A total of 100 male C57BL/6 mice (6–8 weeks old, weighing, 18–20 g) were purchased from The Animal Center of Nanjing Medical University (Nanjing, China), and provided with food and water ad libitu and kept in climate-controlled quarters with a 12 h light/dark cycle with food and water in cages under the germ-free conditions at a temperature of 25°C. The mice were housed for a minimum of one week for environmental adaptation prior to experimentation. All procedures were in accordance with the Regulations of Experimental Animal Administration issued by the Ministry of Science and Technology of the People's Republic of China, and before the animal experiments were performed, the procedures were approved by the Research Ethical Committee of Huai'an First People's Hospital, Nanjing Medical University (Nanjing, China).

C57BL/6 mice were randomly divided into 5 groups as follows (n=20 in each group): i) Control (Con); ii) LPS treatment (LPS); iii) Low dose of LR treatment (LPS treatment + oral gavage of 12.5 mg/kg LR for 3 days, LD-LR); iv) Medium dose of LR treatment (LPS treatment + oral gavage of 25 mg/kg LR for 3 days, MD-LR); and v) High dose of LR treatment (LPS treatment + oral gavage of 50 mg/kg LR for 3 days, HD-LR). The mice from the control and LPS groups received an equal volume of normal saline (0.3 ml) instead of LR. LR was purchased from Fanke Biotech Fanke Biotech Co., Ltd. (http://fanketech.biomart.cn, FA-1701, HPLC ≥98%, Shanghai, China). At 3 h following drug administration on the third day, the mice were slightly anesthetized with inhalation of diethyl ether (1 ml diethyl ether dipping cotton balls), and 10 µg LPS in 50 µl PBS was intranasally instilled to induce lung injury. The mice in the control group were given 50 µl normal saline in the absence of LPS. A total of 24 h later for LPS treatment, all mice were sacrificed through an intraperitoneal injection of 50 mg/kg pentobarbital (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and bronchoalveolar lavage fluid (BALF) and lung tissue samples were harvested for further researches. The ratios of lung wet/dry weight were measured by dividing the wet weight by the dry weight. The middle lobe of the right lung was excised and the wet weight was recorded. Then, the lung was placed in an incubator for 24 h at 80°C to obtain the dry weight.

Thehuman lung epithelial cell line, BEAS-2B, was purchased from Shanghai Haoran Biological Technology Co., Ltd. (Shanghai, China) and kept in DMEM/F12 containing 1% penicillin/streptomycin and 10% FBS (Hyclone; GE Healthcare Life Sciences, Chalfont, UK). Cells were then cultured in a humidified atmosphere with 5% CO₂ and 95% humidity at 37°C in an incubator.

Superoxide dismutase (SOD, A001-3), catalase (CAT, A007-1-1) and glutathione peroxidase (GPx, A005) activities, as well as malondialdehyde (MDA, A003-1) levels in lung tissue sample were determined by commercially available kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions. H₂O₂ levels in lung tissues were measured using a hydrogen peroxide assay kit, (Beyotime Institute of Biotechnology, Haimen, China) following the manufacturer's instructions. O₂⁻ in lung tissues was measured through lucigenin chemiluminescence method. Briefly, lung tissues of mice under different conditions were weighed and homogenized in a homogenization buffer using HEPES and EDTA. Following centrifugation (1,000 × g at 4°C for 10 min), an aliquot of the supernatant was incubated with 5 µM lucigenin in Krebs-HEPES buffer. Light emission was measured with a Tecan Infinite 200 (Tecan Group AG, Männedorf, Switzerland). Specificity for O₂⁻ was evaluated by adding SOD (350 U/ml) to the incubation medium. BCA Protein Quantitative Analysis kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to measure the protein concentration.

A total of 40 male C57BL/6 mice (6–8-weeks old, 18–20 g weight) were pretreated with LR (0, 12.5, 25 and 50 mg/kg) for 3 days by gavage orally. At 3 h following drug administration on the third day, the mice were slightly anesthetized with inhalation of diethyl ether (1 ml diethyl ether dipping cotton balls), and 10 µg LPS in 50 µl PBS was instilled intranasally to induce lung injury. The mice in the control group were given 50 µl of normal saline without LPS. At 24 h following LPS treatment, the mice were sacrificed by an intraperitoneal injection of pentobarbital (50 mg/kg; Sigma-Aldrich; Merck KGaA). The retro-orbital blood samples (1 ml) from each mouse was collected and used to measure the parameters of arterial blood gas, including blood hemoglobin concentration (Hgb), pH, HCO₃, partial pressure of oxygen (PO₂), partial pressure of carbon dioxide (PCO₂), lactate (LAC) and base excess (BE) using Hitachi 8500 automatic analyzer (Hitachi, Ltd., Tokyo, Japan).

For measurement of CD41 using immunofluorescence staining was performed. Briefly, the frozen sections of lung tissue samples were washed with PBS twice and then were blocked with 1% bovine serum albumin at room temperature for 30 min. Then the cells and heart tissue samples were incubated overnight with rabbit anti-CD41 primary antibody (ab63983, 1:50; Abcam, Cambridge, UK). After being washed with PBS for three times, the samples were performed with goat anti-rabbit secondary antibody conjugated to Alexa Fluor 647 (A0468, 1:400) and goat anti-rabbit secondary antibody conjugated to Alexa Fluor 488 (A0423, 1:400) (both from Beyotime Institute of Biotechnology) for 1 h under dark conditions. Then, the cells were counterstained with DAPI (Beyotime Institute of Biotechnology) for 15 min, and the tissue sections were analyzed using an immunofluorescence microscope. Three slides per experimental condition are repeated three times.

BALF was collected by flushing the left lung (0.4 ml, three times). Total BALF cells were measured using a hemocytometer. A part of the BALF was centrifuged for 5 min at 92 × g by cytospin on a microscopic slide at 4°C. BALF cells were stained using Giemsa at room temperature for 10 min. The rest of BALF was filtrated through a 0.22 µm pore-size filter and then stored at −80°C for protein and cytokine detection, while the cell pellet was resuspended in PBS for counting the neutrophils. The concentration of BALF protein was measured using BCA Protein Assay kit.

IL-1β (cat. no. MLB00C), TNF-α (cat. no. MTA00B), IL-10 (cat. no. DY417), IL-6 (cat. no. M6000B), cyclooxygenase (COX)2 (cat. no. DYC4198-2), monocyte chemotactic protein 5 (MCP-5; cat. no. MCC120), macrophage inflammatory protein-1α (MIP-1α) (cat. no. MMA00), thromboxane B2 (TXB2; cat. no. KGE011) and myeloperoxidase (MPO; cat. no. DY3667) levels in BALF were measured using ELISA kits (R&D System Inc., Minneapolis, MN, USA). The procedures were performed following the manufacturer's instructions.

Histopathological evaluation was performed on mice not subjected to BAL. Lungs were inflated and fixed with 10% buffered formalin for 48 h at room temperature and embedded in paraffin (Beyotime Institute of Biotechnology). Tissue sections were cut at 4 µm thickness and stained with hematoxylin and eosin (H&E) following the regular staining method. The sections were deparaffinized in xylene for 10 min. Then, the slides were re-hydrated in absolute alcohol for 5 min each, followed by 95% alcohol for 2 min and 70% alcohol for 2 min and then washing with water. Subsequently, the sections were stained in Harris hematoxylin solution for 8 min at room temperature, followed by washing under a running water for 5 min. The slides were then differentiated in 1% acid alcohol for 30 sec, followed by washing using running tap water for 1 min. Then, the sections were subjected to bluing in 0.2% ammonia water or saturated lithium carbonate solution for 30 sec at room temperature, followed by washing under running water for 5 min and rinsing in 95% alcohol. The sections were counterstained in eosin-phloxine solution for 30 sec at room temperature; the slides were dehydrated via 95% alcohol for 5 min and cleared in xylene for 5 min. Finally, the sections were mounted with xylene-based mounting medium. The severity of injury was judged based on the following criteria: 0, no injury; 1, injury to 25% of the field; 2, injury to 50% of the field; 3, injury to 75% of the field; and 4, diffuse injury. The ultimate score was obtained by adding the aforementioned scores. Immunohistochemistry was performed using the paraffin-embedded tissue sections at 4 µm thickness mounted on glass slides. The slides were then deparaffinized and rehydrated. Then, the lung sections were incubated with primary antibodies against mouse TXNIP (ab210826, dilution: 1:200) and xanthine oxidase (XO, ab176165, dilution: 1:200) (both from Abcam), and then with biotin secondary antibodies (B3640; Sigma-Aldrich; Merck KGaA) at room temperature for 30 min. The number of XO-, and TXNIP-positive cells in lung sections was counted using Pax-it software (version 6; Paxcam, Villa Park, IL, USA).

Whole blood samples from mice were collected and maintained in acid citrate dextrose (Sigma-Aldrich; Merck KGaA). Anti-CD11b eFluor450 (1:100), anti-CD41-allophycocyanin (1:100) (both from eBioscience; Thermo Fisher Scientific, Inc.), anti-Ly6C-PE (1:100; BD Biosciences, Franklin Lakes, NJ, USA) and anti-Ly6G-phycoerythrin (1:100; BD Biosciences) were used to detect leukocyte and platelets antigens. Samples were examined with an LSRII flow cytometer (BD Biosciences) and analyzed using FCS express software (version 3.0, De Novo Software, Glendale, CA, USA). Neutrophils and monocytes were gated through their forward- and side-scatter characteristics and through their Ly-6G⁺/CD11b⁺ (neutrophil) and Ly-6G⁻/CD11b⁺/Ly-6C⁺ (monocyte) expression pattern. Platelet-neutrophil and platelet-monocyte aggregates were calculated using CD41 antibody staining.

Intracellular ROS generation regulated by LPS, ATP and N-Acetylcysteine (NAC) (both from Sigma-Aldrich; Merck KGaA) was assessed by calculating the fluorescence intensity of 2′,7′-dichlorofluorescein diacetate oxidized product (Molecular Probes; Thermo Fisher Scientific, Inc.). The relative fluorescence intensity of oxidized product of 2′,7′-dichlorofluorescein diacetate, was tested at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a Quantus™ Fluorometer (Promega Corporation, Madison, WI, USA). All cells after various treatments were harvested for ROS assessment through ROS Fluorescent Probe-DCF (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China;http://www.vigorousbiol.com/) according to the manufacturer's instructions.

Lung samples were immediately acquired from mice following sacrifice and were used in the extraction of total RNA using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Subsequently, the RNA was purified using the RNeasy mini kit following the manufacturer's instructions (Qiagen, Inc., Valencia, CA, USA). cDNA was synthesized using SuperScript II RNase H-Reverse Transcriptase (Invitrogen; Thermo Fisher Scientific, Inc.). SYBR-Green Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.; 10 µl Master Mix, 0.5 µl forward primer, 0.5 µl reverse primer, 1 µl cDNA and 8 µl ddH₂O) was used, and qPCR was performed in accordance with the manufacturer's protocol. The 7000 Sequence Detection system (Applied Biosystems; Thermo Fisher Scientific, Inc.) was applied to perform qPCR analysis. The specific sequences of primers used in the study for gene amplification were shown in Table I. The cycling conditions (33 cycles) were exhibited as followings: 95°C for 5 min, 95°C for 10 sec, 60°C for 30 sec, 95°C for 15 sec, 60°C for 60 sec and 95°C for 15 sec. The expression levels of targeting mRNAs were normalized to that of housekeeping gene of GAPDH using the 2^−ΔΔCq method (20).

The total protein of lung tissue samples and cells was extracted by addition of cold lysis buffer [10 mM Tris-HCl, 1 mM EDTA, and 250 mM sucrose, pH 7.4, containing 15 µg/ml aprotinin, 5 µg/ml leupeptin, 0.1 mM phenylmeth-anesulfonyl fluoride (PMSF), 1 mM NaF, and 1 mM Na₃VO₄ (Beyotime Institute of Biotechnology)] for 50 min at 4°C. Then, the lysates were centrifuged at 12,000 × g for 20 min at 4°C. The soluble protein concentrations in the lysates were assessed using a BCA Protein Assay kit. Next, 40 µg total protein was separated on a 12% SDS-PAGE gel and was transferred onto a polyvinylidene difluoride membrane. Then, the membrane was blocked in 5% dried milk for 2 h at room temperature and was incubated with specific primary antibodies at 4°C overnight. The membrane was then washed with TBS Tween-20 (TBST, containing 0.1% Tween-20) for three times, followed by incubation with a horseradish peroxidase-conjugated secondary antibody (A0208; Beyotime Institute of Biotechnology) at room temperature for 2 h. Following another round of washing with TBST, the membrane was then developed using enhanced chemiluminescence (Thermo Fisher Scientific, Inc.). The antibodies used in the study are shown below. The staining intensity of the bands was quantitated by densitometry through ImageJ software (version 1.49, National Institute of Health, Bethesda, MD, USA). Triplicate experiments with at least triplicate samples were performed. The primary antibodies: Rabbit anti-NLRP3 (1:1,000, cat. no. 15101), rabbit anti-ASC (1:1,000, cat. no. 67824) (both from Cell Signaling Technology, Inc., Danvers, MA, USA), rabbit anti-caspase-1 (1:1,000, ab1872), rabbit anti-Nrf2 (1:1,000, ab31163) (both from Abcam), rabbit anti-HO-1 (1:1,000, cat. no. 70081), rabbit anti-p-ERK1/2 (1:1,000, cat. no. 4370; Cell Signaling Technology, Inc.), rabbit anti-ERK1/2 (1:1,000, ab17942; Abcam), rabbit anti-p-JNK (1:1,000, cat. no. 9255), rabbit anti-JNK (1:1,000, #9252) (both from Cell Signaling Technology, Inc.), rabbit anti-XO (1:1,000, ab109235), rabbit anti-TXNIP (1:1,000, ab188865) (both from Abcam), rabbit anti-p-p38 (1:1,000, cat. no. 9211), rabbit anti-p38 (1:1,000, cat. no. 8690), rabbit anti-p-IKKα (1:1,000, cat. no. 2697), rabbit anti-IKKα (1:1,000, cat. no. 2682) (all from Cell Signaling Technology, Inc.), anti-NF-κB (1:1,000, ab19870), rabbit anti-p-NF-κB (1:1,000, ab86299), rabbit anti-IκBα (1:1,000, ab32518), rabbit anti-p-IκBα (1:1,000, ab24783) (all from Abcam), rabbit and anti-GAPDH (1:500, sc-293335, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Immunoreactive bands were visualized by ECL Immunoblot Detection system (Pierce; Thermo Fisher Scientific, Inc.) and exposed to Kodak (Rochester, NY, USA) X-ray film. Every protein expression levels will be defined as grey value (ImageJ, Version 1.4.2b, National Institutes of Health) and standardized to housekeeping gene of GAPDH and expressed as a fold of control. All experiments were performed in triplicate and done three times independently.

Data are expressed as the means ± standard error of the mean. Treated cells, tissues and the corresponding controls were compared using GraphPad Prism (GraphPad Software, Inc., La Jolla, CA, USA) by a one-way analysis of variance with Dunn's least significant difference tests. P<0.05 was considered to indicate a statistically significant difference.

Platelets were counted and pulmonary edema was measured with a ratio of lung wet to dry weight. Fig. 1A presents an increased wet/dry ratio in the LPS-treated group and LR reduced this in a dose-dependent manner. In addition, as shown in Table II, PO₂, HCO₃, Hgb, BE and PH increased in the LPS-treated group and these were reduced in LR-pretreated groups. In contrast, LR pretreatment upregulated LAC reduced by LPS but PCO₂ was not significantly different in mice treated as indicated. Arterial blood gas analysis indicated that LR may be protective in LPS-induced ALI in mice. Thromboxane A2 is a marker of platelet activation, and is rapidly hydrolyzed to its inactive stable metabolite, TXB2 (21). Plasma TXB2 were upregulated in mice challenged with LPS and LR treatment dramatically diminished TXB2 expression (Fig. 1B). H&E staining scores were high following LPS treatment indicating lung damage, but this was reversed by LR administration in a dose-dependent manner (Fig. 1C). CD41, a specific platelet marker, can be measured to confirm sequestration of platelets in pulmonary capillaries (22). Platelet activation is involved in ALI (23,24) and immunofluorescence analysis revealed that LR dose-dependently reduced CD41 expression in lung tissues of mice treated with LPS (Fig. 1D). Moreover, RT-qPCR analysis verified downregulated CD41 in LPS-treated lung tissue samples of mice with ALI (Fig. 1E). Thus, LR may have protective effects against LPS-induced ALI.

BALF cytology data show that alveoli undergo dramatic shifts after ALI (25). BAL total protein calculated as an indicator of lung permeability was elevated after LPS instillation (26). Mice given LR had reduced BAL protein compared to the LPS group (Fig. 2A). BAL cells were increased, indicating lung permeability, was markedly increased by LPS treatment, which was reduced by LR administration (Fig. 2B). In addition, BALF macrophages (MACs), BALF polymorphonuclear leukocytes (PMNs) and lymphocytes were increased with LPS induction. However, LR treatment reduced MACs, PMNs and lymphocytes in BALF (Fig. 2C–E). MPO activity, a marker of neutrophil activation (Fig. 2F) was greater in LPS-treated samples compared to controls. MPO activity was reduced in LR-treated groups.

Antibodies against leukocyte-specific surface markers of Ly6G, Ly6C and CD11b were used to measure neutrophil-platelet aggregates (NPA) and monocyte-platelet aggregates (MPAs), defined as Ly6G⁺/CD11b⁺/CD41⁺ and Ly6C⁺/CD11b⁺/CD41⁺, respectively. Whole blood extracted from mice had more NPA and MPA after LPS treatment and this was time-dependent. LR (50 mg/kg) considerably reduced the proportion of NPAs and MPAs after LPS-treatment for 48 h (Fig. 3A and B). NPAs in BAL were elevated after LPS induction and then reduced by LR (Fig. 3C). Furthermore, TXB2 in BAL was greater after LPS treatment and this was downregulated by LR dose-dependently (Fig. 3D). High expression of Ly6G induced by LPS was abolished following LR administration (Fig. 3E). Thus, LR can alleviate LPS-induced ALI via inactivating platelets. LPS treatment increased serum and BALF TNF-α, IL-1β, IL-6, MCP-5, MIP-1α COX2 and IL-10 (Fig. 4) and LR administration reduced all of these except IL-10, which was enhanced, indicating an anti-inflammatory effect of LR.

MDA levels induced LPS were reduced after LR treatment (Fig. 5A). In addition, oxidative stress induced by LPS, as evidenced by elevated levels of H₂O₂ and O₂. in lung tissue samples was suppressed by LR treatment, and SOD, CAT and GPx activity in lung tissue samples (Fig. 5B) were elevated by LPS, and LR treatment increased these levels even further suggesting antioxidant activity. RT-qPCR assays demonstrated that SOD2, SOD1, CAT and glutamate-cysteine ligase (GCLC) expression were enhanced with LPS induction and increased more with LR (Fig. 5C). LPS-triggered increased Nrf2 and HO-1 was augmented by LR dose-dependently (Fig. 5D). Thus, LR exerts an antioxidant effect against LPS-induced ALI in vivo.

The TXNIP/NLRP3 signaling pathway is involved in the progression of oxidative stress (27,28). Therefore, immunohistochemical analysis was used to calculate XO and TXNIP in lung tissue samples. Fig. 6A and B show that XO and TXNIP were significantly induced by LPS compared to controls and LR administration reduced expression of XO and TXNIP. Fig. 6C also shows that LR can reduce LPS-induced increased expression of XO and TXNIP. Western blotting indicated that NLRP3, ASC and caspase-1 protein were markedly increased by LPS treatment, and decreased following LR administration (Fig. 6D). Thus, the TXNIP/NLRP3 pathway are involved in LR-modulated ALI induced by LPS.

MAPKs (p38-MAPK, ERK1/2-MAPK and JNK-MAPK) are associated with oxidative stress (29). Fig. 7A shows that LPS increased phosphorylated p38, ERK1/2 and JNK, and LR considerably reduced this dose-dependently. Inflammation is key to LPS-induced ALI, so western blotting was used to measure the NF-κB pathway, which is important for regulating the secretion of pro-inflammatory cytokines. Fig. 7B data show that LR reduced LPS-induced phosphorylation of IKK-α, IκBα and NF-κB, indicating LR can be used to reduce inflammation by inactivating the NF-κB signaling pathway.

LR may be involved in ameliorating LPS-induced ALI in mice. Fig. 8A presents that pro-inflammatory cytokines TNF-α, IL-1β, IL-6, MCP-5, COX2, inducible nitric oxide synthase (iNOS) and MIP-1α were significantly induced by LPS exposure, and in LR-pretreated groups, these cytokines were downregulated in a dose-dependent manner. IL-10 was stimulated by LPS and enhanced by LR. Western blot analysis indicated that LPS exposure stimulated IKK-α, IκBα and NF-κB phosphorylation (Fig. 8B) and LR dose-dependently reduced NF-κB pathway activation. Therefore, LR had an anti-inflammatory role in LPS-induced BEAS-2B cells.

2′-7′-Dichlorofluorescein (DCF) analysis indicated that LPS triggered high production of ROS, which was reduced by LR in a dose-dependent manner (Fig. 9A and B). In addition, in vitro, LR suppressed LPS-triggered high expression of XO and TXNIP (Fig. 9C). In addition, LR reduced NLRP3, ASC and caspase-1 protein expression induced by LPS. Finally, p38, ERK1/2 and JNK phosphorylation were enhanced by LPS treatment, and this was blunted by LR pretreatment in a dose-dependent manner (Fig. 9D).

Fig. 10A indicates that LR reduced LPS- and ATP-induced ROS production compared to LPS- and ATP-single treatment groups. NAC, an ROS scavenger, reduced LPS-induced production of ROS. LR was similar to NAC in suppressing ROS generation. Fig. 10B shows that high expression of XO, TXNIP, p-p38, p-ERK1/2 and p-JNK induced by LPS were downregulated by LR following ATP addition, indicating that reducing oxidative stress-related signaling pathway expression was implicated in LR-ameliorated lung injury. In addition, LR treatment suppressed activation of IKK-α, IκBα and NF-κB in LPS and ATP co-treatment groups (Fig. 10C). Finally, pro-inflammatory cytokines TNF-α, IL-1β, IL-6, MCP-5, COX2, iNOS and MIP-1α were induced by LPS exposure, and this agrees with data in Fig. 8A. Moreover, after ATP addition, the anti-inflammatory role of LR, especially at the highest dose of 40 µM, was observed compared to the LPS group (Fig. 10D). Thus, LR can reduce LPS-induced ROS, inhibiting inflammation and oxidative stress in human lung epithelial cells.