627160 research-article2016

IJI0010.1177/0394632015627160International Journal of Immunopathology and PharmacologyZhang et al.

Original article

Protective effect of diallyl trisulfide against naphthalene-induced oxidative stress and inflammatory damage in mice

International Journal of Immunopathology and Pharmacology 1­–12 © The Author(s) 2016 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0394632015627160 iji.sagepub.com

Fang Zhang,1 Yongchun Zhang,1,2 Kaiming Wang,1 Guangpu Liu,1 Min Yang,1 Zhongxi Zhao,1,3 Shanzhong Li,3 Jianhua Cai3 and Jimin Cao3

Abstract The aim of this study was to investigate the possible protective effects of diallyl trisulfide (DATS) against naphthaleneinduced oxidative and inflammatory damage in the livers and lungs of mice. Elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels showed significant hepatic damage after the challenge with 100 mg/kg naphthalene. Hepatic malondialdehyde (MDA) contents and the activity of myeloperoxidase (MPO) increased significantly, accompanying a decrease in the hepatic activity of total superoxide dismutase (SOD) and glutathione (GSH) levels after the naphthalene damage. In addition, the serum levels of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 8 (IL-8) increased significantly in the groups damaged with naphthalene. The main parameters related to oxidative stress and inflammatory responses in the lungs, including the NO, MPO, and GSH contents, were determined, and the histopathological and immunohistochemical changes in the lung and liver tissues were also observed. In the DATS-treated groups, all of the oxidative and inflammatory damage in the serum, liver, and lung tissues were significantly prevented. Keywords diallyl trisulfide, inflammatory, myeloperoxidase, naphthalene, nitric oxide, oxidative Date received: 16 June 2015; accepted: 1 December 2015

Introduction Polycyclic aromatic hydrocarbons (PAHs) have been listed as priority pollutants. Naphthalene (NAP), one of the most toxic PAHs, is commonly found in waste water.1,2 Due to its chemical stability and lipophilic nature, NAP biodegradation is difficult, and NAP is easily accumulated and biomagnified in the food chain. The kinetics of NAP metabolism in microsomes and the main metabolites have been described.3 Many studies have been performed in order to investigate the toxicity of NAP and its metabolites in experimental animals by various routes of administration. The ability to cause pulmonary, hematologic, and ocular damage has been confirmed.4–6 In addition, NAP-induced genotoxicity has also been reported.7 Due to its

volatility, NAP might cause irritation following inhalation.8,9 NAP-induced lung injury affects the incidence and severity of several pulmonary diseases.10 The inflammatory response is used to evaluate lung damage.11,12 A previous study characterized 1School

of Pharmaceutical Sciences, Shandong University, Jinan, PR China 2School of Chemistry and Pharmaceutical Engineering, Qilu University of Technology, Jinan, Shandong, PR China 3Jiangsu Shengshi Kangde Biotech Corporation, Lianyungang, Jiangsu, China Corresponding author: Zhongxi Zhao, School of Pharmaceutical Sciences, Shandong University, 44 West Wenhua Road, Jinan, Shandong 250012, PR China. Email: [email protected]

2

the cytotoxicity to the cells of both target (lung, nasal epithelium) and non-target (liver) organs following exposure to high concentrations of NAP.13 Another study demonstrated that NAP exposure results in respiratory tract tumors in mice and rats.14 There is evidence that NAP can induce oxidative stress as shown by measurements of hepatic lipid peroxidation and glutathione (GSH) depletion.15,16 Diallyl trisulfide (DATS) is a major organosulfur compound found in the alliaceae family members, including garlic. It displays a wide variety of internal biological activities.17–19 Some findings indicate that DATS protects against hyperglycemia-induced ROS-mediated apoptosis during diabetes treatment.20 DATS dramatically attenuates acute ethanol-induced liver injury and mitochondrial dysfunction. The increases of hepatic GSH levels and the elevation of the antioxidant enzyme activities account for the preventive effects.21 The anti-inflammatory properties of DATS have been evaluated, and the mechanisms might be related to the inflammatory signaling mediated by NF-κB and STAT3.22 The aim of this study was to evaluate the serum, liver, and lung tissue alterations induced by the toxic effects of NAP and to investigate whether DATS was beneficial in preventing or reducing the various damage. The protective effects of DATS against naphthalene-induced liver injury were examined by testing serum hepatic enzymes of ALT, AST, and LDH, as well as the oxidative enzyme of SOD. Serum cytokines of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 8 (IL-8) were assayed to explore the anti-inflammatory activities of DATS. The prevention potentials of DATS on oxidative damage in the liver tissues were evaluated by the determination of SOD, MDA, MPO, and GSH while MPO, NO, and GSH were assayed for the lung tissues. Finally, the histopathological and immunohistochemical analyses of lung and liver tissue injury were also conducted to detect morphometrical changes in the lung and liver.

Materials and methods Animals and experimental groups Kunming mice (weight range, 18–20 g) of either sex were purchased from the Center for New Drug Evaluation of Shandong University, Jinan, PR China. The mice were housed in a room at a mean

International Journal of Immunopathology and Pharmacology

constant temperature of 25°C with 50–60% relative humidity and a 12-h light/dark cycle, and they had free access to food and water. The mice were maintained under these conditions for at least 1 week before the experiments. These mice were divided into nine groups, each comprising 10 animals (5 females, 5 males): Blank, without any treatment group; DATS, DATS only at a dose of 40 mg/kg p.o.; Pre-1, pre-treated with DATS for 7 days at doses of 20 mg/kg p.o.; Pre-2, 40 mg/kg p.o.; Pre-3, 80 mg/kg p.o.; Nap, NAP only at a dose of 100 mg/kg p.o.; and Co-1, Co-2, and Co-3, DATS at doses of 20, 40, or 80 mg/kg p.o., respectively, after the treatment of naphthalene. DATS and NAP were both dissolved in soybean oil. At 6 h after the NAP challenge, the mice were killed by decapitation, and blood was collected. The serum and tissue (liver and lung) samples were stored at −80°C before the analyses. Part of each lung tissue sample was embedded in paraffin for histological assessment. All animals received humane care, and the study protocols were conducted according to EU Directive 2010/63/EU for animal experiments.

Tissue sample preparation The liver and lung tissue samples were homogenized with ice-cold physiological saline (10%, wt%, 1 g tissue in 10 mL of physiological saline) in an Ultra Turrax tissue homogenizer. The homogenate was centrifuged at 2500 rpm, and the supernatants were diluted to an appropriate concentration with physiological saline. The supernatant was analyzed for total protein using the bicinchoninic acid (BCA) method.

Biochemical analysis The serum AST, ALT, and lactate dehydrogenase (LDH) levels were measured to assess hepatic function. The assays were performed according to the ELISA kit instructions (Nanjing Jiancheng Bioengineering Institute). The mean value was calculated from the measurements performed at least in triplicate.

Determination of SOD activity The SOD activity in the serum samples was determined using the microplate spectrophotometer method, and the serum SOD activity was expressed

3

Zhang et al.

as U/mL. Hepatic SOD was measured using commercial detection kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute). The SOD activity was expressed as U/mg protein.

Determination of MDA The MDA content was determined using the TBA method, which results in the formation of a red compound. The maximum absorbance of the compound was measured at 532 nm. The liver tissue samples were determined without dilution. The MDA levels were assayed as an indication of the products of lipid peroxidation by monitoring thiobarbituric acid-reactive substance formation. The lipid peroxidation as indicated by the MDA results was expressed as nmol/mg protein.

Determination of TNF-α and IL-8 The serum TNF-α and IL-8 levels were determined using commercial ELISA kits (Shanghai Xitang Bioengineering Institute) according to the instructions of the manufacturer. TNF-α and IL-8 concentrations were calculated from standard curves. The mean value was calculated from measurements performed at least in triplicate.

Determination of NO levels The levels of NO were determined by an assay for nitrite. The procedure followed the instructions of the total nitric oxide assay kit (Nanjing Jiancheng Bioengineering Institute). The optical density of the assay samples was measured spectrophotometrically at 540 nm. The serum NO level was determined using the same method. The NO level was expressed as μM/g protein.

Measurement of MPO activity The tissue MPO activity was assessed according to the instructions of the assay kit (Nanjing Jiancheng Bioengineering Institute). One unit of enzyme activity was defined as the amount of MPO present that caused a decomposition of 1 μM H2O2 at 37°C and was expressed in U/g tissue.

Measurement of GSH activity The tissue GSH activity was assessed according to the instructions of the assay kit (Nanjing Jiancheng

Bioengineering Institute). The results are expressed as μmoL/g protein.

Histological preparation Samples of the lung and liver tissues were fixed in 10% formaldehyde and processed routinely for embedding in paraffin. Paraffin sections were stained with hematoxylin and eosin (H&E) and examined under a light microscope. Microscopic analyses were performed by experienced histologists unaware of the treatment to which the animal had been subjected.

Immunohistochemistry Immunohistochemical staining from paraffinembedded lung and liver tissues was performed in accordance with a routine procedure. The sections (10 μm) were mounted on glass slides. The sections were deparaffinized and incubated in 3% H2O2 for 10 min to quench the endogenous peroxidase activity. After blocking with normal goat serum for 20 min, the sections were incubated with anti-MPO or anti-NF-κB p65 primary antibodies (1:100, Beijing Biosynthesis Biotechnology Co., Ltd.) at 4°C overnight, followed by washing with PBS and incubating with goat anti-rabbit antibody at 37°C for 30 min. Then, they were developed using the DAB Envision System according to the manufacturer’s protocol (Nanjing Jiancheng Bioengineering Institute). All incubations were performed in a humidified chamber at room temperature.

Statistical analysis Statistical analysis was carried out using GraphPad Prism 6.0. The groups of data were compared using analysis of one-way ANOVA followed by Tukey’s multiple comparison tests. Values of P

Protective effect of diallyl trisulfide against naphthalene-induced oxidative stress and inflammatory damage in mice.

The aim of this study was to investigate the possible protective effects of diallyl trisulfide (DATS) against naphthalene-induced oxidative and inflam...
565B Sizes 0 Downloads 20 Views