A novel animal model of metabolic syndrome with non-alcoholic fatty liver disease and skin inflammation

Figures & data

Figure 1. (a) Experimental design. (b–c) Effect of oxazolone on ear edema and ear weight in normal and NAFLD mice. (d and e) Effect of oxazolone on change in MPO activity in plasma and ear homogenate in normal and NAFLD mice. Change in MPO activity is expressed as percent relative to the normal + vehicle group (n = 6–9, $$p < 0.01, $$$p < 0.001 as compared with normal + vehicle, **p < 0.01, ***p < 0.001 as compared with NAFLD + vehicle, and #p < 0.05, ##p < 0.01, and ###p < 0.001 as compared with normal + oxazolone, one-way ANOVA followed by the Newman–Keuls multiple comparison test).

Table 1. Effect of oxazolone on body weight, fat pad and biochemical parameters in normal and NAFLD mice.

	Normal + vehicle	Normal + oxazolone	NAFLD + vehicle	NAFLD + oxazolone
Body weight (g)	24.33 ± 1.33	25.05 ± 1.16	33.87 ± 1.29$$	32.83 ± 1.07$$
Epididymal fat (g)	0.52 ± 0.08	0.42 ± 0.03	1.45 ± 0.16$$	1.40 ± 0.15$$
Inguinal fat (g)	0.40 ± 0.07	0.30 ± 0.04	1.23 ± 0.19$$	1.12 ± 0.13$$
Glucose (mg/dl)	160.05 ± 10.44	158.15 ± 3.99	311.71 ± 15.31$$	272.17 ± 13.68$$
Insulin (ng/ml)	0.350 ± 0.30	0.323 ± 0.40	1.599 ± 0.31$$	1.710 ± 0.34$$
Triglyceride (mg/dl)	103.5 ± 13.22	76.83 ± 6.45	87.89 ± 2.32	81.44 ± 5.24
Total cholesterol (mg/dl)	111.67 ± 6.76	112.17 ± 2.06	125.33 ± 5.00	115.56 ± 5.84
ALT (IU/l)	39.65 ± 3.41	48.7 ± 4.32	59.33 ± 12.29	64.49 ± 14.28
AST (IU/l)	100.03 ± 10.78	82.1 ± 7.75	92.63 ± 10.20	80.98 ± 6.03
ALP (IU/l)	228.17 ± 29.41	227.67 ± 9.30	216.33 ± 6.92	204.56 ± 13.57
Total bilirubin (mg/dl)	0.43 ± 0.14	0.27 ± 0.04	0.54 ± 0.08	0.36 ± 0.05
Total protein (g/dl)	5.75 ± 0.11	5.80 ± 0.09	5.58 ± 0.07	5.65 ± 0.07
TNF-α (pg/ml)	12.58 ± 3.16	13.92 ± 3.19	26.17 ± 3.77$$	29.56 ± 4.57$$
IL-6 (pg/ml)	15.36 ± 2.89	19.12 ± 5.11	38.22 ± 3.69$$	45.15 ± 6.58$$

Values are expressed as mean ± SEM (n = 6–9).

$$p < 0.01 as compared with normal + vehicle, one-way ANOVA followed by the Newman–Keuls multiple comparison test.

Figure 2. (a) Elevation in liver triglyceride (TG) levels by dietary manipulation with HFD and HFL diet. (b) Ballooning degenerations and microvacoulations score in liver histopathology indicating induction of NAFLD by HFD and HFL diet. (c) Liver histopathology section of normal chow-fed mice. (d) Liver histopathology section of HFD and HFL-fed mice, showing hepatic steatosis (solid arrow) and ballooning degenerations (dotted arrow) (n = 6–9, **p < 0.01, ***p < 0.001 as compared with normal, the Mann–Whitney U test).

Figure 3. Effect of oxazolone on ear histopathology in normal and NAFLD mice. Mice ears were excised 24 h after oxazolone challenge and stained with hematoxylin and eosin. Mice applied with vehicle showed normal histology (a). Challenging with oxazolone caused significant increase in epidermal thickness (solid arrow) and inflammatory infiltration (dotted arrow) in both normal (b) and NAFLD (c) mice (more pronounced in the NAFLD mice). Ear histopathology also showed the presence of Munro’s microabscess (solid arrow) (d). Epidermal thickness (e) and dermal inflammatory score (f) were higher in the NAFLD + oxazolone group as compared with normal + the oxazolone group (n = 6–9, $p < 0.05, $$p < 0.01 as compared with normal + vehicle, **p < 0.01 as compared with NAFLD + vehicle, and #p < 0.05, as compared with normal + oxazolone, the Kruskal–Wallis test followed by Dunn’s multiple comparison test).

Figure 4. Effect of oxazolone on NF-κB activity in normal and NAFLD mice. (a) Mice ears were excised 24 h after last oxazolone challenge and analyzed for phosphorylation levels of NF-κB. (b) GAPDH was used as a control and relative density after vehicle or oxazolone treatment was expressed from two independent experiments (n = 4, $$$p < 0.001 as compared with normal + vehicle, ***p < 0.001 as compared with NAFLD + vehicle, and ###p < 0.001, as compared with normal + oxazolone, one-way ANOVA followed by the Newman–Keuls multiple comparison test).