using one-way ANOVA and Tukey’s test.

Fig. 3

Graphical presentation of different serum digestive enzymes. Data are expressed as Mean ± SEM, N = 5; the mean difference is significant at: *^ap < 0.05, * *^ap < 0.01; CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01. Significance levels were analysed by using one-way ANOVA and Tukey’s test.

Fig. 4

Photomicrograph of histological alterations in liver (100x and 400x) in different groups. A (CON), B (HFD) and C (THN) were examined under 100x magnification. Whereas, D (CON), E (HFD), F (THN) were examined under 400x magnification. HFD group showed Inflammatory cells (IC), Ballooning cell structure (b), steatosis (S), massive dilation of sinusoids (SD) than other two groups.

Fig. 5

Collagen III distribution in liver tissue indicating the liver fibrosis grade. Magnification 200x (A, B, and C); 400x (D, E, and F). A, D (CON) showing no fibrosis in hepatic tissue, B, E (HFD) showing massive level of fibrosis whereas C, F (THN) showing reduction in collagen tissue distribution. Tissue sections were stained with Masson’s Trichome staining.

Fig. 6

Graphical presentation of liver fibrosis score and NAFLD activity score in hepatic tissue. Values are depicted as Mean ± SEM, N = 5; the mean difference is significant at: *^ap < 0.05, * *^ap < 0.01; CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01. Significance levels were analysed using One-way ANOVA and Tukey’s test.

Fig. 7

Photomicrograph of Sudan Black B staining in different groups. Magnification 400x. Group B (HFD) showing excessive fat accumulation in hepatic tissue in the form of lipid droplet than Group A (CON) and group C (THN). Sudan Black B intensity was statistically analysed using One-way ANOVA. Values are depicted as Mean ± SEM, N = 5; the mean difference is significant at: *^ap < 0.05, * *^ap < 0.01 CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01level.

Fig. 8

Detection of oxidative stress by DCFDA staining and spectrofluorimetric study for quantification of ROS generation in different groups. A (CON), B (HFD), C (THN) were examined under 400x magnification with DCFDA staining. D represented the graphical representation of quantitative study of ROS generation by spectrofluorometer. In both cases HFD group showed increased amount of ROS generation in hepatic tissue.

Fig. 9

Values are presented as Mean ± SEM, N = 5; the mean difference is significant at: *^ap < 0.05, * *^ap < 0.01, CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01 level. Antioxidant status was assessed by SOD, CAT (A) and lipid peroxidation (B) using one-way ANOVA.

Fig. 10

Graphical representation of SREBP-1C and AMPK gene expression in different groups. Values are presented as Mean ± SEM, N = 5; the mean difference is significant at: *^ap < 0.05, * *^ap < 0.01, CON vs HFD; HFD vs NAR, *^bp < 0.05, * *^bp < 0.01 level.

Fig. 11

Transmission electron micrographs of rat liver sections. Control rats (A, B) showing rounded euchromatin nuclei (N) with prompt nuclear membrane and nucleolus (Nu). Normal architecture of double membranous mitochondria (M) encompassed with the cisternae of rough endoplasmic reticulum (RER) as well as ribosomes is distributed throughout the cytoplasm. The HFD group (C, D, E, F) shows various sizes of lipid droplets (LD) throughout the hepatocytes, shrinkage of the nuclear membrane, small nuclei (N) dominated by heterochromatin, and mitochondria (M) in different sizes; some elongated mitochondria (EM), and some mitochondria are fused with lipid droplets (IM). Hepatocytes became vacuolated and also showed swollen rough endoplasmic reticulum (RER), dilated RER (D), and fragmented RER (*). Bile canaliculi show distorted microvilli (curved arrow), presence of electron-dense lysosomes (arrowhead), and collagen deposition (C). The THN group (G, H) shows a prominent nucleolus (Nu) with euchromatin-encompassed nuclei, a well-defined nuclear membrane, presence of autophagosome (#), a distribution of continuous RER throughout the cytoplasm, and some fragmented RER are also present. Normal structure of mitochondria, presence of lipid droplets, but the amount and size are reduced.

Fig. 12

Mechanism of action of naringin to alleviate HFD-induced dyslipidemia, NAFLD and ER stress via downregulating ACC, FAS and SREBP-1C gene and upregulating AMPK gene, as well as inhibiting ROS generation by Nrf-HO-1/NQO-1 pathways. NAFLD: Non-alcoholic fatty liver disease; ER stress: Endoplasmic reticulum stress; FA: Fatty acid; FFA: Free fatty acid; FAS: Fatty acid synthase; ACC: Acetyl-coA Carboxylase; CPT-1: Carnitine palmitoyltransferase I; SREBP-1C: Sterol regulatory element binding protein-1C; AMPK: AMP-activated protein kinase; PARP-α: Poly (ADP-ribose) polymerase-α; ChREBP: Carbohydrate response element binding protein; HO-1: Heme oxygenase-1; NQO-1: NADPH quinone oxidoreductase; Nrf-2: Nuclear factor erythroid 2-related factor 2; TC: Total cholesterol; TG: Triglycerides; LDL-C: Low-density lipoprotein cholesterol; VLDL-C: Very low-density lipoprotein cholesterol; HDL-C: High-density lipoprotein cholesterol; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase.