Contributed equally
Patchouli alcohol (PA) is a tricyclic sesquiterpene isolated from Pogostemon cablin, which exerts anti-inflammatory, anti-influenza and cognitive-enhancing bioactivities. The present study aimed to investigate the protective effects of PA on acute lung injury (ALI) induced by intratracheal instillation of lipopolysaccharide (LPS) in mice. Dexamethasone was used as a positive drug for protection against LPS-induced ALI. The results of the present study demonstrated that pretreatment with PA significantly increased survival rate, attenuated histopathologic damage and lung edema, and decreased the protein content in the bronchoalveolar lavage fluid (BALF) of mice with ALI. Furthermore, PA significantly inhibited the expression levels of proinflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-6 in the BALF, downregulated the levels of myeloperoxidase and malondialdehyde, and upregulated the activity levels of superoxide dismutase and glutathione peroxidase in lung tissue. These results indicated that PA may exert potent protective effects against LPS-induced ALI in mice, the mechanisms of which are possibly associated with the anti-inflammatory and antioxidative activities of PA.
Acute lung injury (ALI) is defined as a complex syndrome associated with an intense pulmonary inflammation (
Lipopolysaccharide (LPS) is the major constituent of the outer membrane of Gram-negative bacteria, and is composed of a polar lipid head group and a chain of repeating disaccharides. LPS has been demonstrated to have an important role in the pathogenesis of ALI (
The aerial parts of P. cablin were obtained from Guangzhou Zhixing Pharmaceutical Co., Ltd. (Guangzhou, China) and authenticated by Professor Lai Xiaoping, an experienced pharmacognosist, at the School of Chinese Materia Medica, Guangzhou University of Chinese Medicine (Guangzhou, China). The voucher specimen was deposited in the herbarium of the School of Chinese Materia Medica, Guangzhou University of Chinese Medicine.
PA was isolated from P. cablin according to methods described in our previous studies (
Male Kunming (KM) mice, 4–5 weeks old and weighing 20–22 g, were obtained from the Guangdong Provincial Medical Animal Experimental Center (certificate no. SCXK2013-0002; Foshan, China). The mice were maintained in microisolator cages with a regular temperature (24±1°C), relative humidity (55±10%) and a 12-h light/dark cycle. All the experimental protocols and schedules involving animals were approved by the Animal Welfare Committee of Guangzhou University of Chinese Medicine.
Dexamethasone (DEX) was purchased from Guangdong Huanan Pharmaceutical Group Co., Ltd. (Dongguan, China). LPS (
For the analysis of mortality rate, 100 mice were randomly divided into five groups (n=20): Sham; LPS; and PA (10, 20 and 40 mg/kg) groups. Mice in the PA groups were intragastrically administered 10, 20 and 40 mg/kg PA, whereas the sham and LPS groups were administered 1% poloxamer 407 once a day for 7 consecutive days. The mice were anesthetized using 3% chloral hydrate (Aladdin Reagent Co., Ltd., Shanghai, China) 1 h after the final administration. Mice in the LPS and PA groups were administered 20 mg/kg LPS via intratracheal instillation, whereas mice in the sham group was administered an equal volume of phosphate-buffered saline (PBS). The mortality rate was recorded for 5 days.
A total of 168 mice were randomly divided into six groups: Sham, LPS, 5 mg/kg DEX, and 10, 20 and 40 mg/kg PA groups. Mice from the sham and LPS groups were administered 1% poloxamer 407, whereas the PA groups were administered 10, 20 or 40 mg/kg PA daily for 7 consecutive days. The DEX group was administered 5 mg/kg DEX daily for 5 consecutive days. The mice were anesthetized 1 h after the final administration. The LPS, PA and DEX groups were administered 5 mg/kg LPS by intratracheal instillation, whereas the sham group was administered an equal volume of PBS. All mice were sacrificed by cervial dislocation after 24 h, and lung tissue and bronchoalveolar lavage fluid (BALF) samples were harvested for further study.
The mice lungs were lavaged with 1.5 ml PBS three times and ~1.35 ml BALF was recovered with ~90±2% recovery rates. The BALF samples were centrifuged at 800 × g for 10 min at 4°C and the supernatants were collected in order to measure the protein content using Coomassie (Bradford) Protein Assay kits (Thermo Fisher Scientific, Inc.).
The lung wet/dry weight (W/D) ratios were determined to evaluate the protective effects of PA on LPS-induced lung edema. Upon completion of the experiments, lung tissues were excised and immediately weighed to record the ‘wet’ weight, to obtain the ‘dry’ weight the tissues were weighed after being heated at 80°C for 48 h.
Lung tissues were fixed in 4% paraformaldehyde, embedded in paraffin and cut into 4 µm sections. The sections were subsequently stained with hematoxylin and eosin according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute), examined, and images were captured using a TE2000-S inverted microscope (Nikon Corporation, Tokyo, Japan).
Lung tissues were homogenized using PBS and centrifuged at 14,167 × g for 10 min at 4°C. Subsequently, the supernatants were collected and the levels of MPO, SOD, GPx and MDA in the lung tissue were examined by the respective assay kits (Nanjing Jiancheng Bioengineering Institute), according to the manufacturer's protocol.
The expression levels of the proinflammatory cytokines TNF-α and IL-6 were examined in the BALF using enzyme-linked immunosorbent assay kits (eBioscience, CA, USA), according to the manufacturer's protocols.
Data are expressed as the mean ± standard error of the mean. Experimental values were analyzed by one-way analysis of variance using SPSS 17.0 statistical analysis software (SPSS, Inc., Chicago, IL, USA). Mortality was presented as Kaplan-Meier curves and differences were assessed by the log-rank test. P<0.05 was considered to indicate a statistically significant difference.
As compared with the sham group, which had a survival rate of 100%, the survival rate of the LPS group was significantly decreased (20%; P<0.01) (
Protein content in the BALF corresponds to the vascular permeability of the lungs. The BALF protein content in the LPS group was significantly increased (P<0.01), as compared with the sham group (
The W/D ratios of the mice with ALI were evaluated, in order to assess the severity of pulmonary edema. The lung W/D weight ratio significantly increased (P<0.01) following LPS stimulation, as compared with the sham group (
In the sham group, the structure of the alveolar wall was normal and minimal inflammation was detected (
In the LPS group, a significant increase in MPO (
The expression levels of TNF-α (
Numerous plant-derived natural products, including polyphenols, chlorogenic acid, ethyl gallate, rutin, magnolol and shikonin (
Pulmonary edema, which is a typical symptom of ALI, is usually assessed by measuring the W/D ratio (
MPO is an enzyme predominantly stored in the primary granules of neutrophils, therefore MPO activity in the parenchyma reflects neutrophil adhesion and margination in the lungs (
Previous experimental and clinical studies have demonstrated that LPS-induced ALI may lead to a rapid overproduction of proinflammatory cytokines, including TNF-α and IL-6, which are characteristic cytokines associated with the inflammatory process of ALI (
Another possible mechanism for the anti-inflammatory effects induced by PA may be associated with its antioxidant activity. Inflammatory stimuli promote the generation of ROS, generated by activated inflammatory cells and circulating enzymatic generators, which contribute to lung pathophysiology (
It has previously been reported that PA has a short elimination half-life (t1/2β), with values of 21.51±8.46 (10 mg/kg, i.g.), 17.81±7.52 (30 mg/kg, i.g.) and 17.82±9.29 h (100 mg/kg, i.g.) detected in rats (
In conclusion, the results of the present study demonstrated that pretreatment with PA improved the survival rate of mice with LPS-induced ALI, and effectively attenuated LPS-induced ALI by inhibiting pulmonary histopathologic alterations. The mechanisms underlying this protective effect included (i) reduced W/D ratio and protein leakage; (ii) reduced MPO activity levels; (iii) reduced lipid peroxidation and MDA formation; (iv) elevated activity of antioxidative enzymes, including SOD and GPx; and (v) decreased secretion of proinflammatory cytokines, including TNF-α and IL-6 (
The present study was supported by grants from: The National Natural Science Foundation of China (grant nos. 81303200 and 81403169); the Natural Science Foundation of Guangdong Province (grant no. S2013010016627); the Administration of Traditional Chinese Medicine Project of Guangdong Province (grant no. 20132142); the Medical Scientific Research Foundation of Guangdong Province (grant no. A2013232); the Combined Program of Ministry of Education of Guangdong Province (grant no. 2012B090600007); the Science and Technology Cooperation Project of Hong Kong, Macao, and Taiwan (grant no. 2014DFH30010); and the Special Funds from Central Finance of China in Support of the Development of Local Colleges and University [grant no. 276 (2014)].
Structure of patchouli alcohol.
Effects of patchouli alcohol (PA) on lipopolysaccharide (LPS)-induced mortality in mice (n=10). The mortality rates of all the groups were recorded for 5 days. Data are presented as Kaplan-Meier curves and comparisons were made using the log rank test. *P<0.05 vs. the LPS group; #P<0.01 vs. the sham group.
Effects of patchouli alcohol (PA) on total protein concentration in bronchoalveolar lavage fluid (BALF) and lung edema. Mice were administered 10, 20 and 40 mg/kg PA, 5 mg/kg dexamethasone (DEX) or an equal volume of vehicle. Mice in the lipopolysaccharide (LPS), PA and DEX groups were administered 5 mg/kg LPS, whereas mice from the sham group received an equal volume of phosphate-buffered saline. Following 24 h, the mice were sacrificed by cervical dislocation, and subsequently, the BALF and lung tissue samples were harvested for the analysis of (A) total protein concentration (n=8) and (B) lung edema (n=6). Data are presented as the mean ± standard error of the mean. ##P<0.01 vs. the sham group; *P<0.05 and **P<0.01 vs. the LPS group. W/D, wet/dry ratio.
Effects of patchouli alcohol (PA) on histopathologic alterations in mice treated with lipopolysaccharide (LPS). Mice were administered 10, 20 and 40 mg/kg PA, 5 mg/kg dexamethasone (DEX) or an equal volume of vehicle. Mice from the LPS, PA and DEX groups were administered 5 mg/kg LPS, whereas mice in the sham group received an equal volume of phosphate-buffered saline. Following 24 h, the mice were sacrificed by cervical dislocation and the lung tissues were subsequently harvested for histological examination. (A) Sham group; (B) LPS group; (C) LPS + 5 mg/kg DEX, (D) LPS + 10 mg/kg PA; (E) LPS + 20 mg/kg PA; (F) LPS + 40 mg/kg PA (magnification, 100×; n=6).
Effects of patchouli alcohol (PA) on myeloperoxidase (MPO), malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels in the lung tissues. Mice were administered 10, 20 and 40 mg/kg PA, 5 mg/kg DEX or an equal volume of vehicle. Mice from the LPS, PA and DEX groups were administered 5 mg/kg LPS, whereas mice in the sham group received an equal volume of phosphate-buffered saline. Following 24 h, the mice were sacrificed by cervical dislocation and the lung tissues were harvested. (A) MPO activity, (B) MDA levels (C) SOD activity and (D) GPx levels in lung tissue. Data are presented as the mean ± standard error of the mean (n=8). ##P<0.01 vs. the sham group; *P<0.05 or **P<0.01 vs. the LPS group.
Effects of patchouli alcohol (PA) on cytokine production in the bronchoalveolar lavage fluid (BALF) of mice challenged with lipopolysaccharide (LPS). Mice were administered 10, 20 and 40 mg/kg PA, 5 mg/kg dexamethasone (DEX) or an equal volume of vehicle. Mice from the LPS, PA and DEX groups were administered 5 mg/kg LPS, whereas mice from sham group was administered an equal volume of phosphate-buffered saline. Following 24 h, the mice were sacrificed by cervical dislocation and the BALF was collected. (A) Expression levels of tumor necrosis factor (TNF)-α in BALF. (B) Expression levels of interleukin (IL)-6 in BALF. Data are presented as the mean ± standard error of the mean (n=8). ##P<0.01 vs. the sham group; *P<0.05 or **P<0.01 vs. the LPS group.
Schematic representation of the mechanisms underlying the protective effects induced by patchouli alcohol (PA) on lipopolysaccharide-induced acute lung injury. The shaded parts indicate the molecules/processes affected by PA. SOD, superoxide dismutase; MPO, myeloperoxidase; GPx, glutathione peroxidase; TNF, tumor necrosis factor; IL, interleukin; MDA, malondialdehyde.