Inflammation ( # 2014) DOI: 10.1007/s10753-014-9839-8

Modulation of LPS-Stimulated Pulmonary Inflammation by Borneol in Murine Acute Lung Injury Model Weiting Zhong,1,2 Yiwen Cui,2 Qinlei Yu,3 Xianxing Xie,2 Yan Liu,2 Miaomiao Wei,2 Xinxin Ci,1,4 and Liping Peng1,4

Abstract—The object of our study is to investigate the protective effects of Borneol on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. To determine the effects of Borneol on the histopathological changes in mice with ALI, inflammatory cell count in bronchoalveolar lavage fluid (BALF) and lung wet/dry weight ratio were measured in LPS-challenged mice, and lung histopathologic changes observed via paraffin section were assessed. Next, cytokine production induced by LPS in BALF and RAW 264.7 cells was measured by enzyme-linked imunosorbent assay (ELISA). To further study the mechanism of Borneol-protective effects on ALI, nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinases (MAPKs) pathways were investigated. In the present study, Borneol obviously alleviated pulmonary inflammation by reducing inflammatory infiltration, histopathological changes, descended cytokine production, and pulmonary edema initiated by LPS. Furthermore, Borneol significantly suppressed phosphorylation of NF-κB/P65, IκBa, p38, JNK, and ERK. Taken together, our results suggest that Borneol suppressed inflammatory responses in LPS-induced acute lung injury through inhibition of the NF-κB and MAPKs signaling pathways. Borneol may be a promising potential preventive agent for acute lung injury treatment. KEY WORDS: borneol; LPS; inflammation; MAPKs; NF-κB.

INTRODUCTION Acute lung injury and acute respiratory distress syndrome (ARDS), commonly observed after sepsis, are characterized by severe hypoxemia, pulmonary edema, and neutrophil accumulation in the lung and apoptosis of the pulmonary epithelial cells, and leads to the destruction of the pulmonary epithelium and impairment of its barrier function [1, 2]. Lipopolysaccharide (LPS), a main component of outer membrane of Gram-negative bacteria, has

Weiting Zhong and Yiwen Cui contributed equally to this work. 1

Department of Respiration, the First Hospital of Jilin University, 71 Xinmin Road, Changchun, 130021, People’s Republic of China 2 College of Veterinary Medicine, Jilin University, Changchun, 130062, People’s Republic of China 3 General Situation of Jilin Provincial Center for Animal Disease Control and Prevention, Changchun, 130062, People’s Republic of China 4 To whom correspondence should be addressed at Department of Respiration, the First Hospital of Jilin University, 71 Xinmin Road, Changchun, 130021, People’s Republic of China. E-mail: [email protected]; E-mail: [email protected]

been referred to be an important risk factor of induced acute lung injury (ALI) and ARDS [3, 4]. LPS-induced ALI was characterized by the disruption of endothelial and epithelial integrity, lung edema, the release of inflammatory mediators, and extensive neutrophil infiltration [5]. In the clinical cases, ALI is a major problem that has a high mortality rate of 30–40 % [6]. Despite various medical therapies that have been applied clinically to improve the survival of ALI/ARDS patients, there are still few effective measures or specific medicines to treat it [7–10]. The nuclear transcription factor nuclear factor-kappaB (NF-κB), which is required for maximal transcription and production of numerous cytokines, including tumor necrosis TNF-α, IL-1β, and IL-6, has been reported to play an important role in the pathogenesis of lung diseases [11]. MAPKs, including ERK, JNK, and P38, are linked to cytokine production in inflammatory diseases. Since expression of proinflammatory mediators is tightly regulated at a transcriptional level via NF-κB and MAPKs signaling pathways, the targeted inhibition of the induction and activity of NF-κB and MAPKs has been identified as a new therapeutic method in inflammatory diseases.

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

Zhong, Cui, Yu, Xie, Liu, Wei, Ci, and Peng Borneol (Fig. 1), a bicyclic monoterpenoid alcohol, one of the valuable medical materials, senior aromatic spices, and chemical materials, has been used in food and also folk medicine in China and India. Previous studies have shown that Borneol has vasorelaxant effect on rat thoracic aorta [12] and neuroprotective effects [13]. It is reported that Borneol could obviously loosen the intercellular tight junction (ICTJ) in blood–brain barrier (BBB), accelerate the transportation of substance through the intercellular passage, it also could increase the number and volume of pinocytosis vesicles in BBB cells, thus to accelerate the transportation of substance by way of cell pinocytosis [14]. Borneol also exhibits high inhibitory effects on hTRPA1 [15]. Three amino acids of hTRPA1, namely, S873, T874, and Y812, were found to be involved in the activity of Borneol. Despite being inserted in pharmaceutical preparations to treat conditions of pulmonary inflammation, few studies have been found investigating the specific role of Borneol in this regard. Thus, in our study, we ought to prove the anti-inflammatory effect of Borneol in LPS-induce ALI model.

MATERIALS AND METHODS Reagents Borneol (purity >96.8 %) was purchased from the National Institute for Food and Drug Control. LPS was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Mouse TNF-α, IL-1β, and IL-6 enzyme-linked imunosorbent assay (ELISA) kits were purchased from Biolegend (San Diego, CA, USA). The myeloperoxidase

(MPO) determination kit was purchased from the Jiancheng Bioengineering Institute of Nanjing (Jiangsu, China). Rabbit mAb ERK, JNK, P38, IκB and NF-κB/ p65, and mouse mAb p-IκB were purchased from Cell Signaling Technology Inc (MA, USA). HRP-conjugated goat anti-rabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). All other chemicals were of reagent grade. In Vitro Study Cell Culture The RAW 264.7 mouse macrophage cell line, purchased from the China Cell Line Bank (Beijing, China), was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 3 mM glutamine, antibiotics (100 U/ml penicillin and 100 U/ml streptomycin), 10 % heat-inactivated fetal bovine serum, at 37 °C under a humidified atmosphere of 5 % CO2. In all experiments, macrophages were incubated in the presence or absence of various concentrations of Borneol which were always added 1 h prior to LPS (1 μg/ml) stimulation. Cell Viability Assay Borneol solubilized in DMSO (less than 0.1 % of total volume of cells) was diluted in DMEM prior to treatment. MTT assay was established to evaluate cell viability. Firstly, cells were seeded onto 96-well plates at a density of 2×105 cells/ml and cultured in a 37 °C, 5 % CO2 incubator for 1 h. After that, Borneol (0–300 μg/ml) was added into the wells to pretreat cells and incubated for 1 h. Then the LPS groups were stimulated with LPS (1 μg/ml). After 18 h, 20 μl of MTT (5 mg/ml) was added to each well, and incubation was continued for 4 h. The supernatant was removed and the formation of formazan was resolved with 150 μl/well of DMSO. The optical density was measured at a wavelength of 570 nm using a microplate reader (TECAN, Austria). Concentrations were determined for three wells of each sample, and each experiment was done in triplicate. Cytokine assay (TNF-α and IL-6)

Fig. 1. The chemical structure of Borneol

RAW 264.7 cells were plated onto 24-well plates (4× 105 cells/well), and incubated in DMEM for 1 h prior to Borneol (25, 50, and 100 μg/ml) pretreatment. One hour after Borneol treatment, LPS (1 μg/ml) was added to stimulated inflammatory reaction. Cell-free supernatants were collected after 12-h incubation, and cytokines were assayed by ELISA kit (R&DS systems) according to the

Modulation of LPS-Stimulated Pulmonary Inflammation manufacturer's instructions (Bio Legend, Inc, Camino Santa Fe, Suite E, San Diego, CA, USA). In Vivo Study Mice All animal experiments were performed in accordance with the guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Male BALB/c mice, 6–8 weeks, weighing approximately 18 to 20 g, were purchased from the Center of Experimental Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were housed in microisolator cages and received food and water. The laboratory temperature was 24±1 °C, and relative humidity was 40–80 %. Mouse Model of LPS-Induced ALI Mice were randomly divided into five groups: control group, LPS group (20 mg/kg), Borneol (20, 40 mg/kg) plus LPS group (20 mg/kg), and dexamethasone (Dex, 5 mg/ kg) plus LPS group. Briefly, after being anesthetized by diethyl ether, mice were administered intranasally with 50 μl LPS (20 μg/ml) or phosphate buffer solution (PBS). Borneol (20, 40 mg/kg) or dexamethasone (Dex, 5 mg/kg) was pretreated 1 h prior to intranasal administration of LPS. Twenty-four hours after LPS administration, animals were euthanized to collect BALF and lung tissue samples. Histopathologic Evaluation Pulmonary tissue samples were fixed in normal 10 % neutral buffered formalin followed by dehydration in ascending series of alcohol and embedding in paraffin wax and sliced. After hematoxylin and eosin (H&E) staining, pathological changes of lung tissues were observed under a light microscope. The severity of microscopic injury was graded from 0 (normal) to 4 (severe) in the following categories: neutroBorneol infiltration, interstitial edema, hemorrhage, hyaline membrane formation, necrosis, and congestion. The total score was calculated by adding up the individual scores of each category [16]. BALF Collection and Cell Count Tracheostomy was performed, and a cannula was inserted into trachea to collect the BALF and inflammatory cells. The lungs were lavaged three times with PBS in a total volume of 1.5 ml. A 0.1-mL aliquot was used for the

total cell count by using a hemocytometer, and the remainder was immediately centrifuged at 800g for 10 min at 4 °C. The levels of TNF-α, IL-1β, and IL-6 in BALF were measured by ELISA kits. All procedures were done according to the manufacturer’s instructions. Pulmonary MPO Activity in Acute Lung Injury Mice One hundred milligram lung tissues were frozen in liquid nitrogen and then homogenized in extraction buffer to obtain 5 % of homogenate. MPO activity was determined by MPO activity kit (Nanjing Jiancheng Bioengineering Institute, China) in accordance with manufacturer’s instructions. The enzymatic activity was determined by measuring the change in absorbance at 460 nm using a 96-well plate reader and expressed as units per gram of protein. Lung Wet-to-Dry Weight (W/D) Ratio After mice were killed, a median sternotomy was performed to expose the lungs. The left lungs were excised, blotted dry, weighed to obtain the “wet” weight, and then placed in an oven at 80 °C for 48 h to obtain the “dry” weight. To quantify edema, the ratio of wet lung to dry lung was calculated. Collection of Lung Tissue Protein and Western Blot Analysis Lung tissue samples were harvested and frozen in liquid nitrogen immediately until homogenization. Cytoplasmic proteins were extracted using Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology; China) according to the manufacturer’s protocol. Protein concentrations were determined by BCA protein assay kit and equal amounts of protein were loaded per well on a 10 % sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane. The membranes were washed in Tris-buffered saline with Tween 20 and incubated in 5 % skim milk (Sigma) at room temperature for 2 h on a rotary shaker and followed by incubations with rabbit polyclonal antibodies specific for ERK(1:1,000 dilution), JNK(1:500 dilution), p38(1:800 dilution), IκB (1:1,000 dilution), NF-κB/p65(1:1,000 dilution), and βactin (1:1,500) in diluents buffer (5 % skim milk in TBST) overnight at 4 °C. Then, the membrane was washed with TBS-T followed by incubation with the peroxidaseconjugated secondary antibody at room temperature for 1 h. Immunoreactive bands were visualized with enhanced

Zhong, Cui, Yu, Xie, Liu, Wei, Ci, and Peng chemiluminescence western blot kit in accordance with the manufacturer’s instructions. The β-actin western blot was performed as the internal control of protein loading. Statistical Analysis All values are expressed as means±SEM. Differences between mean values of normally distributed data were analyzed using one-way ANOVA (Dunnett’s t test) and two-tailed Student’s t test. Statistical significance was accepted at P