International Immunopharmacology 19 (2014) 342–350
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Protective effect of taraxasterol on acute lung injury induced by lipopolysaccharide in mice San Zhihao a,1, Fu Yunhe a,1, Li Wei b, Zhou Ershun a, Li Yimeng a, Song Xiaojing a, Wang Tiancheng a, Tian Yuan a, Wei Zhengkai a, Yao Minjun a, Cao Yongguo a, Zhang Naisheng a,⁎ a b
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, People's Republic of China College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun, Jilin Province 130118, People's Republic of China
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Article history: Received 19 November 2013 Received in revised form 11 January 2014 Accepted 28 January 2014 Available online 15 February 2014 Keywords: Taraxasterol Acute lung injury Myeloperoxidase Nuclear factor-kappaB (NF-κB)
a b s t r a c t Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been reported to have potent antiinflammatory properties. However, the effect of taraxasterol on lipopolysaccharide (LPS)-induced mice acute lung injury has not been investigated. The aims of this study were to investigate whether taraxasterol could ameliorate the inflammation response in LPS-induced acute lung injury and to clarify the possible mechanism. Male BALB/c mice were pretreated with taraxasterol 1 h before intranasal instillation of LPS. 7 h after LPS administration, the myeloperoxidase (MPO) in lung tissues, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NFκB, p46–p54 JNK, p42–p44 ERK, and p38 were determined by western blotting. The results showed that taraxasterol attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), lung wet/ dry ratio, and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL1β) in a dose-dependent manner. Additionally, western blotting results showed that taraxasterol inhibited the phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 caused by LPS. Our data suggest that anti-inflammatory effects of taraxasterol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPK signaling pathways. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Taraxacum officinale is a traditional Chinese herb which has long been used for the treatment of various inflammatory or infectious diseases such as upper respiratory tract infections, pneumonia and mastitis [1]. Taraxasterol, a pentacyclic-triterpene isolated from T. officinale, is responsible for the plant's pharmacological activities. Recently, taraxasterol was found to have anti-inflammatory effects [2]. Studies showed that taraxasterol could inhibit inflammatory cytokines such as TNF-α and IL-1β in LPS-stimulated RAW264.7 macrophages [3]. Meanwhile, taraxasterol was found to have a protective effect on ovalbumin-induced allergic asthma in mice [4]. However, there is no report about the anti-inflammatory effect of taraxasterol on lipopolysaccharide (LPS)-induced lung injury. Acute lung injury (ALI) is characterized by a diffuse inflammatory parenchymal process, often occurring in the context of multisystem organ failure. Its severe form, acute respiratory distress syndrome (ARDS), often results in multi-organ failure with a mortality of ⁎ Corresponding author. Tel./fax: +86 431 87835140. E-mail address: [email protected] (N. Zhang). 1 These two authors contributed equally to this article.
http://dx.doi.org/10.1016/j.intimp.2014.01.031 1567-5769/© 2014 Elsevier B.V. All rights reserved.
approximately 30–50% [5]. LPS is one of the major factors that induce acute lung injury [6]. LPS could activate NF-κB and MAPK pathways and finally results in the release of pro-inflammatory mediators which include TNF-α, IL-1β, IL-6, COX-2, and PGE2 [7–9]. These pro-inflammatory mediators lead to inflammation and various other clinical manifestations [10]. Acute lung injury (ALI) presents high mortality and morbidity clinically, but the specific therapies effective in preventing or reversing the severe pulmonary injury remain inadequate. The aim of this study was to evaluate the anti-inflammatory effects of taraxasterol on LPS-induced acute lung injury in a mouse model, and the results demonstrated that taraxasterol has a protective effect against LPSinduced acute lung injury. 2. Materials and methods 2.1. Animals Male BALB/c mice, weighing approximately 18 to 20 g, were purchased from the Center of Experiment Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were housed in microisolator cages and received food and water ad libitum. The
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laboratory temperature was 24 ± 1 °C, and relative humidity was 40–80%. All animal experiments were performed in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animal. 2.2. Reagents Taraxasterol (purity: N98%) was purchased from Chengdu Preferred Biotechnology Co., Ltd (Chengdu, China). Dexamethasone (DEX, purity: N 99.6%) was purchased from Changle Pharmaceutical Co. (Xinxiang, Henan, China). Mouse TNF-α, IL-6 and IL-1β enzymelinked immunosorbent assay (ELISA) kits were purchased from BioLegend (CA, USA). Mouse monoclonal phospho-specific p38 antibody, mouse monoclonal phospho-specific p42–p44 ERK antibody, mouse monoclonal phospho-specific p46–p54 JNK antibody, mouse mAb Phospho-NF-κB p65, mouse mAb Phospho-IκBα and rabbit mAb IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA). HRP-conjugated goat anti-rabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other chemicals were of reagent grade.
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2.7. Determination of TNF-α, IL-1β and IL-6 levels TNF-α, IL-1β and IL-6 in the BALF were evaluated with the corresponding enzyme-linked immunosorbent assay (ELISA) kits according to the instructions of the manufacturer (BioLegend, Inc., Camino Santa Fe, Suite E, San Diego, CA, USA).
2.8. Pulmonary myeloperoxidase activity in ALI mice MPO activity represents the parenchymal infiltration of neutrophils and macrophages. MPO activity in homogenates of lung tissue was determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions.
2.9. Histopathologic evaluation of the lung tissue Histopathologic examination was performed on mice that were not subjected to BALF collection. Lungs were fixed with 10% buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) stain, pathological changes of lung tissues were observed under a light microscope.
2.3. LPS-induced endotoxemia in mice The 48 healthy male BALB/c mice were randomly classified into four groups and challenged with LPS (20 mg/kg) by i.p. In drug testing, the effect of taraxasterol (2.5, 5, and 10 mg/kg) on LPS-induced mortality was assessed by given taraxasterol 1 h before LPS challenge. Survival in each group was assessed every 12 h for 7 d. 2.4. Experimental design 84 Male BALB/c mice were randomly divided into seven groups: control group, taraxasterol (10 mg/kg) group, LPS group, taraxasterol (2.5, 5, and 10 mg/kg) + LPS group, and dexamethasone (DEX) + LPS group. Before induce acute lung inflammation, taraxasterol (2.5, 5, and 10 mg/kg) dissolved in 50 μl PBS, was given with an intraperitoneal injection (i.p.), while dexamethasone, 0.5 mg/kg, was administrated with an intraperitoneal injection as a positive control. Control and LPS mice were given an equal volume of PBS instead of taraxasterol or DXM. 1 h later, mice were slightly anesthetized with an inhalation of diethyl ether; 10 μg of LPS was instilled intranasally (i.n.) in 50 μl PBS to induce lung injury. Control mice were given a 50 μl PBS i.n. instillation without LPS. To detect the therapeutic effect of taraxasterol on LPS-induced ALI, taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. All the mice were alive after 7 h LPS treatment. Collection of bronchoalveolar lavage fluid (BALF) was performed three times through a tracheal cannula with autoclaved PBS, instilled up to a total volume of 1.3 ml. The chosen doses of these drugs were based on our previous studies and preliminary experiments.
2.10. Western blot analysis Seven hours after the injection of LPS, lung tissues were harvested and frozen in liquid nitrogen immediately until homogenization. Proteins were extracted from the lungs using T-PER Tissue Protein Extraction Reagent Kit according to the manufacturer's instructions. Protein concentrations were determined by BCA protein assay kit. Equal amounts of protein were loaded in each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which subsequently was transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 2 h with 5% skim milk in TBST on the shaker at room temperature and then washed three times for each time 10 min in Tri-Tween buffered saline (TTBS, 20 mM Tris–HCl buffer, pH 7.6, 137 mM NaCl and 0.05% Tween 20). The membrane was placed on primary antibody diluted at a 1:1000 proportion in diluent buffer [5% (w/v)] BSA and 0.1% Tween 20 in TBS] and incubated overnight at 4 °C on the shaker. Then the membrane was washed three times in TBS as above and incubated with secondary antibody diluted at a 1:7000 proportion for 1 h on the shaker at room temperature. The membrane was again washed three times for 10 min each time as above and finally the results were generated by using an enhanced chemiluminescence (ECL) western blotting kit (Fig. 1).
2.5. Lung wet-to-dry weight (W/D) ratio After the mice were euthanized, the lungs were removed and the wet weight was recorded. Lungs were then placed in an incubator at 80 °C for 48 h to obtain the ‘dry’ weight. The ratio of wet lung to dry lung was calculated to assess tissue edema. 2.6. Inflammatory cell counts of BALF The BALF samples were centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for the total cell counts using a hemacytometer. For neutrophil and macrophage counts, cytospins were prepared for differential cell counts by staining with the Wright–Giemsa staining method.
Fig. 1. Chemical structure of taraxasterol.
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added to 100 μl supernatants of BALF, and the solutions were mixed and incubated for 10 min at room temperature. Optical density at 540 nm was measured in a microplate reader. Prostaglandin E2 (PGE2) was determined by commercial EIA using a Prostaglandin E Metabolite Kit (Cayman Chemical, Ann Arbor, MI, USA).
2.12. Statistical analysis All values are expressed as means ± SEM. Differences between mean values of normally distributed data were analyzed using oneway ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted at P b 0.05 or P b 0.01. Fig. 2. Effects of taraxasterol on LPS-induced lethality in mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to LPS challenged. The survival was monitored every 12 h for 7 days. #P b 0.01 vs. control group, ⁎P b 0.05 and ⁎⁎P b 0.01 vs. LPS group.
3. Results 3.1. Effects of taraxasterol on LPS-mediated mortality
2.11. Nitrite and PGE2 assay Extracellular nitrites (NO− 2 ), an indicator of NO synthase activity, were detected by the Griess reaction. Briefly, Griess reagent was
The effect of taraxasterol on LPS-induced mortality was assessed by measuring survival of mice challenged with 20 mg/kg of LPS. As shown in Fig. 2, mice receiving 2.5, 5, and 10 mg/kg carvacrol were 26%, 58% and 78% protective respectively (P b 0.01 or P b 0.05).
Fig. 3. Effects of taraxasterol on histopathological changes in lung tissues in LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. Lungs (n = 4–6) from each experimental group were processed for histological evaluation at 7 h after LPS challenge. Representative histological changes of lung obtained from mice of different groups. A: Control group, B: LPS group, C: LPS + DEX group, D: LPS + taraxasterol (2.5 mg/kg) group, E: LPS + taraxasterol (5 mg/kg) group, F: LPS + taraxasterol (10 mg/kg) group, G: LPS + taraxasterol (2.5 mg/kg) group (1 h later), H: LPS + taraxasterol (5 mg/kg) group (1 h later), I: LPS + taraxasterol (10 mg/kg) group (1 h later) (hematoxylin and eosin staining, magnification 200 ×).
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Some reports had shown that the LD50 value of taraxasterol was 1530 mg/kg i.p. [11]. Thus the doses used in this study were safe. 3.2. Effects of taraxasterol on LPS-mediated lung histopathologic changes To evaluate the effects of taraxasterol on LPS-induced acute lung injury, we first detected the histological changes after taraxasterol treatment. As shown in Fig. 3B, lung showed characteristic histological changes of acute lung injury, including areas of inflammatory infiltration, focal area of fibrosis with collapse of air alveoli and emphysematous, as well as thickening of the alveolar wall and
Fig. 4. Effects of taraxasterol on MPO activity in lung tissues of LPS-induced ALI. (A) Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. MPO activity was determined at 7 h after LPS administration. (B) Taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. MPO activity was determined at 7 h after LPS administration. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
Fig. 5. Effects of taraxasterol on the lung W/D ratio of LPS-induced ALI mice. (A) Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. (B) Taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. The lung W/D ratio was determined at 7 h after LPS challenge. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 vs. LPS group.
Fig. 6. Effects of taraxasterol on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 7 h after LPS administration to measure the number of total cells, neutrophils, and macrophage. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
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Fig. 7. Effects of taraxasterol on the production of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the BALF of LPS-induced ALI mice. (A) Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. (B) Taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. BALF was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1β, and IL-6. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
Fig. 8. Effects of taraxasterol on the production of inflammatory mediators NO, COX-2 and PGE2 levels of LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 7 h following LPS challenge to analyze the inflammatory mediators NO, COX-2 and PGE2 levels. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
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pulmonary congestion. However, LPS-induced pathological changes were significantly attenuated by taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) treatment (Fig. 3C, D, E, F). In addition, LPSinduced pathological changes were also attenuated by taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h (Fig. 3G, H, I). 3.3. Effects of taraxasterol on LPS-induced MPO activity The MPO activity was determined to assess the neutrophil accumulation within pulmonary tissues. As shown in Fig. 4A, the MPO activity of pulmonary tissue increased significantly in the LPS group compared with that of the control group (P b 0.01). This increase in LPS-induced MPO activity was found to be significantly inhibited in the taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) groups (P b 0.05 or P b 0.01). In addition, the inhibition of taraxasterol at the dose of 10 mg/kg was comparable as that of DEX treatment at 5 mg/kg. Meanwhile, LPS-induced MPO activity was also inhibited by taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h (Fig. 4B).
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3.7. Effects of taraxasterol on LPS-induced inflammatory cytokine production in serum To further evaluate anti-inflammatory action by taraxasterol, the production of TNF-α, IL-1β, and IL-6 in serum was analyzed. As shown in Fig. 9, TNF-α, IL-1β and IL-6 levels were found to be significantly increased in the LPS group compared with the normal group. Taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced TNF-α (*P b 0.05), IL-6 (*P b 0.05 or **P b 0.01) and IL-1β (*P b 0.05 or **P b 0.01) production compared to those in the LPS group. 3.8. Effect of taraxasterol on NF-κB and MAPK activation in ALI mice induced by LPS Western blot analysis showed that NF-κB and MAPK signaling pathways were activated 7 h after LPS treatment. Pretreatment with taraxasterol (2.5, 5, and 10 mg/kg) inhibits the phosphorylation of IκB-α, p65 NF-κB, p38, JNK and ERK (Figs. 10 and 11). In all,
3.4. Effects of taraxasterol on LPS-induced lung W/D ratio Seven hours after the intranasal instillation of LPS or normal saline, the lung W/D ratio (Fig. 5A) was evaluated. LPS caused a significant increase in lung W/D ratio (**P b 0.01) compared to the control group. This increase was found to be significantly inhibited in the taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) groups (P b 0.05 or P b 0.01). The inhibition of taraxasterol at the dose of 10 mg/kg was a little weaker than that of DEX treatment at 5 mg/kg. In addition, taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h also inhibited LPS-induced increase in lung W/D ratio (P b 0.05 or P b 0.01) (Fig. 5B). 3.5. Effects of taraxasterol on inflammatory cell count in the BALF of LPS-induced ALI mice Seven hours after administration of LPS, the number of inflammatory cells, such as neutrophils and macrophages, in BALF was analyzed. As shown in Fig. 6, LPS challenge significantly increased the number of total cells, neutrophils and macrophages compared with the control group (P b 0.01). Meanwhile, pretreatment with taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) was found to significantly decrease the number of total cells (P b 0.01), neutrophils (P b 0.01), and macrophages (P b 0.01). 3.6. Effects of taraxasterol on LPS-induced inflammatory mediator production To further evaluate anti-inflammatory action by taraxasterol, the production of TNF-α, IL-1β, IL-6, NO, COX-2 and PGE2 in BALF was analyzed. As shown in Fig. 7A, TNF-α, IL-1β and IL-6 levels were found to be significantly increased in the LPS group compared with the normal group. Taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced TNF-α (*P b 0.05), IL-6 (*P b 0.05 or **P b 0.01) and IL-1β (*P b 0.05 or **P b 0.01) production compared to those in the LPS group. The inhibition of taraxasterol at the dose of 10 mg/kg was weaker than that of DEX treatment at 5 mg/kg. In addition, LPS-induced cytokine production was also suppressed by taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h (*P b 0.05 or **P b 0.01) (Fig. 7B). As shown in Fig. 8, NO, COX-2 and PGE2 levels were found to be significantly increased in the LPS group compared with the normal group. Taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced NO, COX-2 and PGE2 production compared to those in the LPS group.
Fig. 9. Effects of taraxasterol on the production of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum of LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. Serum was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1β, and IL-6. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's ttest. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
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Fig. 10. Taraxasterol pretreatment inhibited LPS-induced activation of NF-κB with western blotting. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 group vs. LPS group.
Fig. 11. Taraxasterol pretreatment inhibited LPS-induced activation of p38, ERK and JNK with western blotting. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 group vs. LPS group.
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these results showed that taraxasterol (2.5, 5, and 10 mg/kg) could simultaneously inhibit NF-κB and MAPK signaling pathways efficiently in mouse model of ALI. 4. Discussion LPS, the outer membrane of Gram-negative bacteria, has been referred to be an important risk factor of acute lung injury (ALI) [11,12]. Intratracheal administration of LPS has gained wide acceptance as a clinically relevant model of severe lung injury [13,14]. Taraxasterol, a pentacyclic-triterpene isolated from T. officinale, has been reported to have anti-inflammatory effects. In the present study, we found that taraxasterol exerted potent anti-inflammatory effects on LPS-induced ALI in mice. Taraxasterol attenuated lung damage, decreased the W/D ratio, pro-inflammatory cytokine production, inflammatory cell migration into the lung, and the activation of NF-κB and MAPK, induced by LPS. MPO activity stands for the number of neutrophils according to a suitable proportion [15]. MPO activity increase reflects neutrophil accumulation in the lung tissues [16]. LPS-induced ALI is characterized by the infiltration of neutrophils in the lung, exhibiting increased MPO activity. MPO activity, an important index of tissue damage, was used to evaluate the protective effects of taraxasterol on LPS-induced ALI [17]. In this study, we found that LPS administration significantly increased the MPO activity and pretreatment with taraxasterol decreased LPS-induced increases in MPO activity in the lungs. Pulmonary edema is one of the major characteristics of ALI [18]. In this study, we evaluated the W/D ratio of the lung to quantify the magnitude of pulmonary edema. Our experiments showed that taraxasterol significantly inhibits edema of the lung. In addition, the histological examination also indicated that taraxasterol had a protective effect on LPS-induce ALI. TNF-α, IL-1β and IL-6 are characterized cytokines involved in the development of acute lung injury [19–21]. Increased of these cytokines have been noted in LPS-induced ALI model [22]. These cytokines amplify the inflammatory response and inflammatory injury. Elevation of these cytokines in humans with ALI has been associated with severe outcome [23]. In the present study, taraxasterol significantly inhibited the production of TNF-α, IL-1β and IL-6 induced by LPS. These results indicate that the protective effects of taraxasterol on ALI may be due to its ability to inhibit inflammatory cytokines. It has been known that the expressions of pro-inflammatory cytokines are modulated by NF-κB and MAPK pathways [24,25]. NF-κB plays a critical role in regulating inflammatory and immune responses to extracellular stimulus. Under normal conditions, NF-κB is present in its inactive cytoplasmic form bound to the repressor of NF-κB (IκBs). Once stimulated by LPS, the degradation and phosphorylation of IκBα will increase, which results in the release of free NF-κB p65 and translocation from the cytoplasm to the nucleus, leading to the transcription of specific target genes, such as TNF-α, IL-1β and IL-6 [26–29]. MAPKs also play an important role in inducing cytokine production [30,31]. The LPS stimulation of murine macrophages has been known to induce phosphorylation and activation of ERK1/2, JNK, and p38 MAPKs [32]. To further illuminate the molecular mechanisms of taraxasterol on LPS-induced ALI, we investigated whether the anti-inflammatory activity of taraxasterol exerted through NF-κB and MAPK signaling pathway. The results suggested that taraxasterol suppressed LPS-induced pro-inflammatory cytokine production by preventing NF-κB and MAPK activation. In our previous studies, we also detected the effects of other compounds on LPS-induced acute lung injury. Based on the results of this study, we found that the anti-inflammatory effect of taraxasterol was better than gossypol in ameliorating inflammatory reaction following ALI. In addition, our results also showed that taraxasterol administered after LPS stimulation for 1 h also inhibited LPS-induced inflammatory response. Though the anti-inflammatory effect of
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taraxasterol is weaker than DEX, it may also be considered as a potential agent in prevention and treatment of acute lung injury. In conclusion, the present study demonstrated that taraxasterol has a protective effect against LPS-induced ALI, which may be related to its suppression of NF-κB and MAPK activation, and subsequently leads to the reduction of pro-inflammatory cytokine expression in lung tissues. It may be considered as a potential agent in prevention and treatment of acute lung injury. Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 31372494). References [1] Ahmad VU, Yasmeen S, Ali Z, Khan MA, Choudhary MI, Akhtar F, et al. Taraxacin, a new guaianolide from Taraxacum wallichii. J Nat Prod 2000;63:1010–1. [2] Unlu S, Onkol T, Dundar Y, Okcelik B, Kupeli E, Yesilada E, et al. 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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effect of taraxasterol on acute lung injury induced by lipopolysaccharide in mice San Zhihao a,1, Fu Yunhe a,1, Li Wei b, Zhou Ershun a, Li Yimeng a, Song Xiaojing a, Wang Tiancheng a, Tian Yuan a, Wei Zhengkai a, Yao Minjun a, Cao Yongguo a, Zhang Naisheng a,⁎ a b
Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Jilin University, Changchun, Jilin Province 130062, People's Republic of China College of Chinese Medicinal Materials, Jilin Agricultural University, Changchun, Jilin Province 130118, People's Republic of China
a r t i c l e
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Article history: Received 19 November 2013 Received in revised form 11 January 2014 Accepted 28 January 2014 Available online 15 February 2014 Keywords: Taraxasterol Acute lung injury Myeloperoxidase Nuclear factor-kappaB (NF-κB)
a b s t r a c t Taraxasterol, a pentacyclic-triterpene isolated from Taraxacum officinale, has been reported to have potent antiinflammatory properties. However, the effect of taraxasterol on lipopolysaccharide (LPS)-induced mice acute lung injury has not been investigated. The aims of this study were to investigate whether taraxasterol could ameliorate the inflammation response in LPS-induced acute lung injury and to clarify the possible mechanism. Male BALB/c mice were pretreated with taraxasterol 1 h before intranasal instillation of LPS. 7 h after LPS administration, the myeloperoxidase (MPO) in lung tissues, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (BALF) were detected. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) in the BALF were measured by ELISA. The extent of phosphorylation of IκB-α, p65 NFκB, p46–p54 JNK, p42–p44 ERK, and p38 were determined by western blotting. The results showed that taraxasterol attenuated the infiltration of inflammatory cells, the activity of myeloperoxidase (MPO), lung wet/ dry ratio, and the expression of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL1β) in a dose-dependent manner. Additionally, western blotting results showed that taraxasterol inhibited the phosphorylation of IκB-α, p65 NF-κB, p46–p54 JNK, p42–p44 ERK, and p38 caused by LPS. Our data suggest that anti-inflammatory effects of taraxasterol against the LPS-induced ALI may be due to its ability of inhibition of the NF-κB and MAPK signaling pathways. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Taraxacum officinale is a traditional Chinese herb which has long been used for the treatment of various inflammatory or infectious diseases such as upper respiratory tract infections, pneumonia and mastitis [1]. Taraxasterol, a pentacyclic-triterpene isolated from T. officinale, is responsible for the plant's pharmacological activities. Recently, taraxasterol was found to have anti-inflammatory effects [2]. Studies showed that taraxasterol could inhibit inflammatory cytokines such as TNF-α and IL-1β in LPS-stimulated RAW264.7 macrophages [3]. Meanwhile, taraxasterol was found to have a protective effect on ovalbumin-induced allergic asthma in mice [4]. However, there is no report about the anti-inflammatory effect of taraxasterol on lipopolysaccharide (LPS)-induced lung injury. Acute lung injury (ALI) is characterized by a diffuse inflammatory parenchymal process, often occurring in the context of multisystem organ failure. Its severe form, acute respiratory distress syndrome (ARDS), often results in multi-organ failure with a mortality of ⁎ Corresponding author. Tel./fax: +86 431 87835140. E-mail address: [email protected] (N. Zhang). 1 These two authors contributed equally to this article.
http://dx.doi.org/10.1016/j.intimp.2014.01.031 1567-5769/© 2014 Elsevier B.V. All rights reserved.
approximately 30–50% [5]. LPS is one of the major factors that induce acute lung injury [6]. LPS could activate NF-κB and MAPK pathways and finally results in the release of pro-inflammatory mediators which include TNF-α, IL-1β, IL-6, COX-2, and PGE2 [7–9]. These pro-inflammatory mediators lead to inflammation and various other clinical manifestations [10]. Acute lung injury (ALI) presents high mortality and morbidity clinically, but the specific therapies effective in preventing or reversing the severe pulmonary injury remain inadequate. The aim of this study was to evaluate the anti-inflammatory effects of taraxasterol on LPS-induced acute lung injury in a mouse model, and the results demonstrated that taraxasterol has a protective effect against LPSinduced acute lung injury. 2. Materials and methods 2.1. Animals Male BALB/c mice, weighing approximately 18 to 20 g, were purchased from the Center of Experiment Animals of Baiqiuen Medical College of Jilin University (Jilin, China). The mice were housed in microisolator cages and received food and water ad libitum. The
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laboratory temperature was 24 ± 1 °C, and relative humidity was 40–80%. All animal experiments were performed in accordance with the National Institutes of Health guide for the Care and Use of Laboratory Animal. 2.2. Reagents Taraxasterol (purity: N98%) was purchased from Chengdu Preferred Biotechnology Co., Ltd (Chengdu, China). Dexamethasone (DEX, purity: N 99.6%) was purchased from Changle Pharmaceutical Co. (Xinxiang, Henan, China). Mouse TNF-α, IL-6 and IL-1β enzymelinked immunosorbent assay (ELISA) kits were purchased from BioLegend (CA, USA). Mouse monoclonal phospho-specific p38 antibody, mouse monoclonal phospho-specific p42–p44 ERK antibody, mouse monoclonal phospho-specific p46–p54 JNK antibody, mouse mAb Phospho-NF-κB p65, mouse mAb Phospho-IκBα and rabbit mAb IκBα were purchased from Cell Signaling Technology Inc. (Beverly, MA). HRP-conjugated goat anti-rabbit and goat-mouse antibodies were provided by GE Healthcare (Buckinghamshire, UK). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other chemicals were of reagent grade.
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2.7. Determination of TNF-α, IL-1β and IL-6 levels TNF-α, IL-1β and IL-6 in the BALF were evaluated with the corresponding enzyme-linked immunosorbent assay (ELISA) kits according to the instructions of the manufacturer (BioLegend, Inc., Camino Santa Fe, Suite E, San Diego, CA, USA).
2.8. Pulmonary myeloperoxidase activity in ALI mice MPO activity represents the parenchymal infiltration of neutrophils and macrophages. MPO activity in homogenates of lung tissue was determined using test kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the instructions.
2.9. Histopathologic evaluation of the lung tissue Histopathologic examination was performed on mice that were not subjected to BALF collection. Lungs were fixed with 10% buffered formalin, imbedded in paraffin and sliced. After hematoxylin and eosin (H&E) stain, pathological changes of lung tissues were observed under a light microscope.
2.3. LPS-induced endotoxemia in mice The 48 healthy male BALB/c mice were randomly classified into four groups and challenged with LPS (20 mg/kg) by i.p. In drug testing, the effect of taraxasterol (2.5, 5, and 10 mg/kg) on LPS-induced mortality was assessed by given taraxasterol 1 h before LPS challenge. Survival in each group was assessed every 12 h for 7 d. 2.4. Experimental design 84 Male BALB/c mice were randomly divided into seven groups: control group, taraxasterol (10 mg/kg) group, LPS group, taraxasterol (2.5, 5, and 10 mg/kg) + LPS group, and dexamethasone (DEX) + LPS group. Before induce acute lung inflammation, taraxasterol (2.5, 5, and 10 mg/kg) dissolved in 50 μl PBS, was given with an intraperitoneal injection (i.p.), while dexamethasone, 0.5 mg/kg, was administrated with an intraperitoneal injection as a positive control. Control and LPS mice were given an equal volume of PBS instead of taraxasterol or DXM. 1 h later, mice were slightly anesthetized with an inhalation of diethyl ether; 10 μg of LPS was instilled intranasally (i.n.) in 50 μl PBS to induce lung injury. Control mice were given a 50 μl PBS i.n. instillation without LPS. To detect the therapeutic effect of taraxasterol on LPS-induced ALI, taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. All the mice were alive after 7 h LPS treatment. Collection of bronchoalveolar lavage fluid (BALF) was performed three times through a tracheal cannula with autoclaved PBS, instilled up to a total volume of 1.3 ml. The chosen doses of these drugs were based on our previous studies and preliminary experiments.
2.10. Western blot analysis Seven hours after the injection of LPS, lung tissues were harvested and frozen in liquid nitrogen immediately until homogenization. Proteins were extracted from the lungs using T-PER Tissue Protein Extraction Reagent Kit according to the manufacturer's instructions. Protein concentrations were determined by BCA protein assay kit. Equal amounts of protein were loaded in each well and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), which subsequently was transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked for 2 h with 5% skim milk in TBST on the shaker at room temperature and then washed three times for each time 10 min in Tri-Tween buffered saline (TTBS, 20 mM Tris–HCl buffer, pH 7.6, 137 mM NaCl and 0.05% Tween 20). The membrane was placed on primary antibody diluted at a 1:1000 proportion in diluent buffer [5% (w/v)] BSA and 0.1% Tween 20 in TBS] and incubated overnight at 4 °C on the shaker. Then the membrane was washed three times in TBS as above and incubated with secondary antibody diluted at a 1:7000 proportion for 1 h on the shaker at room temperature. The membrane was again washed three times for 10 min each time as above and finally the results were generated by using an enhanced chemiluminescence (ECL) western blotting kit (Fig. 1).
2.5. Lung wet-to-dry weight (W/D) ratio After the mice were euthanized, the lungs were removed and the wet weight was recorded. Lungs were then placed in an incubator at 80 °C for 48 h to obtain the ‘dry’ weight. The ratio of wet lung to dry lung was calculated to assess tissue edema. 2.6. Inflammatory cell counts of BALF The BALF samples were centrifuged (4 °C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for the total cell counts using a hemacytometer. For neutrophil and macrophage counts, cytospins were prepared for differential cell counts by staining with the Wright–Giemsa staining method.
Fig. 1. Chemical structure of taraxasterol.
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added to 100 μl supernatants of BALF, and the solutions were mixed and incubated for 10 min at room temperature. Optical density at 540 nm was measured in a microplate reader. Prostaglandin E2 (PGE2) was determined by commercial EIA using a Prostaglandin E Metabolite Kit (Cayman Chemical, Ann Arbor, MI, USA).
2.12. Statistical analysis All values are expressed as means ± SEM. Differences between mean values of normally distributed data were analyzed using oneway ANOVA (Dunnett's t-test) and two-tailed Student's t-test. Statistical significance was accepted at P b 0.05 or P b 0.01. Fig. 2. Effects of taraxasterol on LPS-induced lethality in mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to LPS challenged. The survival was monitored every 12 h for 7 days. #P b 0.01 vs. control group, ⁎P b 0.05 and ⁎⁎P b 0.01 vs. LPS group.
3. Results 3.1. Effects of taraxasterol on LPS-mediated mortality
2.11. Nitrite and PGE2 assay Extracellular nitrites (NO− 2 ), an indicator of NO synthase activity, were detected by the Griess reaction. Briefly, Griess reagent was
The effect of taraxasterol on LPS-induced mortality was assessed by measuring survival of mice challenged with 20 mg/kg of LPS. As shown in Fig. 2, mice receiving 2.5, 5, and 10 mg/kg carvacrol were 26%, 58% and 78% protective respectively (P b 0.01 or P b 0.05).
Fig. 3. Effects of taraxasterol on histopathological changes in lung tissues in LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. Lungs (n = 4–6) from each experimental group were processed for histological evaluation at 7 h after LPS challenge. Representative histological changes of lung obtained from mice of different groups. A: Control group, B: LPS group, C: LPS + DEX group, D: LPS + taraxasterol (2.5 mg/kg) group, E: LPS + taraxasterol (5 mg/kg) group, F: LPS + taraxasterol (10 mg/kg) group, G: LPS + taraxasterol (2.5 mg/kg) group (1 h later), H: LPS + taraxasterol (5 mg/kg) group (1 h later), I: LPS + taraxasterol (10 mg/kg) group (1 h later) (hematoxylin and eosin staining, magnification 200 ×).
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Some reports had shown that the LD50 value of taraxasterol was 1530 mg/kg i.p. [11]. Thus the doses used in this study were safe. 3.2. Effects of taraxasterol on LPS-mediated lung histopathologic changes To evaluate the effects of taraxasterol on LPS-induced acute lung injury, we first detected the histological changes after taraxasterol treatment. As shown in Fig. 3B, lung showed characteristic histological changes of acute lung injury, including areas of inflammatory infiltration, focal area of fibrosis with collapse of air alveoli and emphysematous, as well as thickening of the alveolar wall and
Fig. 4. Effects of taraxasterol on MPO activity in lung tissues of LPS-induced ALI. (A) Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. MPO activity was determined at 7 h after LPS administration. (B) Taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. MPO activity was determined at 7 h after LPS administration. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
Fig. 5. Effects of taraxasterol on the lung W/D ratio of LPS-induced ALI mice. (A) Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. (B) Taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. The lung W/D ratio was determined at 7 h after LPS challenge. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 vs. LPS group.
Fig. 6. Effects of taraxasterol on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 7 h after LPS administration to measure the number of total cells, neutrophils, and macrophage. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
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Fig. 7. Effects of taraxasterol on the production of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the BALF of LPS-induced ALI mice. (A) Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. (B) Taraxasterol (2.5, 5, and 10 mg/kg) was administered after LPS stimulation for 1 h. BALF was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1β, and IL-6. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
Fig. 8. Effects of taraxasterol on the production of inflammatory mediators NO, COX-2 and PGE2 levels of LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. BALF was collected at 7 h following LPS challenge to analyze the inflammatory mediators NO, COX-2 and PGE2 levels. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
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pulmonary congestion. However, LPS-induced pathological changes were significantly attenuated by taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) treatment (Fig. 3C, D, E, F). In addition, LPSinduced pathological changes were also attenuated by taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h (Fig. 3G, H, I). 3.3. Effects of taraxasterol on LPS-induced MPO activity The MPO activity was determined to assess the neutrophil accumulation within pulmonary tissues. As shown in Fig. 4A, the MPO activity of pulmonary tissue increased significantly in the LPS group compared with that of the control group (P b 0.01). This increase in LPS-induced MPO activity was found to be significantly inhibited in the taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) groups (P b 0.05 or P b 0.01). In addition, the inhibition of taraxasterol at the dose of 10 mg/kg was comparable as that of DEX treatment at 5 mg/kg. Meanwhile, LPS-induced MPO activity was also inhibited by taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h (Fig. 4B).
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3.7. Effects of taraxasterol on LPS-induced inflammatory cytokine production in serum To further evaluate anti-inflammatory action by taraxasterol, the production of TNF-α, IL-1β, and IL-6 in serum was analyzed. As shown in Fig. 9, TNF-α, IL-1β and IL-6 levels were found to be significantly increased in the LPS group compared with the normal group. Taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced TNF-α (*P b 0.05), IL-6 (*P b 0.05 or **P b 0.01) and IL-1β (*P b 0.05 or **P b 0.01) production compared to those in the LPS group. 3.8. Effect of taraxasterol on NF-κB and MAPK activation in ALI mice induced by LPS Western blot analysis showed that NF-κB and MAPK signaling pathways were activated 7 h after LPS treatment. Pretreatment with taraxasterol (2.5, 5, and 10 mg/kg) inhibits the phosphorylation of IκB-α, p65 NF-κB, p38, JNK and ERK (Figs. 10 and 11). In all,
3.4. Effects of taraxasterol on LPS-induced lung W/D ratio Seven hours after the intranasal instillation of LPS or normal saline, the lung W/D ratio (Fig. 5A) was evaluated. LPS caused a significant increase in lung W/D ratio (**P b 0.01) compared to the control group. This increase was found to be significantly inhibited in the taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) groups (P b 0.05 or P b 0.01). The inhibition of taraxasterol at the dose of 10 mg/kg was a little weaker than that of DEX treatment at 5 mg/kg. In addition, taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h also inhibited LPS-induced increase in lung W/D ratio (P b 0.05 or P b 0.01) (Fig. 5B). 3.5. Effects of taraxasterol on inflammatory cell count in the BALF of LPS-induced ALI mice Seven hours after administration of LPS, the number of inflammatory cells, such as neutrophils and macrophages, in BALF was analyzed. As shown in Fig. 6, LPS challenge significantly increased the number of total cells, neutrophils and macrophages compared with the control group (P b 0.01). Meanwhile, pretreatment with taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) was found to significantly decrease the number of total cells (P b 0.01), neutrophils (P b 0.01), and macrophages (P b 0.01). 3.6. Effects of taraxasterol on LPS-induced inflammatory mediator production To further evaluate anti-inflammatory action by taraxasterol, the production of TNF-α, IL-1β, IL-6, NO, COX-2 and PGE2 in BALF was analyzed. As shown in Fig. 7A, TNF-α, IL-1β and IL-6 levels were found to be significantly increased in the LPS group compared with the normal group. Taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced TNF-α (*P b 0.05), IL-6 (*P b 0.05 or **P b 0.01) and IL-1β (*P b 0.05 or **P b 0.01) production compared to those in the LPS group. The inhibition of taraxasterol at the dose of 10 mg/kg was weaker than that of DEX treatment at 5 mg/kg. In addition, LPS-induced cytokine production was also suppressed by taraxasterol (2.5, 5, and 10 mg/kg) administered after LPS stimulation for 1 h (*P b 0.05 or **P b 0.01) (Fig. 7B). As shown in Fig. 8, NO, COX-2 and PGE2 levels were found to be significantly increased in the LPS group compared with the normal group. Taraxasterol (2.5, 5, and 10 mg/kg) and DEX (5 mg/kg) significantly reduced NO, COX-2 and PGE2 production compared to those in the LPS group.
Fig. 9. Effects of taraxasterol on the production of inflammatory cytokines TNF-α, IL-1β, and IL-6 in the serum of LPS-induced ALI mice. Mice were given an intraperitoneal injection of taraxasterol (2.5, 5, and 10 mg/kg) 1 h prior to an i.n. administration of LPS. Serum was collected at 7 h following LPS challenge to analyze the inflammatory cytokines TNF-α, IL-1β, and IL-6. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's ttest. #P b 0.01 vs. control group, *P b 0.05, **P b 0.01 vs. LPS group.
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Fig. 10. Taraxasterol pretreatment inhibited LPS-induced activation of NF-κB with western blotting. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 group vs. LPS group.
Fig. 11. Taraxasterol pretreatment inhibited LPS-induced activation of p38, ERK and JNK with western blotting. The values presented are the means ± SEM of three independent experiments and differences between mean values were assessed by Student's t-test. #P b 0.01 vs. control group, *P b 0.05 and **P b 0.01 group vs. LPS group.
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these results showed that taraxasterol (2.5, 5, and 10 mg/kg) could simultaneously inhibit NF-κB and MAPK signaling pathways efficiently in mouse model of ALI. 4. Discussion LPS, the outer membrane of Gram-negative bacteria, has been referred to be an important risk factor of acute lung injury (ALI) [11,12]. Intratracheal administration of LPS has gained wide acceptance as a clinically relevant model of severe lung injury [13,14]. Taraxasterol, a pentacyclic-triterpene isolated from T. officinale, has been reported to have anti-inflammatory effects. In the present study, we found that taraxasterol exerted potent anti-inflammatory effects on LPS-induced ALI in mice. Taraxasterol attenuated lung damage, decreased the W/D ratio, pro-inflammatory cytokine production, inflammatory cell migration into the lung, and the activation of NF-κB and MAPK, induced by LPS. MPO activity stands for the number of neutrophils according to a suitable proportion [15]. MPO activity increase reflects neutrophil accumulation in the lung tissues [16]. LPS-induced ALI is characterized by the infiltration of neutrophils in the lung, exhibiting increased MPO activity. MPO activity, an important index of tissue damage, was used to evaluate the protective effects of taraxasterol on LPS-induced ALI [17]. In this study, we found that LPS administration significantly increased the MPO activity and pretreatment with taraxasterol decreased LPS-induced increases in MPO activity in the lungs. Pulmonary edema is one of the major characteristics of ALI [18]. In this study, we evaluated the W/D ratio of the lung to quantify the magnitude of pulmonary edema. Our experiments showed that taraxasterol significantly inhibits edema of the lung. In addition, the histological examination also indicated that taraxasterol had a protective effect on LPS-induce ALI. TNF-α, IL-1β and IL-6 are characterized cytokines involved in the development of acute lung injury [19–21]. Increased of these cytokines have been noted in LPS-induced ALI model [22]. These cytokines amplify the inflammatory response and inflammatory injury. Elevation of these cytokines in humans with ALI has been associated with severe outcome [23]. In the present study, taraxasterol significantly inhibited the production of TNF-α, IL-1β and IL-6 induced by LPS. These results indicate that the protective effects of taraxasterol on ALI may be due to its ability to inhibit inflammatory cytokines. It has been known that the expressions of pro-inflammatory cytokines are modulated by NF-κB and MAPK pathways [24,25]. NF-κB plays a critical role in regulating inflammatory and immune responses to extracellular stimulus. Under normal conditions, NF-κB is present in its inactive cytoplasmic form bound to the repressor of NF-κB (IκBs). Once stimulated by LPS, the degradation and phosphorylation of IκBα will increase, which results in the release of free NF-κB p65 and translocation from the cytoplasm to the nucleus, leading to the transcription of specific target genes, such as TNF-α, IL-1β and IL-6 [26–29]. MAPKs also play an important role in inducing cytokine production [30,31]. The LPS stimulation of murine macrophages has been known to induce phosphorylation and activation of ERK1/2, JNK, and p38 MAPKs [32]. To further illuminate the molecular mechanisms of taraxasterol on LPS-induced ALI, we investigated whether the anti-inflammatory activity of taraxasterol exerted through NF-κB and MAPK signaling pathway. The results suggested that taraxasterol suppressed LPS-induced pro-inflammatory cytokine production by preventing NF-κB and MAPK activation. In our previous studies, we also detected the effects of other compounds on LPS-induced acute lung injury. Based on the results of this study, we found that the anti-inflammatory effect of taraxasterol was better than gossypol in ameliorating inflammatory reaction following ALI. In addition, our results also showed that taraxasterol administered after LPS stimulation for 1 h also inhibited LPS-induced inflammatory response. Though the anti-inflammatory effect of
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taraxasterol is weaker than DEX, it may also be considered as a potential agent in prevention and treatment of acute lung injury. In conclusion, the present study demonstrated that taraxasterol has a protective effect against LPS-induced ALI, which may be related to its suppression of NF-κB and MAPK activation, and subsequently leads to the reduction of pro-inflammatory cytokine expression in lung tissues. It may be considered as a potential agent in prevention and treatment of acute lung injury. Acknowledgment This work was supported by a grant from the National Natural Science Foundation of China (No. 31372494). References [1] Ahmad VU, Yasmeen S, Ali Z, Khan MA, Choudhary MI, Akhtar F, et al. Taraxacin, a new guaianolide from Taraxacum wallichii. J Nat Prod 2000;63:1010–1. [2] Unlu S, Onkol T, Dundar Y, Okcelik B, Kupeli E, Yesilada E, et al. 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