Inflammation ( # 2017) DOI: 10.1007/s10753-017-0676-4

ORIGINAL ARTICLE

Preventive and Therapeutic Effects of Thymol in a Lipopolysaccharide-Induced Acute Lung Injury Mice Model Limei Wan,1 Dongmei Meng,2 Hong Wang,1 Shanhe Wan,3 Shunjun Jiang,2 Shanshan Huang,2 Li Wei,2,4 and Pengjiu Yu 2,4

Acute lung injury (ALI) is a life-threatening syndrome which causes a high mortality rate worldwide. In traditional medicine, lots of aromatic plants—such as some Thymus species—are used for treatment of various lung diseases including pertussis, bronchitis, and asthma. Thymol, one of the primary active constituent derived from Thymus vulgaris (thyme), has been reported to exhibit potent anti-microbial, anti-oxidant, and antiinflammatory activities in vivo and in vitro. The present study aims to investigate the protective effects of thymol in lipopolysaccharide (LPS)-induced lung injury mice model. In LPS-challenged mice, treatment with thymol (100 mg/kg) before or after LPS challenge significantly improved pathological changes in lung tissues. Thymol also inhibited the LPSinduced inflammatory cells influx, TNF-α and IL-6 releases, and protein concentration in bronchoalveolar lavage fluid (BALF). Additionally, thymol markedly inhibited LPS-induced elevation of MDA and MPO levels, as well as reduction of SOD activity. Further study demonstrated that thymol effectively inhibited the NF-κB activation in the lung. Taken together, these results suggested that thymol might be useful in the therapy of acute lung injury.

Abstract—

KEY WORDS: thymol; acute lung injury; lipopolysaccharide.

INTRODUCTION Acute lung injury (ALI) is a syndrome often posing a great threat to patient’s life [1]. ALI is due to a large 1

Department of Respiratory Medicine, The First Affiliated Hospital of Guangdong Pharmaceutical University, Guangzhou, 510080, China 2 Department of Pharmacy, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, 510120, China 3 Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Science, Southern Medical University, Guangzhou, 510515, China 4 To whom correspondence should be addressed at Department of Pharmacy, The First Affiliated Hospital of Guangzhou Medical U n i v e r s i t y, G u a n g z h o u , 5 1 0 1 2 0 , C h i n a . E - m a i l s : [email protected]; [email protected]

number of neutrophils infiltrating into the lungs, and the release of numerous pro-inflammatory mediators and reactive oxygen species (ROS), leading to the damage of pulmonary microvascular endothelium and epithelium, therefore resulting in refractory hypoxemia [1, 2]. Although significant progress has been made in the pathophysiology and treatment of ALI in the past decade, the mortality rate remains unacceptable [3]. Thus, a novel effective therapy is urgently required. ALI often results from serious infection, shock, aspiration, and blood transfusion [4]. In fact, serious bacterial infection, such as severe pneumonia and sepsis, is the main cause of ALI in humans [4, 5]. Lipopolysaccharide (LPS), the primary constituent of the outer cellular membrane of gram-negative bacteria, has been widely used to establish

0360-3997/17/0000-0001/0 # 2017 Springer Science+Business Media, LLC

Wan, Meng, Wang, Wan, Jiang, Huang, Wei, and Yu experimental ALI model because it yields very reproducible results [6]. Although the precise mechanism of this model is not fully clear, LPS instillation has been confirmed to induce intra-alveolar and interstitial edema, alveolar hemorrhage, and impairment in the alveolar fluid clearance, which imitate the pathophysiologic events observed in patients suffered from acute lung injury [6, 7]. Herbal remedies are now receiving more attention as adjuvant treatments because of their safety, readily available, and cost-effectiveness [8, 9]. A large number of plant species contain a range of bioactive compounds that possess beneficial health properties. In traditional medicine, lots of aromatic plants—such as some Thymus species—are used for treatment of various respiratory diseases including pertussis, bronchitis, and asthma [10–12]. Thymol, a naturally occurring monocyclic phenolic compound, is one of the primary active constituent of the essential oil of Thymus vulgaris (Lamiaceae, thyme) [13]. Recent reports have demonstrated the anti-microbial, antioxidant, and anti-inflammatory effects of thymol in vivo and in vitro [14–17]. It is unknown, however, whether thymol could protect against acute lung injury. In the present study, an LPS-challenged mice model was established to investigate the potential protective effect of thymol on acute lung injury.

MATERIALS AND METHODS Material Thymol (purity > 99% by gas chromatography) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Lipopolysaccharide (Escherichia coli 055:B5) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for phosphorylated p65 and β-actin were obtained from Cell Signaling Technology (Danvers, MA, USA). Enzyme-linked immunosorbent assay (ELISA) kits for determination of mouse TNF-α and IL-6 were obtained from Dakewe Biotech Co. Ltd. (Beijing, China). Kits for examining myeloperoxidase (MPO), malondialdehyde (MDA), and superoxide dismutase (SOD) were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Design LPS-induced acute lung injury was induced as described by our previous study [18]. Male BALB/c mice (8– 9 weeks old; 20–25 g; Experimental Animal Center of Guangdong Province, Guangzhou, Guangdong) were kept

in an acrylic chamber (20 × 30 × 40 cm, 24 L) throughout the LPS exposure period. A solution containing 0.5 mg/mL LPS in saline was aerosolized with an ultrasonic nebulizer (NB-150U; Omron Co., Kyoto, Japan) and air pump coupled to a manometer (flow rate was monitored and adjusted to 4.8 L/min) for 30 min. All procedures were performed in accordance with the General Recommendation and Provisions of the Chinese Experimental Animals Administration Legislation. Thymol dissolved in 1% tween 80 at 3 and 10 mg/mL or the same volume of dissolvent (0.1 mL/10 g body weight) was administered by intraperitoneal injection 0.5 h before or after LPS challenge. The dosages of thymol used in this study were 30 and 100 mg/kg. Six hours after LPS challenge, mice were anesthetized with an overdose of pentobarbital sodium and then killed by exsanguination (for examination of NF-κB activation, mice were executed at 3 h after LPS challenge). Bronchoalveolar Lavage Fluid Collection and Cell Count For the bronchoalveolar lavage fluid (BALF) collection, tracheostomy was performed and a cannula was inserted into the trachea. The cannula was secured with 3-O surgical silk, and the lungs were lavaged three times with phosphate buffer solution in a total volume of 1.5 ml (0.5 mL × 3). The recovery rate of BALF was above 90%. A 0.1 mL aliquot was used for the total cell count by using a hemocytometer, and the remainder was immediately centrifuged at 500g for 10 min at 4 °C. The total protein concentration in the supernatant of BALF was quantified by the BCA method with bovine serum albumin as a standard. The rest of the supernatant was aliquoted and frozen at −70 °C until the cytokines were assessed. Measurement of Pro-inflammatory Cytokines in BALF The levels of TNF-α and IL-6 in BALF were measured with mouse TNF-α and IL-6 ELISA kits according to the manufacturer’s protocol (Dakewe Biotech Co. Ltd., Beijing, China). Histological Evaluation After mice were killed, the lungs were excised. The right lobes were fixed with 10% neutral phosphatebuffered formalin and then embedded in paraffin. Sections 5 μm thick were stained with hematoxylin and eosin. The rest of the lung lobes were flash frozen in liquid nitrogen and stored at − 70 °C until examination of MPO. Lung

Preventive and Therapeutic Effects of Thymol injury was scored according to the following criteria [19]: (1) alveolar congestion, (2) hemorrhage, (3) infiltration or aggregation of neutrophils in airspace or vessel wall, and (4) thickness of the alveolar wall. For each subject, a fivepoint scale was applied: 0, minimal (little) damage; 1+, mild damage; 2+, moderate damage; 3+, severe damage; and 4+, maximal damage. Points were added up and are expressed as median ± range of injury score. Measurement of MPO, MDA, and SOD Lung tissues were frozen in liquid nitrogen and then homogenized in PBS. The homogenate was used to determine MPO, MDA, and SOD according to the manufacturer’s instruction.

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Western Blot After mice were killed, the whole lung tissues were flash frozen in liquid nitrogen immediately after removing and stored at − 70 °C until use. The extraction of total protein from lung tissues was performed. Protein contents in the supernatant of the lysed lungs were determined by the BCA method. Samples were separated on 10% SDS– PAGE and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat milk in TBST (0.1%) for 1 h at room temperature, the membranes were incubated with a primary antibody (phosphorylated p65 or β-actin) and then incubated with the conjugated secondary antibodies. Protein bands were detected by an ECL detection kit (Millipore, Billerica, USA). The protein signals

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Fig. 1. Thymol ameliorated LPS-induced histological changes in lung tissues. Thymol at doses of 30 and 100 mg/kg was administrated 30 min before (preventive) or after (therapeutic) LPS challenge. Mice were killed 6 h after LPS challenge and the lungs were fixed, embedded in paraffin, and cut into 5-μm slices. After H&E staining, histological examination was performed by light microscopy, and lung injury was scored. The preventive (a) and therapeutic (b) effects of thymol on LPS-induced pathological changes in lung tissue were evaluated. All values are mean ± SEM (n = 6). #p < 0.05, significant compared with vehicle-treated control; *p < 0.05, significant compared with LPS alone; **p < 0.01, significant compared with LPS alone.

Wan, Meng, Wang, Wan, Jiang, Huang, Wei, and Yu were quantified by AlphaEaseFC™ software (Alpha Innotech, San Leandro, CA).

RESULTS Thymol Attenuated Lung Histopathology in LPSInduced ALI Mice

Statistical Analysis As shown in Fig. 1, no evident histological alteration was observed in lung specimens of normal mice. However, the inhalation of LPS resulted in significant lung injury, evidenced by the presence of alveolar hemorrhage, a marked swelling of the alveolar walls, and remarkable recruitment of neutrophils into the alveolar spaces. These pathological changes were significantly improved by both

Data are expressed in term of means ± standard error of the mean (SEM). Data were analyzed by using one-way analysis of variance followed by the Student-NewmanKeuls test. Two-tailed p values < 0.05 were considered statistically significant. Statistical analyses were conducted with SPSS 13.0.

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Fig. 2. Thymol inhibited cell infiltration and MPO activity in BALF of LPS-induced ALI mice. Thymol at doses of 30 and 100 mg/kg was administrated 30 min before (preventive) or after (therapeutic) LPS challenge. Mice were killed 6 h after LPS challenge and bronchoalveolar lavage was performed. The numbers of total cells in BALF and MPO activity in lung tissue were examined. a The preventive effect of thymol on cell influx. b The preventive effect of thymol on MPO. c The therapeutic effect of thymol on cell influx. d The therapeutic effect of thymol on MPO. All values are mean ± SEM (n = 6). #p < 0.05, significant compared with vehicle-treated control; *p < 0.05, significant compared with LPS alone; **p < 0.01, significant compared with LPS alone.

Preventive and Therapeutic Effects of Thymol before (preventive) and post (therapeutic) treatment of 100 mg/kg thymol.

cell infiltration. MPO activity in tissues is an indicator of neutrophil infiltration. In parallel with the number of cells, the level of MPO activity was markedly inhibited by thymol. These results demonstrated that thymol significantly decreased the influx of neutrophils into the lungs.

Thymol Suppressed Inflammatory Cell Influx and MPO Activity in LPS-Induced ALI Mice Recruitment of inflammatory cells, especially neutrophils, into the pulmonary compartment is a key event of acute lung injury. As shown in Fig. 2, administration of saline was associated with few cell numbers in BALF, while LPS challenge caused a brisk and strong influx of total cells into BALF. Both preventive and post treatment of 100 mg/kg thymol significantly inhibited LPS-induced

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Thymol Suppressed TNF-α and IL-6 Levels in BALF of LPS-Induced ALI Mice It is well known that inflammatory cytokines, such as TNF-α and IL-6, are important biomarkers of lung injury. The inflammatory cascade induced by these cytokines plays a key role in the development of lung injury. As

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Fig. 3. Thymol suppressed TNF-α and IL-6 levels in BALF of LPS-induced ALI mice. Thymol at doses of 30 and 100 mg/kg was administrated 30 min before (preventive) or after (therapeutic) LPS challenge. Mice were killed 6 h after LPS challenge and bronchoalveolar lavage was performed. The levels of TNF-α and IL-6 in BALF were examined. a The preventive effect of thymol on TNF-α release. b The preventive effect of thymol on IL-6 release. c The therapeutic effect of thymol on TNF-α release. d The therapeutic effect of thymol on IL-6 release. All values are mean ± SEM (n = 6). #p < 0.05, significant compared with vehicle-treated control; *p < 0.05, significant compared with LPS alone; **p < 0.01, significant compared with LPS alone.

Wan, Meng, Wang, Wan, Jiang, Huang, Wei, and Yu shown in Fig. 3, LPS challenge causes significant increase levels of TNF-α and IL-6 in BALF, while these elevations were markedly attenuated by both preventive and post treatment of 100 mg/kg thymol.

Thymol Inhibited NF-κB Activation in LPS-Induced ALI Mice We evaluated phosphorylation of NF-κB p65 level by western blot analysis to investigate the molecular mechanism whereby treatment with thymol attenuated the development of acute lung injury. As shown in Fig. 6, treatment with 100 mg/kg thymol markedly inhibited the expression of phosphorylated NF-κB p65 subunit in the lung tissues.

Thymol Suppressed Total Protein Concentration in BALF of LPS-Induced ALI Mice Vascular leakage is a hallmark of ALI; thus, we examined the total protein concentration in BALF. As shown in Fig. 4, the challenge of LPS resulted in a significant increase of protein level in BALF, while thymol dosedependently inhibited the LPS-induced increase of protein concentration.

DISCUSSION In the early stage of ALI, lots of inflammatory cells, especially neutrophils, are recruited to the site of injury, which play a vital role for the host defense. However, an excess of activated inflammatory cells leads to tissue damage by release of various cytokines, proteinases, and cationic polypeptides [20]. The network of inflammatory cytokines, such as TNF-α and IL-6, has also been shown to involve the occurrence of ALI [21]. These cytokines not only participate in the recruitment of inflammatory cells into the lung and enhance the responses of neutrophils in response to activators, but also cause severe inflammatory cascade injury. Endothelial and epithelial injuries are accompanied by cytokine release and inflammatory cell influx. The damaged epithelial cell barrier results in increased microvascular permeability, facilitating protein

Thymol Decreased MDA Formation and Recovered SOD Activity in LPS-Induced ALI Mice The role of thymol on MDA and SOD in lung tissues was investigated. Significant elevation in MDA content and decrease in SOD activity were observed in the lung tissues of the LPS group, when compared with sham controls. Treatment with 100 mg/kg thymol partly prevented marked elevation in MDA levels and decrease in SOD activity. These data indicated that LPS-induced oxidative damage in lung tissues was significantly alleviated by thymol (Fig. 5).

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Fig. 4. Thymol suppressed total protein concentration in BALF of LPS-induced ALI mice. Thymol at doses of 30 and 100 mg/kg was administrated 30 min before (preventive) or after (therapeutic) LPS challenge. Mice were killed 6 h after LPS challenge and bronchoalveolar lavage was performed. The preventive (a) and therapeutic (b) effects of thymol on total protein concentration in BALF were examined. All values are mean ± SEM (n = 6). #p < 0.05, significant compared with vehicle-treated control; *p < 0.05, significant compared with LPS alone; **p < 0.01, significant compared with LPS alone.

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Fig. 5. Thymol decreased MDA formation and recovered SOD activity in LPS-induced ALI mice. Thymol at doses of 30 and 100 mg/kg was administrated 30 min before (preventive) or after (therapeutic) LPS challenge. Mice were killed 6 h after LPS challenge. The MDA level and SOD activity in lung tissue were examined. a The preventive effect of thymol on MDA level. b The preventive effect of thymol on SOD activity. c The therapeutic effect of thymol on MDA level. d The therapeutic effect of thymol on SOD activity. All values are mean ± SEM (n = 6). #p < 0.05, significant compared with vehicle-treated control; *p < 0.05, significant compared with LPS alone; **p < 0.01, significant compared with LPS alone.

leakage and leading to refractory hypoxemia [22, 23]. Indeed, inflammatory cell influx, releases of cytokines, and protein leakage present the key events of acute pulmonary inflammation in humans and animals [24]. The pharmacological activities of thymol against inflammatory response have been reported a lot recently [25–28]. In the present study, the rapid cell influx induced by LPS was markedly inhibited by thymol. These observations were further confirmed by the results that thymol suppressed MPO activity, an enzyme located mainly in the primary granules of neutrophils which reflects the adhesion and

margination of neutrophils in the lung [29]. In addition, thymol has been found to suppress TNF-α and IL-6 levels in BALF. Because protein extravasation is considered as a pointer of vascular leakage, we examined total protein contents in BALF and detected lower protein levels in thymol-treated mice. These data suggest that the protective effects of thymol against LPS-induced ALI may be due to its potent anti-inflammatory property. The protective effects of thymol against oxidative stress-induced injury have been highlighted in various in vitro and in vivo models [30–32]. In ALI, oxidative

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Fig. 6. Thymol inhibited NF-κB activation in lung tissues of LPS-induced ALI mice. Thymol at doses of 30 and 100 mg/kg was administrated 30 min before (preventive) or after (therapeutic) LPS challenge. Mice were killed 3 h after LPS inhalation and the lungs were removed. The preventive (a) and therapeutic (b) effects of thymol on phosphorylation of NF-κB p65 in lung tissue were examined. All values are mean ± SEM (n = 6). #p < 0.05, significant compared with vehicle-treated control; *p < 0.05, significant compared with LPS alone; **p < 0.01, significant compared with LPS alone.

stress and free radicals represent an extremely aggressive factor to cells, having a direct consequence upon the severity of lung inflammation [33]. MDA is a lipid peroxidation product which has been used as an index of induced oxidative membrane damage [34]. In this study, we examined the MDA level in the lungs of LPS-challenged mice and detected lower MDA contents in thymol-treated animals. The activity of SOD, an endogenous free radical-scavenging agent which

can get rid of oxyradical [35], was also detected. LPS markedly decreased the SOD activity in lung tissue, which is in line with the observation in the ALI patients [36]. Experimental data demonstrated that SOD activities increased in mice treated with thymol when compared with those in the LPS group. These results suggest that thymol could effectively attenuate oxidative stress injury in LPS-induced ALI. NF-κB is a transcription factor regulating various genes involved in inflammatory and immune responses [37]. Clinical research has reported that the degree of NF-κB activation was increased in patients with sepsis or acute lung injury [38, 39]. Inactivation of NF-κB pathway by pharmacological inhibition or gene modification has been demonstrated to decrease the expression of pro-inflammatory mediators and diminish the severity of endotoxemia-induced acute lung injury [40, 41]. In fact, inhibition of phosphorylated NF-κB p65 subunit in lung tissues has been recognized as the underlying mechanism against lung injury in many preclinical experiments for developing candidate drug for ALI [42–45]. In the present study, LPS challenge significantly increased the phosphorylation of NF-κB p65 subunits in the lung, which was consistent with those of previous studies [42, 45]. However, LPS-induced phosphorylation of p65 was suppressed by treatment with thymol. Thus, it is suggested that NF-κB pathway plays an important role in the process of antagonizing acute lung injury by thymol. In conclusion, we have demonstrated the protective effects of thymol on lung injury induced by LPS, which is evidenced by improvement of the pathologic changes in the lung, inhibition of NF-κB activation, and decrease of inflammatory cells infiltration, vascular leakage and cytokines release. Our study suggests that thymol might be useful in the treatment of lung injury. Funding This study was partly supported by grants from Education Bureau of Guangzhou (No. 1201581610), National Natural Science Foundation of China (No. 81402992), and the Open Project of Guangdong Provincial Key Laboratory of New Drug Screening (2016).

COMPLIANCE WITH ETHICAL STANDARDS All procedures in this study were performed in accordance with the General Recommendation and Provisions of the Chinese Experimental Animals Administration Legislation.

Preventive and Therapeutic Effects of Thymol Conflict of Interest. The authors declare that they have no conflict of interest. 16.

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