Inflammation ( # 2014) DOI: 10.1007/s10753-014-9894-1

Galangin Dampens Mice Lipopolysaccharide-Induced Acute Lung Injury Yu-Sheng Shu,1,3,4 Wei Tao,1,2 Qian-Bing Miao,1 Shi-Chun Lu,1 and Ya-Bing Zhu2,3

Abstract—Galangin, an active ingredient of Alpinia galangal, has been shown to possess anti-inflammatory and antioxidant activities. Inflammation and oxidative stress are known to play vital effect in the pathogenesis of acute lung injury (ALI). In this study, we determined whether galangin exerts lung protection in lipopolysaccharide (LPS)-induced ALI. Male BALB/c mice were randomized to receive galangin or vehicle intraperitoneal injection 3 h after LPS challenge. Samples were harvested 24 h post LPS administration. Galangin administration decreased biochemical parameters of oxidative stress and inflammation, and improved oxygenation and lung edema in a dose-dependent manner. These protective effects of galangin were associated with inhibition of nuclear factor (NF)-κB and upregulation of heme oxygenase (HO)-1. Galangin reduces LPS-induced ALI by inhibition of inflammation and oxidative stress. KEY WORDS: inflammation; oxidative stress; galangin; acute lung injury.

INTRODUCTION Inflammation is the hallmark feature of acute lung injury (ALI) [1]. Infiltration of activated inflammatory cells, over released neutrophil elastases, increased pro-inflammatory cytokine levels as well as nuclear factor (NF)-κB activation cause deteriorated oxygenation and lung edema [2–4]. Meanwhile, reactive oxygen species (ROS), an indicator of oxidative stress, are elevated significantly during the pathogenesis of ALI [2, 5]. Pharmaceutical agents with anti-inflammatory or (and) antioxidant features have been shown to have beneficial effects on ALI [2, 3, 5, 6]. Galangin, a member of the flavonol class of flavonoids, is an active ingredient presents in high concentrations in the rhizome of Alpinia officinarum. A. officinarum has been used as a traditional Chinese medicine for a variety of diseases in China and other Asia countries for 1

Department of Thoracic Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou, 225001 Jiangsu, People’s Republic of China 2 Department of Surgery, Yangzhou East Hospital, Yangzhou, 225001 Jiangsu, People’s Republic of China 3 Department of Cardiothoracic Surgery, Hangzhou First People’s Hospital, Hangzhou, 310003 Zhejiang, People’s Republic of China 4 To whom correspondence should be addressed at Department of Thoracic Surgery, Subei People’s Hospital of Jiangsu Province, Yangzhou, 225001 Jiangsu, People’s Republic of China. E-mail: [email protected]

hundreds of years. Ovalbumin-induced airway inflammation has been shown to be reduced by galangin administration [7]. Galangin can suppress ROS production in acute ischemic stroke [8]. These data indicate that galangin has anti-inflammatory and antioxidant activities [7–9]. As there is no effective pharmaceutical therapeutic strategy for ALI up to date [10], we are interested in that whether galangin can be developed as a potential treatment for ALI. The present study was conducted to test the hypothesis that galangin dampens lipopolysaccharide (LPS)-induced ALI via inhibition of inflammation and oxidative stress.

MATERIALS AND METHODS Experimental Protocol All experiments were performed according to the guidelines for the care and use of animals as established by the Animal Ethics Committee of Jiangsu Province. Sixto 8-week-old specific pathogen-free male BALB/c mice (17–20 g, Yangzhou, Jiangsu, China) were used in these studies. Mice were maintained on a 12 h light-dark schedule in a specific pathogen-free environment and received standard laboratory rodent chow ad libitum. The animals were randomly assigned to one of four groups: control group, vehicle group, and galangin high as well as low

0360-3997/14/0000-0001/0 # 2014 Springer Science+Business Media New York

Shu, Tao, Miao, Lu and Zhu dosage group. Mice were anesthetized by intraperitoneal injection of carbrital (40 mg/kg). Then, ALI was induced by LPS (50 μM, Sigma, St. Louis, MO, USA) intratracheal injection as described previously [11]. Equivalent saline was administrated for control group. Three hours after LPS or saline challenge, galangin (1.5 mg/kg (low) or 15 mg/kg (high); Sigma-Aldrich, St. Louis, MO, USA) or vehicle (20 μl dimethylsulphoxide (DMSO) in a total of 200 μl saline) intraperitoneal injection was performed as described elsewhere [7]. Galangin was dissolved in DMSO and stored at −20 °C before use [7]. Animals were reanesthetized 24 h after LPS or saline challenge. A midline incision was made through the sternum and abdominal in the animal of each group. A sample of blood was collected with a heparinized syringe from the abdominal aortic artery and immediately measured with a blood gas analyzer. Then the animal was exsanguinated through the vena cava. The left lung was excised for pulmonary dry/wet ratio analysis and histologic examination. And the right lung was excised, rinsed of blood, then, was homogenized in phosphate buffer solution on ice to make the 10 % pulmonary homogenate, and stored at −70 °C for further analysis.

Lung Water Content Determination The upper left lung was weighed before being dried in an oven at 80 °C for 72 h, and then the dried lung was weighed again to calculate the pulmonary wet/dry ratio.

Measurement of Pulmonary Inflammatory Mediator Levels The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 in pulmonary were determined by using ELISA kits (R&D Systems Inc.) according to the manufacturers’ manual. NF-κB Binding Assay Nuclear extracts of lung homogenates were prepared with a nuclear extract kit (Active Motif North America, Carlsbad, CA, USA). The DNA-binding activity of NF-κB p65 was determined using an ELISA-based NF-κB p65 transcription factor assay kit (Active Motif North America) according to the manufacturers’ manual. Lipid Hydroperoxide Assay The level of lipid hydroperoxide in lung tissues was measured using a lipid hydroperoxide assay kit (Cayman Chemical, Ann Arbor, USA) according to the manufacturers’ manual. Measurement of HO-1 Activity The activity of heme oxygenase (HO)-1 in lung tissues was determined using specific ELISA kits (R&D Systems Inc.) according to the manufacturer’s instructions. Statistical Analyses

The lung sections were stained with H&E and examined with light microscopy. The Murakami technique [12] was employed to grade the degree of lung injury, which is based on the following histologic features: edema, congestion, infiltration of inflammatory cells, and hemorrhage. Each feature was graded as: 0, absent and appears normal; 1, light; 2, moderate; 3, strong; and 4, intense. A total score was calculated for each animal.

All data are presented as mean±SEM. Statistical significance was determined by one-way analysis of variance (ANOVA). The Student-Newman-Keuls method was used for comparison between groups. Kruskal-Wallis one-way analysis of variance on ranks and the Student-Newman-Keuls method were used for statistical evaluation of the histopathologic scores. Survival data were analyzed with the Kaplan-Meier method. P value less than 0.05 was considered to be statistically significant.

Myeloperoxidase Activities Analysis

RESULTS

Histologic Examination

Myeloperoxidase (MPO) activities in lung homogenates were measured according to the manufacturers’ instructions (R&D Systems Inc., Minneapolis, MN, USA) and calculated from the absorbance changes resulting from decomposition of hydrogen peroxide in the presence of odianisidine.

Effects of Galangin on ALI Galangin treatment, with high but not low dosage, significantly reduced lung edema compared with vehicle group (P

Galangin dampens mice lipopolysaccharide-induced acute lung injury.

Galangin, an active ingredient of Alpinia galangal, has been shown to possess anti-inflammatory and antioxidant activities. Inflammation and oxidative...
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