j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
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ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Patchouli alcohol protects against lipopolysaccharide-induced acute lung injury in mice Jin-Long Yu,a Xiao-Shi Zhang, MD,b Xia Xue,a and Rong-Mei Wang, MDa,* a b
Department of Pharmacy, The Second Hospital of Shandong University, Jinan, Shandong, China Department of Clinical Laboratory, Qilu Hospital, Shandong University, Jinan, Shandong, China
article info
abstract
Article history:
Background: Patchouli alcohol (PA), a natural compound isolated from Pogostemon cablin, has
Received 12 August 2014
been reported to possess anti-inflammatory activity. However, the effects of PA on lipo-
Received in revised form
polysaccharide (LPS)-induced acute lung injury (ALI) have not yet been studied. In the
9 October 2014
present study, we investigated in vivo the effect of PA on ALI induced by LPS.
Accepted 17 October 2014
Methods: Mice were administrated intranasally with LPS to induce lung injury. PA was
Available online 22 October 2014
administrated intraperitoneally 1 h before or after the LPS challenge. Results: The results showed that PA significantly decreased the wet-to-dry weight ratio of
Keywords:
lungs and the number of total cells, neutrophils, and macrophages in bronchoalveolar
Patchouli alcohol
lavage fluid at 7 h after the LPS challenge. In addition, PA also suppressed the production of
Acute lung injury
inflammatory cytokines, such as tumor necrosis factor-a, interleukin-1b, and interleukin-6
LPS
in bronchoalveolar lavage fluid. Furthermore, Western blot analysis showed that PA inhibited the phosphorylation of IkB-a and p65 nuclear factor kB (NF-kB) induced by LPS.
NF-kB
Conclusions: Our results suggest that the anti-inflammatory effects of PA against LPS-induced ALI may be due to its ability to inhibit NF-kB signaling pathways. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
The acute respiratory distress syndrome, a clinically important complication of severe acute lung injury (ALI) in humans, is a significant cause of morbidity and mortality in critically ill patients [1]. It is characterized by intense pulmonary inflammatory responses, involving neutrophil recruitment, interstitial edema, disruption of epithelial integrity, and lung parenchymal injury [2,3]. Lipopolysaccharide (LPS), a main component of the outer membrane of gram-negative bacteria, is one of the major factors that induce ALI [4]. Stimulating lung epithelial cells and macrophages by LPS induces the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1b. These
proinflammatory mediators lead to lung inflammation and lung tissue injury [5]. The mouse model of LPS-induced ALI has been used for preliminary pharmacologic studies of potential therapeutic drugs and agents [6,7]. Patchouli alcohol (PA; Fig. 1), a tricyclic sesquiterpene isolated from Pogostemonis Herba, is known to possess a number of pharmacologic activities, such as antioxidant, antitumor, and anti-inflammatory effects [8,9]. PA was found to inhibit TNF-a, IL-1b, and IL-6 production in LPS-stimulated RAW264.7 cells and IL-6 production in TNF-a stimulated HT-29 cells [8,10]. In addition, PA had been reported to have antiinflammatory effects in xylene-induced ear edema in mice and carrageenan-induced paw edema in rats [11]. However, whether PA has the ability to attenuate LPS-induced ALI and
* Corresponding author. Department of Pharmacy, The Second Hospital of Shandong University, Jinan, Shandong 250033, China. Tel.: 0531-85875326; fax: 0531-85875326. E-mail address: [email protected] (R.-M. Wang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.026
538
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
Fig. 1 e Chemical structure of PA.
its underlying molecular mechanisms remains unclear. In this study, we sought to assess the effects of PA on LPS-induced mouse ALI model and elucidate the potential antiinflammatory mechanism.
2.
Materials and methods
2.1.
Materials and chemicals
LPS) þ LPS group, pyrrolidine dithiocarbamate (PDTC) (nuclear factor kB [NF-kB] antagonist) (100 mg/kg) þ LPS group, and PDTC (100 mg/kg) þ PA (40 mg/kg) þ LPS group. DEX was used in this study as a positive control, and the dose of DEX used in this study was based on previous studies [12]. PA (10, 20, and 40 mg/kg), PDTC (100 mg/kg), and DEX (5 mg/kg) were given intraperitoneally. DEX has been reported to have a well protective effect on LPS-induced ALI, and many studies used DEX as a positive control. PA was suspended in 0.1% Tween 80 in phosphate-buffered saline (PBS) [11]. Mice from the control and LPS groups received an equal volume of 0.1% Tween 80 instead of PA or DEX. One hour later, mice were slightly anesthetized with an inhalation of diethyl ether, 10 mg of LPS in 50-mL PBS was instilled intranasally to induce lung injury. Control mice were given 50-mL PBS without LPS. All the mice were alive after 7 h of LPS treatment. Collection of bronchoalveolar lavage fluid (BALF) was performed three times through a tracheal cannula with autoclaved PBS, instilled up to a total volume of 1.3 mL.
2.4.
After the mice were euthanized, the lungs were excised, blotted dry, weighed to obtain the “wet” weight, and then placed in an oven at 60 C for 24 h to obtain the “dry” weight. The ratio of wet lung to dry lung was calculated to assess tissue edema.
2.5. PA was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Enzyme-linked immunosorbent assay (ELISA) kits of TNF-a, IL-6, and IL-1b were purchased from R&D Corporation (R&D Systems Inc, Minneapolis, MN). Anti-pNF-kB p65, anti-NF-kB p65, anti-IkBa, anti-pIkBa, anti-Lamin B, and antib-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other chemicals were of reagent grade.
2.2.
Animals
Male BALB/c mice, 6e8 wk, weighing approximately 18e20 g were purchased from the Experimental Animal Center of Shandong University (Shandong, China). The mice were housed in microisolator cages and received food and water. The laboratory temperature was 24 1 C, and relative humidity was 40%e80%. Mice were housed for 4e6 d to adapt the environment before experimentation. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Inflammatory cell counts of BALF
The fluid recovered from each sample was centrifuged (4 C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemacytometer. Cytospins were prepared for differential cell counts by staining with the WrighteGiemsa staining method.
2.6.
Cytokine ELISA
Levels of TNF-a, IL-6, and IL-1b in the supernatants of the BALF were determined using commercially available ELISA kits according to the manufacturer’s instructions.
2.7.
Pulmonary MPO activity in ALI mice
The accumulation of neutrophils in the lung tissue was assessed by MPO activity. Briefly, the lung tissue samples were frozen and homogenized in cool normal saline (lung tissue to normal saline 1:10). Then, the homogenate was done according to the manufacturer’s instructions. MPO activity was measured with a spectrophotometer at 460 nm.
2.8. 2.3.
Lung wet-to-dry weight ratio measurement
Histologic study
LPS-induced ALI in mice
After adjustment to the environment, 108 male BALB/c mice were randomly divided into eight groups, and each group contained 12 mice as follows: control group, LPS group, PA (10, 20, and 40 mg/kg; 1 h before LPS) þ LPS groups, dexamethasone (DEX; 1 h before LPS) þ LPS group, PA (40 mg/kg; 1 h after
Histopathologic examination was performed on mice that were not subjected to BALF collection. The lung tissues were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. After deparaffinization and dehydration, the lungs were cut into 5-mm sections and stained with hematoxylin and eosin.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
539
Fig. 2 e Effects of PA on the lung W/D ratio of LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. The lung W/D ratio was determined at 7 h after the LPS challenge. The values presented are the means ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
2.9.
Western blot analysis
Lung tissues were harvested and frozen in liquid nitrogen immediately until homogenization 7 h after the injection of LPS. Proteins were extracted from the lungs using T-PER Tissue Protein Extraction Reagent Kit (Thermo, USA) according to the manufacturer’s instructions. Nuclear and cytoplasmic proteins were extracted from the lungs using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo) according to the manufacturer’s protocol. The phosphorylase inhibitor was added. Protein concentrations were determined by the BCA protein assay kit, and equal amounts of protein were loaded per well on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto the polyvinylidene difluoride membrane. The membranes were washed with phosphate belanced solution-T (PBST) and 5% skim milk for 1 h at room temperature. After three washes with PBST, the membranes were incubated with primary antibody diluted 1:1000 in PBST overnight at room temperature. After three further washes, a secondary antibody was added at 1/1000 dilution in PBST and incubated for 1 h at room temperature. The immunoactive proteins were detected using an enhanced chemiluminescence Western blotting detection kit.
2.10.
Statistical analysis
All values are expressed as means standard error of the mean. Differences between multiple treatment groups were evaluated by analysis of variance, followed by post hoc
Fig. 3 e Effects of PA on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. BALF was collected at 7 h after LPS administration to measure the number of total cells (A), neutrophils (B), and macrophage (C). The values presented are the mean ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
540
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
Fig. 5 e Effects of PA on MPO activity in lung tissues of LPSinduced ALI. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. MPO activity was determined at 7 h after LPS administration. The values presented are mean ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS group.
Bonferroni test, and considered significant at P < 0.05 or P < 0.01.
3.
Results
3.1. Effects of PA on LPS-induced lung wet-to-dry weight ratio in mice
Fig. 4 e Effects of PA on the production of inflammatory cytokine TNF-a, IL-1ß, and IL-6 in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. BALF was collected at 7 h after the LPS challenge to analyze the inflammatory cytokines TNF-a (A), IL-1ß (B), and IL-6 (C). The values presented are mean ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
LPS administration produced a significant increase in capillary leakage, as shown by the lung wet-to-dry weight (W/D) ratio. The results showed that the lung W/D ratio increased significantly after the LPS challenge compared with those of the control group. Pretreatment of PA (10, 20, and 40 mg/kg), DEX, and PDTC significantly decreased the lung W/D ratio (P < 0.05) compared with those in the LPS group (Fig. 2). However, pretreatment of both PA and PDTC most strongly inhibited LPSinduced lung W/D ratio. Treatment of PA (40 mg/kg) also inhibited LPS-induced lung W/D ratio, and the inhibition was weaker than pretreatment of PA (40 mg/kg).
3.2. Effects of PA on inflammatory cell counts in BALF of LPS-induced ALI mice Seven hours after the challenge with LPS, the number of inflammatory cells in BALF, such as neutrophils and macrophages, significantly increased compared with the control group (P < 0.01; Fig. 3). Pretreatment of PA (10, 20, and 40 mg/ kg), DEX (5 mg/kg), or PDTC (100 mg/kg) was found to significantly decrease the number of total cells (P < 0.01),
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
541
Fig. 6 e Effects of PA on histopathologic changes in lung tissues in LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. Lungs from each experimental group were processed for histologic evaluation at 7 h after the LPS challenge. Representative histologic changes of lungs obtained from mice of different groups. (A) Control group, (B) LPS group, (C) LPS D DEX group, (D) LPS D PA (10 mg/kg) group, (E) LPS D PA (20 mg/kg) group, (F): LPS D PA (40 mg/kg) group, (G) LPS D PA (40 mg/kg) (1 h after LPS), (H) LPS D PDTC group, (I): LPS D PDTC D PA (hematoxylin and eosin staining, magnification 3200). (Color version of the figure is available online.)
neutrophils (P < 0.01), and macrophages (P < 0.01). Treatment of PA (40 mg/kg) also inhibited LPS-induced inflammatory cells, and the inhibition was weaker than pretreatment of PA (40 mg/kg). Furthermore, pretreatment of both PA and PDTC most strongly inhibited LPS-induced inflammatory cells.
(Fig. 4). We also found that treatment of PA also inhibited LPSinduced inflammatory cytokine production, and the inhibition was weaker than pretreatment of PA (40 mg/kg). In addition, we found that pretreatment of both PA and PDTC most strongly inhibited production of LPS-induced inflammatory cytokines TNF-a, IL-6, and IL-1b.
3.3. Effects of PA on cytokine production in the BALF of LPS-treated ALI mice
3.4.
The concentrations of TNF-a, IL-6, and IL-1b in BALF were measured by ELISA. The levels of TNF-a, IL-6, and IL-1b in BALF were increased by the LPS challenge compared with those in the control group (P < 0.01; Fig. 4). Pretreatment of PA (10, 20, and 40 mg/kg), DEX (5 mg/kg), or PDTC (100 mg/kg) markedly reduced the production of TNF-a (P < 0.01), IL-6, and IL-1b (P < 0.01 or P < 0.05) compared with those in the LPS group
Effects of PA on the MPO activity in mice
To assess the neutrophil accumulation in the lung tissues, MPO activity was measured. As shown in Figure 5, LPS challenge resulted in significant increases in the lung MPO activity compared with the control group (P < 0.01). Pretreatment of PA (10, 20, and 40 mg/kg), DEX (5 mg/kg), or PDTC (100 mg/kg) obviously inhibited the increased MPO activity induced by LPS (P < 0.01). We also found that treatment of PA also inhibited LPS-induced MPO activity, and the inhibition was weaker than
542
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Fig. 7 e PA pretreatment inhibited LPS-induced NF-kB activation with Western blotting. #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
pretreatment of PA (40 mg/kg). In addition, we found that pretreatment of both PA and PDTC most strongly inhibited LPS-induced MPO activity.
3.5. Effects of PA on LPS-mediated lung histopathologic changes To evaluate the histologic changes after PA treatment in LPStreated mice, lung sections were subjected to hematoxylin and eosin staining. As shown in Figure 6, LPS administration after 7 h induced pulmonary edema, infiltration of inflammatory cells in the lungs tissues, and alveolar damage (Fig. 6B). However, LPS-induced pathologic changes were significantly attenuated by DEX (5 mg/kg; Fig. 6C), PA (10, 20, and 40 mg/kg; Fig. 6DeF), PA (40 mg/kg, 1 h after LPS; Fig. 6G), PDTC (Fig. 6H), and PDTC þ PA (Fig. 6I).
3.6. Effect of PA on NF-kB activation in ALI mice induced by LPS Western blot analysis showed that phosphorylation of IkB-a was markedly increased after LPS induction compared with the control group. However, treatment with PA significantly inhibited these upregulations by LPS (Fig. 7). LPS-treated mice also displayed that p65 subunit of NF-kB increased in nucleus and decreased in cytoplasm, and the tendency was reversed by PA (10, 20, and 40 mg/kg) and DEX (5 mg/kg).
4.
Discussion
PA, a natural compound of Pogostemon cablin, has been shown to possess anti-inflammatory effects [8]. In the present study, we demonstrated that pretreatment or treatment with PA prevented LPS-induced ALI in mice. This was the first time to
study the anti-inflammatory effect of PA on LPS-induced lung injury. Moreover, we found that the protective effects of PA on LPS-induced lung injury were due to its ability to inhibit NF-kB signaling pathway. Edema is one of the major characteristics of ALI [13,14]. In this study, lung W/D ratio was used to quantify the magnitude of pulmonary edema. The results demonstrated that PA decreased the lung W/D ratio, indicting that PA significantly inhibited edema of the lung. LPS-induced ALI is characterized by the infiltration of neutrophils in the lungs, exhibiting increased MPO activity [15,16]. Our results showed that pretreatment with PA decreased LPS-induced increases in MPO activity in the lungs. In addition, the lung histologic examination also indicated that lung tissues infused with LPS generated serious inflammatory responses with a large number of infiltrating polymorphonuclear. The treatment group reduced the number of infiltrating polymorphonuclear, especially the treatment group of 40 mg/kg, suggesting that PA could ameliorate the damage of lung tissues. LPS is known to induce the productions of several inflammatory cytokines. Studies showed that proinflammatory cytokines, including TNF-a, IL-1ß, and IL-6, were important mediators in the pathogenesis of ALI [17,18]. Elevated TNF-a, IL-1ß, and IL-6 levels in BALF were observed in patients with ALI or acute respiratory distress syndrome [19,20]. Thus, to explore the protective or treatment effects of PA on LPS-induced ALI, the effects of PA on LPS-induced cytokines were detected. Our results showed that pretreatment or treatment of PA significantly inhibited the production of TNF-a, IL-1b, and IL-6 induced by LPS. The inhibition of inflammatory cytokines by the treatment of PA (40 mg/kg) was a little weaker than that by the pretreatment of PA (40 mg/kg). These results indicated that the protective and treatment effects of PA on LPS-induced ALI may be attributed to the inhibition of inflammatory cytokines production.
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
NF-kB pathway is known to play an important role in the development of ALI [21,22]. Normally, NF-kB is present in the cytoplasm as a heterodimer and is linked to the inhibitory proteins IkBs. Once stimulated by LPS, NF-kB is activated by phosphorylation, enters the nucleus, and regulates the expression of inflammatory cytokines, such as TNF-a, IL-1b, and IL-6 [23,24]. To detect the inhibitory mechanisms of TNFa, IL-1b, and IL-6 production by PA, the effects of PA on NF-kB activation and IkB-a degradation induced by LPS were detected. The results showed that LPS-induced NF-kB activation in the lung tissue was inhibited by pretreatment of PA or PDTC (an NF-kB inhibitor). In addition, we found that pretreatment of both PA and PDTC most strongly inhibited production of LPS-induced inflammatory cytokines TNF-a, IL-6, and IL-1b. These results suggested that the inhibition of NF-kB activation was a mechanism for the anti-inflammatory effects of PA.
5.
Conclusions
[5]
[6]
[7]
[8]
[9]
[10]
In conclusion, we have provided the evidence that pretreatment of PA significantly attenuated LPS-induced ALI in mice, and that the potential mechanism of this action was through its suppression of NF-kB, and subsequently leading to the inhibition of inflammatory cytokines. These evidences suggest that PA has a potential application to treat LPS-induced ALI.
[11]
Acknowledgment
[14]
Authors’ contributions: J.-L.Y. and R.-M.W. contributed to the conception and design. J.-L.Y. and X.-S.Z. did the analysis and interpretation. X.X. did the data collection. J.-L.Y. and R.-M.W. wrote the article, did the critical revision of the article, and obtained the funding.
[12]
[13]
[15] [16]
[17]
Disclosure This study was supported by the Youth Foundation of the Second Hospital of Shandong University (No. Y2013010077) and the science and technology development project of Shandong Administration of traditional Chinese medicine (No.2013-181).
references
[1] Montgomery AB, Stager MA, Carrico CJ, Hudson LD. Causes of mortality in patients with the adult respiratory distress syndrome. Am Rev Respir Dis 1985;132:485. [2] Ware LB. Pathophysiology of acute lung injury and the acute respiratory distress syndrome. Semin Respir Crit Care Med 2006;27:337. [3] Caironi P, Langer T, Gattinoni L. Acute lung injury/acute respiratory distress syndrome pathophysiology: what we have learned from computed tomography scanning. Curr Opin Crit Care 2008;14:64. [4] Kawasaki M, Kuwano K, Hagimoto N, et al. Protection from lethal apoptosis in lipopolysaccharide-induced acute lung
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injury in mice by a caspase inhibitor. Am J Pathol 2000;157: 597. Medzhitov R, Kagan JC. Phosphoinositide-mediated adaptor recruitment controls toll-like receptor signaling. Cell 2006; 125:943. Yunhe F, Bo L, Xiaosheng F, et al. The effect of magnolol on the toll-like receptor 4/nuclear factor kappa B signaling pathway in lipopolysaccharide-induced acute lung injury in mice. Eur J Pharmacol 2012;689:255. Huo MX, Chen N, Chi GF, et al. Traditional medicine alpinetin inhibits the inflammatory response in Raw 264.7 cells and mouse models. Int Immunopharmacol 2012;12:241. Xian YF, Li YC, Ip SP, Lin ZX, Lai XP, Su ZR. Antiinflammatory effect of patchouli alcohol isolated from Pogostemonis Herba in LPS-stimulated RAW264.7 macrophages. Exp Ther Med 2011;2:545. Jeong JB, Choi J, Lou ZY, Jiang XJ, Lee SH. Patchouli alcohol, an essential oil of Pogostemon cablin, exhibits anti-tumorigenic activity in human colorectal cancer cells. Int Immunopharmacol 2013;16:184. Jeong JB, Shin YK, Lee SH. Anti-inflammatory activity of patchouli alcohol in RAW264.7 and HT-29 cells. Food Chem Toxicol 2013;55:229. Li YC, Xian YF, Ip SP, et al. Anti-inflammatory activity of patchouli alcohol isolated from Pogostemonis Herba in animal models. Fitoterapia 2011;82:1295. Huo M, Cui X, Xue J, et al. Anti-inflammatory effects of linalool in RAW 264.7 macrophages and lipopolysaccharideinduced lung injury model. J Surg Res 2013;180:e47. 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. Rana R, Fernandez-Perez ER, Khan SA, et al. Transfusionrelated acute lung injury and pulmonary edema in critically ill patients: a retrospective study. Transfusion 2006;46:1478. Lee WL, Downey GP. Neutrophil activation and acute lung injury. Curr Opin Crit Care 2001;7:1. Zmijewski JW, Lorne E, Zhao X, et al. Antiinflammatory effects of hydrogen peroxide in neutrophil activation and acute lung injury. Am J Respir Crit Care Med 2009;179:694. Goodman RB, Pugin J, Lee JS, Matthay MA. Cytokinemediated inflammation in acute lung injury. Cytokine Growth Factor Rev 2003;14:523. Welbourn CR, Young Y. Endotoxin, septic shock and acute lung injury: neutrophils, macrophages and inflammatory mediators. Br J Surg 1992;79:998. Park WY, Goodman RB, Steinberg KP. Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 2001;164:1896. Ricard JD, Dreyfuss D, Saumon G. Production of inflammatory cytokines in ventilator-induced lung injury: a reappraisal. Am J Respir Crit Care Med 2001;163:1176. Blackwell TS, Debelak JP, Venkatakrishnan A. Acute lung injury after hepatic cryoablation: correlation with NFkappa B activation and cytokine production. Surgery 1999; 126:518. Liu Y, Wu H, Nie YC, Chen JL, Su WW, Li PB. Naringin attenuates acute lung injury in LPS-treated mice by inhibiting NF-kappaB pathway. Int Immunopharmacol 2011; 11:1606. Gilmore TD. Introduction to NF-kappaB: players, pathways, perspectives. Oncogene 2006;25:6680. Diya Z, Lili C, Shenglai L, Zhiyuan G, Jie Y. Lipopolysaccharide (LPS) of Porphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 production by THP-1 cells in a way different from that of Escherichia coli LPS. Innate Immun 2008;14:99.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.JournalofSurgicalResearch.com
Patchouli alcohol protects against lipopolysaccharide-induced acute lung injury in mice Jin-Long Yu,a Xiao-Shi Zhang, MD,b Xia Xue,a and Rong-Mei Wang, MDa,* a b
Department of Pharmacy, The Second Hospital of Shandong University, Jinan, Shandong, China Department of Clinical Laboratory, Qilu Hospital, Shandong University, Jinan, Shandong, China
article info
abstract
Article history:
Background: Patchouli alcohol (PA), a natural compound isolated from Pogostemon cablin, has
Received 12 August 2014
been reported to possess anti-inflammatory activity. However, the effects of PA on lipo-
Received in revised form
polysaccharide (LPS)-induced acute lung injury (ALI) have not yet been studied. In the
9 October 2014
present study, we investigated in vivo the effect of PA on ALI induced by LPS.
Accepted 17 October 2014
Methods: Mice were administrated intranasally with LPS to induce lung injury. PA was
Available online 22 October 2014
administrated intraperitoneally 1 h before or after the LPS challenge. Results: The results showed that PA significantly decreased the wet-to-dry weight ratio of
Keywords:
lungs and the number of total cells, neutrophils, and macrophages in bronchoalveolar
Patchouli alcohol
lavage fluid at 7 h after the LPS challenge. In addition, PA also suppressed the production of
Acute lung injury
inflammatory cytokines, such as tumor necrosis factor-a, interleukin-1b, and interleukin-6
LPS
in bronchoalveolar lavage fluid. Furthermore, Western blot analysis showed that PA inhibited the phosphorylation of IkB-a and p65 nuclear factor kB (NF-kB) induced by LPS.
NF-kB
Conclusions: Our results suggest that the anti-inflammatory effects of PA against LPS-induced ALI may be due to its ability to inhibit NF-kB signaling pathways. ª 2015 Elsevier Inc. All rights reserved.
1.
Introduction
The acute respiratory distress syndrome, a clinically important complication of severe acute lung injury (ALI) in humans, is a significant cause of morbidity and mortality in critically ill patients [1]. It is characterized by intense pulmonary inflammatory responses, involving neutrophil recruitment, interstitial edema, disruption of epithelial integrity, and lung parenchymal injury [2,3]. Lipopolysaccharide (LPS), a main component of the outer membrane of gram-negative bacteria, is one of the major factors that induce ALI [4]. Stimulating lung epithelial cells and macrophages by LPS induces the production of proinflammatory cytokines, such as tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-1b. These
proinflammatory mediators lead to lung inflammation and lung tissue injury [5]. The mouse model of LPS-induced ALI has been used for preliminary pharmacologic studies of potential therapeutic drugs and agents [6,7]. Patchouli alcohol (PA; Fig. 1), a tricyclic sesquiterpene isolated from Pogostemonis Herba, is known to possess a number of pharmacologic activities, such as antioxidant, antitumor, and anti-inflammatory effects [8,9]. PA was found to inhibit TNF-a, IL-1b, and IL-6 production in LPS-stimulated RAW264.7 cells and IL-6 production in TNF-a stimulated HT-29 cells [8,10]. In addition, PA had been reported to have antiinflammatory effects in xylene-induced ear edema in mice and carrageenan-induced paw edema in rats [11]. However, whether PA has the ability to attenuate LPS-induced ALI and
* Corresponding author. Department of Pharmacy, The Second Hospital of Shandong University, Jinan, Shandong 250033, China. Tel.: 0531-85875326; fax: 0531-85875326. E-mail address: [email protected] (R.-M. Wang). 0022-4804/$ e see front matter ª 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2014.10.026
538
j o u r n a l o f s u r g i c a l r e s e a r c h 1 9 4 ( 2 0 1 5 ) 5 3 7 e5 4 3
Fig. 1 e Chemical structure of PA.
its underlying molecular mechanisms remains unclear. In this study, we sought to assess the effects of PA on LPS-induced mouse ALI model and elucidate the potential antiinflammatory mechanism.
2.
Materials and methods
2.1.
Materials and chemicals
LPS) þ LPS group, pyrrolidine dithiocarbamate (PDTC) (nuclear factor kB [NF-kB] antagonist) (100 mg/kg) þ LPS group, and PDTC (100 mg/kg) þ PA (40 mg/kg) þ LPS group. DEX was used in this study as a positive control, and the dose of DEX used in this study was based on previous studies [12]. PA (10, 20, and 40 mg/kg), PDTC (100 mg/kg), and DEX (5 mg/kg) were given intraperitoneally. DEX has been reported to have a well protective effect on LPS-induced ALI, and many studies used DEX as a positive control. PA was suspended in 0.1% Tween 80 in phosphate-buffered saline (PBS) [11]. Mice from the control and LPS groups received an equal volume of 0.1% Tween 80 instead of PA or DEX. One hour later, mice were slightly anesthetized with an inhalation of diethyl ether, 10 mg of LPS in 50-mL PBS was instilled intranasally to induce lung injury. Control mice were given 50-mL PBS without LPS. All the mice were alive after 7 h of LPS treatment. Collection of bronchoalveolar lavage fluid (BALF) was performed three times through a tracheal cannula with autoclaved PBS, instilled up to a total volume of 1.3 mL.
2.4.
After the mice were euthanized, the lungs were excised, blotted dry, weighed to obtain the “wet” weight, and then placed in an oven at 60 C for 24 h to obtain the “dry” weight. The ratio of wet lung to dry lung was calculated to assess tissue edema.
2.5. PA was purchased from Santa Cruz Biotechnology, Inc (Santa Cruz, CA). Enzyme-linked immunosorbent assay (ELISA) kits of TNF-a, IL-6, and IL-1b were purchased from R&D Corporation (R&D Systems Inc, Minneapolis, MN). Anti-pNF-kB p65, anti-NF-kB p65, anti-IkBa, anti-pIkBa, anti-Lamin B, and antib-actin monoclonal antibodies were purchased from Santa Cruz Biotechnology Inc (Santa Cruz, CA). The myeloperoxidase (MPO) determination kit was provided by the Jiancheng Bioengineering Institute of Nanjing (Nanjing, Jiangsu, China). All other chemicals were of reagent grade.
2.2.
Animals
Male BALB/c mice, 6e8 wk, weighing approximately 18e20 g were purchased from the Experimental Animal Center of Shandong University (Shandong, China). The mice were housed in microisolator cages and received food and water. The laboratory temperature was 24 1 C, and relative humidity was 40%e80%. Mice were housed for 4e6 d to adapt the environment before experimentation. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Inflammatory cell counts of BALF
The fluid recovered from each sample was centrifuged (4 C, 3000 rpm, 10 min) to pellet the cells. The cell pellets were resuspended in PBS for total cell counts using a hemacytometer. Cytospins were prepared for differential cell counts by staining with the WrighteGiemsa staining method.
2.6.
Cytokine ELISA
Levels of TNF-a, IL-6, and IL-1b in the supernatants of the BALF were determined using commercially available ELISA kits according to the manufacturer’s instructions.
2.7.
Pulmonary MPO activity in ALI mice
The accumulation of neutrophils in the lung tissue was assessed by MPO activity. Briefly, the lung tissue samples were frozen and homogenized in cool normal saline (lung tissue to normal saline 1:10). Then, the homogenate was done according to the manufacturer’s instructions. MPO activity was measured with a spectrophotometer at 460 nm.
2.8. 2.3.
Lung wet-to-dry weight ratio measurement
Histologic study
LPS-induced ALI in mice
After adjustment to the environment, 108 male BALB/c mice were randomly divided into eight groups, and each group contained 12 mice as follows: control group, LPS group, PA (10, 20, and 40 mg/kg; 1 h before LPS) þ LPS groups, dexamethasone (DEX; 1 h before LPS) þ LPS group, PA (40 mg/kg; 1 h after
Histopathologic examination was performed on mice that were not subjected to BALF collection. The lung tissues were fixed with 4% paraformaldehyde for 24 h and embedded in paraffin. After deparaffinization and dehydration, the lungs were cut into 5-mm sections and stained with hematoxylin and eosin.
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Fig. 2 e Effects of PA on the lung W/D ratio of LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. The lung W/D ratio was determined at 7 h after the LPS challenge. The values presented are the means ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
2.9.
Western blot analysis
Lung tissues were harvested and frozen in liquid nitrogen immediately until homogenization 7 h after the injection of LPS. Proteins were extracted from the lungs using T-PER Tissue Protein Extraction Reagent Kit (Thermo, USA) according to the manufacturer’s instructions. Nuclear and cytoplasmic proteins were extracted from the lungs using NE-PER Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo) according to the manufacturer’s protocol. The phosphorylase inhibitor was added. Protein concentrations were determined by the BCA protein assay kit, and equal amounts of protein were loaded per well on a 10% sodium dodecyl sulfate polyacrylamide gel and transferred onto the polyvinylidene difluoride membrane. The membranes were washed with phosphate belanced solution-T (PBST) and 5% skim milk for 1 h at room temperature. After three washes with PBST, the membranes were incubated with primary antibody diluted 1:1000 in PBST overnight at room temperature. After three further washes, a secondary antibody was added at 1/1000 dilution in PBST and incubated for 1 h at room temperature. The immunoactive proteins were detected using an enhanced chemiluminescence Western blotting detection kit.
2.10.
Statistical analysis
All values are expressed as means standard error of the mean. Differences between multiple treatment groups were evaluated by analysis of variance, followed by post hoc
Fig. 3 e Effects of PA on the number of total cells, neutrophils, and macrophages in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. BALF was collected at 7 h after LPS administration to measure the number of total cells (A), neutrophils (B), and macrophage (C). The values presented are the mean ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
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Fig. 5 e Effects of PA on MPO activity in lung tissues of LPSinduced ALI. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. MPO activity was determined at 7 h after LPS administration. The values presented are mean ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, **P < 0.01 versus LPS group.
Bonferroni test, and considered significant at P < 0.05 or P < 0.01.
3.
Results
3.1. Effects of PA on LPS-induced lung wet-to-dry weight ratio in mice
Fig. 4 e Effects of PA on the production of inflammatory cytokine TNF-a, IL-1ß, and IL-6 in the BALF of LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. BALF was collected at 7 h after the LPS challenge to analyze the inflammatory cytokines TNF-a (A), IL-1ß (B), and IL-6 (C). The values presented are mean ± standard error of the mean (n [ 12 in each group). #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
LPS administration produced a significant increase in capillary leakage, as shown by the lung wet-to-dry weight (W/D) ratio. The results showed that the lung W/D ratio increased significantly after the LPS challenge compared with those of the control group. Pretreatment of PA (10, 20, and 40 mg/kg), DEX, and PDTC significantly decreased the lung W/D ratio (P < 0.05) compared with those in the LPS group (Fig. 2). However, pretreatment of both PA and PDTC most strongly inhibited LPSinduced lung W/D ratio. Treatment of PA (40 mg/kg) also inhibited LPS-induced lung W/D ratio, and the inhibition was weaker than pretreatment of PA (40 mg/kg).
3.2. Effects of PA on inflammatory cell counts in BALF of LPS-induced ALI mice Seven hours after the challenge with LPS, the number of inflammatory cells in BALF, such as neutrophils and macrophages, significantly increased compared with the control group (P < 0.01; Fig. 3). Pretreatment of PA (10, 20, and 40 mg/ kg), DEX (5 mg/kg), or PDTC (100 mg/kg) was found to significantly decrease the number of total cells (P < 0.01),
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Fig. 6 e Effects of PA on histopathologic changes in lung tissues in LPS-induced ALI mice. Mice were given an intraperitoneal injection of PA (10, 20, and 40 mg/kg), DEX, or PDTC 1 h before an intranasal administration of LPS and PA (40 mg/kg) 1 h after LPS administration. Lungs from each experimental group were processed for histologic evaluation at 7 h after the LPS challenge. Representative histologic changes of lungs obtained from mice of different groups. (A) Control group, (B) LPS group, (C) LPS D DEX group, (D) LPS D PA (10 mg/kg) group, (E) LPS D PA (20 mg/kg) group, (F): LPS D PA (40 mg/kg) group, (G) LPS D PA (40 mg/kg) (1 h after LPS), (H) LPS D PDTC group, (I): LPS D PDTC D PA (hematoxylin and eosin staining, magnification 3200). (Color version of the figure is available online.)
neutrophils (P < 0.01), and macrophages (P < 0.01). Treatment of PA (40 mg/kg) also inhibited LPS-induced inflammatory cells, and the inhibition was weaker than pretreatment of PA (40 mg/kg). Furthermore, pretreatment of both PA and PDTC most strongly inhibited LPS-induced inflammatory cells.
(Fig. 4). We also found that treatment of PA also inhibited LPSinduced inflammatory cytokine production, and the inhibition was weaker than pretreatment of PA (40 mg/kg). In addition, we found that pretreatment of both PA and PDTC most strongly inhibited production of LPS-induced inflammatory cytokines TNF-a, IL-6, and IL-1b.
3.3. Effects of PA on cytokine production in the BALF of LPS-treated ALI mice
3.4.
The concentrations of TNF-a, IL-6, and IL-1b in BALF were measured by ELISA. The levels of TNF-a, IL-6, and IL-1b in BALF were increased by the LPS challenge compared with those in the control group (P < 0.01; Fig. 4). Pretreatment of PA (10, 20, and 40 mg/kg), DEX (5 mg/kg), or PDTC (100 mg/kg) markedly reduced the production of TNF-a (P < 0.01), IL-6, and IL-1b (P < 0.01 or P < 0.05) compared with those in the LPS group
Effects of PA on the MPO activity in mice
To assess the neutrophil accumulation in the lung tissues, MPO activity was measured. As shown in Figure 5, LPS challenge resulted in significant increases in the lung MPO activity compared with the control group (P < 0.01). Pretreatment of PA (10, 20, and 40 mg/kg), DEX (5 mg/kg), or PDTC (100 mg/kg) obviously inhibited the increased MPO activity induced by LPS (P < 0.01). We also found that treatment of PA also inhibited LPS-induced MPO activity, and the inhibition was weaker than
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Fig. 7 e PA pretreatment inhibited LPS-induced NF-kB activation with Western blotting. #P < 0.01 versus control group, *P < 0.05, **P < 0.01 versus LPS group.
pretreatment of PA (40 mg/kg). In addition, we found that pretreatment of both PA and PDTC most strongly inhibited LPS-induced MPO activity.
3.5. Effects of PA on LPS-mediated lung histopathologic changes To evaluate the histologic changes after PA treatment in LPStreated mice, lung sections were subjected to hematoxylin and eosin staining. As shown in Figure 6, LPS administration after 7 h induced pulmonary edema, infiltration of inflammatory cells in the lungs tissues, and alveolar damage (Fig. 6B). However, LPS-induced pathologic changes were significantly attenuated by DEX (5 mg/kg; Fig. 6C), PA (10, 20, and 40 mg/kg; Fig. 6DeF), PA (40 mg/kg, 1 h after LPS; Fig. 6G), PDTC (Fig. 6H), and PDTC þ PA (Fig. 6I).
3.6. Effect of PA on NF-kB activation in ALI mice induced by LPS Western blot analysis showed that phosphorylation of IkB-a was markedly increased after LPS induction compared with the control group. However, treatment with PA significantly inhibited these upregulations by LPS (Fig. 7). LPS-treated mice also displayed that p65 subunit of NF-kB increased in nucleus and decreased in cytoplasm, and the tendency was reversed by PA (10, 20, and 40 mg/kg) and DEX (5 mg/kg).
4.
Discussion
PA, a natural compound of Pogostemon cablin, has been shown to possess anti-inflammatory effects [8]. In the present study, we demonstrated that pretreatment or treatment with PA prevented LPS-induced ALI in mice. This was the first time to
study the anti-inflammatory effect of PA on LPS-induced lung injury. Moreover, we found that the protective effects of PA on LPS-induced lung injury were due to its ability to inhibit NF-kB signaling pathway. Edema is one of the major characteristics of ALI [13,14]. In this study, lung W/D ratio was used to quantify the magnitude of pulmonary edema. The results demonstrated that PA decreased the lung W/D ratio, indicting that PA significantly inhibited edema of the lung. LPS-induced ALI is characterized by the infiltration of neutrophils in the lungs, exhibiting increased MPO activity [15,16]. Our results showed that pretreatment with PA decreased LPS-induced increases in MPO activity in the lungs. In addition, the lung histologic examination also indicated that lung tissues infused with LPS generated serious inflammatory responses with a large number of infiltrating polymorphonuclear. The treatment group reduced the number of infiltrating polymorphonuclear, especially the treatment group of 40 mg/kg, suggesting that PA could ameliorate the damage of lung tissues. LPS is known to induce the productions of several inflammatory cytokines. Studies showed that proinflammatory cytokines, including TNF-a, IL-1ß, and IL-6, were important mediators in the pathogenesis of ALI [17,18]. Elevated TNF-a, IL-1ß, and IL-6 levels in BALF were observed in patients with ALI or acute respiratory distress syndrome [19,20]. Thus, to explore the protective or treatment effects of PA on LPS-induced ALI, the effects of PA on LPS-induced cytokines were detected. Our results showed that pretreatment or treatment of PA significantly inhibited the production of TNF-a, IL-1b, and IL-6 induced by LPS. The inhibition of inflammatory cytokines by the treatment of PA (40 mg/kg) was a little weaker than that by the pretreatment of PA (40 mg/kg). These results indicated that the protective and treatment effects of PA on LPS-induced ALI may be attributed to the inhibition of inflammatory cytokines production.
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NF-kB pathway is known to play an important role in the development of ALI [21,22]. Normally, NF-kB is present in the cytoplasm as a heterodimer and is linked to the inhibitory proteins IkBs. Once stimulated by LPS, NF-kB is activated by phosphorylation, enters the nucleus, and regulates the expression of inflammatory cytokines, such as TNF-a, IL-1b, and IL-6 [23,24]. To detect the inhibitory mechanisms of TNFa, IL-1b, and IL-6 production by PA, the effects of PA on NF-kB activation and IkB-a degradation induced by LPS were detected. The results showed that LPS-induced NF-kB activation in the lung tissue was inhibited by pretreatment of PA or PDTC (an NF-kB inhibitor). In addition, we found that pretreatment of both PA and PDTC most strongly inhibited production of LPS-induced inflammatory cytokines TNF-a, IL-6, and IL-1b. These results suggested that the inhibition of NF-kB activation was a mechanism for the anti-inflammatory effects of PA.
5.
Conclusions
[5]
[6]
[7]
[8]
[9]
[10]
In conclusion, we have provided the evidence that pretreatment of PA significantly attenuated LPS-induced ALI in mice, and that the potential mechanism of this action was through its suppression of NF-kB, and subsequently leading to the inhibition of inflammatory cytokines. These evidences suggest that PA has a potential application to treat LPS-induced ALI.
[11]
Acknowledgment
[14]
Authors’ contributions: J.-L.Y. and R.-M.W. contributed to the conception and design. J.-L.Y. and X.-S.Z. did the analysis and interpretation. X.X. did the data collection. J.-L.Y. and R.-M.W. wrote the article, did the critical revision of the article, and obtained the funding.
[12]
[13]
[15] [16]
[17]
Disclosure This study was supported by the Youth Foundation of the Second Hospital of Shandong University (No. Y2013010077) and the science and technology development project of Shandong Administration of traditional Chinese medicine (No.2013-181).
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