LPS (Escherichia coli LPS, 055:B5) was purchased from Sigma Aldrich (St. Louis, MO, USA). The myeloperoxidase (MPO) and malondialdehyde (MDA) kits were purchased from Jiancheng Bioengineering Institute of Nanjing (Nanjing, China). Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 ELISA kits were purchased from USCN Life Science Inc. (Wuhan, China). The fruit body of Auricularia auricular-judae was cultured in the Daxinganling region, Heilongjiang province, China.

AAP was extracted by hot water and ultrasonic-assisted extraction. The concentrated supernatants were subsequently precipitated with 3 volumes of absolute ethanol (95%) and maintained at 4°C overnight. The resulting precipitate was separated by centrifugation, dissolved in deionized water and subsequently dialyzed. The non-dialyzed portion was, in addition, lyophilized to result in a crude polysaccharide extract. The AAP was decolorized by hydrogen peroxide (13).

Adult Sprague-Dawley rats provided by the Laboratory Animal Center of Xinjiang Medical University (Xinjiang, China) were used in all the experiments. All the animal care and experimental procedures were approved by the Animal Care Committee of Xinjiang Medical University. Adult Sprague-Dawley rats were randomly assigned into the control, AAP, LPS and LPS plus AAP groups. The control animals received an equal volume of normal saline at the same time. The LPS group was induced by intraperitoneal injection of 10 mg/kg LPS. Rats in the LPS plus AAP group were treated with AAP for 7 days before LPS administration. Post-LPS infusion (12 h), animals were sacrificed by overdose of ethyl carbamate and blood samples were collected from the abdominal aorta. Blood samples were anticoagulated with EDTA and centrifuged at 3,000 × g for 10 min at 4°C and the plasma was stored at −20°C until measurements were performed. Following death at the end of the protocol, the left lung was lavaged using 500 µl of saline 3 times (total volume, 1.5 ml) for protein leakage. The right lung tissues were divided into 3 pieces, one immersed in 10% formalin solution for histopathological examination, one frozen in liquid nitrogen for quantitative analysis and the remaining part for measurement of the wet/dry (W/D) weight ratio.

The lungs were lavaged with 500 µl of saline 3 times (total volume, 1.5 ml). Retrieval volume was maximized by compression of the thorax following the last lavage. The protein concentration was determined using a protein kit.

The trachea and esophagus were separated from the lungs by blunt dissection and the wet weight of the latter was determined. Subsequently, the lungs were incubated at 60°C for 3–4 days to remove all moisture, the dry weight was measured and the ratio of wet-to-dry weight calculated.

The MPO and MDA activities in the lung tissue were assayed by MPO and MDA kits, respectively, following the manufacturer's instructions.

TNF-α and IL-6 in blood samples were determined with ELISA kits from USCN Life Science Inc., performed according to the manufacturer's instructions. All the measurements were performed in duplicate.

A section of the right lung was removed and put into 10% formaldehyde solution followed by dehydration, paraffin embedding, sectioning and hematoxylin and eosin staining sequentially. The changes of pathology were observed.

Results are presented as mean ± standard deviation. For tests of significance between the groups, one-way analysis of variance was performed. Comparisons between two groups were performed using unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference. All the data were performed in ≥3 independent experiments.

To confirm the efficacy of LPS exposure, the protein concentration in BALF was observed. As shown in Fig. 1, protein concentration in BALF markedly increased in the LPS group compared with the control and AAP groups. However, pretreatment with AAP caused the protein concentration in BALF to decrease compared with the LPS group.

To investigate the effect of AAP on LPS-induced lung edema, W/D weight ratios were detected. As shown in Fig. 2, there were no significant differences between the control and AAP groups, which indicated that AAP had no effect on lung edema in normal rats. LPS injected for 12 h caused a significant increase in the lung W/D weight ratio compared with the control group (P<0.01). As shown in Fig. 3, in the AAP pretreated group the lung W/D weight ratios decreased compared with the LPS groups.

To assess the lung neutrophil burden within pulmonary tissues, lung MPO activity was measured. As shown in Fig. 3, MPO activity increased significantly compared with the control and AAP groups, however, the MPO activity deceased in the LPS plus AAP group. In addition, LPS induced an increase in the MDA level in lung tissues and AAP significantly inhibited the MDA level.

The concentration of TNF-α and IL-6 in the blood represents pro-inflammatory mediators, which were thought to play crucial roles in the development of ALI. As shown in Fig. 4, TNF-α and IL-6 levels increased markedly in the LPS group compared with the control and AAP groups, whereas these levels were decreased by AAP pretreatment.

In order to study the effects of AAP on ALI, the histological changes were determined following AAP treatment in LPS-treated rats. As shown in Fig. 5, in the control and heparin groups, lung tissue showed a normal structure and clear pulmonary alveoli under a light microscope. The changes in the LPS group, such as a large number of neutrophil sequestration and infiltration around the pulmonary vessel and airway, distributed in the alveolar and interstitial were observed. The LPS group pretreated with AAP markedly alleviated the LPS-induced pathological changes of the lung. These results indicated that AAP could protect rats from LPS-induced lung damage.