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Lobeline improves acute lung injury via nuclear factor-κB-signaling pathway and oxidative stress
Respiratory Physiology & Neurobiology 225 (2016) 19–30
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Lobeline improves acute lung injury via nuclear factor-B-signaling pathway and oxidative stress Kun-Cheng Li a,1 , Yu-Ling Ho b,1 , Cing-Yu Chen c , Wen-Tsong Hsieh d , Yuan-Shiun Chang a,e,∗ , Guan-Jhong Huang a,∗ a
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan Department of Nursing, Hungkuang University, Taichung 433, Taiwan c Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan d Department of Pharmacology, School of Medicine, China Medical University, Taichung 404, Taiwan e Chinese Crude Drug Pharmacy, China Medical University Hospital, Taichung 404, Taiwan b
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 9 December 2015 Accepted 10 December 2015 Available online 15 December 2015 Keywords: Lobeline MAPKs NF-B IB␣ AOEs ALI
a b s t r a c t Acute lung injury (ALI) is a severe, life-threatening medical condition whose pathogenesis is linked to neutrophil infiltration of the lung. Activation and recruitment of neutrophils to the lung is mostly attributed to the production of chemokines NO, IL-6, for instance. This study aims to investigate lobeline ability in reducing NO production, and nitric oxide synthase (iNOs) expression. Lobeline was tested by inhibiting phosphorylation of mitogen-activated protein kinases (MAPKs), NF-B and IB␣ in LPS-stimulated RAW 264.7 cells. When RAW 264.7 macrophages were given lobeline with LPS, a significant concentrationdependent inhibition of NO production was detected. In vivo tests, mice were either treated with normal saline, 10 mg/kg dexmethasone or 5, 10, 20 mg/kg lobeline intraperitoneally, and after an hour, the administration of 5 mg/kg of LPS was given intratracheally. External performance, cytokines, MAPK pathways and antioxidative enzymes (AOEs) were also carried out to evaluate the effects of these drugs. This is the first investigation in which lobeline was found to effectively inhibit acute lung edema, which may provide a potential target for treating ALI. Lobeline may utilize MAPKs pathways as well as AOEs activity to attenuate LPS-induced nonspecific pulmonary inflammation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) is a syndrome that can lead to the destruction of the alveolar capillary membrane, pulmonary edema and neutrophil accumulation. The pathophysiological mechanism of ALI is believed to be associated with the chemotactic factor expression and influx of neutrophils into the lungs (Kim et al., 2013; Yu et al., 2015). Not only are there no specific therapies available, but also the disease has no cure. Therefore, it is desirable to identify new molecules that can regulate ALI-associated inflammation (Kim et al., 2013; Sitapara et al., 2014). ARDS/ALI is also characterized as related oxidative damage which is closely linked to neutrophil (PMN) infiltration, secretion of pro-inflammatory cytokines and
∗ Corresponding authors at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan. Fax: +886 4 2208 3362. E-mail addresses: [email protected] (Y.-S. Chang), [email protected] (G.-J. Huang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.resp.2015.12.003 1569-9048/© 2016 Elsevier B.V. All rights reserved.
antioxidants. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) are currently considered the basic oxidative damage stress markers (Kuo et al., 2011; Reddy et al., 2009). Lipopolysacharide (LPS) has been well recognized for studying the pathogenesis of ALI/ARDS. After intratracheal administration, LPS induced nitric oxide synthase (iNOs) and cyclooxygenase-2 (COX-2) in acute lung injury model. Subsequently, the overproduction NO, inflammatory mediator like tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-1 and interleukin-6 (IL-6) are participate in the pathogenesis of inflammation. Responses of proinflammatory mediators is regulated by the activation of the transcription factor nuclear factor (NF-B) which active via mitogen-activated protein kinase (MAPK) pathways (Chen et al., 2015; Huang et al., 2012; Zhang et al., 2015) and it may become part of a novel therapeutic target. Moreover, the expression of iNOs is closely related to the induction of heme oxygenase-1 (HO-1). As murine macrophages generate NO with LPS, one potential system which entails the catabolizing of enzyme HO-1, and brings biliverdin, carbon monoxide (CO) existence can limit synthesis NO production and iNOs expression. (Li et al., 2015; Sung and Lee, 2015; Sung et al.,
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2014; Wang et al., 2014). Thus, enhancing the production of HO-1 reduces the expression of iNOs. ␣-Lobeline (lobeline), a lipophilic nonpyridino, is the predominant alkaloid in a family of structurally-related compounds found in Lobelia chinensis, Indian tobacco (Lim et al., 2004), and has been reported to possibly treat asthma and respiratory illness. However, there has been no report to demonstrate whether or not lobeline has any anti-inflammatory effects on LPS-induced RAW 264.7 in vitro and acute lung injury in vivo. This study explored lobeline’s new uses by LPS-stimulated ALI and demonstrated the molecular mechanism of the regulatory pathway. The results revealed the lobeline can protect against LPS-induced lung injury in mice and possibly exert its activity through the regulation of antioxidant enzyme activity, NF-B, and MAPKs signaling pathways. 2. Materials and methods 2.1. Cell culture A murine macrophage cells (BCRC No. 60001) were purchased from the Bioresources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were propagated and aliquots stored in liquid nitrogen, maintained as adherent cell cultures and passaged 3–25 times after which a new frozen aliquot was used. The cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a CO2 incubator (5% CO2 in air) at 37 ◦ C and subcultured every 3 day at a dilution of 1:5 using 0.05% trypsin—0.02% EDTA in Ca2+ -, Mg2+ - free phosphate-buffered saline (DPBS). 2.2. Cell viability This preliminary experiment consists of two parts. In the first part, we examined the viability of the Raw cells; as for the second portion, the cell medium was examined for NO production. Raw 264.7 cells (2 × 105 per well) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. The cells were cultured with lobeline in the presence of LPS (100 ng/mL) at 37 ◦ C for 24 h. The media were removed and saved for the following NO experiment, while the cells were washed with PBS for another round and incubated with 100 L of 0.5 mg/mL MTT in a humidified atmosphere of 5% CO2 and 95% air for 37 ◦ C 6 h. After incubating, absorbance was read using a microplate reader at 570 nm. The above steps were repeated three times for each concentration. 2.3. NO production NO production was indirectly assessed by measuring the nitrite levels in the cultured media was determined by Griess reaction (Kang et al., 2014). 100 L of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) was added to each sample medium and incubated at room temperature for 10 min. By using sodium nitrite to generate a standard curve, the concentration of nitrite for each sample was measured at the absorbance of 540 nm.
libitum. Animal studies were conducted according to the regulations of Instituted Animal Ethics Committee, and the protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals. After a 1–2 week adaptation period, male ICR mice (25–32 g) were randomly assigned to six groups (n = 12) of the animals in the study. The control group received normal saline (intraperitoneal; i.p.). The other groups included a LPS-treated, a positive control (LPS + Dex) and lobeline administered groups (LPS + lobeline-H, LPS + lobeline-M, LPS + lobeline-L). 2.5. Model of LPS induced ALI Seventy-two healthy male ICR mice were randomly divided into 6 groups (n = 12): control group, LPS group, dexamethasone (Dex) group (10 mg/kg), low dosage lobeline group (5 mg/kg), middle dosage group (10 mg/kg, LPS + lobeline-M) and high dosage group (20 mg/kg, LPS + lobeline-H). Take half of each group for the inflammation protein analysis, slicing, and edema and the rest of mice use for BALF analysis. Mice were intratracheally instilled with 5 mg/kg LPS in 50 L sterile saline or sterile saline alone (control group) (Zhang et al., 2010). In brief, mice were anesthetized with mixed reagent of 10 L/g i.p urethane (0.6 g/mL) and chloral hydrate (0.4 g/mL), followed by Dex (10 mg/kg) or lobeline intraperitoneal injection with individual dose. Six hours later, the mice received sacrifice and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. 2.6. Cell counts, protein levels, TNF-˛, IL-6, and IL-1ˇ assay in BALF Six hours later, mice were exsanguinated after anesthesia. According to the previous report, BALF was collected by the upper part of the trachea, by douche three times with 500 L PBS (pH 7.2). The fluid recovery rate was more than 90%. Lavage sample from each mouse was kept on ice. BALF was centrifuged at 700 × g for 5 min. The sediment cells were resuspended in 2 mL PBS, half of them have used to detect cell counts by cytometer, the rest equally divided into two parts. One has centrifuged again in order to get sediment for extracting proteins with a RIPA solution (radioimmuno-precipitation assay buffer) and the other of BALF supernatant were used to assay the TNF-␣, IL-6 and IL-1 levels using the respective ELISA kits. Total cells were counted double-blindly with flow cytometer, total cell protein extracts were homogenized in cell protein extraction solution and the concentration in the supernatant was determined by Bradford assay. 2.7. Myeloperoxidase (MPO) activity assay The lungs were homogenized, 12,000 × g at 4 ◦ C for 15 min and resuspended in 50 mM KPO4 buffer (PH 6.0) with containing 0.19 mg/mL of o-dianisidine chloride and 0.0005% H2 O2 was a substrate for myeloperoxidase at 460 nm with a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). MPO content was expressed as relative MPO activity (OD460nm /mg protein of lung tissue) (Bani et al., 1998). 2.8. Lung wet/dry weight ratio
2.4. Animals Male ICR mice, 6–7 weeks old, were obtained from BioLASCO Taiwan Co., Ltd. The animals were kept in plexiglass cages at a constant temperature of 22 ± 1 ◦ C, relative humidity 55 ± 5% and with 12 h dark–light cycles. They were given food and water ad
The lungs wet to dry weight ratio (W/D ratio) was measured to evaluate pulmonary edema. The lung tissues were excised, immediately weighed, and then placed in an oven at 80 ◦ C for 48 h to obtain the “dry” weight. The W/D ratio was then calculated (Zhou et al., 2014).
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Fig. 1. The chemical structure of lobeline (A), dexamethasone (Dex), (B) and the effects of lobeline on lipopolysaccharide (LPS)-induced cell viability (C), NO production (D), inhibition of iNOS and COX-2 protein expression (E), MAPK (JNK, p38, and ERK) protein expression (F) IB-␣ and NF-B (G) were evaluated in RAW 264.7 cells. Cells were incubated for 24 h with 100 ng/mL LPS in the absence or presence of lobeline (0, 40, 20, 10, 5, 2.5, and 1.25 g/mL). Lobeline was added 1 h before the incubation with LPS. Cell viability was performed by using MTT assay. Nitrite concentration in the medium was determined by using Griess reagent. Lysed cells were then prepared and subjected to Western blotting by using an antibody specific for iNOS, COX-2, MAPK, IB-␣, NF-B, and -actin was used as an internal control. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ### compared with sample of control group, ** p < 0.01 and *** p < 0.001 were compared with LPS-alone group.
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2.9. H&E staining The left lower lung from each mouse was fixed in 10% formalin, embedded in paraffin, cut into 5 mm sections, stained with. After H&E staining of lung tissue slices, pathological changes were observed. The lung injury score was calculated by assessing the degree of inflammatory cell infiltration, hemorrhage, interstitial and alveolar edema, and the thickness of alveolar septum in five random fields in a blind manner using light microscopy. A score of 0 represented no damage; l represented mild damage; 2 represented moderate damage; 3 represented severe damage and 4 represented very severe histological damage (Parsey et al., 1998). 2.10. Protein lysate preparation and western blot analysis The stimulated murine macrophage cell line RAW2 64.7 cells were washed with PBS and lysed in an ice-cold lysis buffer [10% glycerol, 1% Triton X-100, 1 mM Na3 VO4 , 1 mM EGTA, 10 mM NaF, 1 mM Na4 P2 O7 , 20 mM Tris buffer (pH 7.9), 100 mM bglycerophosphate, 137 mM NaCl, 5 mM EDTA, and one protease inhibitor cocktail tablet (Roche, Indianapolis, IN,USA)] on ice for 1 h, followed by centrifugation at 12,000 × g for 30 min at 4 ◦ C. Soft tissues were removed from individual mice lungs and homogenized in a solution containing PBS and RIPA before grinding. The homogenateswere centrifuged at 12,000 × g for 20 min, and 50 g of protein from the supernatants was then separated on 10% sodium dodecylsulphate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After transfer, the membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). The membranes were probed with primary antibodies (iNOs, COX-2) for phosphorylated and nonphosphorylated forms of p38 MAPK, ERK, IB-␣, JNK, HO-1, p65 and pro-inflammatory enzymes (SOD, GPx, catalase) at 4 ◦ C overnight. The membranes were washed three times and the immunoreactive proteins were detected by enhanced (ECL) by using hyperfilm and ECL reagent (Amersham International plc., Buckinghamshire, U.K.). The results of Western blot analysis. 2.11. Statistical analysis Experimental results were presented as the mean ± standard deviation (SD) of three parallel measurements. IC50 values were estimated using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc., Chicago, IL). Data obtained from animal experiments were expressed as mean standard error (±S.E.M.). Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe’s multiple range tests). Statistical significance is expressed as * P < 0.05, ** P < 0.01 and *** P < 0.001. 3. Results 3.1. Effect of lobeline on LPS-induced cell viability and NO production in macrophages The effect of lobeline on RAW 264.7 cell viability was determined by a MTT assay. Cells cultured with lobeline at concentrations of 0, 1.3, 2.5, 5, 10, 20, and 40 g/mL, was used in the presence of 100 ng/mL LPS for 24 h (Fig. 1C) . It was shown that there were no significant differences between the cell viability of the normal and LPS control groups, indicating that LPS did not induce cell deaths. Additionally, lobeline used within the range of 40–1.3 g/mL did not induce cytotoxicity; the efficacy of these doses, 20 and 40 g/mL, were the same. Therefore, we used lobeline in all animal experiments, with doses below or equal to 20 g/mL. Although LPS did not induce cell death, analyses of culture media
showed that LPS activated human macrophage raw cells to produce considerable NO. Among the experimental groups, the medium which contained lobeline showed the most inhibitory effect on NO production. There was a significant decrease in nitrite production in the group treated with 5 g/mL lobeline (P < 0.001). The IC50 value for inhibition of nitrite production of lobeline was about 2.6 g/mL (Fig. 1D). Based on the results from the MTT and NO production assays, we used lobeline to conduct all subsequent experiments. 3.2. Inhibition of LPS-induced iNOs and COX-2 Protein by lobeline Western blot analyses were performed to look into whether the inhibitory effects of lobeline on the pro-inflammatory mediators NO was related to the modulation of the expressions of iNOs and COX-2. The results showed that incubation with lobeline (0, 5, 10, and 20 g/mL) in being with LPS (100 ng/mL) for 24 h can inhibit iNOs proteins expression in mouse macrophage RAW 264.7 cells, in a dose-dependent manner (Fig. 1E). The detection of bactin was also performed in the same blot as the internal control. The intensity of protein bands were analyzed using Kodak Quantity software in three independent experiments and showed an average of 37.1 and 61.5% down-regulation of iNOs and COX-2 proteins after treatment with lobeline at 20 g/mL, compared with the LPSalone. In general, these results indicate that the inhibitory effect of lobeline on LPS-induced NO production was caused by iNOs and COX-2 suppression. 3.3. Effects of lobeline on the LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) The mitogen-activated protein (MAP) kinases play critical roles in the regulation of cell growth and differentiation, and in controling cellular responses to cytokines and stresses. In particular, they are also known to be important for the activation of NF-B (Achoui et al., 2010; Hsing et al., 2011). To explore whether the inhibition of NF-B activation by lobeline is mediated through the MAPK pathway, MAPK phosphorylation was examined by Western blot in RAW 264.7 cells pretreated with lobeline and then pretreated with LPS. As shown in Fig. 1F, pretreatment with lobeline had an effect on the phosphorylation of JNK and p38 (but not ERK1/2). These results suggest that the phosphorylation of JNK and p38 may be involved in the inhibitory effects of lobeline on LPS-stimulated NF-kB binding in RAW 264.7 cells. 3.4. Inhibition of LPS-induced IkB-˛ and NF-kB proteins by lobeline The effect of NF-kB and IkB-␣ expression by lobeline in the presence of LPS for 1 h was assessed by Western blot. The intensity of protein bands showed an increase of NF Anti kB protein with the average of 41.1% and a decrease of phosphoryl IkB-␣ protein of 42.3% after treatment with lobeline at 20 g/mL, compared with the LPS-alone (Fig. 1G). Therefore, it can be concluded that lobeline is capable of inhibiting iNOs expression in LPS-induced RAW 264.7 cells via attenuation of NF-B signaling by p38, JNK and IkB-␣. 3.5. Histological examination We used H&E stain and lung injury score to evaluate the effects of therapeutic lobeline treatment on the worse alveolar collapsed state and perivascular and peribronchial edema of lung inflammation. Histologically, lung biopsies of mice treated with lobeline (5 mg/kg) showed that the inflammatory cells were reduced in number, confined to the surroundings of the vascular areas and that the intercellular spaces did not show any cellular infiltrates. In addition, neutrophils increased with LPS treatment (Fig. 2B)
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Fig. 2. Histological appearances of mouse lung after individual injecting 10% formalin then stained with H&E stain demonstrates a representative view from each group and severity of lung injury was analyzed by the lung injury scoring system. [A: Control, B: LPS, C: LPS + Dex D: LPS + lobeline-H, E: LPS + lobeline-M, F: LPS + lobeline-L]. Severity of lung injury was analyzed by the lung injury scoring system (G). The Figure demonstrates a representative view (X200) from each group; each bar represents the mean ± S.E.M. of 6 mice.
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and the treatment group Dex and lobeline (5 mg/kg) were able to decrease the neutrophil numbers as opposed to the LPS-treated group (Fig. 2C–F). At the same time, in Fig. 2G, the lung injury score stands at around 4 points after LPS injection, which is absolutely much higher than that of the other three groups with different amounts of lobeline, with the highest value of only 3 points. 3.6. Lobeline attenuates pulmonary inflammation and pulmonary edema in vivo Because lobeline effectively inhibited iNOs inductions in macrophages, our study was extended to determine whether or not lobeline affected acute inflammation in animal models. In this study, LPS-treated mice exhibited the typical pathological changes of ALI, including notably, inflammatory neutrophil infiltration, interstitial edema, interalveolar septal thickening and intra-alveolar, interstitial patchy hemorrhage. As for pulmonary edema and alveolar fluid removal efficiency in LPS- treated mice, the W/D ratio was employed (Fig. 3A) . The results of pulmonary inflammation and pulmonary edema were found to be dose dependent and the W/D weight ratio in lobeline 20 mg/kg group was found to be significantly different from the LPS group (Fig. 3A) (P < 0.05). 3.7. Lobeline reduces cellular counts and proteins in BALF Bronchoalveolar lavage (BAL) fluid protein was quantified after insult. To identify the anti-inflammatory properties of lobeline, cellular counts and proteins of PMNs in BALF were measured in our study. After LPS injection of six hours, we evaluated the leukocyte changes in the lung of LPS-administered mice, with or without lobeline pretreatment. As shown in Fig. 4A, B administration of LPS caused extensive leukocyte infiltration, whereas with pretreatment of lobeline, the LPS-induced leukocyte infiltration was suppressive in a concentration-dependent manner. A significant inhibitory effect began at 10 mg/kg (P < 0.05). 3.8. Lobeline down-regulates TNF-˛, IL-6, and IL-1ˇ in BALF The BAL fluid: plasma protein ratio was substantially augmented in animals at this proinflammatory cytokines stage, TNF-␣, IL-6, and IL-1 in BALF were measured by ELISA. TNF-␣, IL-6 and IL-1 in BALF in the mice with LPS IT were markedly higher than in the mice in the control group. However, cytokines in BALF in the mice with lobeline and Dex were decreased (Fig. 4C). The above results indicated that lobeline reduced the expression of proinflammatory cytokines, which in turn, improved lung damage caused by LPSinduced ALI. 3.9. Effects of lobeline on MPO activity and antioxidative enzymes in LPS-induced ALI The accumulation of activated neutrophils was assessed by measuring MPO activity. Oxidative stress exerted by activated PMNs is critically important in the pathophysiology of ALI, such as SOD, CAT, and GPx, which are consumed during the amelioration of ALI (Hosakote et al., 2009). This antioxidative enzyme can decrease inflammatory activities in LPS-treated mice. Lung MPO activity was markedly increased in the mice 6 h after LPS IT, and LPS animals also showed a marked increase (Fig. 4D, P < 0. 001), whereas lobeline animals exhibited a trend toward lower levels of lung MPO activity when compared with the control animals; and the upregulation of MPO activity and was dose-dependent prevented in animals pretreated with lobeline. In contrast, pretreatment with lobeline and Dex lead to recovery in LPS-induced inflammation when activated
by these enzymes SOD, CAT, and GPx. These enzymes have significantly different contents with the LPS groups (P < 0.001; Fig. 4E). 3.10. Inhibition of LPS-induced iNOs and COX-2 protein by lobeline in lung tissues iNOs and COX-2 play critical roles in the pathology of LPSinduced ALI. The effect of lobeline on iNOS and COX-2 expressions in the lung tissue was analyzed using Western blot assay. As shown in Fig. 5A, the level of iNOs and COX-2 proteins were significantly decreased in mice challenged with lobeline + LPS compared with the LPS groups (P < 0.001). 3.11. Effects of lobeline on MAPK activation in LPS-induced ALI MAPKs play critical roles in the regulation of cell growth and differentiation, and in the control of cellular responses to cytokines and stresses (Khan et al., 2013). The effect of lobeline on phosphorylation of ERK, JNK and p38MAPK in LPS-induced ALI was analyzed by Western blot. The results showed that LPS stimulation significantly increased MAPK phosphorylation and lobeline, which significantly inhibited the LPS-induced phosphorylation of p38 MAPK, JNK not ERK, and JNK in the lobeline group decreased more significantly than the Dex group. The significant inhibitory effect started at 20 mg/kg (P < 0.001; Fig. 5B). In addition, lobeline also inhibited LPS-induced phosphorylation of p38MAPK, but had slightly lower inhibitory effect than the Dex group. 3.12. Inhibition of LPS-induced IB˛ and NF-kB proteins activation by lobeline NF-B is activated by a variety of stimuli including LPS, TNF␣, and IL-1. The effect of lobeline on NF-B expression in lung tissue of LPS-induced ALI mice was assessed by Western blot. The expression of p-IB␣ evoked by LPS was significantly suppressed at 20 mg/kg by lobeline pretreatment. On the contrary, the expression of NF-B p65 in cytoplasm was significantly reduced by LPS administration (P < 0.001; Fig. 5C), and these changes were inhibited by lobeline pretreatment. The signal of NF-B inhibition is regulated by the interaction with inhibitory IB␣ proteins. 3.13. Effects of lobeline on HO-1 expression in LPS-induced ALI Increased HO-1 induction has a direct effect on iNOs expression. Therefore, we investigated the inhibitory effects of lobeline on iNOs expression to see if it was associated with increased HO-1 production. We assessed HO-1 induction in the pretreatment of lobeline in LPS- induced ALI and analyzed by using Western blot. The effects of LPS elicited on HO-1 expression, lobeline enhanced LPS-induced HO-1expression at 20 mg/kg (P < 0.001; Fig. 6). This result suggests that lobeline has the ability to reduce LPS-induced NO and iNOs production by increasing HO-1 induction. 3.14. Comparison of the effects of lobeline and dexamethasone on LPS-induced ALI Dex reduces oxidative stress and supplies useful effects on LPS-induced ALI (Jing et al., 2015). Although LPS-induced PMN infiltration was reduced by both lobeline and Dex, lobeline’s lung damage data lower than Dex (Fig. 2G). Moreover lobeline could reduce not only the secretion of MPO but also increase the activity of CAT, whereas Dex had no effect (Fig. 4C and E). Nevertheless, SOD and GPx activities were recovered by lobeline and Dex. These results suggested the improvement by lobeline is better than that by Dex on LPS-induced ALI.
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Fig. 3. Lobeline improves pulmonary edema in vivo (A). Six hours after LPS injection with or without lobeline pretreatments, mice were exsanguinated and their right lower lungs were obtained. The right lower lungs weighed and then dehydrated at 60 ◦ C for 72 h in an oven. Each value represents as mean ± S.E.M. of 6 mice. ### compared with sample of control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group.
4. Discussion ALI/ARDS might be a continuous phenomenon that depends on the level and time of stress and strain application, and not a switch process. It is important to consider therapeutic maneuvers that minimize injury in the early postoperative period.The clinical prevent of ALI/ARDS syndromes especially at its more severe form, is greater than the treatment effect. Recent evidence indicates that prevention plays a key point to improve outcome of patients at risk of ALI/ARDS (Huang et al., 2014; Litell et al., 2011). The data presented demonstrate that lobeline pretreatment in both in vitro and in vivo reduces LPS-induced RAW 264.7 macrophages and a murine model of ALI, respectively. Lobeline is one of the most effective alkaloids from the plant L. chinensis, which grows in the wild in China and other Asian countries, and has been used as a smoking cessation aid, and has successfully been shown to reduce inflammation of ALI (Chen et al., 2014). However, no report has been published on the anti-inflammatory effects of lobeline in LPS acute lung injury. Thus, this study aimed to evaluate the antiinflammatory effects of lobeline by screening the effects of lobeline in LPS-induced pro-inflammatory molecules in vitro and in acute phase inflammation in vivo cases. We also assessed the mechanisms of lobeline on NF-B expressions associated with oxidative enzyme and phosphorylation of MAPK and IB␣.signaling pathways in the anti-inflammation. LPS is (when something is true or is a fact, you use IS) found in the outer membrane of gram-negative bacteria, and is frequently used to induce lung injury in mice as a model for studying ALI. After LPS IT, inducing macrophage and PMNs activation and membrane protein mainly expressed like iNOs and COX-2. Meanwhile, macrophage and PMNs infiltrated into the lung tissues and releasing enzymes and phagocytizing the pathogen. Meantime, iNOs and COX-2 are the main cytotoxic and pro-apoptotic mechanisms participating in the innate response of many mammals via inflammatory mediators (Hidalgo et al., 2005; Oh et al.,
2009). Examination of the cytotoxicity of lobeline in RAW 264.7 macrophages using MTT assay has indicated that lobeline, even at 40 g/mL, did not affect the viability of RAW 264.7 cells (Fig. 1C). In a murine model of LPS-induced ALI, lobeline reduction inhibited iNOs expression in LPS-stimulated macrophages and has been linked to neutrophil infiltration release of NO. The results indicate that lobeline inhibits LPS-induced NO through modulating iNOs expression and COX-2 enzyme activity (Fig. 1E). These findings suggest that lobeline could potentially be an alternative treatment of immunological disorders by interfering with the endogenous factors (Kang et al., 2011). In response to LPS, it is well-known that LPS induces the endogenous transcription of NF-B in order to regulate many of the pro-inflammatory cytokine genes. LPS stimulation elicits a cascade leading to the activation of IB and various MAPKs, like JNK, ERK, and p38 kinase signaling molecules which involves the regulation of cell growth and differentiation and in the control of cellular responses to cytokines and stressors (Lee et al., 2014). In the present study, we have demonstrated that the phosphorylation of IB and MAPKs can be induced by LPS on macrophage and ALI models. Our inflammatory analysis showed that after treatment of lobeline, the phosphorylation level of p38, JNK and IB were significantly reduced at 20 mg/kg dosage (29.21%, 41.76% and 42.29%, respectively) in RAW 264.7 cells. (Fig. 1F). The lobeline lung samples collected from animals decreased at (73.18%, 20.02% and 19.29% respectively) (Fig. 5B) compared to the LPS group. Therefore, this suggests that the treatment of lobeline blocked the activation of JNK p38MAPK and IB, suggesting that lobeline suppresses LPS-induced NF-kB translocation by inhibiting the activation of these intracellular signaling cascades and decreases the protein level of iNOs in vivo and in vitro. In ALI, PMNs are rapidly activated and migrate into the alveolar space and inter-alveolar septum in response to intratracheal administration of LPS. In this regard, it has been proposed that oxidative stress may be related to corticosteroid resistance and may cause severe inflammatory response, including ALI/ARDS. LPS-
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Fig. 4. Six hour after LPS injection with or without lobeline pre-treatments, mice were sacrificed, their lungs were lavage and the BALF were collected. Cellular counts (A), total protein (B), TNF-␣, IL-6 and IL-1 (C) in BALF, myeloperoxidase activity (MPO) (D) in vivo were detected. Lobeline down-regulates antioxidative enzyme activation presented in western blotting (E). The antioxidative enzymes are represented SOD, CAT, and GPx which were performed at 6 h after LPS challenge. Each value represents as mean ± S.E.M. ### compared with sample of control group. The data were presented as mean ± S.E.M. for the three different experiments performed in triplicate. *** p < 0.001 were compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple range test). Values are expressed as the mean ± SD;* p < 0.05, ** p < 0.01, *** p < 0.001 were compared with LPS-alone group.
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Fig. 5. Effects of lobeline on LPS-induced iNOs, COX-2 expression in lung (A), MAPK phosphorylation (B), IB-␣, and NF-B protein expression (C) in ALI mice. Mice were pretreated with different concentrations of lobeline for 1 h and stimulated with LPS. The Western blotting by using an antibody specific were used for the detection of IB-␣ phosphorylated, NF-B nuclear and cytosol, and total forms of three MAPK molecules, ERK, p38, and JNK. The fold change in protein expression between the treated and the control groups was calculated. A representative Western blot from two separate experiments is shown. ### compared with sample of control group. The data were presented as mean ± S.E.M. for the three different experiments performed in triplicate. *** P < 0.001 were compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple range test). Values are expressed as the mean ± SD;* P < 0.05, ∗∗ P < 0.01, *** P < 0.001 were compared with LPS-alone group.
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Fig. 6. Effects of lobeline on LPS-induced HO-1 expression in lung. Tissue suspended were then prepared and subjected to Western blotting by using an antibody specific for HO-1. -actin was used as an internal control. The fold change in HO-1 expression between the treated and the control groups was calculated. ### compared with sample of control group. The data were presented as mean ± S.E.M. for the three different experiments performed in triplicate. *** P < 0.001 were compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple range test). Values are expressed as the mean ± S.E.M.; * P < 0.05, ** P < 0.01, *** P < 0.001 were compared with LPS-alone group.
evoked ALI is characterized by neutrophil infiltration in lung tissues that have reduced antioxidative enzymes (AOEs) activities and increased MPO activities (Huang et al., 2013; Shi et al., 2010; Wan et al., 2013). It was supposed that LPS-induced ALI stimulates neutrophil migration into BALF and the expression of LPS induced pro-inflammatory mediators. This paper suggests that lobeline potently inhibited neutrophil infiltration, MPO activity, edema formation and increased AOEs. In terms of MPO as a detector, it is released via PMN degranulation, which can catalyze hydrogen peroxide to induce the formation of TNF-␣, IL-6, and IL-1. Not only are these induced compounds but they also over-produce NO from iNOs, which has been implicated as an important mediator in the pathogenesis of inflammation. Our current studies also have reported that lobeline can decrease iNOs and COX-2 protein expression in in vitro and in vivo (Figs. 1E and 5A). Our results also indicate that IL-1, IL6, and TNF-␣ all have a direct target of lobeline and suppresses the downstream signaling pathways in decreasing inflammatory protein expression.
Furthermore, LPS induced inflammation has been linked to neutrophils infiltration and the production of neutrophils-derived free radicals like reactive oxygen species (ROS). ROS activation has a mutual relationship with NF-B signal pathway and many researchers demonstrated that antioxidative enzymes (AOEs) such as SOD, CAT, and GPx, can improve the oxidative damage induced by LPS. Superoxide anions are converted to hydrogen peroxide by SOD, which is then metabolized to water by CAT or GPx (Annapurna et al., 2013; Chien et al., 2014). In this study, there were significantly decreases in iNOs and COX-2 protein expression level with lobeline treatment. Furthermore, there was a significant increase in CAT, SOD, and GPx activities with lobeline treatment. We assume that the suppression of NO production is probably due to the increase of CAT, SOD, and GPx activities (Fig. 4E). Heat shock protein-32 (HSP32) is a strong negative regulator in the development of oxidative stress and responds to lung inflammation during LPS-induced ALI (Chen et al., 2013). The induction of HO-1 expression was due to a direct effect on iNOs expression (Chen et al., 2013). Recently, studies have shown that LPS-induced inflam-
Fig. 7. Proposed mechanism of lobeline inhibition of LPS induced inflammation in RAW 264.7 cells. Lobeline abrogates the phosphorylation of MAPKs/IKB and subsequently inactivates NF-kB, which may result from lobeline down-regulation of iNOS and COX-2. Arrows indicate the main inflammatory pathway activated by LPS stimulation.
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matory responses such as lung permeability and lung alveolitis was associated with the expression of HO-1 via MAPK pathway (SaradyAndrews et al., 2005). Our data demonstrates that lobeline induced a significant upregulation of HO-1 expression (Fig. 6). These observations suggest that lobeline is capable of reducing serious lung damage through HO-1 and the MAPK pathway. In summary, pretreatment with lobeline could up-regulate HO1 and AOEs, which attenuate lung injury during ALI. The mechanism may be through the up-regulation or activation of NF-B via signal transductions of the MAPK and IB pathway in ALI. (Fig. 7). These results suggest that lobeline represents a potential antiinflammatory agent and this new beneficial effect may expand future researches on anti-inflammatory properties of lobeline in vitro and in vivo. 5. Conclusion The present study reports the anti-inflammatory effects of lobeline in LPS-stimulated acute lung injury and relates detailed molecular mechanisms for the first time. Exploiting new uses for old drugs has important implications in the development of new drugs. We consider lobeline to have an important role as a natural product with few side effects and low toxicity in the food, so lobeline may be a promising therapeutic candidate for various lung inflammatory disorders, such as lung diseases and obstructive pulmonary diseases. Conflict of interests The authors declare that they have no conflict of interests. Acknowledgment The authors want to thank the financial supports from the National Science Council (NSC101-2313-B-039-002-MY3 and MOST 103-2320-B-468-002-), China Medical University (CMU) (CMU103-ASIA-22, and ASIA104-CMUH-06), and this study also is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212133019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.resp.2015.12.003. References Achoui, M., Appleton, D., Abdulla, M.A., Awang, K., Mohd, M.A., Mustafa, M.R., 2010. In vitro and in vivo anti-inflammatory activity of 17-O-acetylacuminolide through the inhibition of cytokines, NF-kappaB translocation and IKKbeta activity. PLoS One 5, e15105. Annapurna, A., Ansari, M.A., Manjunath, P.M., 2013. Partial role of multiple pathways in infarct size limiting effect of quercetin and rutin against cerebral ischemia-reperfusion injury in rats. Eur. Rev. Med. Pharmacol. Sci. 17, 491–500. Bani, D., Masini, E., Bello, M.G., Bigazzi, M., Sacchi, T.B., 1998. Relaxin protects against myocardial injury caused by ischemia and reperfusion in rat heart. Am. J. Pathol. 152, 1367–1376. Chen, M.W., Chen, W.R., Zhang, J.M., Long, X.Y., Wang, Y.T., 2014. Lobelia chinensis: chemical constituents and anticancer activity perspective. Chin. J. Nat. Med. 12, 103–107. Chen, T.Y., Sun, H.L., Yao, H.T., Lii, C.K., Chen, H.W., Chen, P.Y., Li, C.C., Liu, K.L., 2013. Suppressive effects of indigofera suffruticosa mill extracts on lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages. Food Chem. Toxicol. 55, 257–264. Chen, X., Tang, S.A., Lee, E., Qiu, Y., Wang, R., Quan Duan, H., Dan, S., Jin, M., Kong, D., 2015. IVSE, isolated from Inula japonica, suppresses LPS-induced NO production via NF-kappaB and MAPK inactivation in RAW 264.7 cells. Life Sci. Chien, S.C., Tseng, Y.H., Hsu, W.N., Chu, F.H., Chang, S.T., Kuo, Y.H., Wang, S.Y., 2014. Anti-inflammatory and anti-oxidative activities of polyacetylene from dendropanax dentiger. Nat. Prod. Commun. 9, 1589–1590.
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Respiratory Physiology & Neurobiology journal homepage: www.elsevier.com/locate/resphysiol
Lobeline improves acute lung injury via nuclear factor-B-signaling pathway and oxidative stress Kun-Cheng Li a,1 , Yu-Ling Ho b,1 , Cing-Yu Chen c , Wen-Tsong Hsieh d , Yuan-Shiun Chang a,e,∗ , Guan-Jhong Huang a,∗ a
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan Department of Nursing, Hungkuang University, Taichung 433, Taiwan c Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan d Department of Pharmacology, School of Medicine, China Medical University, Taichung 404, Taiwan e Chinese Crude Drug Pharmacy, China Medical University Hospital, Taichung 404, Taiwan b
a r t i c l e
i n f o
Article history: Received 5 May 2015 Received in revised form 9 December 2015 Accepted 10 December 2015 Available online 15 December 2015 Keywords: Lobeline MAPKs NF-B IB␣ AOEs ALI
a b s t r a c t Acute lung injury (ALI) is a severe, life-threatening medical condition whose pathogenesis is linked to neutrophil infiltration of the lung. Activation and recruitment of neutrophils to the lung is mostly attributed to the production of chemokines NO, IL-6, for instance. This study aims to investigate lobeline ability in reducing NO production, and nitric oxide synthase (iNOs) expression. Lobeline was tested by inhibiting phosphorylation of mitogen-activated protein kinases (MAPKs), NF-B and IB␣ in LPS-stimulated RAW 264.7 cells. When RAW 264.7 macrophages were given lobeline with LPS, a significant concentrationdependent inhibition of NO production was detected. In vivo tests, mice were either treated with normal saline, 10 mg/kg dexmethasone or 5, 10, 20 mg/kg lobeline intraperitoneally, and after an hour, the administration of 5 mg/kg of LPS was given intratracheally. External performance, cytokines, MAPK pathways and antioxidative enzymes (AOEs) were also carried out to evaluate the effects of these drugs. This is the first investigation in which lobeline was found to effectively inhibit acute lung edema, which may provide a potential target for treating ALI. Lobeline may utilize MAPKs pathways as well as AOEs activity to attenuate LPS-induced nonspecific pulmonary inflammation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Acute lung injury (ALI) is a syndrome that can lead to the destruction of the alveolar capillary membrane, pulmonary edema and neutrophil accumulation. The pathophysiological mechanism of ALI is believed to be associated with the chemotactic factor expression and influx of neutrophils into the lungs (Kim et al., 2013; Yu et al., 2015). Not only are there no specific therapies available, but also the disease has no cure. Therefore, it is desirable to identify new molecules that can regulate ALI-associated inflammation (Kim et al., 2013; Sitapara et al., 2014). ARDS/ALI is also characterized as related oxidative damage which is closely linked to neutrophil (PMN) infiltration, secretion of pro-inflammatory cytokines and
∗ Corresponding authors at: Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, College of Pharmacy, China Medical University, Taichung 404, Taiwan. Fax: +886 4 2208 3362. E-mail addresses: [email protected] (Y.-S. Chang), [email protected] (G.-J. Huang). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.resp.2015.12.003 1569-9048/© 2016 Elsevier B.V. All rights reserved.
antioxidants. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) are currently considered the basic oxidative damage stress markers (Kuo et al., 2011; Reddy et al., 2009). Lipopolysacharide (LPS) has been well recognized for studying the pathogenesis of ALI/ARDS. After intratracheal administration, LPS induced nitric oxide synthase (iNOs) and cyclooxygenase-2 (COX-2) in acute lung injury model. Subsequently, the overproduction NO, inflammatory mediator like tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-1 and interleukin-6 (IL-6) are participate in the pathogenesis of inflammation. Responses of proinflammatory mediators is regulated by the activation of the transcription factor nuclear factor (NF-B) which active via mitogen-activated protein kinase (MAPK) pathways (Chen et al., 2015; Huang et al., 2012; Zhang et al., 2015) and it may become part of a novel therapeutic target. Moreover, the expression of iNOs is closely related to the induction of heme oxygenase-1 (HO-1). As murine macrophages generate NO with LPS, one potential system which entails the catabolizing of enzyme HO-1, and brings biliverdin, carbon monoxide (CO) existence can limit synthesis NO production and iNOs expression. (Li et al., 2015; Sung and Lee, 2015; Sung et al.,
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2014; Wang et al., 2014). Thus, enhancing the production of HO-1 reduces the expression of iNOs. ␣-Lobeline (lobeline), a lipophilic nonpyridino, is the predominant alkaloid in a family of structurally-related compounds found in Lobelia chinensis, Indian tobacco (Lim et al., 2004), and has been reported to possibly treat asthma and respiratory illness. However, there has been no report to demonstrate whether or not lobeline has any anti-inflammatory effects on LPS-induced RAW 264.7 in vitro and acute lung injury in vivo. This study explored lobeline’s new uses by LPS-stimulated ALI and demonstrated the molecular mechanism of the regulatory pathway. The results revealed the lobeline can protect against LPS-induced lung injury in mice and possibly exert its activity through the regulation of antioxidant enzyme activity, NF-B, and MAPKs signaling pathways. 2. Materials and methods 2.1. Cell culture A murine macrophage cells (BCRC No. 60001) were purchased from the Bioresources Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were propagated and aliquots stored in liquid nitrogen, maintained as adherent cell cultures and passaged 3–25 times after which a new frozen aliquot was used. The cells were cultured in plastic dishes containing Dulbecco’s Modified Eagle Medium (DMEM, Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS, Sigma, USA) in a CO2 incubator (5% CO2 in air) at 37 ◦ C and subcultured every 3 day at a dilution of 1:5 using 0.05% trypsin—0.02% EDTA in Ca2+ -, Mg2+ - free phosphate-buffered saline (DPBS). 2.2. Cell viability This preliminary experiment consists of two parts. In the first part, we examined the viability of the Raw cells; as for the second portion, the cell medium was examined for NO production. Raw 264.7 cells (2 × 105 per well) were cultured in 96-well plate containing DMEM supplemented with 10% FBS for 1 day to become nearly confluent. The cells were cultured with lobeline in the presence of LPS (100 ng/mL) at 37 ◦ C for 24 h. The media were removed and saved for the following NO experiment, while the cells were washed with PBS for another round and incubated with 100 L of 0.5 mg/mL MTT in a humidified atmosphere of 5% CO2 and 95% air for 37 ◦ C 6 h. After incubating, absorbance was read using a microplate reader at 570 nm. The above steps were repeated three times for each concentration. 2.3. NO production NO production was indirectly assessed by measuring the nitrite levels in the cultured media was determined by Griess reaction (Kang et al., 2014). 100 L of Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylenediamine dihydrochloride and 5% phosphoric acid) was added to each sample medium and incubated at room temperature for 10 min. By using sodium nitrite to generate a standard curve, the concentration of nitrite for each sample was measured at the absorbance of 540 nm.
libitum. Animal studies were conducted according to the regulations of Instituted Animal Ethics Committee, and the protocol was approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals. After a 1–2 week adaptation period, male ICR mice (25–32 g) were randomly assigned to six groups (n = 12) of the animals in the study. The control group received normal saline (intraperitoneal; i.p.). The other groups included a LPS-treated, a positive control (LPS + Dex) and lobeline administered groups (LPS + lobeline-H, LPS + lobeline-M, LPS + lobeline-L). 2.5. Model of LPS induced ALI Seventy-two healthy male ICR mice were randomly divided into 6 groups (n = 12): control group, LPS group, dexamethasone (Dex) group (10 mg/kg), low dosage lobeline group (5 mg/kg), middle dosage group (10 mg/kg, LPS + lobeline-M) and high dosage group (20 mg/kg, LPS + lobeline-H). Take half of each group for the inflammation protein analysis, slicing, and edema and the rest of mice use for BALF analysis. Mice were intratracheally instilled with 5 mg/kg LPS in 50 L sterile saline or sterile saline alone (control group) (Zhang et al., 2010). In brief, mice were anesthetized with mixed reagent of 10 L/g i.p urethane (0.6 g/mL) and chloral hydrate (0.4 g/mL), followed by Dex (10 mg/kg) or lobeline intraperitoneal injection with individual dose. Six hours later, the mice received sacrifice and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. 2.6. Cell counts, protein levels, TNF-˛, IL-6, and IL-1ˇ assay in BALF Six hours later, mice were exsanguinated after anesthesia. According to the previous report, BALF was collected by the upper part of the trachea, by douche three times with 500 L PBS (pH 7.2). The fluid recovery rate was more than 90%. Lavage sample from each mouse was kept on ice. BALF was centrifuged at 700 × g for 5 min. The sediment cells were resuspended in 2 mL PBS, half of them have used to detect cell counts by cytometer, the rest equally divided into two parts. One has centrifuged again in order to get sediment for extracting proteins with a RIPA solution (radioimmuno-precipitation assay buffer) and the other of BALF supernatant were used to assay the TNF-␣, IL-6 and IL-1 levels using the respective ELISA kits. Total cells were counted double-blindly with flow cytometer, total cell protein extracts were homogenized in cell protein extraction solution and the concentration in the supernatant was determined by Bradford assay. 2.7. Myeloperoxidase (MPO) activity assay The lungs were homogenized, 12,000 × g at 4 ◦ C for 15 min and resuspended in 50 mM KPO4 buffer (PH 6.0) with containing 0.19 mg/mL of o-dianisidine chloride and 0.0005% H2 O2 was a substrate for myeloperoxidase at 460 nm with a spectrophotometer (Molecular Devices, Sunnyvale, CA, USA). MPO content was expressed as relative MPO activity (OD460nm /mg protein of lung tissue) (Bani et al., 1998). 2.8. Lung wet/dry weight ratio
2.4. Animals Male ICR mice, 6–7 weeks old, were obtained from BioLASCO Taiwan Co., Ltd. The animals were kept in plexiglass cages at a constant temperature of 22 ± 1 ◦ C, relative humidity 55 ± 5% and with 12 h dark–light cycles. They were given food and water ad
The lungs wet to dry weight ratio (W/D ratio) was measured to evaluate pulmonary edema. The lung tissues were excised, immediately weighed, and then placed in an oven at 80 ◦ C for 48 h to obtain the “dry” weight. The W/D ratio was then calculated (Zhou et al., 2014).
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Fig. 1. The chemical structure of lobeline (A), dexamethasone (Dex), (B) and the effects of lobeline on lipopolysaccharide (LPS)-induced cell viability (C), NO production (D), inhibition of iNOS and COX-2 protein expression (E), MAPK (JNK, p38, and ERK) protein expression (F) IB-␣ and NF-B (G) were evaluated in RAW 264.7 cells. Cells were incubated for 24 h with 100 ng/mL LPS in the absence or presence of lobeline (0, 40, 20, 10, 5, 2.5, and 1.25 g/mL). Lobeline was added 1 h before the incubation with LPS. Cell viability was performed by using MTT assay. Nitrite concentration in the medium was determined by using Griess reagent. Lysed cells were then prepared and subjected to Western blotting by using an antibody specific for iNOS, COX-2, MAPK, IB-␣, NF-B, and -actin was used as an internal control. The data were presented as mean ± S.D. for three different experiments performed in triplicate. ### compared with sample of control group, ** p < 0.01 and *** p < 0.001 were compared with LPS-alone group.
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2.9. H&E staining The left lower lung from each mouse was fixed in 10% formalin, embedded in paraffin, cut into 5 mm sections, stained with. After H&E staining of lung tissue slices, pathological changes were observed. The lung injury score was calculated by assessing the degree of inflammatory cell infiltration, hemorrhage, interstitial and alveolar edema, and the thickness of alveolar septum in five random fields in a blind manner using light microscopy. A score of 0 represented no damage; l represented mild damage; 2 represented moderate damage; 3 represented severe damage and 4 represented very severe histological damage (Parsey et al., 1998). 2.10. Protein lysate preparation and western blot analysis The stimulated murine macrophage cell line RAW2 64.7 cells were washed with PBS and lysed in an ice-cold lysis buffer [10% glycerol, 1% Triton X-100, 1 mM Na3 VO4 , 1 mM EGTA, 10 mM NaF, 1 mM Na4 P2 O7 , 20 mM Tris buffer (pH 7.9), 100 mM bglycerophosphate, 137 mM NaCl, 5 mM EDTA, and one protease inhibitor cocktail tablet (Roche, Indianapolis, IN,USA)] on ice for 1 h, followed by centrifugation at 12,000 × g for 30 min at 4 ◦ C. Soft tissues were removed from individual mice lungs and homogenized in a solution containing PBS and RIPA before grinding. The homogenateswere centrifuged at 12,000 × g for 20 min, and 50 g of protein from the supernatants was then separated on 10% sodium dodecylsulphate-polyacrylamide gel (SDS-PAGE) and transferred to polyvinylidene difluoride membranes. After transfer, the membrane was blocked for 2 h at room temperature with 5% skim milk in Tris-buffered saline-Tween (TBST; 20 mM Tris, 500 mM NaCl, pH 7.5, 0.1% Tween 20). The membranes were probed with primary antibodies (iNOs, COX-2) for phosphorylated and nonphosphorylated forms of p38 MAPK, ERK, IB-␣, JNK, HO-1, p65 and pro-inflammatory enzymes (SOD, GPx, catalase) at 4 ◦ C overnight. The membranes were washed three times and the immunoreactive proteins were detected by enhanced (ECL) by using hyperfilm and ECL reagent (Amersham International plc., Buckinghamshire, U.K.). The results of Western blot analysis. 2.11. Statistical analysis Experimental results were presented as the mean ± standard deviation (SD) of three parallel measurements. IC50 values were estimated using a non-linear regression algorithm (SigmaPlot 8.0; SPSS Inc., Chicago, IL). Data obtained from animal experiments were expressed as mean standard error (±S.E.M.). Statistical evaluation was carried out by one-way analysis of variance (ANOVA followed by Scheffe’s multiple range tests). Statistical significance is expressed as * P < 0.05, ** P < 0.01 and *** P < 0.001. 3. Results 3.1. Effect of lobeline on LPS-induced cell viability and NO production in macrophages The effect of lobeline on RAW 264.7 cell viability was determined by a MTT assay. Cells cultured with lobeline at concentrations of 0, 1.3, 2.5, 5, 10, 20, and 40 g/mL, was used in the presence of 100 ng/mL LPS for 24 h (Fig. 1C) . It was shown that there were no significant differences between the cell viability of the normal and LPS control groups, indicating that LPS did not induce cell deaths. Additionally, lobeline used within the range of 40–1.3 g/mL did not induce cytotoxicity; the efficacy of these doses, 20 and 40 g/mL, were the same. Therefore, we used lobeline in all animal experiments, with doses below or equal to 20 g/mL. Although LPS did not induce cell death, analyses of culture media
showed that LPS activated human macrophage raw cells to produce considerable NO. Among the experimental groups, the medium which contained lobeline showed the most inhibitory effect on NO production. There was a significant decrease in nitrite production in the group treated with 5 g/mL lobeline (P < 0.001). The IC50 value for inhibition of nitrite production of lobeline was about 2.6 g/mL (Fig. 1D). Based on the results from the MTT and NO production assays, we used lobeline to conduct all subsequent experiments. 3.2. Inhibition of LPS-induced iNOs and COX-2 Protein by lobeline Western blot analyses were performed to look into whether the inhibitory effects of lobeline on the pro-inflammatory mediators NO was related to the modulation of the expressions of iNOs and COX-2. The results showed that incubation with lobeline (0, 5, 10, and 20 g/mL) in being with LPS (100 ng/mL) for 24 h can inhibit iNOs proteins expression in mouse macrophage RAW 264.7 cells, in a dose-dependent manner (Fig. 1E). The detection of bactin was also performed in the same blot as the internal control. The intensity of protein bands were analyzed using Kodak Quantity software in three independent experiments and showed an average of 37.1 and 61.5% down-regulation of iNOs and COX-2 proteins after treatment with lobeline at 20 g/mL, compared with the LPSalone. In general, these results indicate that the inhibitory effect of lobeline on LPS-induced NO production was caused by iNOs and COX-2 suppression. 3.3. Effects of lobeline on the LPS-stimulated activation of mitogen-activated protein kinases (MAPKs) The mitogen-activated protein (MAP) kinases play critical roles in the regulation of cell growth and differentiation, and in controling cellular responses to cytokines and stresses. In particular, they are also known to be important for the activation of NF-B (Achoui et al., 2010; Hsing et al., 2011). To explore whether the inhibition of NF-B activation by lobeline is mediated through the MAPK pathway, MAPK phosphorylation was examined by Western blot in RAW 264.7 cells pretreated with lobeline and then pretreated with LPS. As shown in Fig. 1F, pretreatment with lobeline had an effect on the phosphorylation of JNK and p38 (but not ERK1/2). These results suggest that the phosphorylation of JNK and p38 may be involved in the inhibitory effects of lobeline on LPS-stimulated NF-kB binding in RAW 264.7 cells. 3.4. Inhibition of LPS-induced IkB-˛ and NF-kB proteins by lobeline The effect of NF-kB and IkB-␣ expression by lobeline in the presence of LPS for 1 h was assessed by Western blot. The intensity of protein bands showed an increase of NF Anti kB protein with the average of 41.1% and a decrease of phosphoryl IkB-␣ protein of 42.3% after treatment with lobeline at 20 g/mL, compared with the LPS-alone (Fig. 1G). Therefore, it can be concluded that lobeline is capable of inhibiting iNOs expression in LPS-induced RAW 264.7 cells via attenuation of NF-B signaling by p38, JNK and IkB-␣. 3.5. Histological examination We used H&E stain and lung injury score to evaluate the effects of therapeutic lobeline treatment on the worse alveolar collapsed state and perivascular and peribronchial edema of lung inflammation. Histologically, lung biopsies of mice treated with lobeline (5 mg/kg) showed that the inflammatory cells were reduced in number, confined to the surroundings of the vascular areas and that the intercellular spaces did not show any cellular infiltrates. In addition, neutrophils increased with LPS treatment (Fig. 2B)
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Fig. 2. Histological appearances of mouse lung after individual injecting 10% formalin then stained with H&E stain demonstrates a representative view from each group and severity of lung injury was analyzed by the lung injury scoring system. [A: Control, B: LPS, C: LPS + Dex D: LPS + lobeline-H, E: LPS + lobeline-M, F: LPS + lobeline-L]. Severity of lung injury was analyzed by the lung injury scoring system (G). The Figure demonstrates a representative view (X200) from each group; each bar represents the mean ± S.E.M. of 6 mice.
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and the treatment group Dex and lobeline (5 mg/kg) were able to decrease the neutrophil numbers as opposed to the LPS-treated group (Fig. 2C–F). At the same time, in Fig. 2G, the lung injury score stands at around 4 points after LPS injection, which is absolutely much higher than that of the other three groups with different amounts of lobeline, with the highest value of only 3 points. 3.6. Lobeline attenuates pulmonary inflammation and pulmonary edema in vivo Because lobeline effectively inhibited iNOs inductions in macrophages, our study was extended to determine whether or not lobeline affected acute inflammation in animal models. In this study, LPS-treated mice exhibited the typical pathological changes of ALI, including notably, inflammatory neutrophil infiltration, interstitial edema, interalveolar septal thickening and intra-alveolar, interstitial patchy hemorrhage. As for pulmonary edema and alveolar fluid removal efficiency in LPS- treated mice, the W/D ratio was employed (Fig. 3A) . The results of pulmonary inflammation and pulmonary edema were found to be dose dependent and the W/D weight ratio in lobeline 20 mg/kg group was found to be significantly different from the LPS group (Fig. 3A) (P < 0.05). 3.7. Lobeline reduces cellular counts and proteins in BALF Bronchoalveolar lavage (BAL) fluid protein was quantified after insult. To identify the anti-inflammatory properties of lobeline, cellular counts and proteins of PMNs in BALF were measured in our study. After LPS injection of six hours, we evaluated the leukocyte changes in the lung of LPS-administered mice, with or without lobeline pretreatment. As shown in Fig. 4A, B administration of LPS caused extensive leukocyte infiltration, whereas with pretreatment of lobeline, the LPS-induced leukocyte infiltration was suppressive in a concentration-dependent manner. A significant inhibitory effect began at 10 mg/kg (P < 0.05). 3.8. Lobeline down-regulates TNF-˛, IL-6, and IL-1ˇ in BALF The BAL fluid: plasma protein ratio was substantially augmented in animals at this proinflammatory cytokines stage, TNF-␣, IL-6, and IL-1 in BALF were measured by ELISA. TNF-␣, IL-6 and IL-1 in BALF in the mice with LPS IT were markedly higher than in the mice in the control group. However, cytokines in BALF in the mice with lobeline and Dex were decreased (Fig. 4C). The above results indicated that lobeline reduced the expression of proinflammatory cytokines, which in turn, improved lung damage caused by LPSinduced ALI. 3.9. Effects of lobeline on MPO activity and antioxidative enzymes in LPS-induced ALI The accumulation of activated neutrophils was assessed by measuring MPO activity. Oxidative stress exerted by activated PMNs is critically important in the pathophysiology of ALI, such as SOD, CAT, and GPx, which are consumed during the amelioration of ALI (Hosakote et al., 2009). This antioxidative enzyme can decrease inflammatory activities in LPS-treated mice. Lung MPO activity was markedly increased in the mice 6 h after LPS IT, and LPS animals also showed a marked increase (Fig. 4D, P < 0. 001), whereas lobeline animals exhibited a trend toward lower levels of lung MPO activity when compared with the control animals; and the upregulation of MPO activity and was dose-dependent prevented in animals pretreated with lobeline. In contrast, pretreatment with lobeline and Dex lead to recovery in LPS-induced inflammation when activated
by these enzymes SOD, CAT, and GPx. These enzymes have significantly different contents with the LPS groups (P < 0.001; Fig. 4E). 3.10. Inhibition of LPS-induced iNOs and COX-2 protein by lobeline in lung tissues iNOs and COX-2 play critical roles in the pathology of LPSinduced ALI. The effect of lobeline on iNOS and COX-2 expressions in the lung tissue was analyzed using Western blot assay. As shown in Fig. 5A, the level of iNOs and COX-2 proteins were significantly decreased in mice challenged with lobeline + LPS compared with the LPS groups (P < 0.001). 3.11. Effects of lobeline on MAPK activation in LPS-induced ALI MAPKs play critical roles in the regulation of cell growth and differentiation, and in the control of cellular responses to cytokines and stresses (Khan et al., 2013). The effect of lobeline on phosphorylation of ERK, JNK and p38MAPK in LPS-induced ALI was analyzed by Western blot. The results showed that LPS stimulation significantly increased MAPK phosphorylation and lobeline, which significantly inhibited the LPS-induced phosphorylation of p38 MAPK, JNK not ERK, and JNK in the lobeline group decreased more significantly than the Dex group. The significant inhibitory effect started at 20 mg/kg (P < 0.001; Fig. 5B). In addition, lobeline also inhibited LPS-induced phosphorylation of p38MAPK, but had slightly lower inhibitory effect than the Dex group. 3.12. Inhibition of LPS-induced IB˛ and NF-kB proteins activation by lobeline NF-B is activated by a variety of stimuli including LPS, TNF␣, and IL-1. The effect of lobeline on NF-B expression in lung tissue of LPS-induced ALI mice was assessed by Western blot. The expression of p-IB␣ evoked by LPS was significantly suppressed at 20 mg/kg by lobeline pretreatment. On the contrary, the expression of NF-B p65 in cytoplasm was significantly reduced by LPS administration (P < 0.001; Fig. 5C), and these changes were inhibited by lobeline pretreatment. The signal of NF-B inhibition is regulated by the interaction with inhibitory IB␣ proteins. 3.13. Effects of lobeline on HO-1 expression in LPS-induced ALI Increased HO-1 induction has a direct effect on iNOs expression. Therefore, we investigated the inhibitory effects of lobeline on iNOs expression to see if it was associated with increased HO-1 production. We assessed HO-1 induction in the pretreatment of lobeline in LPS- induced ALI and analyzed by using Western blot. The effects of LPS elicited on HO-1 expression, lobeline enhanced LPS-induced HO-1expression at 20 mg/kg (P < 0.001; Fig. 6). This result suggests that lobeline has the ability to reduce LPS-induced NO and iNOs production by increasing HO-1 induction. 3.14. Comparison of the effects of lobeline and dexamethasone on LPS-induced ALI Dex reduces oxidative stress and supplies useful effects on LPS-induced ALI (Jing et al., 2015). Although LPS-induced PMN infiltration was reduced by both lobeline and Dex, lobeline’s lung damage data lower than Dex (Fig. 2G). Moreover lobeline could reduce not only the secretion of MPO but also increase the activity of CAT, whereas Dex had no effect (Fig. 4C and E). Nevertheless, SOD and GPx activities were recovered by lobeline and Dex. These results suggested the improvement by lobeline is better than that by Dex on LPS-induced ALI.
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Fig. 3. Lobeline improves pulmonary edema in vivo (A). Six hours after LPS injection with or without lobeline pretreatments, mice were exsanguinated and their right lower lungs were obtained. The right lower lungs weighed and then dehydrated at 60 ◦ C for 72 h in an oven. Each value represents as mean ± S.E.M. of 6 mice. ### compared with sample of control group. * p < 0.05, ** p < 0.01, and *** p < 0.001 were compared with LPS-alone group.
4. Discussion ALI/ARDS might be a continuous phenomenon that depends on the level and time of stress and strain application, and not a switch process. It is important to consider therapeutic maneuvers that minimize injury in the early postoperative period.The clinical prevent of ALI/ARDS syndromes especially at its more severe form, is greater than the treatment effect. Recent evidence indicates that prevention plays a key point to improve outcome of patients at risk of ALI/ARDS (Huang et al., 2014; Litell et al., 2011). The data presented demonstrate that lobeline pretreatment in both in vitro and in vivo reduces LPS-induced RAW 264.7 macrophages and a murine model of ALI, respectively. Lobeline is one of the most effective alkaloids from the plant L. chinensis, which grows in the wild in China and other Asian countries, and has been used as a smoking cessation aid, and has successfully been shown to reduce inflammation of ALI (Chen et al., 2014). However, no report has been published on the anti-inflammatory effects of lobeline in LPS acute lung injury. Thus, this study aimed to evaluate the antiinflammatory effects of lobeline by screening the effects of lobeline in LPS-induced pro-inflammatory molecules in vitro and in acute phase inflammation in vivo cases. We also assessed the mechanisms of lobeline on NF-B expressions associated with oxidative enzyme and phosphorylation of MAPK and IB␣.signaling pathways in the anti-inflammation. LPS is (when something is true or is a fact, you use IS) found in the outer membrane of gram-negative bacteria, and is frequently used to induce lung injury in mice as a model for studying ALI. After LPS IT, inducing macrophage and PMNs activation and membrane protein mainly expressed like iNOs and COX-2. Meanwhile, macrophage and PMNs infiltrated into the lung tissues and releasing enzymes and phagocytizing the pathogen. Meantime, iNOs and COX-2 are the main cytotoxic and pro-apoptotic mechanisms participating in the innate response of many mammals via inflammatory mediators (Hidalgo et al., 2005; Oh et al.,
2009). Examination of the cytotoxicity of lobeline in RAW 264.7 macrophages using MTT assay has indicated that lobeline, even at 40 g/mL, did not affect the viability of RAW 264.7 cells (Fig. 1C). In a murine model of LPS-induced ALI, lobeline reduction inhibited iNOs expression in LPS-stimulated macrophages and has been linked to neutrophil infiltration release of NO. The results indicate that lobeline inhibits LPS-induced NO through modulating iNOs expression and COX-2 enzyme activity (Fig. 1E). These findings suggest that lobeline could potentially be an alternative treatment of immunological disorders by interfering with the endogenous factors (Kang et al., 2011). In response to LPS, it is well-known that LPS induces the endogenous transcription of NF-B in order to regulate many of the pro-inflammatory cytokine genes. LPS stimulation elicits a cascade leading to the activation of IB and various MAPKs, like JNK, ERK, and p38 kinase signaling molecules which involves the regulation of cell growth and differentiation and in the control of cellular responses to cytokines and stressors (Lee et al., 2014). In the present study, we have demonstrated that the phosphorylation of IB and MAPKs can be induced by LPS on macrophage and ALI models. Our inflammatory analysis showed that after treatment of lobeline, the phosphorylation level of p38, JNK and IB were significantly reduced at 20 mg/kg dosage (29.21%, 41.76% and 42.29%, respectively) in RAW 264.7 cells. (Fig. 1F). The lobeline lung samples collected from animals decreased at (73.18%, 20.02% and 19.29% respectively) (Fig. 5B) compared to the LPS group. Therefore, this suggests that the treatment of lobeline blocked the activation of JNK p38MAPK and IB, suggesting that lobeline suppresses LPS-induced NF-kB translocation by inhibiting the activation of these intracellular signaling cascades and decreases the protein level of iNOs in vivo and in vitro. In ALI, PMNs are rapidly activated and migrate into the alveolar space and inter-alveolar septum in response to intratracheal administration of LPS. In this regard, it has been proposed that oxidative stress may be related to corticosteroid resistance and may cause severe inflammatory response, including ALI/ARDS. LPS-
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Fig. 4. Six hour after LPS injection with or without lobeline pre-treatments, mice were sacrificed, their lungs were lavage and the BALF were collected. Cellular counts (A), total protein (B), TNF-␣, IL-6 and IL-1 (C) in BALF, myeloperoxidase activity (MPO) (D) in vivo were detected. Lobeline down-regulates antioxidative enzyme activation presented in western blotting (E). The antioxidative enzymes are represented SOD, CAT, and GPx which were performed at 6 h after LPS challenge. Each value represents as mean ± S.E.M. ### compared with sample of control group. The data were presented as mean ± S.E.M. for the three different experiments performed in triplicate. *** p < 0.001 were compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple range test). Values are expressed as the mean ± SD;* p < 0.05, ** p < 0.01, *** p < 0.001 were compared with LPS-alone group.
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Fig. 5. Effects of lobeline on LPS-induced iNOs, COX-2 expression in lung (A), MAPK phosphorylation (B), IB-␣, and NF-B protein expression (C) in ALI mice. Mice were pretreated with different concentrations of lobeline for 1 h and stimulated with LPS. The Western blotting by using an antibody specific were used for the detection of IB-␣ phosphorylated, NF-B nuclear and cytosol, and total forms of three MAPK molecules, ERK, p38, and JNK. The fold change in protein expression between the treated and the control groups was calculated. A representative Western blot from two separate experiments is shown. ### compared with sample of control group. The data were presented as mean ± S.E.M. for the three different experiments performed in triplicate. *** P < 0.001 were compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple range test). Values are expressed as the mean ± SD;* P < 0.05, ∗∗ P < 0.01, *** P < 0.001 were compared with LPS-alone group.
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Fig. 6. Effects of lobeline on LPS-induced HO-1 expression in lung. Tissue suspended were then prepared and subjected to Western blotting by using an antibody specific for HO-1. -actin was used as an internal control. The fold change in HO-1 expression between the treated and the control groups was calculated. ### compared with sample of control group. The data were presented as mean ± S.E.M. for the three different experiments performed in triplicate. *** P < 0.001 were compared with the LPS-alone group (one-way ANOVA followed by Scheffe’s multiple range test). Values are expressed as the mean ± S.E.M.; * P < 0.05, ** P < 0.01, *** P < 0.001 were compared with LPS-alone group.
evoked ALI is characterized by neutrophil infiltration in lung tissues that have reduced antioxidative enzymes (AOEs) activities and increased MPO activities (Huang et al., 2013; Shi et al., 2010; Wan et al., 2013). It was supposed that LPS-induced ALI stimulates neutrophil migration into BALF and the expression of LPS induced pro-inflammatory mediators. This paper suggests that lobeline potently inhibited neutrophil infiltration, MPO activity, edema formation and increased AOEs. In terms of MPO as a detector, it is released via PMN degranulation, which can catalyze hydrogen peroxide to induce the formation of TNF-␣, IL-6, and IL-1. Not only are these induced compounds but they also over-produce NO from iNOs, which has been implicated as an important mediator in the pathogenesis of inflammation. Our current studies also have reported that lobeline can decrease iNOs and COX-2 protein expression in in vitro and in vivo (Figs. 1E and 5A). Our results also indicate that IL-1, IL6, and TNF-␣ all have a direct target of lobeline and suppresses the downstream signaling pathways in decreasing inflammatory protein expression.
Furthermore, LPS induced inflammation has been linked to neutrophils infiltration and the production of neutrophils-derived free radicals like reactive oxygen species (ROS). ROS activation has a mutual relationship with NF-B signal pathway and many researchers demonstrated that antioxidative enzymes (AOEs) such as SOD, CAT, and GPx, can improve the oxidative damage induced by LPS. Superoxide anions are converted to hydrogen peroxide by SOD, which is then metabolized to water by CAT or GPx (Annapurna et al., 2013; Chien et al., 2014). In this study, there were significantly decreases in iNOs and COX-2 protein expression level with lobeline treatment. Furthermore, there was a significant increase in CAT, SOD, and GPx activities with lobeline treatment. We assume that the suppression of NO production is probably due to the increase of CAT, SOD, and GPx activities (Fig. 4E). Heat shock protein-32 (HSP32) is a strong negative regulator in the development of oxidative stress and responds to lung inflammation during LPS-induced ALI (Chen et al., 2013). The induction of HO-1 expression was due to a direct effect on iNOs expression (Chen et al., 2013). Recently, studies have shown that LPS-induced inflam-
Fig. 7. Proposed mechanism of lobeline inhibition of LPS induced inflammation in RAW 264.7 cells. Lobeline abrogates the phosphorylation of MAPKs/IKB and subsequently inactivates NF-kB, which may result from lobeline down-regulation of iNOS and COX-2. Arrows indicate the main inflammatory pathway activated by LPS stimulation.
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matory responses such as lung permeability and lung alveolitis was associated with the expression of HO-1 via MAPK pathway (SaradyAndrews et al., 2005). Our data demonstrates that lobeline induced a significant upregulation of HO-1 expression (Fig. 6). These observations suggest that lobeline is capable of reducing serious lung damage through HO-1 and the MAPK pathway. In summary, pretreatment with lobeline could up-regulate HO1 and AOEs, which attenuate lung injury during ALI. The mechanism may be through the up-regulation or activation of NF-B via signal transductions of the MAPK and IB pathway in ALI. (Fig. 7). These results suggest that lobeline represents a potential antiinflammatory agent and this new beneficial effect may expand future researches on anti-inflammatory properties of lobeline in vitro and in vivo. 5. Conclusion The present study reports the anti-inflammatory effects of lobeline in LPS-stimulated acute lung injury and relates detailed molecular mechanisms for the first time. Exploiting new uses for old drugs has important implications in the development of new drugs. We consider lobeline to have an important role as a natural product with few side effects and low toxicity in the food, so lobeline may be a promising therapeutic candidate for various lung inflammatory disorders, such as lung diseases and obstructive pulmonary diseases. Conflict of interests The authors declare that they have no conflict of interests. Acknowledgment The authors want to thank the financial supports from the National Science Council (NSC101-2313-B-039-002-MY3 and MOST 103-2320-B-468-002-), China Medical University (CMU) (CMU103-ASIA-22, and ASIA104-CMUH-06), and this study also is supported in part by Taiwan Ministry of Health and Welfare Clinical Trial and Research Center of Excellence (MOHW105-TDU-B-212133019). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.resp.2015.12.003. References Achoui, M., Appleton, D., Abdulla, M.A., Awang, K., Mohd, M.A., Mustafa, M.R., 2010. In vitro and in vivo anti-inflammatory activity of 17-O-acetylacuminolide through the inhibition of cytokines, NF-kappaB translocation and IKKbeta activity. PLoS One 5, e15105. Annapurna, A., Ansari, M.A., Manjunath, P.M., 2013. Partial role of multiple pathways in infarct size limiting effect of quercetin and rutin against cerebral ischemia-reperfusion injury in rats. Eur. Rev. Med. Pharmacol. Sci. 17, 491–500. Bani, D., Masini, E., Bello, M.G., Bigazzi, M., Sacchi, T.B., 1998. Relaxin protects against myocardial injury caused by ischemia and reperfusion in rat heart. Am. J. Pathol. 152, 1367–1376. Chen, M.W., Chen, W.R., Zhang, J.M., Long, X.Y., Wang, Y.T., 2014. Lobelia chinensis: chemical constituents and anticancer activity perspective. Chin. J. Nat. Med. 12, 103–107. Chen, T.Y., Sun, H.L., Yao, H.T., Lii, C.K., Chen, H.W., Chen, P.Y., Li, C.C., Liu, K.L., 2013. Suppressive effects of indigofera suffruticosa mill extracts on lipopolysaccharide-induced inflammatory responses in murine RAW 264.7 macrophages. Food Chem. Toxicol. 55, 257–264. Chen, X., Tang, S.A., Lee, E., Qiu, Y., Wang, R., Quan Duan, H., Dan, S., Jin, M., Kong, D., 2015. IVSE, isolated from Inula japonica, suppresses LPS-induced NO production via NF-kappaB and MAPK inactivation in RAW 264.7 cells. Life Sci. Chien, S.C., Tseng, Y.H., Hsu, W.N., Chu, F.H., Chang, S.T., Kuo, Y.H., Wang, S.Y., 2014. Anti-inflammatory and anti-oxidative activities of polyacetylene from dendropanax dentiger. Nat. Prod. Commun. 9, 1589–1590.
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