目錄:
中英文摘要及關鍵詞………..Ⅱ 報告內容………..1 參考文獻………..11 成果報告自評表………..23
中英文摘要及關鍵詞
Abstract
We investigated the effect of sesamol on systemic lipopolysaccharide (LPS)-induced lung inflammation in rats. Sesamol decreased lung edema and injury, significantly decreased LPS-induced cell counts, protein concentration, tumor necrosis factor (TNF)-α, and nitrite levels in bronchoalveolar lavage fluid, and decreased the TNF-α, nitrite, and inducible nitric oxide synthase protein expression in lung tissue. Further, sesamol significantly inhibited LPS-induced TNF-α, nitrite, inducible nitric oxide synthase expression, and nuclear factor- κB activation levels in primary alveolar macrophages. We hypothesize that sesamol attenuates systemic LPS-induced lung inflammation by inhibiting the alveolar macrophage inflammatory response in rats.
Keywords: Alveolar macrophage; lipopolysaccharide; lung inflammation; nuclear factor-κB;
nitric oxide; sesamol
中文摘要
本篇研究探討芝麻酚對脂多醣所誘發的大鼠肺臟發炎反應之影響。結果發現芝麻酚可 降低由脂多醣誘發後所引起的肺臟水腫和傷害情形，降低支氣管沖出液中發炎細胞數 量、蛋白質濃度、TNF-α及亞硝酸鹽濃度，並降低肺臟中 TNF-α、亞硝酸鹽及 iNOS
蛋白表現。由此可知芝麻酚可以抑制由脂多醣誘發傷害後產生的 TNF-α、亞硝酸鹽及
iNOS 蛋白表現，此外在初代培養肺泡巨噬細胞中發現芝麻酚可以抑制 NF-κB 的活化 表現。因此本研究假設芝麻酚可以藉由抑制肺泡巨噬細胞減緩由脂多醣誘發的大鼠肺 臟發炎反應。
關鍵字: 肺泡巨噬細胞、脂多醣、肺臟發炎、NF-κB、一氧化氮、芝麻酚
Abbreviations: LPS, lipopolysaccharide; TNF, tumor necrosis factor; PBS, phosphate buffered saline; BALF, bronchoalveolar lavage fluid; iNOS, inducible nitric oxide synthase;
NF-κB, nuclear factor-κB; LWC, lung water content; TBST, Tris-buffered saline Tween-20;
ANOVA, analysis of variance.
報告內容
1. Introduction
During endotoxemia, severe lung inflammation causes acute lung injury, an important clinical problem with significant mortality (Bone, 1991; Astiz and Rackow, 1998). Lung inflammation is characterized by increased pulmonary inflammatory cell sequestration and the production of pro-inflammatory mediators, which leads to the development of protein leakage in alveolar space, reduced lung compliance, and, finally, in impaired lung function (Bone, 1991; Matthay and Zimmerman, 2005; Braude et al., 1986; Tomashefski, 1990;
Sinclair et la., 1994).
Nitric oxide produced by inducible nitric oxide synthase (iNOS) plays an important role in the pathogenesis of endotoxemia-associated lung inflammation and injury (Cross et al., 2006; MacMinckig et al., 1997; Hollenberg et al., 2000; Wu et al., 2003). Nitric oxide up- regulates inflammatory cytokines, such as TNF-α, production and amplifies the inflammatory response during inflammation (Van Dervort et al., 1994; Zinetti et al., 1995; Borges et al., 1998; Zhang and Feng, 2010). Although iNOS can be produced in various phagocytes including neutrophil, dendritic cell (Förstermann et al., 1994), alveolar macrophage plays a critical role in iNOS activation and nitric oxide production during pulmonary inflammation in sepsis (Tavaf-Motamen et a., 1998; Fujii et al., 1998; Farley et al., 2006). Further, iNOS gene expression can be regulated by modulating nuclear factor (NF)-κB, a transcriptional factor, activation (Jung et al., 2007; Liu and Malik, 2006). After stimulated by bacterial endotoxin, NF-κB translocates into nucleus and binds to NF-κB DNA binding site which leads to iNOS gene expression (Uwe, 2008).
Sesamol (3,4-methylenedioxyphenol), a potent antioxidant in sesame seed oil, is also an
anti-inflammatory agent (Parihar et al., 2006). Sesamol attenuates organ injury and mortality
in septic (Hsu et al., 2006a) and endotoxemic (Hsu et al., 2006b) rats. However, the effects of
sesamol on acute pulmonary inflammation and injury during endotoxemia have never been investigated. Therefore, we investigated the effects of sesamol on systemic endotoxin- induced acute pulmonary inflammation and injury in rats.
2. Materials and Methods 2.1. Materials
Endotoxin (lipopolysaccharide [LPS], derived from Escherichia coli serotype O55:B5) and sesamol (purity ≥ 99%; no preservatives added) were purchased from Sigma-Aldrich (St.
Louis, MO).
2.2. Animals
Male Sprague-Dawley rats (weight: 200-300 g) were purchased from our institution’s Laboratory Animal Center and housed individually in a room with 12-h dark/light cycle and central air conditioning (25°C, 70% humidity). The rats were allowed free access to tap water and were given a rodent diet (Richmond Standard, PMI Feeds, Inc., St. Louis, MO). The animal care and experimental protocols were in accord with nationally approved guidelines.
2.3. Isolating and culturing primary alveolar macrophages
The rats were given a pulmonary lavage, using 10-ml syringes with 22G needles, with sterile phosphate buffered saline (PBS; 5 × 10 ml). The pooled post-lavage fluids were centrifuged. The resulting cell pellet was suspended in RPMI medium (Gibco BRL, Life Technologies, Inc., Grand Island, NY) with 10% fetal bovine serum. The cells (1 × 10
6/ml) were then seeded in 24-well plates and 10-cm dishes. After 2 h pre-culture, non-adherent cells were removed before treatment by washing them three times with ice-cold PBS. More than 95% of the attached cells were macrophages (Lasbury et al., 2006).
2.4. Experimental design
Experiment I. The effects of sesamol on LPS-induced acute lung injury in rats. Thirty
rats were divided into five groups of six. Control (C)-group rats were intraperitoneally (i.p.) and subcutaneously (s.c.) injected with saline alone (1 ml/kg body weight); LPS-group rats were injected with LPS (10 mg/kg, dissolved in saline; i.p.) alone. The LS0.3-, LS1-, and LS3-group rats were injected with sesamol (0.3, 1, and 3 mg/kg, respectively, dissolved in saline; s.c.) immediately after the LPS injection. Cell counts and protein concentration as well as TNF-α and nitrite levels in bronchoalveolar lavage fluid (BALF) were assessed 4 h post-injection. Lung edema, histology, and TNF-α, nitrite, and iNOS levels in lung tissue were assessed 4 h after saline or LPS administration.
Experiment II. The effects of sesamol on the LPS-induced inflammatory response in rat primary alveolar macrophages. Rat alveolar macrophages were divided into six groups. C- group cells were treated with PBS only; LPS-group cells were treated with LPS (1 ng/ml) only; and LS30-, LS100-, LS300-, and LS1000-group cells were treated with LPS plus sesamol (30, 100, 300, and 1000 μM, respectively). TNF-α and nitrite in medium, as well as iNOS expression and NF-κB activation in macrophages were assessed 24 h after PBS or LPS administration.
2.5. Assessing pulmonary edema
The rats’ lungs were excised, immediately weighed, and then placed in an oven at 85°C for 48 h, at which point their dry weight was determined. Lung edema was evaluated using lung water content (LWC) derived using a formula (Ohkuda et al., 1982):
LWC (mg/mg dry lung weight) = ([wet lung weight] − [dry lung weight])/dry lung weight
2.6. Histological examination
Lung inflammation was further assessed using a histological examination. Briefly, lung
tissue was fixed in 4% formaldehyde buffered with a phosphate solution (0.1 M, pH 7.4) at
room temperature. Lung fragments were washed in PBS, dehydrated in graded concentrations
of ethanol, and then embedded in paraffin. From each tissue fragment, 4-μm-thin sections
were obtained and stained with hematoxylin and eosin to evaluate lung morphology (Sewerynek et al., 1995).
2.7. Collecting BALF
The lung was lavaged with 10 ml of PBS, through a 10-ml syringe with a 22G needle inserted into the trachea. The fluids were slowly instilled and withdrawn three times. Then the fluid was harvested by gentle aspiration. BALF was centrifuged (350 g for 10 min, 4°C) and the protein concentrations in supernatants were measured using a Coomassie (Bradford) protein assay kit (Bio-Rad Laboratories, Hercules, CA). The cell pellet was resuspended, and viable cells were counted using trypan blue dye exclusion.
2.8. Measuring TNF-
αlevels
TNF-α levels in BALF, lung tissue, and alveolar macrophage medium were
quantitatively measured using enzyme-linked immunosorbent assay (ELISA) kits (DuoSet;
R&D Systems Inc., Minneapolis, MN). Briefly, sample was incubated with biotinylated rabbit anti-rat TNF-α antibody for 2 h, and then streptavidine-conjugated horseradish peroxidase was added for 20 min. The peroxidase reaction was initiated by adding 3,3’,5,5’- tetramethylbenzidine/H
2O
2(R&D Systems) for 30 min, and then stopped by adding 0.5 M H
2SO
4. The absorbance was measured at 450 nm (Jinbo et al., 2002).
2.9. Measuring nitrite concentrations
Lung tissue was homogenized in Milli-Q water (1:5, w/v) and then centrifuged (12,000 g at 4°C for 30 min). The amounts of nitrite were measured after the Griess reaction by incubating 100 μl of BALF, tissue homogenate, or medium with 100 μl of Griess reagent (Sigma-Aldrich). The absorbance was measured at 550 nm.
2.10. Western Blotting
Fifty micrograms of protein was loaded on SDS-PAGE, and then transferred to
nitrocellulose sheets (NEN Life Science Products, Inc., Boston, MA). After blocking, the
blots were incubated with iNOS or G3PDH antibody (dilution 1:1000) (Santa Cruz
Biotechnology, Inc, Santa Cruz, CA) in 5% non-fat skim milk (using G3PDH as a loading control). After washed, the blots were incubated with anti-rabbit IgG conjugated with alkaline phosphatase (dilution 1:3000) (Jackson ImmunoResearch Laboratories, Inc., Philadelphia, PA). Immunoblots were developed using bromochloroindolyl
phosphate/nitroblue tetrazolium solution (Kirkegaard & Perry Laboratories, Inc., Baltimore, MD).
2.11. Assessing iNOS mRNA expression
Total cellular RNA was isolated using an RNA extraction kit (Pierce, Rockford, IL) according to the manufacturer’s instructions. RNA was reverse-transcribed (RT) from each sample using reverse transcriptase (ImProm-II; Promega, Madison, WI), dNTP, and oligo (dT15). Polymerase chain reaction (PCR) analyses were done on aliquots of the cDNA preparations to detect iNOS (using β-actin as an internal standard) gene expression, using a thermal cycler (Perkin Elmer Cetus, Foster City, CA). PCR was done using Taq DNA
polymerase, dNTP, reaction buffer, and primers. After they had been denatured at 95°C for 2 min, 30 amplification cycles were done for iNOS and β-actin (denaturation at 95°C for 1 min, annealing at 55°C for 1 min, and extension at 72°C for 1 min). The PCR primers used in this study are listed below:
sense strand iNOS, 5’-TTG GGT CTT AGC CTA GTC-3’;
anti-sense strand iNOS, 5’-TGT GCA GTC CCA GTG AGG AAC-3’;
sense strand β-actin, 5’-GAC TAC CTC ATG AAG ATC CT-3’;
antisense strand β-actin, 5’-CCA CAT CTG CTG GAA GGT GG-3’.
2.12. Measuring NF-
κB activation
Nuclear protein extraction kit (Sigma, Inc, St. Louis, MO) was used to isolate nuclear
protein. NF-κB was detected by the chemiluminescent NF-κB assay kits (Thermo Scientific,
Inc, Rockford, USA). In brief, nuclear protein was loaded to the 96-well plate and bound to the biotin-Duplex. After incubation, the primary antibody and the secondary antibody-HRP were added. And then, chemiluminescent substrate was added, and the luminescence was detected with Fluoroskan Ascent FL (Thermo Fisher Scientific Inc, Waltham, MA).
2.13. Statistical analysis
Data are means ± SD. One-way analysis of variance (ANOVA) and then Tukey’s Honestly Significant Difference post-hoc analysis were used to make comparisons between the treatments. Statistical significance was set at P < 0.05.
3. Results
3.1. The effects of sesamol on pulmonary injury in LPS-treated rats
To examine the effects of sesamol on systemic LPS-induced lung injury, pulmonary edema and histological changes were assessed. The weight gain in the lung tissue of rats treated with LPS only was significant compared with the gain in rats treated with 1- and 3- mg/kg doses of sesamol, which inhibited the weight gain (Fig. 1A). A histological
examination of lung tissue from the LPS group showed severe intra-alveolar infiltrates;
however, sesamol inhibited effect of the infiltration of LPS-induced inflammatory cells in the alveolar space in a dose response manner (Fig. 1B).
3.2. The effects of sesamol on the inflammatory indicators in BALF in LPS-treated rats
To assess the role of inflammation in sesamol-associated pulmonary protection, the cell
infiltration, protein leakage, and cytokine expression in BALF were assessed in LPS-treated
rats. Cell counts (Fig. 2A), and protein (Fig. 2B), TNF-α (Fig. 2C), and nitrite levels (Fig. 2D)
were significantly higher in the LPS group than in the C group and the three sesamol-treated
groups (except for the nitrite level in the LS0.3 group). This means that sesamol (0.3, 1, and 3
mg/kg) significantly inhibited inflammatory cell infiltration, protein leakage, and TNF-α
expression (Figs. 2A-2C), and that the two higher doses of sesamol (1 and 3 mg/kg) inhibited nitrite production (Fig. 2D).
3.3. The effects of sesamol on lung inflammation in LPS-treated rats
To further confirm the effects of sesamol on systemic LPS-induced lung inflammation, TNF-α, nitrite, and iNOS expression were determined in lung tissue. TNF-α (Fig. 3A), nitrite (Fig. 3B), and iNOS (Fig. 3C) expression levels were significantly higher in the LPS group than in controls. Sesamol significantly and dose-dependently inhibited these proinflammatory mediators (Fig. 3A-C)
3.4. The effects of sesamol on the expression of proinflammatory mediators in LPS-treated alveolar macrophages
To examine the possible role of alveolar macrophages in sesamol’s inhibitory effect on LPS-induced lung inflammation, we assessed TNF-α and nitrite levels in cell culture medium, as well as the expression of iNOS protein and mRNA in macrophages. TNF-α (Fig. 4A), nitrite (Fig. 4B), iNOS protein (Fig. 4C), and iNOS mRNA (Fig. 4D) expression levels were significantly higher in the LPS groups than in the C groups. Sesamol dose-dependently inhibited all tested parameters (Figs. 4A-4D). Further, LPS-induced NF-κB activation was significantly inhibited by sesamol (Fig. 5).
4. Discussion
We showed that sesamol inhibited LPS-induced pulmonary inflammation and injury in endotoxemic rats. It also reduced cell infiltration and protein leakage in BALF, and
downregulated TNF-α and nitric oxide production in both BALF and lung tissue.
Furthermore, sesamol downregulated TNF-α and nitric oxide release as well as the expression of iNOS protein and mRNA in LPS-treated alveolar macrophages.
The inhibition of lung inflammation may be crucial for protecting against exdotoxemia-
associated lung injury. Lung inflammatory responses and edema are positively correlated with pulmonary function, including airway pressure and the oxygenation index
(Mukhopadhyay et al., 2006; Cayabyab et al., 2007; Kullowatz et al., 2008). TNF-α and nitric oxide are important inflammatory mediators involved in the pathogenesis of pulmonary inflammation and injury in sepsis (Su et al., 2007; Togbe et al., 2007). Sesamol decreased LPS-induced lung edema, alveolar damage, as well as pulmonary TNF-α and nitric oxide production. We suggest that sesamol attenuates systemic LPS-induced pulmonary injury by inhibiting the pulmonary inflammatory response.
Inhibiting alveolar macrophage-associated nitric oxide production may be involved in sesamol’s anti-inflammatory effect in systemic LPS-induced lung inflammation and injury.
Inhibiting nitric oxide production attenuates lung inflammation and injury in various models (Kristof et al., 1998; Baron et al., 2004; Farley et al., 2008; Westphal et al., 2008; Kao and Chen, 2008). Further, very low level of lung inflammation was observed in LPS-challenged iNOS knock out mice compared with wild-type mice (Shanley et al., 2002). In the present study, sesamol significantly decreased iNOS expression and nitric oxide production in primary alveolar macrophages. It is likely that sesamol decreased LPS-induced lung
inflammation by inhibiting alveolar macrophage-associated iNOS activation and nitric oxide production, at least partially.
Inhibiting NF-κB activity in alveolar macrophages may contribute to the sesamol's inhibitory effect on iNOS expression. NF-κB is a dominant transcription factor responsible for inflammation. Once activated, NF-κB binds to DNA and transcripts various pro-
inflammatory genes, including cytokines and iNOS (Jung et al., 2007; Liu and Malik, 2006;
Jarrar et al., 2002). Sesamol significantly decreased LPS-induced NF-κB activation and
iNOS mRNA expression. Therefore, sesamol may decrease iNOS expression and nitric oxide
production by inhibiting NF-κB signal pathway. We conclude that sesamol may decrease
endotoxin-induced pulmonary inflammatory response and injury by inhibiting alveolar
macrophage NF-κB activation in rats. However, more investigation is needed to confirm this.
Acknowledgments
This research was supported by NSC 96-2628-B-006-038-MY3 from the National
Science Council, Taiwan.
參考文獻
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Figure legends
Fig. 1. Sesamol (SM) attenuated pulmonary edema and lung injury in lipopolysaccharide (LPS)-treated rats. Rats were divided into five groups of six: C-group rats were injected with saline only; LPS-group rats were injected with LPS (10 mg/kg; i.p.) only; LS0.3-, LS1-, and LS3-group rats were injected with LPS plus sesamol (0.3, 1, and 3 mg/kg, respectively; s.c.) immediately after the LPS injection. Lung edema and histology were assessed 4 h post- injection. Data are means ± standard deviation (SD). Significant differences between measurement traits were analyzed using one-way ANOVA. Different letters above the bars indicate statistically significant (P < 0.05) differences.
Fig. 2. Sesamol (SM) decreased inflammatory indicator levels in bronchoalveolar lavage fluid (BALF) in LPS-treated rat. Rats were divided into five groups of six: C-group rats were injected with saline only; LPS-group rats were injected with LPS (10 mg/kg; i.p.) only;
LS0.3-, LS1-, and LS3-group rats were injected with LPS plus sesamol (0.3, 1, and 3 mg/kg, respectively; s.c.) immediately after the LPS injection. BALF cell counts, and protein, tumor necrosis factor (TNF)-α, and nitrite levels were assessed 4 h post-injection. Data are means ± SD. Significant differences between measurement traits were analyzed using one-way
ANOVA. Different letters above the bars indicate statistically significant (P < 0.05) differences.
Fig. 3. Sesamol (SM) decreased inflammatory indicator levels in the lungs of LPS-treated rat.
Thirty rats were divided into five groups of six: C-group rats were injected with saline only;
LPS-group rats were injected with LPS (10 mg/kg; i.p.) only; LS0.3-, LS1-, and LS3-group
rats were injected with LPS plus sesamol (0.3, 1, and 3 mg/kg, respectively; s.c.) immediately
after the LPS injection. Tumor necrosis factor (TNF)-α, nitrite, and inducible nitric oxide
synthase (iNOS) expression levels in lung tissue were assessed 4 h post-injection. Data are
means ± SD. Significant differences between measurement traits were analyzed using one-
way ANOVA. Different letters above the bars indicate statistically significant (P < 0.05) differences.
Fig. 4. Sesamol (SM) attenuated the inflammatory response in LPS-treated alveolar macrophages. Primary alveolar macrophages were divided into six groups of six: C-group macrophages were treated with PBS only; LPS-group macrophages were treated with LPS (1 ng/ml) only; LS30-, LS100-, LS300-, and LS1000-group macrophages were treated with LPS plus sesamol (30, 100, 300, and 1000 μM, respectively). Tumor necrosis factor (TNF)-α and nitrite levels in medium, as well as inducible nitric oxide synthase (iNOS) protein and mRNA expression levels in cells, were assessed 24 h post-treatment. Data are means ± SD.
Significant differences between measurement traits were analyzed using one-way ANOVA.
Different letters above the bars indicate statistically significant (P < 0.05) differences.
Fig. 5. Sesamol (SM) decreased the LPS-induced nuclear factor (NF)-κB activation in
alveolar macrophages. Primary alveolar macrophages were divided into six groups of five: C-
group macrophages were treated with PBS only; LPS-group macrophages were treated with
LPS (1 ng/ml) only; LS30-, LS100-, LS300-, and LS1000-group macrophages were treated
with LPS plus sesamol (30, 100, 300, and 1000 μM, respectively). NF-κB activation was
assessed 24 h post-treatment. Data are means ± standard error of the mean. Significant
differences between measurement traits were analyzed using one-way ANOVA. Different
letters above the bars indicate statistically significant (P < 0.05) differences.
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
國科會補助專題研究計畫成果報告自評表
請就研究內容與原計畫相符程度、達成預期目標情況、研究成果之學術或 應用價值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能 性）、是否適合在學術期刊發表或申請專利、主要發現或其他有關價值 等，作一綜合評估。
1. 請就研究內容與原計畫相符程度、達成預期目標情況作一綜合評估
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說明：本研究的成果與原計畫相符，研究結果達成目標。
2. 研究成果在學術期刊發表或申請專利等情形：
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其他：本研究的成果與原計畫相符，研究結果達成目標。
3. 請依學術成就、技術創新、社會影響等方面，評估研究成果之學術或應 用價值（簡要敘述成果所代表之意義、價值、影響或進一步發展之可能 性）（以 500 字為限）
本人所進行的以芝麻酚對抗細菌內毒素所引發的敗血症，是一系列強 調 從 環 境 教 育 到 臨 床 治 療 的 多 元 主 題 。 芝 麻 酚 抑 制 pro-inflammatory cytokine 的作用可能來自於其與 LPS binding protein (LBP)的競爭性拮抗，
LBP 在敗血症的發生過程中扮演相當重要的角色，減少 LBP 與細菌的內 毒素的結合，可使得內毒素的作用降低而達到保護作用。芝麻酚也可以透 過抑制巨噬細胞的 nuclear factor-kappa B，而降低 LPS 引發的發炎物質產 生。本計畫成果豐富、研究思路清晰、能達成預期目標，也順利將三年計 畫完成並發表在國際期刋上。本人的研究成果，陸續被發表在國際期刊 上。此計畫工作的主軸除了環繞在環境毒物研究之外，更發展出獨特並兼 具創新性的實證醫學研究，尤其以產官學合作為目標，以做出具有獨特性 的研究，並有十足的信心要在臺灣完成優秀的研究。
