http://informahealthcare.com/phb ISSN 1388-0209 print/ISSN 1744-5116 online Editor-in-Chief: John M. Pezzuto Pharm Biol, Early Online: 1–8 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/13880209.2014.960944
ORIGINAL ARTICLE
A novel animal model of metabolic syndrome with non-alcoholic fatty liver disease and skin inflammation
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Nagaraj M. Kulkarni1,2, Mallikarjun S. Jaji1, Pranesha Shetty1, Yeshwant V. Kurhe1, Shilpee Chaudhary1, G. Vijaykant1, J. Raghul1, Santosh L. Vishwakarma1, B. Navin Rajesh1, Jeyamurugan Mookkan1, Uma Maheswari Krishnan2, and Shridhar Narayanan2 1
Department of Biology, Drug Discovery Research, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, Tamil Nadu, India and 2Centre for Nanotechnology and Advanced Biomaterials (CeNTAB), School of Chemical and Biotechnology, Sastra University, Thanjavur, Tamil Nadu, India Abstract
Keywords
Context: Metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) are the emerging co-morbidities of skin inflammation. Occurrence of skin inflammation such as psoriasis is substantially higher in NAFLD patients than normal. Currently, there are no animal models to study the interaction between these co-morbidities. Objective: The present study seeks to develop a simple mouse model of NAFLD-enhanced skin inflammation and to study the effect of NAFLD on different parameters of skin inflammation. Materials and method: Metabolic syndrome and NAFLD were induced in C57BL/6 mice by feeding high-fat diet (HFD, 60% kcal) and high fructose liquid (HFL, 40% kcal) in drinking water. Skin inflammation was induced by repeated application of oxazolone (1% sensitization and repeated 0.5% challenge) in both normal and NAFLD mice and various parameters of skin inflammation and NAFLD were measured. Results: HFD and HFL diet induced obesity, hyperglycemia, hyperinsulinemia, and histological features of NAFLD in mice. Oxazolone challenge significantly increased ear thickness, ear weight, MPO activity, NF-kB activity, and histological features of skin inflammation in NAFLD mice as compared with normal mice. Overall, induction of oxazolone-induced skin inflammation was more prominent in NAFLD mice than normal mice. Hence, HFD and HFL diet followed by topical oxazolone application develops metabolic syndrome, NAFLD, and enhanced skin inflammation in mice. Discussion and conclusion: This simple model can be utilized to evaluate a therapeutic strategy for the treatment of metabolic syndrome and NAFLD with skin inflammation and also to understand the nexus between these co-morbidities.
Animal model, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), nuclear factor-kappa B, oxazolone, skin inflammation
Introduction Non-alcoholic fatty liver disease (NAFLD) refers to a histological spectrum of liver damage from simple steatosis to steatohepatitis in those without a history of excessive alcohol consumption (520 g/d) (Angulo et al., 2002). NAFLD is considered as the manifestation of metabolic syndrome in the liver. The metabolic syndrome is a condition characterized by a cluster of alterations including insulin resistance, obesity, dyslipidemia, hypertension, a pro-inflammatory, and prothrombotic state (Almeda-Valdes et al., 2009). The development of NAFLD is strongly associated with metabolic syndrome as reflected by the fact that approximately 90% of the patients with NAFLD have more than one feature of metabolic syndrome and about 33% have three or more criteria (Marchesini et al., 2003). Increasing severity of NAFLD Correspondence: Dr. Shridhar Narayanan, Vice President and Head, Infection iScience, AstraZeneca India Pvt. Ltd., Off Bellary Road, Hebbal, Bangalore 560024, Karnataka, India. Tel: +91 9611598805. E-mail: [email protected]
History Received 16 July 2014 Revised 27 August 2014 Accepted 28 August 2014 Published online 27 November 2014
represents worsening inflammatory and insulin-resistant state with poorer metabolic outcome (Bhatia et al., 2012). There are a number of observations in the literature linking NAFLD and metabolic syndrome to systemic inflammation (Bhatia et al., 2012; Hamminga et al., 2006). The liver is a key metabolic organ and plays important role in the regulation of systemic inflammation. Liver disease like NAFLD increases various inflammatory mediators like TNF-a, IL-6, C-reactive protein, glucose, and plasminogen activator inhibitor-1 (Bhatia et al., 2012). Obesity and diabetes are associated with chronic low-grade inflammatory state through release of pro-inflammatory cytokines including TNF-a, and IL-6 (Hamminga et al., 2006). In rodents, a high-fat diet (HFD) results in NAFLD and up-regulation of NF-kB activity, which leads to hepatic production of various proinflammatory cytokines and activation of Kupffer cells and macrophages (Bhatia et al., 2012; Cia et al., 2005). Interestingly, several recent studies have found prevalence of NAFLD and metabolic syndrome in skin inflammation like psoriasis (Gisondi et al., 2009; Matsumoto et al., 2004).
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Gisondi et al. (2009) reported that occurrence of NAFLD is substantially more in psoriasis patients than in controls (47% versus 28%, p50.001). Several medications used to treat skin inflammation are known to cause liver damage, which can affect the outcome of drugs used to treat metabolic syndrome, especially NAFLD. Drugs like methotrexate cause NAFLD-like histological changes in liver and hence may worsen the pre-existing NAFLD in patients with skin inflammation (Wenk et al., 2010). Moreover, no specific therapy is approved for the treatment of NAFLD. Animal models of human disease are necessary to allow the study of and understand the efficacy and safety of drugs at real-life level. The ideal disease model should include all the characteristics of the disease along with the co-morbidities. HFD with high-fructose liquid (HFL) in drinking water has been shown to induce NAFLD in mice (Tetri et al., 2008). Chronic oxazolone-induced experimental skin inflammation is being used as a preliminary model for screening of novel drugs for skin inflammation like dermatitis and psoriasis in drug discovery (Peterson, 2006; Yeom et al., 2012). The aim of the present study was to develop a simple mouse model of NAFLD-enhanced skin inflammation and to study the effect of NAFLD on different parameters of skin inflammation.
Materials and methods Animals Male C57BL/6 mice of 4–5 weeks age and 13–18 g body weight were procured from the National Institute of Nutrition, Hyderabad, India. All mice were maintained in 12 h light/dark cycle with free access to standard laboratory chow diet and water ad libitum in controlled environment (23 ± 2 C). Mice were handled according to the guidelines of experimental animal care issued by the committee for the purpose of control and supervision of experiments on animals (CPCSEA) and the experimental protocol was approved by the institutional animal ethical committee (IAEC) (Protocol no. 04/ IAEC-01/PCP/2011). Materials Normal chow diet was procured from Nutri LabÕ Rodent (Tetragon Chemie Pvt. Ltd., Bangalore, India) and 60 kcal% HFD procured from Open Source diets (Cat# D12492, New Brunswik, NJ). Fructose was procured from Sisco Research Laboratories (Mumbai, India). Oxazolone was purchased from Sigma (St Louis, MO). All other reagents were of the highest commercially available grade. Methodology Male C57BL/6 mice were divided into two groups. The first group (n ¼ 22) was fed with normal chow feed and the second group (n ¼ 29) fed with 60 kcal% HFD and HFL 40 % for 60 d. On the 45th day, two animals from each group were sacrificed; liver histopathology examination was carried out to confirm the induction of NAFLD. At the end of induction period (day 60), the remaining animals were grouped as follows. Mice fed with normal chow diet (n ¼ 20) were further divided into three groups; normal untreated control (n ¼ 8), normal + vehicle (n ¼ 6), and normal + oxazolone (n ¼ 6).
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Mice fed with HFD and HFL (n ¼ 27) were further divided into three groups (n ¼ 9) as untreated NAFLD control, NAFLD + vehicle, and NAFLD + oxazolone. Basal ear thickness was measured and animals were sensitized with 1% oxazolone or vehicle (acetone:olive oil, 4:1) on both ears (20 ml each). Seven days later, the animals were challenged with 0.5% oxazolone or vehicle for 2 weeks as shown in Figure 1(a). Ear thickness was measured with a digital micrometer (Mitutoyo, Kanagawa, Japan) before and 24 h post each challenge. On 15th day, animals were kept for overnight fasting and, on 16th day, blood was collected from retroorbital sinus under light isoflurane anesthesia and plasma separated by centrifugation at 6000 rpm for 10 min for biochemical analysis. Following blood collection, animals were sacrificed by cervical dislocation and liver, ear, and fat pad were collected, weighed, and frozen (80 C) for further analysis. Part of liver and ear tissues were kept in 10% formalin for histopathology examination. Biochemical estimations in plasma Plasma glucose, triglyceride (TG), cholesterol (TC), total bilirubin, total protein, alanine aminotransaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured from plasma by random access clinical chemistry analyzer Erba XL 300 using commercially available ERBA diagnostics kit (OpenSource Diets, New Brunswik, NJ). Plasma insulin was estimated by using ELISA kits following manufacturer’s instructions (Millipore, Billerica, MA). Cytokines in serum and ear homogenates Briefly, the treated mouse ear tissue extracts were prepared by tissue lysis with T-PER buffer (Pierce, Rockford, IL) supplemented with 1 Protease inhibitor cocktail (Calbiochem, San Diego, CA) and 1 phosphatase inhibitor cocktail (Calbiochem, San Diego, CA) using a tissue homogenizer. Homogenates were then centrifuged at 14 000 rpm for 30 min. IL-6 and TNF-a were measured in the supernatant (tissue homogenate) or serum using ELISA kits following manufacturer’s instructions (GE Healthcare, Little Chalfont, UK). Myeloperoxidase (MPO) activity in plasma and ear homogenates MPO activity was measured in plasma and ear homogenates (homogenized as described above for assessment of cytokines) (De Vry et al., 2005). Briefly, 50 ml of plasma or supernatant from ear homogenate was added to 200 ml of an assay reaction mixture containing 0.5% hexadecyltrimethylmmonium bromide (in 50 mM potassium phosphate, pH 6.4), 0.165 mg/ml o-dianisidine hydrochloride and 0.0015% H2O2. Absorbance was measured at 460 nm. Liver triglycerides The liver lipids were extracted using a modified Folch extraction protocol (Folch et al., 1957). Briefly, approximately 100 mg of liver tissue was homogenized with methanol (1 ml) then the homogenate was centrifuged at 4000 rpm for 5 min and supernatant transferred into separate
Novel animal model of NAFLD and skin inflammation
DOI: 10.3109/13880209.2014.960944
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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 ¼ 6–9, $$p50.01, $$$p50.001 as compared with normal + vehicle, **p50.01, ***p50.001 as compared with NAFLD + vehicle, and #p50.05, ##p50.01, and ###p50.001 as compared with normal + oxazolone, one-way ANOVA followed by the Newman– Keuls multiple comparison test).
tubes (15 ml). Then the pellet was re-suspended in chloroform:methanol (2:1) solution for 2 min. The homogenate was then centrifuged and supernatant separated and mixed with first supernatant. About 0.1 M potassium chloride was added to supernatant and mixed well by vortexing. After centrifugation of the mixture, bottom phase (organic phase) was removed to a new tube (2 ml). The samples were evaporated in Turbovap LV evaporator (GE Healthcare, Little Chalfont, UK). Residue was reconstituted in 400 ml of mixture of N-butyl alcohol:Triton X-100:methanol (3:1:1) and mixed properly by vortexing. The samples were used for the triglyceride estimation using commercially available kit (Erba Diagnostics).
Ear tissues were analyzed quantitatively for epidermal thickness and inflammatory infiltrate by an investigator blinded to treatment as described previously (Boehncke et al., 2001). Briefly, maximal epidermal thickness was measured from the tip of the rete ridges to the border of the viable dorsal or ventral epidermis. The mean value of six such rete ridges was measured using the ocular micrometer of the microscope at 400 each division measuring 2.45 mm. The inflammatory grading was done based on the presence of dermal inflammation without Munro’s abscess (score 0.5), mild inflammation with Munro’s abscess (score 1), moderate inflammation with Munro’s abscess (score 2), and marked inflammation with Munro’s abscess (score 3).
Histopathology
Western blot analysis
Liver and ear tissue samples were fixed in 10% formalin and embedded in paraffin. Sections measuring 3–5 mm in thickness were cut and stained with hematoxylin and eosin (H&E). Liver sections were examined using a light microscope (NIKON, ECLIPSE-E200, Tokyo, Japan) and graded by the in-house method based on Hubscher (2006) and Brunt and Tiniakos (2002). It included scoring of microvacuolation, namely 1 (mild), 2 (moderate), and ballooning degeneration as 1 (mild), 2 (moderate), 3 (marked), and 4 (severe).
Briefly, the treated mice ear tissue extracts were prepared by tissue lysis as described earlier for the cytokine estimation. The protein content was determined using a bicinchoninic acid assay (BCAÔ protein assay kit, Pierce, Appleton, WI) and samples were prepared in SDS, bromophenol blue, and 100 mg/mL DTT, and boiled for 5 min at 99 C. The 50 mg of protein was resolved on a 10% SDS-PAGE and blotted on to a nitrocellulose membrane and the blots were blocked for 1 h at room temperature with 5% non-fat milk protein in
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Tris-buffered saline (TBS) containing 0.1% Tween 20, then blots were probed with antibodies against anti-rabbit pNF-kB (Cell Signaling Technology, Danvers, MA) and anti-rabbit GAPDH (Cell Signaling Technology, Danvers, MA) overnight at 4 C. Horseradish peroxidase-conjugated secondary antibody was subsequently incubated with blot for 1 h room temperature. The ECL (Enhanced chemiluminescence; Pierce, Appleton, WI) was used to visualize signal (Li et al., 2012).
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Statistical analysis All values are expressed as mean ± standard error of the mean (SEM). The Graphs were generated using Graph-Pad PrismÕ (Version 4, GraphPad Software, La Jolla, CA). Statistical analysis was performed by Student ‘t’ test or one-way ANOVA followed by the Newman–Keuls multiple comparison test or two-way ANOVA followed by the Bonferroni post test as applicable. For histopathology, non-parametric analysis was done by the Mann–Whitney U test or the Kruskal Wallis test followed by Dunn’s multiple comparison test as applicable. Results were considered statistically significant at p50.05.
Results There was no significant difference observed between normal control versus normal + vehicle and NAFLD control versus NAFLD + vehicle, indicating vehicle did not affect any of the parameters in both normal and NAFLD mice. Hence, the data for normal control and NAFLD control are not shown in the results. Induction of obesity and NAFLD with dietary manipulation Induction of obesity and NAFLD in mice was achieved by feeding HFD and HFL diet to C57BL/6 mice. Feeding mice with HFD and HFL significantly increased body weight (p50.01), epididymal (p50.01), and inguinal fat (p50.01) as compared with animals on the normal chow diet (Table 1). On 45th day of dietary manipulation, two animals were sacrificed and induction of NAFLD in HFD- and HFLfed mice was confirmed by the histopathological examination for the presence of ballooning degenerations and
microvacoulations (data not shown). Oxazolone or vehicle application did not affect the body weight and fat pad in both normal and HFD- and HFL-fed mice. Induction of hyperglycemia and hyperinsulinemia with dietary manipulationHyperglycemia and hyperinsulinemia are important criteria of metabolic disorder. Mice fed with HFD and HFL diet showed a significant increase in fasting plasma glucose (p50.01) and insulin (p50.01) as compared to mice on normal chow diet (Table 1). Mice fed with HFD and HFL caused elevation in plasma ALT levels indicating the induction of NAFLD in this model. Dietary manipulation did not cause any significant difference in other biochemical parameters tested compared to normal chow-fed mice (Table 1). Oxazolone application in both normal and NAFLD mice did not affect any of the biochemical parameters as compared with their respective controls (Table 1). Induction of ear edema in normal and NAFLD mice Ear edema was induced by topical application of oxazolone to both ears of normal and NAFLD mice as shown in Figure 1(b–e). Ear thickness was measured before and 24 h post each oxazolone challenge. Ear thickness increased significantly from the second challenge onwards in NAFLD + oxazolone mice as compared with NAFLD + vehicle mice (p50.001) and remained till the end of the experiment (p50.001) (Figure 1b). Whereas oxazolone application to the normal mice showed significant increase in ear thickness from third challenge onwards and remained till the end of the experiment (p50.001). Ear thickness in NAFLD + oxazolone mice was significantly higher than normal + oxazolone mice (p50.001). This increase in ear thickness was statistically significant from the second challenge onwards till the end of the experiment, indicating increased inflammatory response in NAFLD mice. At the end of the study, animals were sacrificed and ear weight was recorded. Oxazolone application significantly increased ear weight in both normal and NAFLD mice (p50.01) (Figure 1c). Ear weight of NAFLD + oxazolone mice was significantly higher than normal + oxazolone mice (p50.01).
Table 1. Effect of oxazolone on body weight, fat pad and biochemical parameters in normal and NAFLD mice.
Body weight (g) Epididymal fat (g) Inguinal fat (g) Glucose (mg/dl) Insulin (ng/ml) Triglyceride (mg/dl) Total cholesterol (mg/dl) ALT (IU/l) AST (IU/l) ALP (IU/l) Total bilirubin (mg/dl) Total protein (g/dl) TNF-a (pg/ml) IL-6 (pg/ml)
Normal + vehicle
Normal + oxazolone
NAFLD + vehicle
NAFLD + oxazolone
24.33 ± 1.33 0.52 ± 0.08 0.40 ± 0.07 160.05 ± 10.44 0.350 ± 0.30 103.5 ± 13.22 111.67 ± 6.76 39.65 ± 3.41 100.03 ± 10.78 228.17 ± 29.41 0.43 ± 0.14 5.75 ± 0.11 12.58 ± 3.16 15.36 ± 2.89
25.05 ± 1.16 0.42 ± 0.03 0.30 ± 0.04 158.15 ± 3.99 0.323 ± 0.40 76.83 ± 6.45 112.17 ± 2.06 48.7 ± 4.32 82.1 ± 7.75 227.67 ± 9.30 0.27 ± 0.04 5.80 ± 0.09 13.92 ± 3.19 19.12 ± 5.11
33.87 ± 1.29$$ 1.45 ± 0.16$$ 1.23 ± 0.19$$ 311.71 ± 15.31$$ 1.599 ± 0.31$$ 87.89 ± 2.32 125.33 ± 5.00 59.33 ± 12.29 92.63 ± 10.20 216.33 ± 6.92 0.54 ± 0.08 5.58 ± 0.07 26.17 ± 3.77$$ 38.22 ± 3.69$$
32.83 ± 1.07$$ 1.40 ± 0.15$$ 1.12 ± 0.13$$ 272.17 ± 13.68$$ 1.710 ± 0.34$$ 81.44 ± 5.24 115.56 ± 5.84 64.49 ± 14.28 80.98 ± 6.03 204.56 ± 13.57 0.36 ± 0.05 5.65 ± 0.07 29.56 ± 4.57$$ 45.15 ± 6.58$$
Values are expressed as mean ± SEM (n ¼ 6–9). $$p50.01 as compared with normal + vehicle, one-way ANOVA followed by the Newman–Keuls multiple comparison test.
Novel animal model of NAFLD and skin inflammation
DOI: 10.3109/13880209.2014.960944
MPO activity, an indicator of polymorphonuclear leukocyte influx, was measured in both plasma and ear homogenate. Oxazolone application significantly increased MPO activity in plasma and ear homogenate in both normal and NAFLD mice (p50.01) as compared with their respective control (Figure 1d). In accordance with ear thickness and ear weight, MPO activity in plasma and ear homogenate was significantly higher in the NAFLD + oxazolone group than the normal + oxazolone group (p50.05, p50.01) (Figure 1d and e). TNF-a and IL-6 levels were measured in serum and ear homogenate. Serum TNF-a, and IL-6 levels were significantly elevated in NAFLD mice as compared with normal mice (Table 1). In ear homogenate, there was no significant difference observed in TNF-a levels in both normal and NAFLD mice (data not shown). While IL-6 levels significantly increased in normal + oxazolone (285.48 ± 17.61 pg/ml versus 558.10 ± 53.88 pg/ml) and NAFLD + oxazolone groups (260.71 ± 18.60 pg/ml versus 504.52 ± 54.47 pg/ml) as compared with their respective controls, no significant difference was observed between normal + oxazolone and NAFLD + oxazolone groups. Effect of dietary manipulation on liver triglyceride levels and liver histopathology Liver triglyceride levels were significantly higher in NAFLD mice than normal mice (p50.01) indicating accumulation of lipid in the liver with HFD and HFL diet (Figure 2a). Microvacuolations and ballooning degenerations were scored by a pathologist blinded to treatment. Liver sections of HFDand HFL-fed mice showed significant induction of ballooning
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degenerations and microvacuolations (Figure 2b and d). Animals fed with normal chow diet did not show any pathology of NAFLD (Figure 2c). Application of oxazolone on ears did not affect the liver histopathology in both normal and NAFLD mice. Effect of oxazolone on ear histopathology in normal and NAFLD mice Application of oxazolone to ears showed psoriasis and dermatitis like histopathology in both normal and NAFLD mice (Figure 3). Munro’s microabscesses were present in both normal + oxazolone and NAFLD + oxazolone groups (Figure 3d). Epidermal thickness, as measured using an ocular micrometer by an investigator blinded to treatment, was significantly higher in both normal + oxazolone and NAFLD + oxazolone groups as compared with their respective controls (p50.01) (Figure 3e). Epidermal thickness in the NAFLD + oxazolone group was greater than the normal + oxazolone group. Dermal inflammatory score was significantly more in the NAFLD + oxazolone group than the normal + oxazolone group (Figure 3f). Inflammation was not observed in un-induced normal and NAFLD mice (Figure 3a). Overall, induction of skin histopathology resembling psoriasis and dermatitis was more prominent in the NAFLD + oxazolone group than the normal + oxazolone group (Figure 3b and c). Effect of oxazolone on NF-kB expression in normal and NAFLD mice NF-kB is a key regulatory element in a variety of immune and inflammatory pathways and hence is a crucial mediator involved in the pathogenesis of various skin inflammatory
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Effect of oxazolone on MPO and cytokine levels in normal and NAFLD mice
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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 ¼ 6–9, **p50.01, ***p50.001 as compared with normal, the Mann–Whitney U test).
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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 ¼ 6–9, $p50.05, $$p50.01 as compared with normal + vehicle, **p50.01 as compared with NAFLD + vehicle, and #p50.05, as compared with normal + oxazolone, the Kruskal–Wallis test followed by Dunn’s multiple comparison test).
conditions. Chronic oxazolone treatment in mice strongly stimulated the phosphorylation of NF-kB in both normal and NAFLD mice. Phosphorylation of NF-kB was significantly higher in NAFLD mice than in normal mice treated with oxazolone (p50.01). However, there was no significant difference observed between normal and NAFLD mice treated with vehicle (Figure 4).
Discussion This is the first attempt to evaluate the effect of NAFLD on skin inflammation and eventually to develop an animal model. Recent literature indicates that the association among skin inflammation, metabolic syndrome, and NAFLD is growing in patient population (Gerdes et al., 2010; Gottlieb et al., 2008; Saraceno et al., 2008; Wenk et al., 2010). However, the exact pathogenic mechanism between these comorbidities is still unclear. Moreover, several medications used to treat skin inflammation are also known to induce liver diseases. Lack of proper animal model has hampered the understanding of the nexus between such co-morbidities and
also evaluation of potential pharmacological interventions. Hence, the animal model developed in the present study will enable us to study the interaction among NAFLD, metabolic syndrome, and skin inflammation and also to evaluate different therapeutic strategies for the treatment of such co-morbidities. In the present study, we induced metabolic syndrome and NAFLD first by combination of HFD and HFL in drinking water to C57BL/6 mice for 2 months. Combination of HFD and HFL is associated with the development of obesity, increased de novo lipogenesis in the liver, hepatic steatosis, impaired glucose tolerance, insulin resistance, and hypertension (Tetri et al., 2008). Mice fed with HFD and HFL experienced rapid gain in body weight and increased fat pad weight as compared with normal chow-fed mice. Histopathological examination of liver clearly demonstrated that the HFD- and HFL-fed mice showed ballooning degenerations and microvacuolations indicating the development of NAFLD. The induction of NAFLD was further confirmed by increased liver triglyceride levels and plasma level of ALT. NAFLD mice also showed significant elevation of plasma
Novel animal model of NAFLD and skin inflammation
DOI: 10.3109/13880209.2014.960944
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Figure 4. Effect of oxazolone on NF-kB activity in normal and NAFLD mice. (a) Mice ears were excised 24 h after last oxazolone challenge and analyzed for phosphorylation levels of NF-kB. (b) GAPDH was used as a control and relative density after vehicle or oxazolone treatment was expressed from two independent experiments (n ¼ 4, $$$p50.001 as compared with normal + vehicle, ***p50.001 as compared with NAFLD + vehicle, and ###p50.001, as compared with normal + oxazolone, one-way ANOVA followed by the Newman–Keuls multiple comparison test).
glucose and insulin levels along with the elevation of circulating cytokines like TNF-a, and IL-6 in serum than normal mice. These data suggest that the current animal model displayed the most important criteria of metabolic syndrome like obesity and insulin resistance along with NAFLD. For pharmacological screening of new drugs for skin inflammation, acute and chronic oxazolone-induced skin inflammation is being used as the preliminary screening model in drug discovery for years. This model is particularly useful for the evaluation of broad acting anti-inflammatory drugs (Peterson, 2006). Oxazolone application to ears causes histopathological changes similar to that of psoriasis and atopic dermatitis. Moreover, standard drugs like hydrocortisone, phosphodiesterase inhibitors, and dexamethasone show significant inhibitory activity in this model (Peterson, 2006). In the present study, application of oxazolone to the ears of normal C57BL/6 mice caused significant increase in ear thickness, ear weight, MPO activity, and NF-kB expression. Histopathology of ear displayed epidermal thickness and infiltration of inflammatory cells along with the presence of Munro’s microabscess, a characteristic histopathological feature of psoriasis (Boehncke et al., 2001). A similar application of oxazolone to NAFLD mice improved the induction of disease compared with that of normal mice. Ear thickness, ear weight, MPO activity, and NF-kB activity were significantly higher in NAFLD mice than that of normal mice. Incidence of microabscess, epidermal thickness, and dermal inflammatory score were also higher in NAFLD mice than normal mice. The data from this study clearly show that the presence of co-morbidities like obesity, insulin resistance, and
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NAFLD increased the severity of oxazolone-induced skin inflammation. These results are in accordance with clinical reports where the incidence of skin inflammation was found higher in patients with metabolic disorder and NAFLD (Gisondi et al., 2009; Saraceno et al., 2008; Wenk et al., 2010). Elevated levels of pro-inflammatory cytokines, especially TNF-a in adipose tissue, are an important feature of obesity and contribute significantly to insulin resistance and development of NAFLD (Baker et al., 2011; Wenk et al., 2010). Accumulation of free fatty acids within hepatocytes causes hepatic TNF-a production and further increasing the inflammatory condition in metabolic syndrome. Adipocyte and hepatic TNF-a may act on skin to worsen skin inflammation by promoting keratinocyte proliferation, increased inflammation, and the upregulation of vascular adhesion molecules and transcription factor NF-kB (Bhatia et al., 2012; Wenk et al., 2010). In the present study, serum levels of TNF-a and IL-6 were significantly higher in NAFLD mice indicating the presence of chronic low-grade systemic inflammation. This increased systemic inflammation may be responsible for enhanced skin inflammation in NAFLD mice. Notably, several studies have indicated that the increase in the activation of factor NF-kB has an important role in the development and maintenance of cutaneous inflammatory diseases, including psoriasis, contact dermatitis, and atopic dermatitis (Bell et al., 2003). In the skin, the transcription of cytokines, such as IL-1, IL-6, and TNF-a, and many of the effectors of cytokine action, such as vascular cell adhesion molecule-1, and cyclo-oxygenase-2, are regulated by NF-kB (De Vry et al., 2005). In this study, oxazolone treatment increased NF-kB activation in ear tissue of both normal and NAFLD mice, with pronounced increase in NAFLD mice treated with oxazolone than normal mice. These data clearly shows that NAFLD-enhanced oxazolone-induced skin inflammation may be mediated by increased NF-kB activation. Further, MPO activity, an indicator of neutrophil accumulation and a characteristic feature of cutaneous inflammation, was significantly higher in plasma and ear tissues of NAFLD mice than normal mice, indicating the increased severity of inflammation in NAFLD mice. Most of the drugs used for the treatment of skin inflammation are known to cause liver damage and there are no formal guidelines regarding systemic treatment of skin inflammation in NAFLD patients. Methotrexate induced liver damage resembles that of NAFLD and may worsen the condition if used in such patient population (Wenk et al., 2010). The present animal model can be used to study the effect of anti-inflammatory drugs in the presence of both metabolic syndrome and NAFLD. In our preliminary studies, orally administered methotrexate caused severe mortality at 1 mg/kg in this model and worsened NAFLD, whereas it was well tolerated in normal mice (unpublished data). This finding encourages us to further utilize this model to study the effect of such drugs alone and in combination with drugs used in the management of metabolic syndrome on NAFLD and skin inflammation. Hence, pharmacotherapy of skin inflammation and NAFLD in the presence of co-morbidities can be established using this model.
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In conclusion, the present study is an attempt to develop an animal model of metabolic syndrome and NAFLD-enhanced skin inflammation. Dietary manipulation followed by topical oxazolone application increases systemic inflammation and NF-kB activation in skin resulting in metabolic syndrome and NAFLD-enhanced skin inflammation in mice. This simple animal model can be utilized to evaluate therapeutic strategy for the treatment of metabolic syndrome and NAFLD with skin inflammation like psoriasis or dermatitis and also to understand the nexus between these co-morbidities.
Declaration of interest
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
References Almeda-Valdes P, Cuevas-Ramos D, Anguilar-Salinas CA. (2009). Metabolic syndrome and non-alcoholic fatty liver disease. Ann Hepatol 8:S18–14. Angulo P. (2002). Nonalcoholic fatty liver disease. N Engl J Med 346: 1221–31. Baker RG, Hayden MS, Ghosh S. (2011). NF-kB, inflammation and metabolic disease. Cell Metab 13:11–22. Bell S, Degitz K, Quirling M, et al. (2003). Involvement of NF-kappaB signalling in skin physiology and disease. Cell Signal 15:1–7. Bhatia LS, Curzen NP, Calder PC, Byrne CD. (2012). Non-alcoholic fatty liver disease: A new and important cardiovascular risk factor? Eur Heart J 33:1190–200. Boehncke W, Hardt-Weinelt K, Nilsson H, et al. (2001). Anatagnoistic effects of the Staphylococcal Enterotoxin A mutant, SEA on psoriasis in SCID-hu xenogeneic transplantation model. J Invest Dermatol 116: 596–601. Brunt EM, Tiniakos DG. (2002). Pathology of steatohepatitis. Best Pract Res Clin Gastroenterol 16:691–707. Cia D, Yuan M, Frantz DF, et al. (2005). Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NFkappaB. Nat Med 11:183–90. De Vry CG, Valdez M, Lazarov M, et al. (2005). Topical application of a novel immunomodulatory peptide, RDP58, reduces skin inflammation
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in the phorbol ester-induced dermatitis model. J Invest Dermatol 125: 473–81. Folch J, Lees M, Stanley GHS. (1957). A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 226: 497–509. Gerdes S, Rostami-Yazdi M, Mrowietz U. (2010). Adipokines and psoriasis. Exp Dermatol 20:81–7. Gisondi P, Targher G, Zoppini G, Girolomoni G. (2009). Non-alcoholic fatty liver disease in patients with chronic plaque psoriasis. J Hepatol 51:758–64. Gottlieb AB, Chao C, Dann F. (2008). Psoriasis comorbidities. J Dermatol Treat 19:5–21. Hamminga EA, Van Der Lely AJ, Neumann HAM, Thio HB. (2006). Chronic inflammation in psoriasis and obesity: Implications for therapy. Med Hypotheses 67:768–73. Hubscher SG. (2006). Histological assessment of non-alcoholic fatty liver disease. Histopathology 49:450–65. Li M, Zhong X, He Z, et al. (2012). Effect of erythromycin on cigaretteinduced histone deacetylase protein expression and nuclear factor-kB activity in human macrophages in vitro. Int Immunopharmacol 12: 643–50. Marchesini G, Bugianesi E, Forlani G, et al. (2003). Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 37: 917–23. Matsumoto T, Suziki N, Watanabe H, et al. (2004). Nonalcoholic steatohepatitis associated with psoriasis vulgaris. J Gastroenterol 39: 1102–5. Peterson TK. (2006). In vivo pharmacological disease models for psoriasis and atopic dermatitis in drug discovery. Basic Clin Pharmacol Toxicol 99:104–15. Saraceno R, Ruzzetti M, De Martino MU, et al. (2008). Does metabolic syndrome influence psoriasis? Eur Rev Med Pharmacol Sci 12: 339–41 Tetri LH, Basaranoglu M, Brunt EM, et al. (2008). Severe NAFLD with hepatic necroinflammatory changes in mice fed trans fats and a highfructose corn syrup equivalent. Am J Physiol Gastrointest Liver Physiol 295:G987–95. Wenk KS, Arrington KC, Ehrlich A. (2010). Psoriasis and nonalcoholic fatty liver disease. J Eur Acad Dermatol Venereol 25: 383–91. Yeom M, Kim S, Lee B, et al. (2012). Oral administration of glucosylceramide ameliorates inflammatory dry-skin condition in chronic oxazolone-induced irritant contact dermatitis in the mouse ear. J Dermatol Sci 67:101–10.
ORIGINAL ARTICLE
A novel animal model of metabolic syndrome with non-alcoholic fatty liver disease and skin inflammation
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Nagaraj M. Kulkarni1,2, Mallikarjun S. Jaji1, Pranesha Shetty1, Yeshwant V. Kurhe1, Shilpee Chaudhary1, G. Vijaykant1, J. Raghul1, Santosh L. Vishwakarma1, B. Navin Rajesh1, Jeyamurugan Mookkan1, Uma Maheswari Krishnan2, and Shridhar Narayanan2 1
Department of Biology, Drug Discovery Research, Orchid Chemicals and Pharmaceuticals Ltd., Chennai, Tamil Nadu, India and 2Centre for Nanotechnology and Advanced Biomaterials (CeNTAB), School of Chemical and Biotechnology, Sastra University, Thanjavur, Tamil Nadu, India Abstract
Keywords
Context: Metabolic syndrome and non-alcoholic fatty liver disease (NAFLD) are the emerging co-morbidities of skin inflammation. Occurrence of skin inflammation such as psoriasis is substantially higher in NAFLD patients than normal. Currently, there are no animal models to study the interaction between these co-morbidities. Objective: The present study seeks to develop a simple mouse model of NAFLD-enhanced skin inflammation and to study the effect of NAFLD on different parameters of skin inflammation. Materials and method: Metabolic syndrome and NAFLD were induced in C57BL/6 mice by feeding high-fat diet (HFD, 60% kcal) and high fructose liquid (HFL, 40% kcal) in drinking water. Skin inflammation was induced by repeated application of oxazolone (1% sensitization and repeated 0.5% challenge) in both normal and NAFLD mice and various parameters of skin inflammation and NAFLD were measured. Results: HFD and HFL diet induced obesity, hyperglycemia, hyperinsulinemia, and histological features of NAFLD in mice. Oxazolone challenge significantly increased ear thickness, ear weight, MPO activity, NF-kB activity, and histological features of skin inflammation in NAFLD mice as compared with normal mice. Overall, induction of oxazolone-induced skin inflammation was more prominent in NAFLD mice than normal mice. Hence, HFD and HFL diet followed by topical oxazolone application develops metabolic syndrome, NAFLD, and enhanced skin inflammation in mice. Discussion and conclusion: This simple model can be utilized to evaluate a therapeutic strategy for the treatment of metabolic syndrome and NAFLD with skin inflammation and also to understand the nexus between these co-morbidities.
Animal model, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), nuclear factor-kappa B, oxazolone, skin inflammation
Introduction Non-alcoholic fatty liver disease (NAFLD) refers to a histological spectrum of liver damage from simple steatosis to steatohepatitis in those without a history of excessive alcohol consumption (520 g/d) (Angulo et al., 2002). NAFLD is considered as the manifestation of metabolic syndrome in the liver. The metabolic syndrome is a condition characterized by a cluster of alterations including insulin resistance, obesity, dyslipidemia, hypertension, a pro-inflammatory, and prothrombotic state (Almeda-Valdes et al., 2009). The development of NAFLD is strongly associated with metabolic syndrome as reflected by the fact that approximately 90% of the patients with NAFLD have more than one feature of metabolic syndrome and about 33% have three or more criteria (Marchesini et al., 2003). Increasing severity of NAFLD Correspondence: Dr. Shridhar Narayanan, Vice President and Head, Infection iScience, AstraZeneca India Pvt. Ltd., Off Bellary Road, Hebbal, Bangalore 560024, Karnataka, India. Tel: +91 9611598805. E-mail: [email protected]
History Received 16 July 2014 Revised 27 August 2014 Accepted 28 August 2014 Published online 27 November 2014
represents worsening inflammatory and insulin-resistant state with poorer metabolic outcome (Bhatia et al., 2012). There are a number of observations in the literature linking NAFLD and metabolic syndrome to systemic inflammation (Bhatia et al., 2012; Hamminga et al., 2006). The liver is a key metabolic organ and plays important role in the regulation of systemic inflammation. Liver disease like NAFLD increases various inflammatory mediators like TNF-a, IL-6, C-reactive protein, glucose, and plasminogen activator inhibitor-1 (Bhatia et al., 2012). Obesity and diabetes are associated with chronic low-grade inflammatory state through release of pro-inflammatory cytokines including TNF-a, and IL-6 (Hamminga et al., 2006). In rodents, a high-fat diet (HFD) results in NAFLD and up-regulation of NF-kB activity, which leads to hepatic production of various proinflammatory cytokines and activation of Kupffer cells and macrophages (Bhatia et al., 2012; Cia et al., 2005). Interestingly, several recent studies have found prevalence of NAFLD and metabolic syndrome in skin inflammation like psoriasis (Gisondi et al., 2009; Matsumoto et al., 2004).
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Gisondi et al. (2009) reported that occurrence of NAFLD is substantially more in psoriasis patients than in controls (47% versus 28%, p50.001). Several medications used to treat skin inflammation are known to cause liver damage, which can affect the outcome of drugs used to treat metabolic syndrome, especially NAFLD. Drugs like methotrexate cause NAFLD-like histological changes in liver and hence may worsen the pre-existing NAFLD in patients with skin inflammation (Wenk et al., 2010). Moreover, no specific therapy is approved for the treatment of NAFLD. Animal models of human disease are necessary to allow the study of and understand the efficacy and safety of drugs at real-life level. The ideal disease model should include all the characteristics of the disease along with the co-morbidities. HFD with high-fructose liquid (HFL) in drinking water has been shown to induce NAFLD in mice (Tetri et al., 2008). Chronic oxazolone-induced experimental skin inflammation is being used as a preliminary model for screening of novel drugs for skin inflammation like dermatitis and psoriasis in drug discovery (Peterson, 2006; Yeom et al., 2012). The aim of the present study was to develop a simple mouse model of NAFLD-enhanced skin inflammation and to study the effect of NAFLD on different parameters of skin inflammation.
Materials and methods Animals Male C57BL/6 mice of 4–5 weeks age and 13–18 g body weight were procured from the National Institute of Nutrition, Hyderabad, India. All mice were maintained in 12 h light/dark cycle with free access to standard laboratory chow diet and water ad libitum in controlled environment (23 ± 2 C). Mice were handled according to the guidelines of experimental animal care issued by the committee for the purpose of control and supervision of experiments on animals (CPCSEA) and the experimental protocol was approved by the institutional animal ethical committee (IAEC) (Protocol no. 04/ IAEC-01/PCP/2011). Materials Normal chow diet was procured from Nutri LabÕ Rodent (Tetragon Chemie Pvt. Ltd., Bangalore, India) and 60 kcal% HFD procured from Open Source diets (Cat# D12492, New Brunswik, NJ). Fructose was procured from Sisco Research Laboratories (Mumbai, India). Oxazolone was purchased from Sigma (St Louis, MO). All other reagents were of the highest commercially available grade. Methodology Male C57BL/6 mice were divided into two groups. The first group (n ¼ 22) was fed with normal chow feed and the second group (n ¼ 29) fed with 60 kcal% HFD and HFL 40 % for 60 d. On the 45th day, two animals from each group were sacrificed; liver histopathology examination was carried out to confirm the induction of NAFLD. At the end of induction period (day 60), the remaining animals were grouped as follows. Mice fed with normal chow diet (n ¼ 20) were further divided into three groups; normal untreated control (n ¼ 8), normal + vehicle (n ¼ 6), and normal + oxazolone (n ¼ 6).
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Mice fed with HFD and HFL (n ¼ 27) were further divided into three groups (n ¼ 9) as untreated NAFLD control, NAFLD + vehicle, and NAFLD + oxazolone. Basal ear thickness was measured and animals were sensitized with 1% oxazolone or vehicle (acetone:olive oil, 4:1) on both ears (20 ml each). Seven days later, the animals were challenged with 0.5% oxazolone or vehicle for 2 weeks as shown in Figure 1(a). Ear thickness was measured with a digital micrometer (Mitutoyo, Kanagawa, Japan) before and 24 h post each challenge. On 15th day, animals were kept for overnight fasting and, on 16th day, blood was collected from retroorbital sinus under light isoflurane anesthesia and plasma separated by centrifugation at 6000 rpm for 10 min for biochemical analysis. Following blood collection, animals were sacrificed by cervical dislocation and liver, ear, and fat pad were collected, weighed, and frozen (80 C) for further analysis. Part of liver and ear tissues were kept in 10% formalin for histopathology examination. Biochemical estimations in plasma Plasma glucose, triglyceride (TG), cholesterol (TC), total bilirubin, total protein, alanine aminotransaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured from plasma by random access clinical chemistry analyzer Erba XL 300 using commercially available ERBA diagnostics kit (OpenSource Diets, New Brunswik, NJ). Plasma insulin was estimated by using ELISA kits following manufacturer’s instructions (Millipore, Billerica, MA). Cytokines in serum and ear homogenates Briefly, the treated mouse ear tissue extracts were prepared by tissue lysis with T-PER buffer (Pierce, Rockford, IL) supplemented with 1 Protease inhibitor cocktail (Calbiochem, San Diego, CA) and 1 phosphatase inhibitor cocktail (Calbiochem, San Diego, CA) using a tissue homogenizer. Homogenates were then centrifuged at 14 000 rpm for 30 min. IL-6 and TNF-a were measured in the supernatant (tissue homogenate) or serum using ELISA kits following manufacturer’s instructions (GE Healthcare, Little Chalfont, UK). Myeloperoxidase (MPO) activity in plasma and ear homogenates MPO activity was measured in plasma and ear homogenates (homogenized as described above for assessment of cytokines) (De Vry et al., 2005). Briefly, 50 ml of plasma or supernatant from ear homogenate was added to 200 ml of an assay reaction mixture containing 0.5% hexadecyltrimethylmmonium bromide (in 50 mM potassium phosphate, pH 6.4), 0.165 mg/ml o-dianisidine hydrochloride and 0.0015% H2O2. Absorbance was measured at 460 nm. Liver triglycerides The liver lipids were extracted using a modified Folch extraction protocol (Folch et al., 1957). Briefly, approximately 100 mg of liver tissue was homogenized with methanol (1 ml) then the homogenate was centrifuged at 4000 rpm for 5 min and supernatant transferred into separate
Novel animal model of NAFLD and skin inflammation
DOI: 10.3109/13880209.2014.960944
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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 ¼ 6–9, $$p50.01, $$$p50.001 as compared with normal + vehicle, **p50.01, ***p50.001 as compared with NAFLD + vehicle, and #p50.05, ##p50.01, and ###p50.001 as compared with normal + oxazolone, one-way ANOVA followed by the Newman– Keuls multiple comparison test).
tubes (15 ml). Then the pellet was re-suspended in chloroform:methanol (2:1) solution for 2 min. The homogenate was then centrifuged and supernatant separated and mixed with first supernatant. About 0.1 M potassium chloride was added to supernatant and mixed well by vortexing. After centrifugation of the mixture, bottom phase (organic phase) was removed to a new tube (2 ml). The samples were evaporated in Turbovap LV evaporator (GE Healthcare, Little Chalfont, UK). Residue was reconstituted in 400 ml of mixture of N-butyl alcohol:Triton X-100:methanol (3:1:1) and mixed properly by vortexing. The samples were used for the triglyceride estimation using commercially available kit (Erba Diagnostics).
Ear tissues were analyzed quantitatively for epidermal thickness and inflammatory infiltrate by an investigator blinded to treatment as described previously (Boehncke et al., 2001). Briefly, maximal epidermal thickness was measured from the tip of the rete ridges to the border of the viable dorsal or ventral epidermis. The mean value of six such rete ridges was measured using the ocular micrometer of the microscope at 400 each division measuring 2.45 mm. The inflammatory grading was done based on the presence of dermal inflammation without Munro’s abscess (score 0.5), mild inflammation with Munro’s abscess (score 1), moderate inflammation with Munro’s abscess (score 2), and marked inflammation with Munro’s abscess (score 3).
Histopathology
Western blot analysis
Liver and ear tissue samples were fixed in 10% formalin and embedded in paraffin. Sections measuring 3–5 mm in thickness were cut and stained with hematoxylin and eosin (H&E). Liver sections were examined using a light microscope (NIKON, ECLIPSE-E200, Tokyo, Japan) and graded by the in-house method based on Hubscher (2006) and Brunt and Tiniakos (2002). It included scoring of microvacuolation, namely 1 (mild), 2 (moderate), and ballooning degeneration as 1 (mild), 2 (moderate), 3 (marked), and 4 (severe).
Briefly, the treated mice ear tissue extracts were prepared by tissue lysis as described earlier for the cytokine estimation. The protein content was determined using a bicinchoninic acid assay (BCAÔ protein assay kit, Pierce, Appleton, WI) and samples were prepared in SDS, bromophenol blue, and 100 mg/mL DTT, and boiled for 5 min at 99 C. The 50 mg of protein was resolved on a 10% SDS-PAGE and blotted on to a nitrocellulose membrane and the blots were blocked for 1 h at room temperature with 5% non-fat milk protein in
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Tris-buffered saline (TBS) containing 0.1% Tween 20, then blots were probed with antibodies against anti-rabbit pNF-kB (Cell Signaling Technology, Danvers, MA) and anti-rabbit GAPDH (Cell Signaling Technology, Danvers, MA) overnight at 4 C. Horseradish peroxidase-conjugated secondary antibody was subsequently incubated with blot for 1 h room temperature. The ECL (Enhanced chemiluminescence; Pierce, Appleton, WI) was used to visualize signal (Li et al., 2012).
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Statistical analysis All values are expressed as mean ± standard error of the mean (SEM). The Graphs were generated using Graph-Pad PrismÕ (Version 4, GraphPad Software, La Jolla, CA). Statistical analysis was performed by Student ‘t’ test or one-way ANOVA followed by the Newman–Keuls multiple comparison test or two-way ANOVA followed by the Bonferroni post test as applicable. For histopathology, non-parametric analysis was done by the Mann–Whitney U test or the Kruskal Wallis test followed by Dunn’s multiple comparison test as applicable. Results were considered statistically significant at p50.05.
Results There was no significant difference observed between normal control versus normal + vehicle and NAFLD control versus NAFLD + vehicle, indicating vehicle did not affect any of the parameters in both normal and NAFLD mice. Hence, the data for normal control and NAFLD control are not shown in the results. Induction of obesity and NAFLD with dietary manipulation Induction of obesity and NAFLD in mice was achieved by feeding HFD and HFL diet to C57BL/6 mice. Feeding mice with HFD and HFL significantly increased body weight (p50.01), epididymal (p50.01), and inguinal fat (p50.01) as compared with animals on the normal chow diet (Table 1). On 45th day of dietary manipulation, two animals were sacrificed and induction of NAFLD in HFD- and HFLfed mice was confirmed by the histopathological examination for the presence of ballooning degenerations and
microvacoulations (data not shown). Oxazolone or vehicle application did not affect the body weight and fat pad in both normal and HFD- and HFL-fed mice. Induction of hyperglycemia and hyperinsulinemia with dietary manipulationHyperglycemia and hyperinsulinemia are important criteria of metabolic disorder. Mice fed with HFD and HFL diet showed a significant increase in fasting plasma glucose (p50.01) and insulin (p50.01) as compared to mice on normal chow diet (Table 1). Mice fed with HFD and HFL caused elevation in plasma ALT levels indicating the induction of NAFLD in this model. Dietary manipulation did not cause any significant difference in other biochemical parameters tested compared to normal chow-fed mice (Table 1). Oxazolone application in both normal and NAFLD mice did not affect any of the biochemical parameters as compared with their respective controls (Table 1). Induction of ear edema in normal and NAFLD mice Ear edema was induced by topical application of oxazolone to both ears of normal and NAFLD mice as shown in Figure 1(b–e). Ear thickness was measured before and 24 h post each oxazolone challenge. Ear thickness increased significantly from the second challenge onwards in NAFLD + oxazolone mice as compared with NAFLD + vehicle mice (p50.001) and remained till the end of the experiment (p50.001) (Figure 1b). Whereas oxazolone application to the normal mice showed significant increase in ear thickness from third challenge onwards and remained till the end of the experiment (p50.001). Ear thickness in NAFLD + oxazolone mice was significantly higher than normal + oxazolone mice (p50.001). This increase in ear thickness was statistically significant from the second challenge onwards till the end of the experiment, indicating increased inflammatory response in NAFLD mice. At the end of the study, animals were sacrificed and ear weight was recorded. Oxazolone application significantly increased ear weight in both normal and NAFLD mice (p50.01) (Figure 1c). Ear weight of NAFLD + oxazolone mice was significantly higher than normal + oxazolone mice (p50.01).
Table 1. Effect of oxazolone on body weight, fat pad and biochemical parameters in normal and NAFLD mice.
Body weight (g) Epididymal fat (g) Inguinal fat (g) Glucose (mg/dl) Insulin (ng/ml) Triglyceride (mg/dl) Total cholesterol (mg/dl) ALT (IU/l) AST (IU/l) ALP (IU/l) Total bilirubin (mg/dl) Total protein (g/dl) TNF-a (pg/ml) IL-6 (pg/ml)
Normal + vehicle
Normal + oxazolone
NAFLD + vehicle
NAFLD + oxazolone
24.33 ± 1.33 0.52 ± 0.08 0.40 ± 0.07 160.05 ± 10.44 0.350 ± 0.30 103.5 ± 13.22 111.67 ± 6.76 39.65 ± 3.41 100.03 ± 10.78 228.17 ± 29.41 0.43 ± 0.14 5.75 ± 0.11 12.58 ± 3.16 15.36 ± 2.89
25.05 ± 1.16 0.42 ± 0.03 0.30 ± 0.04 158.15 ± 3.99 0.323 ± 0.40 76.83 ± 6.45 112.17 ± 2.06 48.7 ± 4.32 82.1 ± 7.75 227.67 ± 9.30 0.27 ± 0.04 5.80 ± 0.09 13.92 ± 3.19 19.12 ± 5.11
33.87 ± 1.29$$ 1.45 ± 0.16$$ 1.23 ± 0.19$$ 311.71 ± 15.31$$ 1.599 ± 0.31$$ 87.89 ± 2.32 125.33 ± 5.00 59.33 ± 12.29 92.63 ± 10.20 216.33 ± 6.92 0.54 ± 0.08 5.58 ± 0.07 26.17 ± 3.77$$ 38.22 ± 3.69$$
32.83 ± 1.07$$ 1.40 ± 0.15$$ 1.12 ± 0.13$$ 272.17 ± 13.68$$ 1.710 ± 0.34$$ 81.44 ± 5.24 115.56 ± 5.84 64.49 ± 14.28 80.98 ± 6.03 204.56 ± 13.57 0.36 ± 0.05 5.65 ± 0.07 29.56 ± 4.57$$ 45.15 ± 6.58$$
Values are expressed as mean ± SEM (n ¼ 6–9). $$p50.01 as compared with normal + vehicle, one-way ANOVA followed by the Newman–Keuls multiple comparison test.
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DOI: 10.3109/13880209.2014.960944
MPO activity, an indicator of polymorphonuclear leukocyte influx, was measured in both plasma and ear homogenate. Oxazolone application significantly increased MPO activity in plasma and ear homogenate in both normal and NAFLD mice (p50.01) as compared with their respective control (Figure 1d). In accordance with ear thickness and ear weight, MPO activity in plasma and ear homogenate was significantly higher in the NAFLD + oxazolone group than the normal + oxazolone group (p50.05, p50.01) (Figure 1d and e). TNF-a and IL-6 levels were measured in serum and ear homogenate. Serum TNF-a, and IL-6 levels were significantly elevated in NAFLD mice as compared with normal mice (Table 1). In ear homogenate, there was no significant difference observed in TNF-a levels in both normal and NAFLD mice (data not shown). While IL-6 levels significantly increased in normal + oxazolone (285.48 ± 17.61 pg/ml versus 558.10 ± 53.88 pg/ml) and NAFLD + oxazolone groups (260.71 ± 18.60 pg/ml versus 504.52 ± 54.47 pg/ml) as compared with their respective controls, no significant difference was observed between normal + oxazolone and NAFLD + oxazolone groups. Effect of dietary manipulation on liver triglyceride levels and liver histopathology Liver triglyceride levels were significantly higher in NAFLD mice than normal mice (p50.01) indicating accumulation of lipid in the liver with HFD and HFL diet (Figure 2a). Microvacuolations and ballooning degenerations were scored by a pathologist blinded to treatment. Liver sections of HFDand HFL-fed mice showed significant induction of ballooning
40 30 20 10
degenerations and microvacuolations (Figure 2b and d). Animals fed with normal chow diet did not show any pathology of NAFLD (Figure 2c). Application of oxazolone on ears did not affect the liver histopathology in both normal and NAFLD mice. Effect of oxazolone on ear histopathology in normal and NAFLD mice Application of oxazolone to ears showed psoriasis and dermatitis like histopathology in both normal and NAFLD mice (Figure 3). Munro’s microabscesses were present in both normal + oxazolone and NAFLD + oxazolone groups (Figure 3d). Epidermal thickness, as measured using an ocular micrometer by an investigator blinded to treatment, was significantly higher in both normal + oxazolone and NAFLD + oxazolone groups as compared with their respective controls (p50.01) (Figure 3e). Epidermal thickness in the NAFLD + oxazolone group was greater than the normal + oxazolone group. Dermal inflammatory score was significantly more in the NAFLD + oxazolone group than the normal + oxazolone group (Figure 3f). Inflammation was not observed in un-induced normal and NAFLD mice (Figure 3a). Overall, induction of skin histopathology resembling psoriasis and dermatitis was more prominent in the NAFLD + oxazolone group than the normal + oxazolone group (Figure 3b and c). Effect of oxazolone on NF-kB expression in normal and NAFLD mice NF-kB is a key regulatory element in a variety of immune and inflammatory pathways and hence is a crucial mediator involved in the pathogenesis of various skin inflammatory
NAFLD
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Balloning degenerations/ Microvacuolations
Normal
(a) Liver TG (mg/ g of tissue)
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Effect of oxazolone on MPO and cytokine levels in normal and NAFLD mice
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4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5
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0 Balloniong degenerations
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Microvacuolations
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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 ¼ 6–9, **p50.01, ***p50.001 as compared with normal, the Mann–Whitney U test).
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Normal + Oxazolone NAFLD + Oxazolone **
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Normal + vehicle NAFLD + vehicle
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3.5 3.0 2.5 2.0 1.5 1.0 0.5
Normal+oxazolone NAFLD+oxazolone # ***
$
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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 ¼ 6–9, $p50.05, $$p50.01 as compared with normal + vehicle, **p50.01 as compared with NAFLD + vehicle, and #p50.05, as compared with normal + oxazolone, the Kruskal–Wallis test followed by Dunn’s multiple comparison test).
conditions. Chronic oxazolone treatment in mice strongly stimulated the phosphorylation of NF-kB in both normal and NAFLD mice. Phosphorylation of NF-kB was significantly higher in NAFLD mice than in normal mice treated with oxazolone (p50.01). However, there was no significant difference observed between normal and NAFLD mice treated with vehicle (Figure 4).
Discussion This is the first attempt to evaluate the effect of NAFLD on skin inflammation and eventually to develop an animal model. Recent literature indicates that the association among skin inflammation, metabolic syndrome, and NAFLD is growing in patient population (Gerdes et al., 2010; Gottlieb et al., 2008; Saraceno et al., 2008; Wenk et al., 2010). However, the exact pathogenic mechanism between these comorbidities is still unclear. Moreover, several medications used to treat skin inflammation are also known to induce liver diseases. Lack of proper animal model has hampered the understanding of the nexus between such co-morbidities and
also evaluation of potential pharmacological interventions. Hence, the animal model developed in the present study will enable us to study the interaction among NAFLD, metabolic syndrome, and skin inflammation and also to evaluate different therapeutic strategies for the treatment of such co-morbidities. In the present study, we induced metabolic syndrome and NAFLD first by combination of HFD and HFL in drinking water to C57BL/6 mice for 2 months. Combination of HFD and HFL is associated with the development of obesity, increased de novo lipogenesis in the liver, hepatic steatosis, impaired glucose tolerance, insulin resistance, and hypertension (Tetri et al., 2008). Mice fed with HFD and HFL experienced rapid gain in body weight and increased fat pad weight as compared with normal chow-fed mice. Histopathological examination of liver clearly demonstrated that the HFD- and HFL-fed mice showed ballooning degenerations and microvacuolations indicating the development of NAFLD. The induction of NAFLD was further confirmed by increased liver triglyceride levels and plasma level of ALT. NAFLD mice also showed significant elevation of plasma
Novel animal model of NAFLD and skin inflammation
DOI: 10.3109/13880209.2014.960944
(a)
Normal + Vehicale
NAFLD + Vehicale
Normal + oxazolone
NAFLD + oxazolone
NF–kB p65 kDa
GAPDH 3– kDa
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Normal+vehicle
NAFLD+vehicle
NAFLD+oxazolone
Normal+oxazolone ## ***
0.35
Relative Density
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0.30
$$$
0.25 0.20 0.15 0.10 0.05 0.00
Figure 4. Effect of oxazolone on NF-kB activity in normal and NAFLD mice. (a) Mice ears were excised 24 h after last oxazolone challenge and analyzed for phosphorylation levels of NF-kB. (b) GAPDH was used as a control and relative density after vehicle or oxazolone treatment was expressed from two independent experiments (n ¼ 4, $$$p50.001 as compared with normal + vehicle, ***p50.001 as compared with NAFLD + vehicle, and ###p50.001, as compared with normal + oxazolone, one-way ANOVA followed by the Newman–Keuls multiple comparison test).
glucose and insulin levels along with the elevation of circulating cytokines like TNF-a, and IL-6 in serum than normal mice. These data suggest that the current animal model displayed the most important criteria of metabolic syndrome like obesity and insulin resistance along with NAFLD. For pharmacological screening of new drugs for skin inflammation, acute and chronic oxazolone-induced skin inflammation is being used as the preliminary screening model in drug discovery for years. This model is particularly useful for the evaluation of broad acting anti-inflammatory drugs (Peterson, 2006). Oxazolone application to ears causes histopathological changes similar to that of psoriasis and atopic dermatitis. Moreover, standard drugs like hydrocortisone, phosphodiesterase inhibitors, and dexamethasone show significant inhibitory activity in this model (Peterson, 2006). In the present study, application of oxazolone to the ears of normal C57BL/6 mice caused significant increase in ear thickness, ear weight, MPO activity, and NF-kB expression. Histopathology of ear displayed epidermal thickness and infiltration of inflammatory cells along with the presence of Munro’s microabscess, a characteristic histopathological feature of psoriasis (Boehncke et al., 2001). A similar application of oxazolone to NAFLD mice improved the induction of disease compared with that of normal mice. Ear thickness, ear weight, MPO activity, and NF-kB activity were significantly higher in NAFLD mice than that of normal mice. Incidence of microabscess, epidermal thickness, and dermal inflammatory score were also higher in NAFLD mice than normal mice. The data from this study clearly show that the presence of co-morbidities like obesity, insulin resistance, and
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NAFLD increased the severity of oxazolone-induced skin inflammation. These results are in accordance with clinical reports where the incidence of skin inflammation was found higher in patients with metabolic disorder and NAFLD (Gisondi et al., 2009; Saraceno et al., 2008; Wenk et al., 2010). Elevated levels of pro-inflammatory cytokines, especially TNF-a in adipose tissue, are an important feature of obesity and contribute significantly to insulin resistance and development of NAFLD (Baker et al., 2011; Wenk et al., 2010). Accumulation of free fatty acids within hepatocytes causes hepatic TNF-a production and further increasing the inflammatory condition in metabolic syndrome. Adipocyte and hepatic TNF-a may act on skin to worsen skin inflammation by promoting keratinocyte proliferation, increased inflammation, and the upregulation of vascular adhesion molecules and transcription factor NF-kB (Bhatia et al., 2012; Wenk et al., 2010). In the present study, serum levels of TNF-a and IL-6 were significantly higher in NAFLD mice indicating the presence of chronic low-grade systemic inflammation. This increased systemic inflammation may be responsible for enhanced skin inflammation in NAFLD mice. Notably, several studies have indicated that the increase in the activation of factor NF-kB has an important role in the development and maintenance of cutaneous inflammatory diseases, including psoriasis, contact dermatitis, and atopic dermatitis (Bell et al., 2003). In the skin, the transcription of cytokines, such as IL-1, IL-6, and TNF-a, and many of the effectors of cytokine action, such as vascular cell adhesion molecule-1, and cyclo-oxygenase-2, are regulated by NF-kB (De Vry et al., 2005). In this study, oxazolone treatment increased NF-kB activation in ear tissue of both normal and NAFLD mice, with pronounced increase in NAFLD mice treated with oxazolone than normal mice. These data clearly shows that NAFLD-enhanced oxazolone-induced skin inflammation may be mediated by increased NF-kB activation. Further, MPO activity, an indicator of neutrophil accumulation and a characteristic feature of cutaneous inflammation, was significantly higher in plasma and ear tissues of NAFLD mice than normal mice, indicating the increased severity of inflammation in NAFLD mice. Most of the drugs used for the treatment of skin inflammation are known to cause liver damage and there are no formal guidelines regarding systemic treatment of skin inflammation in NAFLD patients. Methotrexate induced liver damage resembles that of NAFLD and may worsen the condition if used in such patient population (Wenk et al., 2010). The present animal model can be used to study the effect of anti-inflammatory drugs in the presence of both metabolic syndrome and NAFLD. In our preliminary studies, orally administered methotrexate caused severe mortality at 1 mg/kg in this model and worsened NAFLD, whereas it was well tolerated in normal mice (unpublished data). This finding encourages us to further utilize this model to study the effect of such drugs alone and in combination with drugs used in the management of metabolic syndrome on NAFLD and skin inflammation. Hence, pharmacotherapy of skin inflammation and NAFLD in the presence of co-morbidities can be established using this model.
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In conclusion, the present study is an attempt to develop an animal model of metabolic syndrome and NAFLD-enhanced skin inflammation. Dietary manipulation followed by topical oxazolone application increases systemic inflammation and NF-kB activation in skin resulting in metabolic syndrome and NAFLD-enhanced skin inflammation in mice. This simple animal model can be utilized to evaluate therapeutic strategy for the treatment of metabolic syndrome and NAFLD with skin inflammation like psoriasis or dermatitis and also to understand the nexus between these co-morbidities.
Declaration of interest
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The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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