Protective Effect of Nicotine on Lipopolysaccharide-Induced Acute Lung Injury in Mice

Background: Recently, nicotine administration has been shown to be a potent inhibitor of a variety of innate immune responses, including endotoxin-induced sepsis. Objective: It was the aim of this study to evaluate the effect of nicotine on attenuating lung injury and improving the survival in mice with lipopolysaccharide (LPS)-induced acute lung injury (ALI). Methods: ALI was induced in mice by intratracheal instillation of LPS (3 mg/ml). The mice received intratracheal instillation of nicotine (50, 250 and 500  g/kg) before or after LPS administration. Pulmonary histological changes were evaluated by hematoxylin-eosin stain, and lung wet/dry weight ratios were observed. Concentrations of tumor necrosis factor (TNF), interleukin (IL)-1 and high mobility group box (HMGB)-1, as well as myeloperoxidase (MPO) activity were measured by enzyme-linked immunosorbent assay. The mortality rate was recorded and analyzed by the Kaplan-Meier method. Results: Nicotine pretreatment significantly attenuated the severity of lung injury and inhibitReceived: February 9, 2010 Accepted after revision: May 24, 2010 Published online: July 21, 2010 Yun Jie Wang Department of Thoracic Surgery, Tangdu Hospital The Fourth Military Medical University Xi’an 710038 (China) Tel./Fax +86 29 8477 7827, E-Mail yjwmd @ yahoo.com © 2010 S. Karger AG, Basel 0025–7931/11/0811–0039$38.00/0 Accessible online at: www.karger.com/res Y.F.N. and F.T. contributed equally to this work. For editorial comment see p. 6 D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 8 :2 2: 58 P M Ni /Tian /Lu /Yang /Fu /Wang /Yan /Zhao / Wang /Jiang Respiration 2011;81:39–46 40 that the network of inflammatory cytokines and chemokines plays a major role in mediating, amplifying and perpetuating the lung injury process [2] . The proinflammatory cytokines, tumor necrosis factor (TNF) and interleukin (IL)-1 , can stimulate the production of a host of other cytokines and eventually result in lung injury. The reduction in the mortality rate in animal sepsis models has been observed in experimental therapies against TNF and IL-1 [3, 4] . However, these therapies have proved difficult clinically, in part, because TNFand IL-1 are released within minutes after bacterial challenge [5, 6] . Therefore, we need to search for late proinflammatory cytokines that may offer a wider therapeutic window. High mobility group box (HMGB)-1, a nuclear nonhistone DNA-binding protein, has been identified as a ‘late’ mediator of endotoxin lethality [7] . HMGB-1 is detected in the serum of patients with sepsis, and serum HMGB-1 levels are significantly increased in patients with poor prognosis [7] . In addition, high levels of HMGB-1 were associated with neutrophil-mediated diseases including lung inflammation and ALI [7, 8] . Unlike the ‘early’ cytokines, such as TNFand IL-1 , HMGB-1 is released days after lipopolysaccharide (LPS) exposure that provides a wider time frame for clinical intervention against progressive inflammatory disorders [9] . Therefore, HMGB-1 is a potential novel therapeutic target for inflammatory diseases. Nicotine, a small organic alkaloid, is a major component of cigarette smoke and acts as an agonist on the nicotinic acetylcholine receptors (nAChRs) found mainly in the central and peripheral nervous systems and on many other tissue cells throughout the body, including immune cells [10, 11] . Recently, a study showed that nicotine acted on the 7nAChR, inhibited the nuclear factor B pathway and suppressed HMGB-1 release from human macrophages; in vivo, treatment with nicotine attenuated serum HMGB-1 levels and improved survival in ‘established’ sepsis, even when treatment was initiated after the onset of the disease [12] . In addition, nicotine protected the kidney from renal ischemia/perfusion injury by preventing neutrophil infiltration and decreasing productions of keratinocyte-derived chemokine, TNF and HMGB-1 [13] . Although the anti-inflammatory effects of nicotine have been showed in different animal models, the protective effect of nicotine on ALI is controversial. Therefore, the present study was designed to investigate whether nicotine could be beneficial in mice with LPSinduced ALI. Materials and Methods Animals and Reagents One hundred and eighty male BALB/C mice weighing 20– 25 g were purchased from the Animal Center of the Fourth Military Medical University (Xi’an, China). All animals were allowed to take food and tap water ad libitum. All procedures were in accordance with the Declaration of Helsinki of the World Medical Association. The protocols were also approved by the Institutional Animal Care and Use Committee of the Fourth Military Medical University Tangdu Hospital. LPS ( Escherichia coli lipopolysaccharide, 055:B5) and nicotine were obtained from Sigma Chemical Company, St. Louis, Mo., USA. Enzyme-linked immunosorbent assay (ELISA) kits of TNF , IL-1 , myeloperoxidase (MPO) and HGMB-1 were purchased from the Wuhan USCN Sciences Co., Ltd., Wuhan, China. Animal Model of ALI Before the induction of ALI, mice were fasted overnight but allowed water ad libitum. Animals were anesthetized with intraperitoneal pentobarbital (50 mg/kg). LPS (3 mg/kg) was instilled intratracheally to induce ALI. Sham-operated mice underwent the same procedure with intratracheal instillation of saline. Experimental Protocol In the first set of experiments, animals were randomly divided into 4 groups (n = 15 for each group). Group 1 (control group) received an intratracheal instillation of saline, group 2 (nicotine group) received an intranasal instillation of nicotine (500  g/kg), group 3 (LPS group) received an intratracheal instillation of LPS (3 mg/kg), and group 4 (LPS + nicotine group) received an intranasal instillation of nicotine (500 g/kg) 30 min before LPS administration. Twelve hours after LPS administration, animals were anesthetized and serum was collected. Bronchoalveolar lavage (BAL) was performed through the left lung. The superior lobe of the right lung was excised for histopathologic examination. The middle lobe was excised for analysis of the lung wet/dry weight ratio. The lower lobe was homogenized and frozen in a cold phosphate solution at –80 ° C for MPO analysis. The second set of experiments was designed to evaluate the effect on mice survival of treatment with variable doses of nicotine (50, 250 and 500  g/kg). Nicotine treatment was started 30 min before LPS administration, and thereafter, nicotine was administered twice a day for 3 days. Animals were randomly divided into 4 groups (n = 20 for each group). The mice in these groups were treated with nicotine at 50, 250 and 500  g/kg intranasally, and the control mice received the same volume of saline. Mortality was recorded up to a week after the procedure. The third set of experiments was designed to investigate whether nicotine could improve survival in ‘established’ ALI. Mice were randomly divided into 2 groups (n = 20 for each group). Delayed nicotine treatment (500  g/kg) started 24 h later, and thereafter, was administered twice a day for 3 days intranasally. Control mice received the same volume of saline. Mortality was recorded up to a week after LPS administration. Bronchoalveolar Lavage Animals were anesthetized with intraperitoneal pentobarbital (50 mg/kg). A median sternotomy allowed for exposure of both of the lungs. The trachea was exposed and inserted with an intraveD ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 8 :2 2: 58 P M Protective Effect of Nicotine on LPS-Induced ALI in Mice Respiration 2011;81:39–46 41 nous infusion needle. After ligating the hilum of the right lung, the left lung was lavaged 5 times with 0.5 ml ice-cold phosphatebuffered saline. The recovery ratio of the fluid was about 90%. The BAL fluid (BALF) was immediately centrifuged at 500 g for 10 min at 4 ° C, and the cell-free supernatant was stored at –80 ° C for analysis of cytokines. Cytokine Measurement Concentrations of TNFand IL-1 in BALF, and concentrations of HGMB-1 in serum were measured by using ELISA kits. All procedures were done in accordance with the manufacturer’s instructions. MPO Assays To carry out the assays, tissue samples were subjected to 3 further freeze-thaw cycles and centrifuged at 12,000 g for 10 min at 4 ° C. The supernatant was assayed for MPO activity with ELISA kits. All procedures were done in accordance with the manufacturer’s instructions. Lung Wet/Dry Weight Ratio As an index of lung edema, the amount of extravascular lung water was calculated. The middle lobe of the right lung was excised and the wet weight was recorded. The lobe was then placed in an incubator at 80 ° C for 24 h to obtain the dry weight. And the wet/dry weight ratios were calculated by dividing the wet weight by the dry weight. Pulmonary Histopathology The superior lobe of the right lung was harvested 12 h after LPS administration and fixed with an intratracheal instillation of 1 ml buffered formalin (10%, pH 7.2). The lobe was further fixed in 10% neutral buffered formalin for 24 h at 4 ° C. The tissues were embedded in paraffin and cut into 5 m sections. Hematoxylineosin stains were performed using the standard protocol. Statistical Analyses Data were entered into a database and analyzed using SPSS software and are expressed as means 8 SD. On a preliminary 3,500