- Email: [email protected]
Protective effects of Isofraxidin against lipopolysaccharide-induced acute lung injury in mice
International Immunopharmacology 24 (2015) 432–439
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of Isofraxidin against lipopolysaccharide-induced acute lung injury in mice Xiaofeng Niu, Yu Wang, Weifeng Li ⁎, Qingli Mu, Huani Li, Huan Yao, Hailin Zhang School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, PR China
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 27 November 2014 Accepted 5 December 2014 Available online 13 January 2015 Keywords: Isofraxidin Acute lung injury Inflammatory cytokines Cyclooxygenase-2 Prostaglandin E2
a b s t r a c t Acute lung injury (ALI) is a life-threatening disease characterized by serious lung inflammation and increased capillary permeability, which presents a high mortality worldwide. Isofraxidin (IF), a Coumarin compound isolated from the natural medicinal plants such as Sarcandra glabra and Acanthopanax senticosus, has been reported to have definite anti-bacterial, anti-oxidant, and anti-inflammatory activities. However, the effects of IF against lipopolysaccharide-induced ALI have not been clarified. The aim of the present study is to explore the protective effects and potential mechanism of IF against LPS-induced ALI in mice. In this study, We found that pretreatment with IF significantly lowered LPS-induced mortality and lung wet-to-dry weight (W/D) ratio and reduced the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in serum and bronchoalveolar lavage fluid (BALF). We also found that total cells, neutrophils and macrophages in BALF, MPO activity in lung tissues were markedly decreased. Besides, IF obviously inhibited lung histopathological changes and cyclooxygenase-2 (COX-2) protein expression. These results suggest that IF has a protective effect against LPSinduced ALI, and the protective effect of IF seems to result from the inhibition of COX-2 protein expression in the lung, which regulates the production of PGE2. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inflammation is a self-defensive immune process in which the body reacts to external stimuli, infection, or injury [1]. The development of inflammation is associated with the production of inflammatory cytokines by neutrophils and macrophages, tissue edema caused by the leakage of fluid and proteins, and the infiltration of leucocytes at the site of inflammation [2,3]. Acute lung injury (ALI), a common inflammatory disease in the clinic, shows the characteristics of interstitial edema, neutrophil recruitment, disruption of epithelial integrity, and lung parenchymal injury [4]. Despite significant advances in clinical research and therapeutic trials made in the past several decades, ALI still presents a high mortality in the diseases of shock, sepsis, ischemia reperfusion and viral pneumonia [5]. Therefore, it is extremely urgent to discover effective drugs and clinical therapies for ALI. Lipopolysaccharide (LPS) is widely used for induction of animal models of ALI for its similar characteristics of human ALI [6]. ⁎ Corresponding author at: School of Pharmacy, Xi'an Jiaotong University, No. 76 Western Yanta Road, Xi'an City, Shaanxi Province 710061, PR China. Tel.: + 86 29 82655139; fax: +86 29 82655138. E-mail addresses: [email protected] (X. Niu), [email protected] (W. Li).
http://dx.doi.org/10.1016/j.intimp.2014.12.041 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Ingestion of LPS stimulates vascular permeability, promotes inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from blood into lung tissues and activates numerous inflammatory cells such as neutrophils and macrophages [7]. In macrophages, LPS challenge induces the transcription of gene encoding pro-inflammatory protein, which leads to cytokine release and synthesis of enzymes, such as cyclooxygenase-2 (COX2) [8]. COX-2 usually can't be found in normal tissues, but widely induced by pro-inflammatory stimuli, such as cytokines, endotoxins, and growth factors [9]. COX-2 plays a vital role in the regulation of inflammatory process by modulating the production of prostaglandin E2 (PGE2). PGE2, induced by cytokines and other initiator, is an inflammatory mediator which is produced in the regulation of COX-2. Previous researches demonstrated that inhibition of COX-2 produced a dramatically anti-inflammatory effect with little gastrointestinal toxicity [10]. Therefore, inhibition of COX-2 protein expression has far-reaching significance in the treatment of ALI. Isofraxidin (IF, 7-hydroxy-6,8-dimethoxycoumarin, shown in Fig. 1) is a Coumarin compound that widely exists in natural plants such as Sarcandra glabra and Acanthopanax senticosus, both of which are traditional Chinese herbs usually used for anti-tumor, anti-bacterial, anti-oxidant, and anti-inflammatory treatments [11]. IF is a bioactive component with definite anti-bacterial and
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
433
performed through the left lungs. The superior lobe of the right lung was removed for histopathological analysis and immunohistochemical study. The middle lobe of the right lung was removed for the evaluation of lung wet-to-dry weight (W/D) ratio. The lower lobe of the right lung was removed and rapidly cut into two parts. One part of the lower lobe was used for MPO analysis, and the other part was used for Western blot analysis. 2.4. LPS-induced mortality in ALI mice Fig. 1. Chemical structure of Isofraxidin.
anti-oxidant activities [12]. The previous studies have also shown that IF possesses anti-inflammatory activity [13,14]. However, there is no report about the protective effects of IF on LPS-induced ALI. The purpose of the present study is to explore the protective effects of IF against LPS-induced ALI in mice.
The normal control group and the negative control group were given an equal volume of vehicle according to experimental design. All other groups received intraperitoneal injection of IF (5, 10, 15 mg/kg) or DEX (5 mg/kg). An hour later, mice received intraperitoneal injection of LPS (20 mg/kg). Within 72 h after LPS injection, the mortality of mice was observed every 12 h in each group (n = 12/group).
2. Materials and methods
2.5. Lung W/D ratio
2.1. Reagents
After the mice were euthanized by cervical dislocation, the middle lobe of the right lungs were excised and weighed to record the “wet” weight. Then, the lung tissues were arranged in an oven at 80 °C for 48 h to obtain the stable “dry” weight. The lung W/D ratios were computed to assess the severity of pulmonary edema.
IF (purity ≥ 98%) was purchased from Xi'an Honson Biotechnology Co., Ltd. (Xi'an, China) and confirmed by the Pharmacognosy Laboratory, School of Medicine, Xi'an Jiaotong University (Xi'an, China). Dexamethasone (DEX), as a positive control, was supplied by Xi'an Lijun Pharmaceutical Company Limited (Shanxi, China). LPS (Escherichia coli serotype O55:B5) was obtained from Sigma (St. Louis, MO). The enzyme linked immunosorbent assay (ELISA) kit for mouse TNF-α, IL-6, and PGE 2 was purchased from R&D Systems (Minneapolis, MN, USA). The kit for biochemical analysis of myeloperoxidase (MPO) was provided by Jiancheng Bioengineering Institute (Nanjing, China). Histostain-Plus kits, DAB (3, 3′-diaminobenzidine) staining kit and rabbit anti-cyclooxygenase-2 were supplied by Beijing Biosynthesis Biotechnology Co., Ltd. RPMI-1640 was purchased from Gibco (Gibco-BRL, Gaithersburg, MD, USA). Antibody against COX-2 (UniProt: P35354) were supplied by Epitomics Inc. (an Abcam Company, U.S.A.). Antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyvinylidene fluoride (PVDF) membranes were provided by Pall Gelman Laboratory (Ann Arbor, MI, USA). All other reagents used in the study were of analytical grade.
2.6. Collection of serum and BALF Six hours after LPS administration, blood samples were collected from the retro-orbital plexus and immediately centrifuged at 3000 rpm for 10 min at 4 °C to obtained the serum. The left lung was lavaged through a tracheal cannula with 1.5 mL autoclaved phosphate buffered saline (PBS) three times in each group. The recovery rate of BALF was better than 90%. BALF was immediately centrifuged at 1500 rpm for 10 min for pelleting the cells. 2.7. Measurement of the levels of TNF-α, IL-6, and PGE2 in serum and BALF The BALF was centrifuged at 1500 rpm for 10 min at 4 °C, and the supernatant of BALF was used for determining the concentrations of TNF-α, IL-6, and PGE2. The levels of TNF-α, IL-6, and PGE2 in serum and BALF were evaluated by mouse ELISA kits according to the manufacturer's directions. The absorbance was read at 450 nm and the samples were detected three times.
2.2. Animals 2.8. Inflammatory cell counts of BALF All male Kunming mice (22–25 g) were supplied by the Experimental Animal Center, Xi'an Jiaotong University (Xi'an, China). Animals were raised under standard conditions with a 12 h day/night cycle and acclimatized to their environment for at least one week before the beginning of animal experiments. All animal experiments were performed in line with the National Institute of Health guidelines. 2.3. Mouse model of LPS-induced ALI The mice were divided into six groups randomly (n = 12/group): Control group; LPS group (LPS, 5 mg/kg, i.p.); LPS + DEX group (5 mg/kg, i.p.); LPS + IF groups (5, 10, 15 mg/kg, i.p.). The doses and administration route of IF and DEX were confirmed based on preliminary tests. IF or DEX were administrated intraperitoneally 1 h prior to LPS challenge. The control and LPS groups were given an equal volume of vehicle according to experimental design. The severity of LPS-induced pulmonary injury was assessed at 6 h after LPS administration. Blood samples were collected from the retro-orbital plexus, and then all mice were euthanized. Bronchoalveolar lavage fluid (BALF) was
The BALF were centrifuged at 1500 rpm for 10 min at 4 °C to pellet the cells and the cell pellets were resuspended in PBS. A hemacytometer was used for the total cell counts and cytospins were essential for different cell counts via staining with the Wright–Giemsa staining method. 2.9. MPO activity in the lung tissues MPO activity is an important index of neutrophils accumulation in inflammatory tissues. After the lungs were removed, one part of the lower lobe were homogenized in PBS by a homogenizer. The mixtures were centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatants were used for MPO analysis. MPO activity were measured with an MPO activity kit based on the manufacturer's instructions. MPO activities were expressed as units per gram of protein. 2.10. Histopathological study of the lung tissues LPS-induced ALI mice possess the characteristics of congestion, edema, and the infiltration of inflammatory cells. To assess the lung
434
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
histopathological changes induced by LPS, The superior lobe of the right lungs were fixed in Bouin, dehydrated in graded alcohol, embedded in paraffin, sliced with 5 μm thick, mounted on slides and then stained with hematoxylin and eosin. Histopathological changes of lung tissues were observed using a light microscope. 2.11. Immunohistochemical study of the lung tissues In immunohistochemistry study, the lung sections were deparaffinized in xylene, rehydrated in graded alcohol, soaked in citrate buffer (0.1 M, pH 6.0) for antigen retrieval by microwave oven. After cooling, the sections were washed in PBS three times and situated in 3% hydrogen peroxide at 37 °C for 10 min to wipe out the endogenous peroxidase. After that, the sections were incubated with goat serum at 37 °C for 20 min to reduce non-specific antibody-binding and then were incubated with the primary COX-2 antibody at a dilution of 1:200 in PBS (v/v) overnight at 4 °C. In the next day, the sections were sealed with the rabbit anti-mouse secondary antibody at 37 °C for 30 min and then stained with 3, 3′-diaminobenzidine (DAB) in the dark for 5 min. The sections were stained with hematoxylin again and were dehydrated in graded ethanol. At last, the sections were sealed in neutral gum. The immunohistochemical results of the lung tissues were observed by a light microscope. 2.12. Western blot analysis The lung tissues were homogenized in cold RIPA lysis buffer (150 mM NaCl, 25 mM Tris–HCl pH 7.6, 1% NP-40, 1% sodium deoxycholate, 1% SDS) containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF) for 10 min. The homogenates were centrifuged at 12,000 rpm at 4 °C for 10 min and the supernatants were collected for Western blot analysis. The protein concentrations were determined by a bicinchoninic acid (BCA) protein assay kit according to the manufacturer's protocols. An equivalent amount of total protein from each group was loaded in each well on a 10% sodium dodecyl sulfatepolyacrylamidgel (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in a blocking buffer (5% non-fat dry milk in 1 × TBS solution containing 0.1% Tween-20) for 2 h at room temperature to decrease non-specific binding, and then incubated with primary antibodies diluted 1:10,000 in antibody dilution buffer (0.1% Tween-20 in 1 × TBS) overnight at 4 °C. After washing three times with TBST, the membranes were incubated with a secondary antibody (1:40,000 dilutions) conjugated with horseradish peroxidase at room temperature for 1 h and then washed again three times with TBST. Immunoreactive bands were visualized by enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Western blot analyses were repeated three times.
Fig. 2. The effects of IF on LPS-induced mortality in ALI mice (n = 12/group). IF (5, 10, 15 mg/kg, i.p.) or DEX (5 mg/kg, i.p.) were given to mice 1 h prior to LPS challenge. The mortalities were observed at 0, 12, 24, 36, 48, 60, and 72 h. ###P b 0.001 when compared with the control group; *P b 0.05, **P b 0.01, and ***P b 0.001 when compared with the LPS group.
evidently lower than that in LPS group (91.67%). In addition, DEX also significantly reduced LPS-induced death compared with the LPS group.
3.2. Effects of IF on LPS-induced lung W/D ratio Pulmonary edema is the typical characteristic of ALI. The lung W/D ratio was evaluated to quantify the magnitude of pulmonary edema. As shown in Fig. 3, LPS challenge produced a remarkable increase in the lung W/D ratio compared with the control group. However, pretreatment with IF or DEX significantly decreased the lung W/D ratios, thus relieved the degree of pulmonary edema.
3.3. Effects of IF on the production of TNF-α, IL-6, and PGE2 in serum and BALF The effects of IF on TNF-α, IL-6, and PGE2 concentrations in serum and BALF were analyzed 6 h after LPS stimulation by ELISA. As shown in Figs. 4 and 5, the concentrations of TNF-α, IL-6, and PGE2 were obviously elevated in LPS group compared with those in control group both in serum and BALF. Pretreatment with IF efficiently decreased the levels of IL-6 and PGE2 at the low dose. For TNF-α, the medium and high doses significantly decreased the elevation of TNF-α. DEX also performed significant inhibitory effect on TNF-α, IL-6, and PGE2 secretion into the serum and BALF.
2.13. Statistical analysis All data were presented as means ± SEM. The comparison of mean values in different groups were performed by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test. Statistical analyses and column charts were finished by GraphPad Software (California, USA). The difference between survival rates of two groups were evaluated by a Kaplan–Meier curve and log-rank test with SPSS software. Statistical significance was accepted at P b 0.05. 3. Results 3.1. Effects of IF on LPS-induced mortality in ALI mice As shown in Fig. 2, the mortalities of IF treatment groups (5, 10, 15 mg/kg) were 41.67%, 25%, and 16.67% respectively, which were
Fig. 3. The effects of IF on LPS-induced lung W/D ratio (n = 12/group). Mice were given an intraperitoneal injection of IF (5, 10, 15 mg/kg) or DEX (5 mg/kg) 1 h before an intraperitoneal administration of LPS. The lung W/D ratio was determined at 6 h after LPS challenge. Data presented were mean ± SEM. ##P b 0.01 compared with the control group; *P b 0.05 and **P b 0.01 compared with the LPS group.
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 4. The effects of IF on the production of TNF-α, IL-6, and PGE2 in serum (n = 12/ group). Mice were pretreated with IF (5, 10, 15 mg/kg) or DEX (5 mg/kg) 1 h prior to LPS injection. Serum were collected at 6 h after LPS injection to analyze the levels of TNF-α (Fig. 4A), IL-6 (Fig. 4B), and PGE2 (Fig. 4C). Data presented were mean ± SEM. ### P b 0.001 versus the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 versus the LPS group.
3.4. Effects of IF on inflammatory cell counts in BALF Six hours after LPS challenge, BALF were collected and the number of total inflammatory cells, neutrophils, and macrophages in BALF were analyzed. Compared with the control group, LPS markedly increased the amounts of total inflammatory cells (Fig. 6A), neutrophils (Fig. 6B), and macrophages (Fig. 6C) in BALF. However, IF significantly reduced the number of total inflammatory cells, neutrophils, and macrophages in BALF in a dose-dependent manner. DEX also obviously decreased the amounts of total inflammatory cells, neutrophils, and macrophages in BALF.
3.5. Effect of IF on MPO activity in lung tissues The MPO activity was measured to evaluate the effects of IF on neutrophil accumulation in pulmonary tissues. As shown in Fig. 7, LPS
435
Fig. 5. The effects of IF on the production of TNF-α, IL-6, and PGE2 in BALF (n = 12/group). BALF was collected 6 h after LPS challenge and the concentrations of TNF-α (Fig. 5A), IL-6 (Fig. 5B), and PGE2 (Fig. 5C) in BALF were determined. Data presented were mean ± SEM. ## P b 0.01, ###P b 0.001 compared with the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the LPS group.
stimulation lead to significant increases in lung MPO activity compared with the control group. However, these increases were markedly reduced when pretreated with IF and DEX.
3.6. Effects of IF on LPS-induced lung histopathological changes in ALI mice As shown in Fig. 8, lung sections from the control group showed a normal structure without histopathological changes. In the LPS group, Lung sections showed obvious histopathological changes, including inflammatory cell infiltration, alveolar wall thickening caused by edema, alveolus collapse, and alveolar hemorrhage. However, the histopathological changes were significantly decreased by IF and DEX pretreatment. Furthermore, a similar inhibitory effect was recorded in semiquantitative analysis, which facilitated the evaluation of severity and the extent of inflammatory change. These results suggested that IF attenuated the histopathological changes of LPS-induced ALI in mice.
436
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 7. The effects of IF on MPO activity in lung tissues (n = 12/group). Mice were euthanized 6 h after the injection of LPS and the lung tissues were harvested for MPO assay. The values of MPO activity were expressed as units per gram of protein. Data presented were mean ± SEM. ##P b 0.01 versus the control group; *P b 0.05, **P b 0.01 versus the LPS group.
expression in lung tissues by Western blot assay. As shown in Fig. 10, in the LPS group, COX-2 protein expression was apparently higher than that in the control group. However, pretreatment with IF significantly reduced COX-2 protein expression compared with the LPS group and the DEX group showed the similar effects. The results indicated that IF pretreatment significantly decreased the COX-2 protein expression in the lung. 4. Discussion
Fig. 6. The effects of IF on inflammatory cell counts in BALF (n = 12/group). Mice were given a pretreatment with IF or DEX 1 h prior to LPS administration. BALF were performed at 6 h after LPS administration to measure the number of total cells, neutrophils, and macrophage. Data presented were mean ± SEM. ##P b 0.01, ###P b 0.001 compared with the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the LPS group.
3.7. Effects of IF on COX-2 protein expression in lung tissues of ALI mice The effects of IF on COX-2 protein expression in the lung were assessed 6 h after LPS challenge by immunohistochemical study. As shown in Fig. 9, The COX-2 specific immunolabeling in the surface epithelium and mononuclear cells of mucosa were scarcely discovered in the control group. In the LPS group, LPS challenge considerably increased COX-2 protein expression and abundant COX-2 positive cells were found. Pretreatment with IF and DEX notably diminished the COX-2 protein expression. 3.8. IF inhibits COX-2 protein expression in lung tissues of ALI mice To further explore the anti-inflammatory mechanism of IF in LPSinduced ALI mice, we investigated the effects of IF on COX-2 protein
LPS, the major component of the outer cell wall of Gramnegative bacteria, can interact with circulating LPS-binding protein and CD14 in the cell membrane and trigger a series of inflammatory responses [15]. LPS challenge in mice can induce a clinically relevant model of ALI characterized by the parenchymal infiltration of neutrophils and macrophages, the release of inflammatory cytokines such as TNF-α and IL-6, the disruption of endothelial and epithelial integrity and other pulmonary inflammation [16]. IF is a bioactive component extracted from natural plants such as Chloranthaceae and Umbelliferae and possesses anti-oxidant, antibacterial, and anti-inflammatory effects [11]. In this study, we selected a mouse model to evaluate the protective effects of IF against LPS-induced ALI. The experimental results demonstrated that pretreatment with IF effectively attenuated LPS-induced ALI in mice. The conclusion can be supported by the following results: Pretreatment with IF markedly reduced LPS-induced mortality and lung edema. Lung histopathological changes, MPO activity in lung tissues and the number of total cells, neutrophils, and macrophages in BALF were significantly inhibited in mice treated with IF. Moreover, IF notably decreased inflammatory cytokines production of TNF-α and IL-6 in serum and BALF, COX-2 protein expression in the lung, and inflammatory mediator concentration of PGE 2 in serum and BALF. LPS challenge can cause acute and chronic inflammations in mice, even lead to death. In the present study, just as the previous reports, mice in LPS group exhibited a high mortality. However, pretreatment with IF significantly reduced the mortality. It indicated that pretreatment with IF could protect the mice from LPSinduced death. edema is the typical feature of ALI [17], we assessed the lung W/D ratio to quantify the magnitude of pulmonary edema. Experimental results showed that IF treatment obviously decreased the lung W/D ratio compared with the LPS group, which provided powerful evidence for the protective effect of IF on LPS-induced ALI. The inflammatory cells, including macrophages and neutrophils, play a vital role in the process of ALI. Macrophages produced most of the inflammatory cytokines such as TNF-α and IL-6 [18].
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
437
Fig. 8. The effects of IF on LPS-induced lung histopathological changes in ALI mice (magnification × 400). Lung tissues were stained with hematoxylin and eosin for histopathological evaluation. (A) Control group, normal structure. (B) LPS group, obvious inflammatory infiltration, alveolar wall thickness. (C) LPS + DEX (5 mg/kg) group, showing an obvious inhibition of histopathological changes compared with the LPS group. (D) LPS + IF (5 mg/kg) group, (E) LPS + IF (10 mg/kg) group, (F) LPS + IF (15 mg/kg) group, histopathological changes were significantly decreased compared with the LPS group. The arrows indicated prominent inflammatory cell infiltration and alveolar wall thickness. (G) The effects of IF on LPS-induced lung morphology. The slides were histopathologically assessed using a semi-quantiative scoring method. Lung injury was graded from 0 (normal) to 4 (severe) in four categories: inflammatory cell infiltration, edema, congestion and interstitial inflammation. The total lung injury score was calculated by adding up the individual scores of each category. Data presented were mean ± SEM. ###P b 0.001 when compared with the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 when compared with the LPS group.
Neutrophils play a main role in the inflammatory cells and act as the first reactor in self-defense mechanism to fight infection [19]. Our study revealed that pretreatment with IF notably diminished the number of the total cells, neutrophils, and macrophages in BALF compared with the LPS group. In human and animal models, neutrophil influx and vascular leakage at the site of injury are the common features of ALI [20]. MPO, an enzyme located mostly in the primary granules of neutrophils, is proportional with the number of neutrophils in lung tissues. In our study, mice exposed to LPS showed an elevated level of MPO activity while pretreatment with IF significantly reduced the MPO activity in lung tissues. In the histopathological analyses, the LPS group showed evident histopathological abnormalities, while IF treatment markedly ameliorated the symptoms of neutrophil infiltration, alveolar wall thickening,
alveolus collapse, and alveolar hemorrhage in the lung. Overall, these findings further demonstrated that IF had a protective effect against LPS-induced ALI in mice. Excessive release of pro-inflammatory cytokines such as TNF-α and IL-6 is an important inducement of ALI and causes unmanageable inflammation followed by various pathologic responses. TNF-α is a representative and pleiotropic inflammatory cytokine in the development of inflammation. It stimulates the production of COX-2 and iNOS [21,22] and trigger injury, inflammation and carcinogenesis in various tissues [23,24]. It has already been confirmed that the concentration of TNF-α enhanced in BALF from patients with ALI [25]. Moreover, the previous research has proven that anti-TNF-α antibodies were considered to ameliorate pulmonary injury [26]. Another crucial inflammatory cytokine is IL-6, which is generated by
438
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 9. The effects of IF on immunohistochemical localization of COX-2 in ALI mice (magnification × 400). After the mice were euthanized, the lung tissues were collected for immunohistochemical study. (A) Control group, normal lung tissue. (B) LPS group, abundant COX-2 protein expression in alveolar macrophages and epithelial cells. (C) LPS + DEX (5 mg/kg) group, COX-2 protein expression was evidently reduced when compared with the LPS group. (D) LPS + IF (5 mg/kg) group, (E) LPS + IF (10 mg/kg) group, (F) LPS + IF (15 mg/kg) group, showing a significant inhibition of COX-2 protein expression compared with the LPS group. The arrows indicated abundant COX-2 protein expression in lung tissues. (G) Densitometry analysis of photographs of COX-2 immunohistochemistry from lung tissues. Image-Pro was used for quantifying the expression of COX-2 protein in each group. Grade represents the mean density, which is the concentration of COX-2 protein per unit area of the lung sections. Grade is proportional to the expression of COX-2 protein in lung tissues. Data presented were mean ± SEM. ### P b 0.001 compared with the control group; *P b 0.05, **P b 0.01 compared with the LPS group.
different cells and possesses pleiotropic effects on various tissues. IL6 regulates genes expression involved in cell cycle progression and suppression of apoptosis [27]. On the basis of the previous studies, we discovered an increasing level of IL-6 in inflammatory diseases such as sepsis [28], fever [29], mesangial glomerulonephritis [30], ulcerative colitis [31], and rheumatoid arthritis [32]. Therefore, inhibition of TNF-α and IL-6 production played an important role in the treatment of ALI. In the present study, the concentrations of TNF-α and IL-6 were obviously increased in LPS group compared with those in control group both in serum and BALF. However, IF pretreatment efficiently decreased the level of TNF-α at the medium (10 mg/kg) and high (15 mg/kg) doses in serum and BALF. For IL-6, pretreatment with IF significantly decreased the concentration of
IL-6 at all doses (5, 10, 15 mg/kg) in serum and BALF. Above results demonstrated that IF had the protective effects on LPS-induced ALI by inhibiting the production of TNF-α and IL-6. In the present study, COX-2 protein expression in lung tissues increased after LPS challenge. COX-2 scarcely exists in normal tissues, but widely induced by pro-inflammatory stimulations and plays a significant role in inflammation [33]. PGE2, an indirect index of COX-2 activity, generally releases from the part of lesion and dominates in the spinal ruled nociception [34]. The previous research revealed that abundant PGE2 produced under the stimulation of LPS through the pathway of COX-2 [35]. Therefore, inhibition of COX-2 protein expression and PGE2 production has showed potent anti-inflammatory effects in inflammatory diseases. In present study, COX-2 protein expression in lung tissues was analyzed
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 10. The effects of IF on COX-2 protein expression in lung tissues of ALI mice. Mice were killed 6 h after LPS challenge and the lung tissues were removed. The extraction of nuclear and cytoplasmic proteins from lung was performed. The COX-2 protein expression was determined by Western blot assay. Quantification of protein expression was normalized to GAPDH using a densitometer. Data presented were mean ± SEM. ###P b 0.001 versus the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 versus the LPS group.
by immunohistochemical study to verify the results of PGE2 production in serum and BALF. We found that IF pretreatment significantly attenuated PGE2 production in serum and BALF corresponded with the suppression of COX-2 protein expression in lung tissues, which were consistent with the previous research. In Western blot assay, the present study showed that COX-2 protein expression was markedly increased in the LPS group compared with the control group. However, IF pretreatment significantly inhibited LPS-induced COX-2 protein expression. In summary, these results indicated that the protective effects of IF against LPS-induced ALI may be achieved by inhibition of COX-2 protein expression, which regulates PGE2 production. 5. Conclusion In the present study, pretreatment with IF significantly decreased LPS-induced mortality, pulmonary edema, inflammatory cells infiltration, MPO activity, the concentrations of TNF-α, IL-6, and PGE2 in serum and BALF, and histopathological changes, COX-2 protein expression in the lung. These results demonstrated that IF possessed the protective effects against LPS-induced ALI, and the protective effects may result from the suppression of COX-2 protein expression, which causes the reduction of PGE2 production. Above findings suggest that IF may be an efficacious agent for preventing and treating ALI, but further researches are still needed to confirm the protective mechanism of IF. Acknowledgments This work was supported by a research grant (no.: 2013KW26-02) from the Natural Science Foundation of International Cooperation Projects (Shaanxi Province, PR China). References [1] Niu X, Yao H, Li W, Mu Q, Li H, Hu H, et al. δ-Amyrone, a specific inhibitor of cyclooxygenase-2, exhibits anti-inflammatory effects in vitro and in vivo of mice. Int Immunopharmacol 2014;21:112–8. [2] Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121:2381–6. [3] O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses. Immunity 2008;28:477–87.
439
[4] Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000; 342:1334–49. [5] Raghavendran K, Napolitano LM. Definition of ALI/ARDS. Crit Care Clin 2011;27: 429–37. [6] Marshall HE, Potts EN, Kelleher ZT, Stamler JS, Foster WM, Auten RL. Protection from lipopolysaccharide-induced lung injury by augmentation of airway S-nitrosothiols. Am J Respir Crit Care Med 2009;180:11–8. [7] Kagan JC, Medzhitov R. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 2006;125:943–55. [8] Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, et al. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem 1992;267(36):25934–8. [9] Kim SO, Kundu JK, Shin YK, Park JH, Cho MH, Kim TY, et al. [6]-Gingerol inhibits COX2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene 2005;24(15):2558–67. [10] Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, Vane JR. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci U S A 1999;96:7563–8. [11] Niu X, Xing W, Li W, Fan T, Hu H, Li Y. Isofraxidin exhibited anti-inflammatory effects in vivo and inhibited TNF-α production in LPS-induced mouse peritoneal macrophages in vitro via the MAPK pathway. Int Immunopharmacol 2012;14:164–71. [12] Khan S, Riaz N, Afza N, Malik A, Aziz-ur-Rehman, Iqbal L, et al. Antioxidant constituents from Cotoneaster racemiflora. J Asian Nat Prod Res 2009;11:44–8. [13] Yamazaki T, Shimosaka S, Sasaki H, Matsumura T, Tukiyama T, Tokiwa T. (+)Syringaresinol-di-O-β-d-glucoside, a phenolic compound from Acanthopanax senticosus Harms, suppresses proinflammatory mediators in SW982 human synovial sarcoma cells by inhibiting activating protein-1 and/or nuclear factor-κB activities. Toxicol Vitro 2007;21:1530–7. [14] Nakamura T, Kodama N, Oda M, Tsuchiya S, Arai Y, Kumamoto T, et al. The structure–activity relationship between oxycoumarin derivatives showing inhibitory effects on iNOS in mouse macrophage RAW264.7 cells. J Nat Med 2009;63:15–20. [15] Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274: 10689–92. [16] Suda K, Tsuruta M, Eom J, Or C, Mui T, Jaw J-E, et al. Acute lung injury induces cardiovascular dysfunction: effects of IL-6 and budesonide/formoterol. Am J Respir Cell Mol Biol 2011;45:510–6. [17] Zhang X, Song K, Xiong H, Li H, Chu X, Deng X. Protective effect of florfenicol on acute lung injury induced by lipopolysaccharide in mice. Int Immunopharmacol 2009;9:1525–9. [18] Cavaillon J-M, Adib-Conquy M. Monocytes/macrophages and sepsis. Crit Care Med 2005;33:S506–9. [19] Xie X, Sun S, Zhong W, Soromou LW, Zhou X, Wei M, et al. Zingerone attenuates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol 2014;19:103–9. [20] Zarbock A, Singbartl K, Ley K. Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation. J Clin Invest 2006;116: 3211–9. [21] Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res 2004:S27–36. [22] Mastbergen SC, Lafeber F, Bijlsma JWJ. Selective COX-2 inhibition prevents proinflammatory cytokine-induced cartilage damage. Rheumatology 2002;41:801–8. [23] Faubion WA, Gores GJ. Death receptors in liver biology and pathobiology. Hepatology 1999;29:1–4. [24] Diamond JR, Pesek I. Glomerular tumor-necrosis-factor and interleukin-1 during acute aminonucleoside nephrosis — an immunohistochemical study. Lab Invest 1991;64:21–8. [25] Zhang B, Liu ZY, Li YY, Luo Y, Liu ML, Dong H-Y, et al. Antiinflammatory effects of matrine in LPS-induced acute lung injury in mice. Eur J Pharm Sci 2011;44:573–9. [26] Feng Q, Ren Y, Wang Y, Ma H, Xu J, Zhou C, et al. Anti-inflammatory effect of SQCbeta-CD on lipopolysaccharide-induced acute lung injury. J Ethnopharmacol 2008; 118:51–8. [27] Lin WW, Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest 2007;117:1175–83. [28] Baker SS, Campbell CL. Rat enterocyte injury by oxygen-dependent processes. Gastroenterology 1991;101:716–20. [29] Baeuerle PA, Henkel T. Function and activation of NF-KAPPA-B in the immunesystem. Annu Rev Immunol 1994;12:141–79. [30] Autschbach F, Braunstein J, Helmke B, Zuna I, Schurmann G, Niemir ZI, et al. In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease. Am J Pathol 1998;153:121–30. [31] Atreya I, Atreya R, Neurath MF. NF-kappa B in inflammatory bowel disease. J Intern Med 2008;263:591–6. [32] Akgun E, Caliskan C, Celik HA, Ozutemiz AO, Tuncyurek M, Aydin HH. Effects of Nacetylcysteine treatment on oxidative stress in acetic acid-induced experimental colitis in rats. J Int Med Res 2005;33:196–206. [33] Hussain T, Gupta S, Mukhtar H. Cyclooxygenase-2 and prostate carcinogenesis. Cancer Lett 2003;191(2):125–35. [34] Hunter DA, Barr GA, Shivers KY, Amador N, Jenab S, Inturrisi C, et al. Interactions of estradiol and NSAIDS on carrageenan-induced hyperalgesia. Brain Res 2011;1382: 181–8. [35] Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 2004;56:387–437.
Contents lists available at ScienceDirect
International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Protective effects of Isofraxidin against lipopolysaccharide-induced acute lung injury in mice Xiaofeng Niu, Yu Wang, Weifeng Li ⁎, Qingli Mu, Huani Li, Huan Yao, Hailin Zhang School of Pharmacy, Xi'an Jiaotong University, Xi'an 710061, PR China
a r t i c l e
i n f o
Article history: Received 1 September 2014 Received in revised form 27 November 2014 Accepted 5 December 2014 Available online 13 January 2015 Keywords: Isofraxidin Acute lung injury Inflammatory cytokines Cyclooxygenase-2 Prostaglandin E2
a b s t r a c t Acute lung injury (ALI) is a life-threatening disease characterized by serious lung inflammation and increased capillary permeability, which presents a high mortality worldwide. Isofraxidin (IF), a Coumarin compound isolated from the natural medicinal plants such as Sarcandra glabra and Acanthopanax senticosus, has been reported to have definite anti-bacterial, anti-oxidant, and anti-inflammatory activities. However, the effects of IF against lipopolysaccharide-induced ALI have not been clarified. The aim of the present study is to explore the protective effects and potential mechanism of IF against LPS-induced ALI in mice. In this study, We found that pretreatment with IF significantly lowered LPS-induced mortality and lung wet-to-dry weight (W/D) ratio and reduced the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and prostaglandin E2 (PGE2) in serum and bronchoalveolar lavage fluid (BALF). We also found that total cells, neutrophils and macrophages in BALF, MPO activity in lung tissues were markedly decreased. Besides, IF obviously inhibited lung histopathological changes and cyclooxygenase-2 (COX-2) protein expression. These results suggest that IF has a protective effect against LPSinduced ALI, and the protective effect of IF seems to result from the inhibition of COX-2 protein expression in the lung, which regulates the production of PGE2. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Inflammation is a self-defensive immune process in which the body reacts to external stimuli, infection, or injury [1]. The development of inflammation is associated with the production of inflammatory cytokines by neutrophils and macrophages, tissue edema caused by the leakage of fluid and proteins, and the infiltration of leucocytes at the site of inflammation [2,3]. Acute lung injury (ALI), a common inflammatory disease in the clinic, shows the characteristics of interstitial edema, neutrophil recruitment, disruption of epithelial integrity, and lung parenchymal injury [4]. Despite significant advances in clinical research and therapeutic trials made in the past several decades, ALI still presents a high mortality in the diseases of shock, sepsis, ischemia reperfusion and viral pneumonia [5]. Therefore, it is extremely urgent to discover effective drugs and clinical therapies for ALI. Lipopolysaccharide (LPS) is widely used for induction of animal models of ALI for its similar characteristics of human ALI [6]. ⁎ Corresponding author at: School of Pharmacy, Xi'an Jiaotong University, No. 76 Western Yanta Road, Xi'an City, Shaanxi Province 710061, PR China. Tel.: + 86 29 82655139; fax: +86 29 82655138. E-mail addresses: [email protected] (X. Niu), [email protected] (W. Li).
http://dx.doi.org/10.1016/j.intimp.2014.12.041 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Ingestion of LPS stimulates vascular permeability, promotes inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) from blood into lung tissues and activates numerous inflammatory cells such as neutrophils and macrophages [7]. In macrophages, LPS challenge induces the transcription of gene encoding pro-inflammatory protein, which leads to cytokine release and synthesis of enzymes, such as cyclooxygenase-2 (COX2) [8]. COX-2 usually can't be found in normal tissues, but widely induced by pro-inflammatory stimuli, such as cytokines, endotoxins, and growth factors [9]. COX-2 plays a vital role in the regulation of inflammatory process by modulating the production of prostaglandin E2 (PGE2). PGE2, induced by cytokines and other initiator, is an inflammatory mediator which is produced in the regulation of COX-2. Previous researches demonstrated that inhibition of COX-2 produced a dramatically anti-inflammatory effect with little gastrointestinal toxicity [10]. Therefore, inhibition of COX-2 protein expression has far-reaching significance in the treatment of ALI. Isofraxidin (IF, 7-hydroxy-6,8-dimethoxycoumarin, shown in Fig. 1) is a Coumarin compound that widely exists in natural plants such as Sarcandra glabra and Acanthopanax senticosus, both of which are traditional Chinese herbs usually used for anti-tumor, anti-bacterial, anti-oxidant, and anti-inflammatory treatments [11]. IF is a bioactive component with definite anti-bacterial and
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
433
performed through the left lungs. The superior lobe of the right lung was removed for histopathological analysis and immunohistochemical study. The middle lobe of the right lung was removed for the evaluation of lung wet-to-dry weight (W/D) ratio. The lower lobe of the right lung was removed and rapidly cut into two parts. One part of the lower lobe was used for MPO analysis, and the other part was used for Western blot analysis. 2.4. LPS-induced mortality in ALI mice Fig. 1. Chemical structure of Isofraxidin.
anti-oxidant activities [12]. The previous studies have also shown that IF possesses anti-inflammatory activity [13,14]. However, there is no report about the protective effects of IF on LPS-induced ALI. The purpose of the present study is to explore the protective effects of IF against LPS-induced ALI in mice.
The normal control group and the negative control group were given an equal volume of vehicle according to experimental design. All other groups received intraperitoneal injection of IF (5, 10, 15 mg/kg) or DEX (5 mg/kg). An hour later, mice received intraperitoneal injection of LPS (20 mg/kg). Within 72 h after LPS injection, the mortality of mice was observed every 12 h in each group (n = 12/group).
2. Materials and methods
2.5. Lung W/D ratio
2.1. Reagents
After the mice were euthanized by cervical dislocation, the middle lobe of the right lungs were excised and weighed to record the “wet” weight. Then, the lung tissues were arranged in an oven at 80 °C for 48 h to obtain the stable “dry” weight. The lung W/D ratios were computed to assess the severity of pulmonary edema.
IF (purity ≥ 98%) was purchased from Xi'an Honson Biotechnology Co., Ltd. (Xi'an, China) and confirmed by the Pharmacognosy Laboratory, School of Medicine, Xi'an Jiaotong University (Xi'an, China). Dexamethasone (DEX), as a positive control, was supplied by Xi'an Lijun Pharmaceutical Company Limited (Shanxi, China). LPS (Escherichia coli serotype O55:B5) was obtained from Sigma (St. Louis, MO). The enzyme linked immunosorbent assay (ELISA) kit for mouse TNF-α, IL-6, and PGE 2 was purchased from R&D Systems (Minneapolis, MN, USA). The kit for biochemical analysis of myeloperoxidase (MPO) was provided by Jiancheng Bioengineering Institute (Nanjing, China). Histostain-Plus kits, DAB (3, 3′-diaminobenzidine) staining kit and rabbit anti-cyclooxygenase-2 were supplied by Beijing Biosynthesis Biotechnology Co., Ltd. RPMI-1640 was purchased from Gibco (Gibco-BRL, Gaithersburg, MD, USA). Antibody against COX-2 (UniProt: P35354) were supplied by Epitomics Inc. (an Abcam Company, U.S.A.). Antiglyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Polyvinylidene fluoride (PVDF) membranes were provided by Pall Gelman Laboratory (Ann Arbor, MI, USA). All other reagents used in the study were of analytical grade.
2.6. Collection of serum and BALF Six hours after LPS administration, blood samples were collected from the retro-orbital plexus and immediately centrifuged at 3000 rpm for 10 min at 4 °C to obtained the serum. The left lung was lavaged through a tracheal cannula with 1.5 mL autoclaved phosphate buffered saline (PBS) three times in each group. The recovery rate of BALF was better than 90%. BALF was immediately centrifuged at 1500 rpm for 10 min for pelleting the cells. 2.7. Measurement of the levels of TNF-α, IL-6, and PGE2 in serum and BALF The BALF was centrifuged at 1500 rpm for 10 min at 4 °C, and the supernatant of BALF was used for determining the concentrations of TNF-α, IL-6, and PGE2. The levels of TNF-α, IL-6, and PGE2 in serum and BALF were evaluated by mouse ELISA kits according to the manufacturer's directions. The absorbance was read at 450 nm and the samples were detected three times.
2.2. Animals 2.8. Inflammatory cell counts of BALF All male Kunming mice (22–25 g) were supplied by the Experimental Animal Center, Xi'an Jiaotong University (Xi'an, China). Animals were raised under standard conditions with a 12 h day/night cycle and acclimatized to their environment for at least one week before the beginning of animal experiments. All animal experiments were performed in line with the National Institute of Health guidelines. 2.3. Mouse model of LPS-induced ALI The mice were divided into six groups randomly (n = 12/group): Control group; LPS group (LPS, 5 mg/kg, i.p.); LPS + DEX group (5 mg/kg, i.p.); LPS + IF groups (5, 10, 15 mg/kg, i.p.). The doses and administration route of IF and DEX were confirmed based on preliminary tests. IF or DEX were administrated intraperitoneally 1 h prior to LPS challenge. The control and LPS groups were given an equal volume of vehicle according to experimental design. The severity of LPS-induced pulmonary injury was assessed at 6 h after LPS administration. Blood samples were collected from the retro-orbital plexus, and then all mice were euthanized. Bronchoalveolar lavage fluid (BALF) was
The BALF were centrifuged at 1500 rpm for 10 min at 4 °C to pellet the cells and the cell pellets were resuspended in PBS. A hemacytometer was used for the total cell counts and cytospins were essential for different cell counts via staining with the Wright–Giemsa staining method. 2.9. MPO activity in the lung tissues MPO activity is an important index of neutrophils accumulation in inflammatory tissues. After the lungs were removed, one part of the lower lobe were homogenized in PBS by a homogenizer. The mixtures were centrifuged at 3000 rpm for 10 min at 4 °C, and the supernatants were used for MPO analysis. MPO activity were measured with an MPO activity kit based on the manufacturer's instructions. MPO activities were expressed as units per gram of protein. 2.10. Histopathological study of the lung tissues LPS-induced ALI mice possess the characteristics of congestion, edema, and the infiltration of inflammatory cells. To assess the lung
434
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
histopathological changes induced by LPS, The superior lobe of the right lungs were fixed in Bouin, dehydrated in graded alcohol, embedded in paraffin, sliced with 5 μm thick, mounted on slides and then stained with hematoxylin and eosin. Histopathological changes of lung tissues were observed using a light microscope. 2.11. Immunohistochemical study of the lung tissues In immunohistochemistry study, the lung sections were deparaffinized in xylene, rehydrated in graded alcohol, soaked in citrate buffer (0.1 M, pH 6.0) for antigen retrieval by microwave oven. After cooling, the sections were washed in PBS three times and situated in 3% hydrogen peroxide at 37 °C for 10 min to wipe out the endogenous peroxidase. After that, the sections were incubated with goat serum at 37 °C for 20 min to reduce non-specific antibody-binding and then were incubated with the primary COX-2 antibody at a dilution of 1:200 in PBS (v/v) overnight at 4 °C. In the next day, the sections were sealed with the rabbit anti-mouse secondary antibody at 37 °C for 30 min and then stained with 3, 3′-diaminobenzidine (DAB) in the dark for 5 min. The sections were stained with hematoxylin again and were dehydrated in graded ethanol. At last, the sections were sealed in neutral gum. The immunohistochemical results of the lung tissues were observed by a light microscope. 2.12. Western blot analysis The lung tissues were homogenized in cold RIPA lysis buffer (150 mM NaCl, 25 mM Tris–HCl pH 7.6, 1% NP-40, 1% sodium deoxycholate, 1% SDS) containing a protease inhibitor phenylmethanesulfonyl fluoride (PMSF) for 10 min. The homogenates were centrifuged at 12,000 rpm at 4 °C for 10 min and the supernatants were collected for Western blot analysis. The protein concentrations were determined by a bicinchoninic acid (BCA) protein assay kit according to the manufacturer's protocols. An equivalent amount of total protein from each group was loaded in each well on a 10% sodium dodecyl sulfatepolyacrylamidgel (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in a blocking buffer (5% non-fat dry milk in 1 × TBS solution containing 0.1% Tween-20) for 2 h at room temperature to decrease non-specific binding, and then incubated with primary antibodies diluted 1:10,000 in antibody dilution buffer (0.1% Tween-20 in 1 × TBS) overnight at 4 °C. After washing three times with TBST, the membranes were incubated with a secondary antibody (1:40,000 dilutions) conjugated with horseradish peroxidase at room temperature for 1 h and then washed again three times with TBST. Immunoreactive bands were visualized by enhanced chemiluminescence system (GE Healthcare, Little Chalfont, Buckinghamshire, UK). Western blot analyses were repeated three times.
Fig. 2. The effects of IF on LPS-induced mortality in ALI mice (n = 12/group). IF (5, 10, 15 mg/kg, i.p.) or DEX (5 mg/kg, i.p.) were given to mice 1 h prior to LPS challenge. The mortalities were observed at 0, 12, 24, 36, 48, 60, and 72 h. ###P b 0.001 when compared with the control group; *P b 0.05, **P b 0.01, and ***P b 0.001 when compared with the LPS group.
evidently lower than that in LPS group (91.67%). In addition, DEX also significantly reduced LPS-induced death compared with the LPS group.
3.2. Effects of IF on LPS-induced lung W/D ratio Pulmonary edema is the typical characteristic of ALI. The lung W/D ratio was evaluated to quantify the magnitude of pulmonary edema. As shown in Fig. 3, LPS challenge produced a remarkable increase in the lung W/D ratio compared with the control group. However, pretreatment with IF or DEX significantly decreased the lung W/D ratios, thus relieved the degree of pulmonary edema.
3.3. Effects of IF on the production of TNF-α, IL-6, and PGE2 in serum and BALF The effects of IF on TNF-α, IL-6, and PGE2 concentrations in serum and BALF were analyzed 6 h after LPS stimulation by ELISA. As shown in Figs. 4 and 5, the concentrations of TNF-α, IL-6, and PGE2 were obviously elevated in LPS group compared with those in control group both in serum and BALF. Pretreatment with IF efficiently decreased the levels of IL-6 and PGE2 at the low dose. For TNF-α, the medium and high doses significantly decreased the elevation of TNF-α. DEX also performed significant inhibitory effect on TNF-α, IL-6, and PGE2 secretion into the serum and BALF.
2.13. Statistical analysis All data were presented as means ± SEM. The comparison of mean values in different groups were performed by one-way analysis of variance (ANOVA) followed by Student–Newman–Keuls test. Statistical analyses and column charts were finished by GraphPad Software (California, USA). The difference between survival rates of two groups were evaluated by a Kaplan–Meier curve and log-rank test with SPSS software. Statistical significance was accepted at P b 0.05. 3. Results 3.1. Effects of IF on LPS-induced mortality in ALI mice As shown in Fig. 2, the mortalities of IF treatment groups (5, 10, 15 mg/kg) were 41.67%, 25%, and 16.67% respectively, which were
Fig. 3. The effects of IF on LPS-induced lung W/D ratio (n = 12/group). Mice were given an intraperitoneal injection of IF (5, 10, 15 mg/kg) or DEX (5 mg/kg) 1 h before an intraperitoneal administration of LPS. The lung W/D ratio was determined at 6 h after LPS challenge. Data presented were mean ± SEM. ##P b 0.01 compared with the control group; *P b 0.05 and **P b 0.01 compared with the LPS group.
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 4. The effects of IF on the production of TNF-α, IL-6, and PGE2 in serum (n = 12/ group). Mice were pretreated with IF (5, 10, 15 mg/kg) or DEX (5 mg/kg) 1 h prior to LPS injection. Serum were collected at 6 h after LPS injection to analyze the levels of TNF-α (Fig. 4A), IL-6 (Fig. 4B), and PGE2 (Fig. 4C). Data presented were mean ± SEM. ### P b 0.001 versus the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 versus the LPS group.
3.4. Effects of IF on inflammatory cell counts in BALF Six hours after LPS challenge, BALF were collected and the number of total inflammatory cells, neutrophils, and macrophages in BALF were analyzed. Compared with the control group, LPS markedly increased the amounts of total inflammatory cells (Fig. 6A), neutrophils (Fig. 6B), and macrophages (Fig. 6C) in BALF. However, IF significantly reduced the number of total inflammatory cells, neutrophils, and macrophages in BALF in a dose-dependent manner. DEX also obviously decreased the amounts of total inflammatory cells, neutrophils, and macrophages in BALF.
3.5. Effect of IF on MPO activity in lung tissues The MPO activity was measured to evaluate the effects of IF on neutrophil accumulation in pulmonary tissues. As shown in Fig. 7, LPS
435
Fig. 5. The effects of IF on the production of TNF-α, IL-6, and PGE2 in BALF (n = 12/group). BALF was collected 6 h after LPS challenge and the concentrations of TNF-α (Fig. 5A), IL-6 (Fig. 5B), and PGE2 (Fig. 5C) in BALF were determined. Data presented were mean ± SEM. ## P b 0.01, ###P b 0.001 compared with the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the LPS group.
stimulation lead to significant increases in lung MPO activity compared with the control group. However, these increases were markedly reduced when pretreated with IF and DEX.
3.6. Effects of IF on LPS-induced lung histopathological changes in ALI mice As shown in Fig. 8, lung sections from the control group showed a normal structure without histopathological changes. In the LPS group, Lung sections showed obvious histopathological changes, including inflammatory cell infiltration, alveolar wall thickening caused by edema, alveolus collapse, and alveolar hemorrhage. However, the histopathological changes were significantly decreased by IF and DEX pretreatment. Furthermore, a similar inhibitory effect was recorded in semiquantitative analysis, which facilitated the evaluation of severity and the extent of inflammatory change. These results suggested that IF attenuated the histopathological changes of LPS-induced ALI in mice.
436
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 7. The effects of IF on MPO activity in lung tissues (n = 12/group). Mice were euthanized 6 h after the injection of LPS and the lung tissues were harvested for MPO assay. The values of MPO activity were expressed as units per gram of protein. Data presented were mean ± SEM. ##P b 0.01 versus the control group; *P b 0.05, **P b 0.01 versus the LPS group.
expression in lung tissues by Western blot assay. As shown in Fig. 10, in the LPS group, COX-2 protein expression was apparently higher than that in the control group. However, pretreatment with IF significantly reduced COX-2 protein expression compared with the LPS group and the DEX group showed the similar effects. The results indicated that IF pretreatment significantly decreased the COX-2 protein expression in the lung. 4. Discussion
Fig. 6. The effects of IF on inflammatory cell counts in BALF (n = 12/group). Mice were given a pretreatment with IF or DEX 1 h prior to LPS administration. BALF were performed at 6 h after LPS administration to measure the number of total cells, neutrophils, and macrophage. Data presented were mean ± SEM. ##P b 0.01, ###P b 0.001 compared with the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 compared with the LPS group.
3.7. Effects of IF on COX-2 protein expression in lung tissues of ALI mice The effects of IF on COX-2 protein expression in the lung were assessed 6 h after LPS challenge by immunohistochemical study. As shown in Fig. 9, The COX-2 specific immunolabeling in the surface epithelium and mononuclear cells of mucosa were scarcely discovered in the control group. In the LPS group, LPS challenge considerably increased COX-2 protein expression and abundant COX-2 positive cells were found. Pretreatment with IF and DEX notably diminished the COX-2 protein expression. 3.8. IF inhibits COX-2 protein expression in lung tissues of ALI mice To further explore the anti-inflammatory mechanism of IF in LPSinduced ALI mice, we investigated the effects of IF on COX-2 protein
LPS, the major component of the outer cell wall of Gramnegative bacteria, can interact with circulating LPS-binding protein and CD14 in the cell membrane and trigger a series of inflammatory responses [15]. LPS challenge in mice can induce a clinically relevant model of ALI characterized by the parenchymal infiltration of neutrophils and macrophages, the release of inflammatory cytokines such as TNF-α and IL-6, the disruption of endothelial and epithelial integrity and other pulmonary inflammation [16]. IF is a bioactive component extracted from natural plants such as Chloranthaceae and Umbelliferae and possesses anti-oxidant, antibacterial, and anti-inflammatory effects [11]. In this study, we selected a mouse model to evaluate the protective effects of IF against LPS-induced ALI. The experimental results demonstrated that pretreatment with IF effectively attenuated LPS-induced ALI in mice. The conclusion can be supported by the following results: Pretreatment with IF markedly reduced LPS-induced mortality and lung edema. Lung histopathological changes, MPO activity in lung tissues and the number of total cells, neutrophils, and macrophages in BALF were significantly inhibited in mice treated with IF. Moreover, IF notably decreased inflammatory cytokines production of TNF-α and IL-6 in serum and BALF, COX-2 protein expression in the lung, and inflammatory mediator concentration of PGE 2 in serum and BALF. LPS challenge can cause acute and chronic inflammations in mice, even lead to death. In the present study, just as the previous reports, mice in LPS group exhibited a high mortality. However, pretreatment with IF significantly reduced the mortality. It indicated that pretreatment with IF could protect the mice from LPSinduced death. edema is the typical feature of ALI [17], we assessed the lung W/D ratio to quantify the magnitude of pulmonary edema. Experimental results showed that IF treatment obviously decreased the lung W/D ratio compared with the LPS group, which provided powerful evidence for the protective effect of IF on LPS-induced ALI. The inflammatory cells, including macrophages and neutrophils, play a vital role in the process of ALI. Macrophages produced most of the inflammatory cytokines such as TNF-α and IL-6 [18].
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
437
Fig. 8. The effects of IF on LPS-induced lung histopathological changes in ALI mice (magnification × 400). Lung tissues were stained with hematoxylin and eosin for histopathological evaluation. (A) Control group, normal structure. (B) LPS group, obvious inflammatory infiltration, alveolar wall thickness. (C) LPS + DEX (5 mg/kg) group, showing an obvious inhibition of histopathological changes compared with the LPS group. (D) LPS + IF (5 mg/kg) group, (E) LPS + IF (10 mg/kg) group, (F) LPS + IF (15 mg/kg) group, histopathological changes were significantly decreased compared with the LPS group. The arrows indicated prominent inflammatory cell infiltration and alveolar wall thickness. (G) The effects of IF on LPS-induced lung morphology. The slides were histopathologically assessed using a semi-quantiative scoring method. Lung injury was graded from 0 (normal) to 4 (severe) in four categories: inflammatory cell infiltration, edema, congestion and interstitial inflammation. The total lung injury score was calculated by adding up the individual scores of each category. Data presented were mean ± SEM. ###P b 0.001 when compared with the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 when compared with the LPS group.
Neutrophils play a main role in the inflammatory cells and act as the first reactor in self-defense mechanism to fight infection [19]. Our study revealed that pretreatment with IF notably diminished the number of the total cells, neutrophils, and macrophages in BALF compared with the LPS group. In human and animal models, neutrophil influx and vascular leakage at the site of injury are the common features of ALI [20]. MPO, an enzyme located mostly in the primary granules of neutrophils, is proportional with the number of neutrophils in lung tissues. In our study, mice exposed to LPS showed an elevated level of MPO activity while pretreatment with IF significantly reduced the MPO activity in lung tissues. In the histopathological analyses, the LPS group showed evident histopathological abnormalities, while IF treatment markedly ameliorated the symptoms of neutrophil infiltration, alveolar wall thickening,
alveolus collapse, and alveolar hemorrhage in the lung. Overall, these findings further demonstrated that IF had a protective effect against LPS-induced ALI in mice. Excessive release of pro-inflammatory cytokines such as TNF-α and IL-6 is an important inducement of ALI and causes unmanageable inflammation followed by various pathologic responses. TNF-α is a representative and pleiotropic inflammatory cytokine in the development of inflammation. It stimulates the production of COX-2 and iNOS [21,22] and trigger injury, inflammation and carcinogenesis in various tissues [23,24]. It has already been confirmed that the concentration of TNF-α enhanced in BALF from patients with ALI [25]. Moreover, the previous research has proven that anti-TNF-α antibodies were considered to ameliorate pulmonary injury [26]. Another crucial inflammatory cytokine is IL-6, which is generated by
438
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 9. The effects of IF on immunohistochemical localization of COX-2 in ALI mice (magnification × 400). After the mice were euthanized, the lung tissues were collected for immunohistochemical study. (A) Control group, normal lung tissue. (B) LPS group, abundant COX-2 protein expression in alveolar macrophages and epithelial cells. (C) LPS + DEX (5 mg/kg) group, COX-2 protein expression was evidently reduced when compared with the LPS group. (D) LPS + IF (5 mg/kg) group, (E) LPS + IF (10 mg/kg) group, (F) LPS + IF (15 mg/kg) group, showing a significant inhibition of COX-2 protein expression compared with the LPS group. The arrows indicated abundant COX-2 protein expression in lung tissues. (G) Densitometry analysis of photographs of COX-2 immunohistochemistry from lung tissues. Image-Pro was used for quantifying the expression of COX-2 protein in each group. Grade represents the mean density, which is the concentration of COX-2 protein per unit area of the lung sections. Grade is proportional to the expression of COX-2 protein in lung tissues. Data presented were mean ± SEM. ### P b 0.001 compared with the control group; *P b 0.05, **P b 0.01 compared with the LPS group.
different cells and possesses pleiotropic effects on various tissues. IL6 regulates genes expression involved in cell cycle progression and suppression of apoptosis [27]. On the basis of the previous studies, we discovered an increasing level of IL-6 in inflammatory diseases such as sepsis [28], fever [29], mesangial glomerulonephritis [30], ulcerative colitis [31], and rheumatoid arthritis [32]. Therefore, inhibition of TNF-α and IL-6 production played an important role in the treatment of ALI. In the present study, the concentrations of TNF-α and IL-6 were obviously increased in LPS group compared with those in control group both in serum and BALF. However, IF pretreatment efficiently decreased the level of TNF-α at the medium (10 mg/kg) and high (15 mg/kg) doses in serum and BALF. For IL-6, pretreatment with IF significantly decreased the concentration of
IL-6 at all doses (5, 10, 15 mg/kg) in serum and BALF. Above results demonstrated that IF had the protective effects on LPS-induced ALI by inhibiting the production of TNF-α and IL-6. In the present study, COX-2 protein expression in lung tissues increased after LPS challenge. COX-2 scarcely exists in normal tissues, but widely induced by pro-inflammatory stimulations and plays a significant role in inflammation [33]. PGE2, an indirect index of COX-2 activity, generally releases from the part of lesion and dominates in the spinal ruled nociception [34]. The previous research revealed that abundant PGE2 produced under the stimulation of LPS through the pathway of COX-2 [35]. Therefore, inhibition of COX-2 protein expression and PGE2 production has showed potent anti-inflammatory effects in inflammatory diseases. In present study, COX-2 protein expression in lung tissues was analyzed
X. Niu et al. / International Immunopharmacology 24 (2015) 432–439
Fig. 10. The effects of IF on COX-2 protein expression in lung tissues of ALI mice. Mice were killed 6 h after LPS challenge and the lung tissues were removed. The extraction of nuclear and cytoplasmic proteins from lung was performed. The COX-2 protein expression was determined by Western blot assay. Quantification of protein expression was normalized to GAPDH using a densitometer. Data presented were mean ± SEM. ###P b 0.001 versus the control group; *P b 0.05, **P b 0.01 and ***P b 0.001 versus the LPS group.
by immunohistochemical study to verify the results of PGE2 production in serum and BALF. We found that IF pretreatment significantly attenuated PGE2 production in serum and BALF corresponded with the suppression of COX-2 protein expression in lung tissues, which were consistent with the previous research. In Western blot assay, the present study showed that COX-2 protein expression was markedly increased in the LPS group compared with the control group. However, IF pretreatment significantly inhibited LPS-induced COX-2 protein expression. In summary, these results indicated that the protective effects of IF against LPS-induced ALI may be achieved by inhibition of COX-2 protein expression, which regulates PGE2 production. 5. Conclusion In the present study, pretreatment with IF significantly decreased LPS-induced mortality, pulmonary edema, inflammatory cells infiltration, MPO activity, the concentrations of TNF-α, IL-6, and PGE2 in serum and BALF, and histopathological changes, COX-2 protein expression in the lung. These results demonstrated that IF possessed the protective effects against LPS-induced ALI, and the protective effects may result from the suppression of COX-2 protein expression, which causes the reduction of PGE2 production. Above findings suggest that IF may be an efficacious agent for preventing and treating ALI, but further researches are still needed to confirm the protective mechanism of IF. Acknowledgments This work was supported by a research grant (no.: 2013KW26-02) from the Natural Science Foundation of International Cooperation Projects (Shaanxi Province, PR China). References [1] Niu X, Yao H, Li W, Mu Q, Li H, Hu H, et al. δ-Amyrone, a specific inhibitor of cyclooxygenase-2, exhibits anti-inflammatory effects in vitro and in vivo of mice. Int Immunopharmacol 2014;21:112–8. [2] Federico A, Morgillo F, Tuccillo C, Ciardiello F, Loguercio C. Chronic inflammation and oxidative stress in human carcinogenesis. Int J Cancer 2007;121:2381–6. [3] O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses. Immunity 2008;28:477–87.
439
[4] Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000; 342:1334–49. [5] Raghavendran K, Napolitano LM. Definition of ALI/ARDS. Crit Care Clin 2011;27: 429–37. [6] Marshall HE, Potts EN, Kelleher ZT, Stamler JS, Foster WM, Auten RL. Protection from lipopolysaccharide-induced lung injury by augmentation of airway S-nitrosothiols. Am J Respir Crit Care Med 2009;180:11–8. [7] Kagan JC, Medzhitov R. Phosphoinositide-mediated adaptor recruitment controls Toll-like receptor signaling. Cell 2006;125:943–55. [8] Lee SH, Soyoola E, Chanmugam P, Hart S, Sun W, Zhong H, et al. Selective expression of mitogen-inducible cyclooxygenase in macrophages stimulated with lipopolysaccharide. J Biol Chem 1992;267(36):25934–8. [9] Kim SO, Kundu JK, Shin YK, Park JH, Cho MH, Kim TY, et al. [6]-Gingerol inhibits COX2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene 2005;24(15):2558–67. [10] Warner TD, Giuliano F, Vojnovic I, Bukasa A, Mitchell JA, Vane JR. Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: a full in vitro analysis. Proc Natl Acad Sci U S A 1999;96:7563–8. [11] Niu X, Xing W, Li W, Fan T, Hu H, Li Y. Isofraxidin exhibited anti-inflammatory effects in vivo and inhibited TNF-α production in LPS-induced mouse peritoneal macrophages in vitro via the MAPK pathway. Int Immunopharmacol 2012;14:164–71. [12] Khan S, Riaz N, Afza N, Malik A, Aziz-ur-Rehman, Iqbal L, et al. Antioxidant constituents from Cotoneaster racemiflora. J Asian Nat Prod Res 2009;11:44–8. [13] Yamazaki T, Shimosaka S, Sasaki H, Matsumura T, Tukiyama T, Tokiwa T. (+)Syringaresinol-di-O-β-d-glucoside, a phenolic compound from Acanthopanax senticosus Harms, suppresses proinflammatory mediators in SW982 human synovial sarcoma cells by inhibiting activating protein-1 and/or nuclear factor-κB activities. Toxicol Vitro 2007;21:1530–7. [14] Nakamura T, Kodama N, Oda M, Tsuchiya S, Arai Y, Kumamoto T, et al. The structure–activity relationship between oxycoumarin derivatives showing inhibitory effects on iNOS in mouse macrophage RAW264.7 cells. J Nat Med 2009;63:15–20. [15] Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem 1999;274: 10689–92. [16] Suda K, Tsuruta M, Eom J, Or C, Mui T, Jaw J-E, et al. Acute lung injury induces cardiovascular dysfunction: effects of IL-6 and budesonide/formoterol. Am J Respir Cell Mol Biol 2011;45:510–6. [17] Zhang X, Song K, Xiong H, Li H, Chu X, Deng X. Protective effect of florfenicol on acute lung injury induced by lipopolysaccharide in mice. Int Immunopharmacol 2009;9:1525–9. [18] Cavaillon J-M, Adib-Conquy M. Monocytes/macrophages and sepsis. Crit Care Med 2005;33:S506–9. [19] Xie X, Sun S, Zhong W, Soromou LW, Zhou X, Wei M, et al. Zingerone attenuates lipopolysaccharide-induced acute lung injury in mice. Int Immunopharmacol 2014;19:103–9. [20] Zarbock A, Singbartl K, Ley K. Complete reversal of acid-induced acute lung injury by blocking of platelet-neutrophil aggregation. J Clin Invest 2006;116: 3211–9. [21] Goldring SR, Goldring MB. The role of cytokines in cartilage matrix degeneration in osteoarthritis. Clin Orthop Relat Res 2004:S27–36. [22] Mastbergen SC, Lafeber F, Bijlsma JWJ. Selective COX-2 inhibition prevents proinflammatory cytokine-induced cartilage damage. Rheumatology 2002;41:801–8. [23] Faubion WA, Gores GJ. Death receptors in liver biology and pathobiology. Hepatology 1999;29:1–4. [24] Diamond JR, Pesek I. Glomerular tumor-necrosis-factor and interleukin-1 during acute aminonucleoside nephrosis — an immunohistochemical study. Lab Invest 1991;64:21–8. [25] Zhang B, Liu ZY, Li YY, Luo Y, Liu ML, Dong H-Y, et al. Antiinflammatory effects of matrine in LPS-induced acute lung injury in mice. Eur J Pharm Sci 2011;44:573–9. [26] Feng Q, Ren Y, Wang Y, Ma H, Xu J, Zhou C, et al. Anti-inflammatory effect of SQCbeta-CD on lipopolysaccharide-induced acute lung injury. J Ethnopharmacol 2008; 118:51–8. [27] Lin WW, Karin M. A cytokine-mediated link between innate immunity, inflammation, and cancer. J Clin Invest 2007;117:1175–83. [28] Baker SS, Campbell CL. Rat enterocyte injury by oxygen-dependent processes. Gastroenterology 1991;101:716–20. [29] Baeuerle PA, Henkel T. Function and activation of NF-KAPPA-B in the immunesystem. Annu Rev Immunol 1994;12:141–79. [30] Autschbach F, Braunstein J, Helmke B, Zuna I, Schurmann G, Niemir ZI, et al. In situ expression of interleukin-10 in noninflamed human gut and in inflammatory bowel disease. Am J Pathol 1998;153:121–30. [31] Atreya I, Atreya R, Neurath MF. NF-kappa B in inflammatory bowel disease. J Intern Med 2008;263:591–6. [32] Akgun E, Caliskan C, Celik HA, Ozutemiz AO, Tuncyurek M, Aydin HH. Effects of Nacetylcysteine treatment on oxidative stress in acetic acid-induced experimental colitis in rats. J Int Med Res 2005;33:196–206. [33] Hussain T, Gupta S, Mukhtar H. Cyclooxygenase-2 and prostate carcinogenesis. Cancer Lett 2003;191(2):125–35. [34] Hunter DA, Barr GA, Shivers KY, Amador N, Jenab S, Inturrisi C, et al. Interactions of estradiol and NSAIDS on carrageenan-induced hyperalgesia. Brain Res 2011;1382: 181–8. [35] Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 2004;56:387–437.