Wogonin prevents lipopolysaccharide-induced acute lung injury and inflammation in mice via peroxisome proliferator-activated receptor gamma-mediated attenuation of the nuclear factor-kappaB pathway

Abstract

Acute lung injury (ALI) from a variety of clinical disorders, characterized by diffuse inflammation, is a cause of acute respiratory failure that develops in patients of all ages. Previous studies reported that wogonin, a flavonoid-like chemical compound which was found in Scutellaria baicalensis, has anti-inflammatory effects in several inflammation models, but not in ALI. Here, the in vivo protective effect of wogonin in the amelioration of lipopolysaccharide (LPS) -induced lung injury and inflammation was assessed. In addition, the in vitro effects and mechanisms of wogonin were studied in the mouse macrophage cell lines Ana-1 and RAW264.7. In vivo results indicated that wogonin attenuated LPS-induced histological alterations. Peripheral blood leucocytes decreased in the LPS-induced group, which was ameliorated by wogonin. In addition, wogonin inhibited the production of several inflammatory cytokines, including tumour necrosis factor-α, interleukin-1β (IL-1β) and IL-6, in the bronchoalveolar lavage fluid and lung tissues after LPS challenge, while the peroxisome proliferator-activated receptor γ (PPARγ) inhibitor GW9662 reversed these effects. In vitro results indicated that wogonin significantly decreased the secretion of IL-6, IL-1β and tumour necrosis factor-α in Ana-1 and RAW264.7 cells, which was suppressed by transfection of PPARγ small interfering RNA and GW9662 treatment. Moreover, wogonin activated PPARγ, induced PPARγ-mediated attenuation of the nuclear translocation and the DNA-binding activity of nuclear factor-κB in vivo and in vitro. In conclusion, all of these results showed that wogonin may serve as a promising agent for the attenuation of ALI-associated inflammation and pathology by regulating the PPARγ-involved nuclear factor-κB pathway.

Introduction

Inflammatory lung diseases, including acute lung injury (ALI) and its severe form, acute respiratory distress syndrome, pneumonia and chronic obstructive pulmonary disease, cause significant morbidity and mortality worldwide and have a major impact on public health.¹ ALI is highly associated with infectious aetiologies,² which are characterized by a systemic inflammatory state in response to circulating microbes or microbial toxins.³ Despite extensive investigation revealing the pathogenetic factors of ALI, current treatments do not significantly reduce lung injury and mortality.^4,5 Therefore, new and effective strategies are still required for ALI patients. Animal pulmonary inflammation models induced by the experimental administration of lipopolysaccharide (LPS) have been used extensively for testing new anti-inflammatory drugs.^6,7

The infiltration of inflammatory cells, especially neutrophils, into airspaces is a key event in the development of LPS-induced ALI.^7,8 In particular, activated alveolar macrophages and neutrophils cause the excessive production of pro-inflammatory cytokines, including tumour necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), which attract neutrophils and cause the release of further cytokines and chemokines.^9,10 High levels of TNF-α and nitric oxide (NO) production can aggravate lung injuries in patients with acute respiratory distress syndrome and endotoxaemic animals.^9,11 Hence, these pro-inflammatory mediators and the upstream nuclear factor-κB (NF-κB) signalling pathway may play critical roles in the pathogenesis of ALI.

Peroxisome proliferator-activated receptor γ (PPARγ), a member of the nuclear hormone receptor family, is prominently involved in many feedback loops that normally limit inflammation. The anti-inflammatory effects attributed to PPARγ activation include decreases in cytokines, chemokines, reactive oxygen species and adhesion molecules.¹² Notably, PPARγ also inhibits the activation and nuclear translocation of NF-κB,¹³ while it enhances the transcription of anti-inflammatory and anti-oxidant genes.¹⁴

Flavonoids, the major bioactive ingredients in radix scutellariae, comprise a group of naturally occurring and low-molecular-weight plant products including wogonin, oroxylin A, baicalein and baicalin. They are well-known for their various biological activities such as anti-inflammatory activities.^15,16 It is reported that wogonin alleviates inflammatory processes by inhibiting the production of NO, prostaglandin E₂, cyclooxygenase-2 and monocyte chemoattractant protein-1.^16–18 Increasing evidence has also demonstrated that wogonin possesses anti-inflammatory activity in vivo in several animal models of inflammation, including dextran sulphate sodium-induced colitis in the mouse¹⁸ and 12-O-tetradecanoylphorbol-13-acetate-induced skin inflammation in mice,¹⁷ however, the anti-inflammatory effect of wogonin on ALI has not been studied. Therefore, the present study examined the effect and mechanisms of wogonin on ALI induced by LPS in mice.

Materials and methods

Materials

The LPS (Escherichia coli: Serotype O55:B5; Sigma Chemical Co., St Louis, MO) was administered intravenously (20 mg/kg) to induce acute pulmonary inflammation. Wogonin was provided by Dr Zhiyu Li in the College of Pharmacy at China Pharmaceutical University. In the in vivo study, wogonin (25 mg/bottle) was made from freeze-dried powder by Dr Xue Ke in the College of Pharmacy at China Pharmaceutical University, and administered (30 mg/kg, intravenous) 3 hr before LPS in mice. The doses of drugs used were based on previous studies^19,20 and preliminary experiments. In the in vitro study, wogonin was dissolved in DMSO as a stock solution (100 mm), stored at −20°, then freshly diluted with Dulbecco's modified Eagle medium (Gibco, Invitrogen, Carlsbad, CA) to its final concentration. GW9662 (Sigma-Aldrich, St Louis, MO), a PPARγ antagonist, was dissolved in 10% DMSO and administered at a dose of 1 mg/kg by tail vein injection 45 min before wogonin injection. The dose and timing of GW9662 administration were based on previous work.²¹ Pentobarbital was purchased from Ceva Sante Animale (Maassluis, the Netherlands).

The PPARγ small interfering RNA (siRNA) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were used for Western blot analysis: IκBα (6A920), NF-κB p65 (C-20), PPARγ (E-8), and Lamin A (H-102) antibodies (Santa Cruz Biotechnology) at 1 : 400 dilution; NOS2 (V1131) polyclonal antibody and Histone H3 (L20) polyclonal antibody (Bioworld Technology Inc., St. Louis Park, MN) at 1 : 800 dilution; β-actin antibodies (Boster, Wuhan, China) at 1 : 2000 dilution.

Cell culture

The mouse macrophage cell lines Ana-1 and RAW 264.7 were purchased from the Cell Bank of Shanghai Institute of Biochemistry & Cell Biology at the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle medium (Gibco) supplemented with 10% (volume/volume) heat-inactivated fetal bovine serum (Gibco, Paisley, UK), 100 U/ml penicillin G, and 100 μg/ml streptomycin at 37° with 5% CO₂.

Model of LPS-induced acute lung inflammation and grouping

Female C57BL/6 mice, 6–8 weeks old, weighing 18–22 g, were supplied by the Shanghai Laboratory Animal Centre at the China Academy of Sciences (Certificate No. 122). The mice were raised in an air-conditioned room under controlled lighting (12 : 12 hr light : day) with food and water provided ad libitum. Animal care and surgery protocols were approved through the Animal Care Committees of China Pharmaceutical University. All animal studies were appropriately conducted and animals were used in a scientifically valid and ethical manner.

Mice were randomly divided into six groups (n = 8 per group): a control group receiving saline, a group receiving wogonin without LPS, a group receiving LPS only, an experimental group receiving wogonin followed by LPS, a group receiving GW9662, and a group receiving GW9662 followed by wogonin and finally LPS. The first four groups served to determine the protective effects of wogonin in LPS-induced ALI. The last two groups, which received GW9662, served to explore whether the effects of wogonin occurred via the PPARγ pathway.

Mice were killed at 6, 12 and 24 hr after LPS administration under anaesthesia by intraperitoneal injection of 30 mg/kg pentobarbital to collect bronchoalveolar lavage fluid (BALF), blood plasma and tissue samples.

Histological analysis and immunohistochemical staining (IHC)

To characterize the histological alterations, lungs from four animals in each experimental group were immersed in 4% formaldehyde (pH 7·4) fixative for 24 hr, embedded in paraffin, cut into sections 4 mm thick, and stained with haematoxylin and eosin using standard histological techniques. Alveolar congestion, haemorrhage, infiltration or aggregation of inflammatory cells in airspaces or vessel walls, and the thickness of the alveolar walls were assessed. Immunohistochemistry against NF-κB p65 was performed with standard techniques.

Determination of protein concentration and cell counts of BALF

After the trachea of mice was cannulated, and the chest cavity was opened via a midline incision, the lung was lavaged three times with 1 ml of ice-cold sterile saline. Total cell counts were determined using a haemocytometer. The remaining BALF was centrifuged at 1000 g for 5 min at 4°, and the cell-free supernatant was stored at −80° for protein concentration analysis using the BCA Protein Assay kit (Thermo Scientific, Rockford, IL) according to manufacturer's instructions.

Measurement of complete blood count

Complete blood count in whole blood samples (100 μl, containing 50 U/ml heparin), which provides detailed information about white blood cells, was counted with an automatic multi-parameter blood cell counter (model Sysmex KX-21, Hyogo, Japan).

Single-cell preparation and FACS analysis

Whole blood from the abdominal aorta of killed mice was depleted of red blood cells using a red blood cell lysing buffer (eBioscience, San Diego, CA). Total nucleated cells in peripheral blood were isolated after erythrocyte lysis. For BALF, it was centrifuged at 350 g for 5 min at 4° to collected total cells. Single lung cells were obtained by disaggregating the lung. For single-cell suspension preparation from lung tissues, lung tissue was minced and ground into a lung homogenate, filtered through a 300-mesh stainless steel mesh, and resuspended in 1 ml PBS. Repeated centrifugations were performed, removing the supernatant each time. The cell suspensions were then diluted down to 5 × 10⁶ to 10 × 10⁶ cells per ml. For FACS analysis, single-cell suspensions were stained with anti-mouse CD45 Peridinin chlorophyll protein-Cy5.5, CD11b-allophycocyanin, Ly-6G (Gr-1)-phycoerythrin, or F4/80-FITC antibodies (eBioscience) and detected using an LSRII flow cytometer (Becton Dickinson, Franklin Lakes, NJ). Data were analysed using FlowJo7 software (Tree Star, Ashland, OR). In this model, CD11b⁺ F4/80⁺ cells appeared to be monocytes/macrophages, and CD11b⁺ Gr-1⁺ cells appeared to be neutrophils, which accords with the previous study.^22,23

To detect the expression of PPARγ in inflammatory cells, cells were stained with CD11b-allophycocyanin antibodies at 4° for 1 hr and washed with PBS containing 0·1% BSA three times. After incubation with 1 × Fixation/Permeabilization buffer (eBioscience) for 30 min and 1 × Permeabilization buffer (eBioscience) washes, cells were incubated with anti-mouse CD16/CD32 antibody (eBioscience) at 4° for 1 hr, and washed with PBS containing 0·1% BSA. Rabbit anti-PPARγ FITC antibody (BIOSS, Beijing, China) was incubated with cells at 4° for 1 hr and washed with PBS/BSA (0·1% BSA) three times, and then detected using an LSRII flow cytometer. Data were analysed using FlowJo7 software. CD11b⁺ cells appear to be inflammatory cells.^24,25

Measurement of myeloperoxidase activity

The infiltration of inflammatory cells, including neutrophils and macrophages, into lung tissue was assessed through measuring the activity of myeloperoxidase (MPO). Frozen lung tissues were thawed, weighed and homogenized on ice. After centrifugation at 10 000 g at 4° for 20 min, the supernatant was collected and detected by the MPO Assay Kit (Genmed Scientifics Inc., Arlington, MA) according to the manufacturer's instructions.

Cytokine quantification by ELISA

Interleukin-6, IL-1β, TNF-α and macrophage inflammatory protein 2 (MIP-2) secretion was measured using an ELISA Kit (KeyGEN, Nanjing, China) according to the manufacturer's recommendations. The experiments were repeated three times.

Determination of nitrite

The NO production was measured by the Griess reaction using Total Nitric Oxide Assay Kit (Beyotime Institute of Biotechnology, Nanjing, China).²⁶

Measurement of inducible NO synthase enzyme activity

The inducible NO synthase (iNOS) activity is detected by the Nitric Oxide Synthase Assay Kit (Beyotime Institute of Biotechnology), which contains cell-permeable probe DAF-FM DA (3-amino-aminomethyl–2′, 4′- 7′-difluorescein, diacetate) and sufficient substrate l-arginine. Specific experimental process is as follows: intracellular iNOS enzyme catalyses l-arginine to l-citrulline and NO, and non-fluorescent DAF-FM DA is transformed to fluorescent benzotriazole derivative by NO under certain conditions; then the OD values can be measured to detect iNOS activity.

Preparation of cytosolic and nuclear extracts and whole cell lysates

Nuclear and cytosolic protein extracts were prepared according to a modified method previously described.²⁷ One part of the cytosolic and nuclear fractions was subjected to Western blots. For DNA binding activity, 8 μg of the nuclear extract was used for analysis using the electrophoretic mobility-shift assay (EMSA). The whole cell lysates were prepared as described elsewhere.²⁸

Western blot analysis

After cytosolic nuclear extracts and whole cell lysates were prepared, Western blot analysis was performed as previously described.²⁷ Protein samples were separated by SDS–PAGE and transferred onto the PVDF membranes (Millipore, Billerica, MA). The blots were incubated with specific antibodies against the indicated primary antibodies overnight at 4° followed by IRDyeTM800-conjugated secondary antibody for 1 hr at 37°. Detection was performed by the Odyssey Infrared Imaging System (LI-COR Inc., Lincoln, MT). All blots were stripped and reprobed with polyclonal anti-β-actin antibody to ascertain equal loading of proteins.

Immunofluorescence confocal microscopy

The immunofluorescence assays were performed according to a method previously described.²⁷ The images were captured with an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan).

EMSA

Nuclear extracts were isolated as described above. The EMSA was then performed with a non-radioactive (biotin label) gel shift assay according to the manufacturer's protocol. The NF-κB consensus oligonucleotide probes (5′-AGTTGAGGGGACTTTCCCAGGC-3′ and 3′-TCAACTCCCCTGAAAGGGTCCG-5′) were end labelled with biotin using terminal deoxynucleotidyl transferase. The binding reaction was detected as previously described.²⁷

siRNA-mediated knockdown of PPARγ

PPARγ siRNA was delivered by a lipid-based method using Lipofectamine 2000 (Invitrogen Life Technologies, Grand Island, NY) at a final siRNA concentration of 30 μm. After formation of the siRNA–liposome complexes, the mixture was added to cell-free supernatant for 4 hr. The medium was aspirated, and complete medium was added. Transfection efficiency was monitored by Western blot.

Statistical analysis

All results in the text are expressed as means ± SD from triplicate experiments performed in a parallel manner unless otherwise indicated. Statistically significant differences (one-way analysis of variance and post hoc tests) were analysed using GraphPad Prism software (GraphPad Software Inc., Avenida, CA). Details of each statistical analysis tool are provided in the figure legends. Differences with P values < 0·05 were considered statistically significant.

Results

Wogonin alters the number of blood leucocytes, monocytes and neutrophils

After LPS was administered in the earlier (6 hr after LPS), middle (12 hr after LPS), and later (24 hr after LPS) stages, the numbers of leucocytes were detected using a cell counter. The results showed that leucocyte numbers decreased by administering LPS, an effect partially reversed by wogonin at three time-points (Fig. 1a). Next, we first used CD45 gated on all events, ensured that CD45⁺ cell numbers stayed the same and then further detected the frequencies of monocytes and neutrophils using FACS analysis. We found that the frequencies of CD11b⁺ F4/80⁺ monocytes and CD11b⁺ Gr-1⁺ neutrophils in CD45⁺ leucocytes significantly decreased after LPS challenge, but wogonin prevented the LPS-induced decrease (Fig. 1b,c). As shown as Table 1, wogonin could facilitate the recovery of neutrophils in peripheral blood.

Figure 1.

Effects of wogonin on lipopolysaccharide (LPS) -induced changes in circulating leucocytes and the frequencies of monocytes and neutrophils. (a) The effect of wogonin (30 mg/kg) on the number of circulating leucocytes after LPS administration was detected using the cell counter. Data represent the mean ± SD of six animals. **P < 0·01 versus the LPS group. (b) The frequencies of monocytes and neutrophils in blood were measured using FACS. Data represent the mean ± SD of six animals. *P < 0·05 versus the saline group. ^#P < 0·05 versus the LPS group. (c) Representative FACS blots for detecting the distribution of myeloid cells in peripheral blood, including CD11b⁺F4/80⁺ monocytes and CD11b⁺Gr-1⁺ neutrophils.

Table 1.

Effects of wogonin on blood counts of neutrophils at 24 hr after lipopolysaccharide (LPS) injection in mice

	Parameters
Treatments	Neutrophils (%)
Saline	12·05 ± 1·01
LPS	5·94 ± 0·771
LPS + wogonin	18·14 ± 2·412
Wogonin	13·45 ± 0·61

Each data point represents the mean ± SD of six animals for each group.

¹P < 0·05 versus basal levels of saline group.

²P < 0·05 versus LPS group.

Wogonin alleviates LPS-induced acute lung injury

Lung tissue samples were collected after administrated with LPS 24 hr and sections stained with haematoxylin & eosin to determine the degree of lung injury (Fig. 2a). Lung tissues from the experimental group administered LPS alone were significantly damaged with interstitial oedema and haemorrhage, thickening of the alveolar wall and infiltration of neutrophils and macrophages in the alveolar wall. Wogonin effectively relieved these symptoms.

Figure 2.

Effects of wogonin on lipopolysaccharide (LPS) -induced lung injury. (a) Lung tissue samples were collected at 24 hr after LPS administration, and sections were stained with haematoxylin & eosin. Mice treated with the following: saline, LPS, LPS+wogonin, LPS+wogonin+GW9662, wogonin and GW9662. Arrows show venous/alveolar congestion, and infiltrating macrophages and neutrophils in lung tissues. (b) The frequencies of CD11b⁺ F4/80⁺ macrophages and CD11b⁺ Gr-1⁺ neutrophils in lung tissues were measured by FACS. Data are the mean ± SD of six mice. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group. (c) Representative FACS blots for detecting CD11b⁺ F4/80⁺ macrophages and CD11b⁺ Gr-1⁺ neutrophils in lung tissues. (d) Effect of wogonin on myeloperoxidase (MPO) activity in LPS-challenged mice. Data represent the mean ± SD of six animals. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group. (e) Effect of wogonin on the expression of macrophage inflammatory protein 2 (MIP-2) in the LPS-treated mice. Data represent the mean ± SD of six animals. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group.

Next, the infiltration of inflammatory cells was examined in the lung. There were significant changes in the frequencies of CD11b⁺ F4/80⁺ macrophages and CD11b⁺ Gr-1⁺ neutrophils in the lung of ALI mice compared with control mice, whereas there was a reduction after the administration of wogonin (Fig. 2b,c). It is known that MPO was released from the cytoplasmic granules of monocytes/macrophages and neutrophils, which correlates with neutrophil infiltration into lung parenchyma or alveolar spaces.^29,30 The MPO activity was much higher in the LPS group compared with the control group, whereas this increase was significantly reduced by the pre-administration of wogonin (Fig. 2d). Additionally, LPS-induced secretion of MIP-2 was decreased by wogonin in lung tissue at 24 hr (Fig. 2e).

The PPARγ transcription factor family has recently been the focus of much interest for its possible roles in the regulation of inflammation and immune responses.^32,31 To determine the role of PPARγ in the anti-inflammatory effect of wogonin, the degree of inflammatory injury with the administration of GW9662 was examined. GW9662 prevented the effect of wogonin on LPS-induced acute lung injury, inflammatory cell infiltration, MPO activity and MIP-2 secretion (Fig. 2).

All the results above demonstrated that wogonin attenuated LPS-induced lung injury, which was related with its effect on PPARγ activity.

Wogonin reduces BALF protein concentration, total cell number, and inflammatory cell number in LPS-induced ALI mice

After LPS was intravenously administered, the BALF protein concentration and total cell number significantly increased; however, these effects were attenuated by wogonin (Fig. 3a,b). Wogonin significantly reduced the LPS-induced increase of CD11b⁺ Gr-1⁺ neutrophils and CD11b⁺ F4/80⁺ macrophages in BALF using FACS (Fig. 3c). Administering GW9662 partially reversed the wogonin-induced reduction in protein concentration and the numbers of total cells, macrophages and neutrophils.

Figure 3.

Effect of wogonin on protein concentration, total cell counts, and the frequencies of neutrophils and macrophages in bronchoalveolar lavage fluid (BALF) from lipopolysaccharide (LPS) -induced acute lung injury (ALI) mice. BALF samples were collected at 6, 12 and 24 hr after LPS administration. Wogonin reduced the protein concentration (a) and numbers of total cells (b) in BALF and these effects were partially reversed by GW9662. Data represent the mean ± SD of six animals. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group. (c) Representative FACS blots for detecting CD11b⁺ Gr-1⁺ neutrophils and CD11b⁺ F4/80⁺ macrophages in BALF.

Wogonin suppresses the secretion and expression of inflammatory mediators in LPS-induced ALI mice

To determine the anti-inflammatory activity of wogonin, the concentrations of inflammatory cytokines were first detected using the ELISA. For BALF, the concentrations of TNF-α, IL-6 and IL-1β were significantly elevated by LPS at three time-points (6, 12, and 24 hr), but IL-6 and IL-1β peaked at 24 hr and TNF-α peaked at 6 hr. However, wogonin inhibited the increased secretion of inflammatory cytokines at the three time-points and GW9662 partially reversed the wogonin-induced down-regulation (Fig. 4a–c). Levels of TNF-α, IL-1β and IL-6 in lung tissues were significantly increased in ALI mice, whereas wogonin blocked this up-regulation (Fig. 4d–f). A number of previous studies suggested that many chemical compounds inhibit LPS-induced iNOS expression by blocking the NF-κB signalling pathway.^33–35 Our results demonstrated that wogonin blocked the LPS-stimulated up-regulation of NO production in lung tissue (Fig. 4g). Similar effects were observed on the expression and activity of iNOS, and GW9662 also blocked these effects of wogonin (Fig. 4h,i). Overall, these results indicated that wogonin inhibited the expression and activation of inflammatory mediators and that the inhibition was reversed by GW9662.

Figure 4.

Effect of wogonin on the production of lipopolysaccharide (LPS) -induced inflammatory mediators in LPS-induced acute lung injury (ALI) mice. The secretion of tumour necrosis factor-α (TNF-α) (a), interleukin-1β (IL-1β) (b) and IL-6 (c) in bronchoalveolar lavage fluid (BALF) after administration of LPS for 6, 12 and 24 hr were detected using ELISA. Data represent the mean ± SD of six animals. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group. After administration of LPS for 24 hr, the lung tissue was minced, ground into a homogenate, and then the supernatant of lung tissue was collected. Expression of TNF-α (d), IL-1β (e) and IL-6 (f) in lung tissue was determined using ELISA. Data represent the mean ± SD of six animals. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group. The nitric oxide (NO) concentration (g) and inducible nitric oxide synthase (iNOS) activity (h) were deteced in lung tissue. Data represent the mean ± SD of six animals. *P < 0·05, **P < 0·01 versus the LPS group. ^#P < 0·05, ^##P < 0·01 versus the LPS+wogonin group. (i) The expression of iNOS protein was detected using Western blots. (j) The relative expression of iNOS protein was shown by densitometric analysis. The results are representative of three independent experiments and expressed as means ± SD. *P < 0·05, **P < 0·01 compared with LPS alone; ^#P < 0·05, ^##P < 0·01 compared with the LPS+wogonin group.

As we know, NF-κB is responsible for the transcription of the genes encoding these pro-inflammatory mediators.^36,37 For this reason, we next examined the effect of wogonin on PPARγ-regulated NF-κB signalling.

Wogonin promotes PPARγ expression in inflammatory cells in vivo

To check the PPARγ expression in the infiltrating inflammatory cells, we used FACS analysis to detect the frequencies of inflammatory cell expresseion of PPARγ. As shown in Fig. 5(a,b), we found that wogonin increased the numbers of CD11b- and PPARγ-positive cells at three time-points (6, 12, and 24 hr), and a higher expression of PPARγ in inflammatory cells was presented at 24 hr.

Figure 5.

Effect of wogonin on peroxisome proliferator-activated receptor γ (PPARγ) expression in inflammatory cells of lung tissues. Lung tissues were collected at, 6, 12 and 24 hr after lipopolysaccharide (LPS) administration. (a) Representative FACS blots for detecting inflammatory cells positive for CD11b and PPARγ in lung tissues. (b) The relative ratios of CD11b and PPARγ-positive cells to CD11b-positive cells. Data represent the mean ± SD of six animals. **P < 0·01 versus the LPS group.

Wogonin regulates PPARγ-involved NF-κB signalling in vivo

To further understand the role of PPARγ/NF-κB signalling on wogonin-induced anti-inflammation in vivo, the nuclear expression of PPARγ in lung tissues was detected using Western blot analysis. The results showed that wogonin increased the nuclear level of PPARγ, which was partially reversed by GW9662 (Fig. 6a,b).

Figure 6.

Effect of wogonin on peroxisome proliferator-activated receptor γ (PPARγ) and nuclear factor-κB (NF-κB) nuclear localization and DNA-binding ability in lung tissues of lipopolysaccharide (LPS) -induced acute lung injury (ALI) mice. (a) Nuclear expression level of PPARγ detected by Western blots. (b) The relative ratios of PPARγ protein were represented by densitometric analysis. The results are representative of three independent experiments and expressed as means ± SD. *P < 0·05, **P < 0·01 compared with the LPS-alone group; ^#P < 0·05, ^##P < 0·01 compared with the LPS+wogonin group. (c) Expression level of IκBα detected by Western blots. (d) Densitometric analysis and the relative ratios of IκBα protein. The results are representative of three independent experiments and expressed as means ± SD. *P < 0·05, **P < 0·01 compared with the LPS+wogonin group; ^#P < 0·05, ^##P < 0·01 compared with the LPS+wogonin group. Nuclear expression of NF-κB was detected using Western blots (e), immunohistochemistry (g) and immunofluorescence (h). (f) Densitometric analysis of Western blots and the relative ratios of NF-κB protein in the cytoplasm and nucleus. The results are representative of three independent experiments and expressed as means ± SE. *P < 0·05, **P < 0·01 compared with LPS alone; ^#P < 0·05, ^##P < 0·01 compared with the LPS+wogonin group. (i) The effects of wogonin on LPS-activated NF-κB DNA-binding ability in lung tissue were assessed by EMSA.

The activation of the NF-κB signalling pathway in the lung tissue was examined next. The results from Western blot assay showed that the cytosol content of IκBα was reduced after LPS administration. In contrast, in wogonin-treated mice, the IκBα content was significantly higher, which suggested that wogonin inhibited IκBα degradation (Fig. 6c,d). Moreover, wogonin decreased the nuclear translocation of NF-κB p65 (Fig. 6e,f). Using immunohistochemical staining and immunofluorescence, strong NF-κB p65 staining was observed in lung tissues of ALI mice, compared with rare p65-positive cells in wogonin-treated mice (Fig. 6g,h), suggesting that the anti-inflammatory effect of wogonin may result from the down-regulation of NF-κB. The DNA-binding activity of the NF-κB complex in nuclear extracts was evaluated by EMSA and the results demonstrated that wogonin suppressed NF-κB DNA-binding activity (Fig. 6i).

Based on the above results, we found that wogonin decreased the nuclear translocation and DNA-binding activity of NF-κB, and these effects were also inhibited with GW9662 pre-treatment (Fig. 6). Overall, the inhibition of NF-κB activity and nuclear localization by wogonin was found to partially depend upon the increasing activity and expression of PPARγ.

Wogonin inhibits the secretion of inflammatory cytokines through PPARγ in vitro

Based on the results above, we proposed that wogonin exerted its anti-inflammatory effect by increasing the expression and activity of PPARγ. GW9662 and PPARγ siRNA were applied as tools to examine this hypothesis in vitro.

The survival rates of Ana-1 and RAW 264.7 cells transfected with PPARγ siRNA or treated with GW9662, and then administered with wogonin were examined. These treatments did not affect cell survival (Fig. 7a). The results from Western blot analysis showed that PPARγ expression partly decreased after transfection of PPARγ siRNA in Ana-1 and RAW 264.7 cells (Fig. 7b,c). A decrease in the LPS-induced secretion of TNF-α, IL-1β and IL-6 in the two cell lines after administrated of wogonin was found, whereas the inhibition was reversed in the presence of GW9662 and PPARγ siRNA transfection (Fig. 7d and g). This suggested that PPARγ was involved in the wogonin-induced inhibition of inflammatory cytokines.

Figure 7.

Administration of GW9662 and transfection with peroxisome proliferator-activated receptor γ (PPARγ) small interfering RNA (siRNA) reverses the wogonin-induced decrease of inflammatory cytokines in vitro. (a) The survival rates of Ana-1 and RAW 264.7 cells transfected with PPARγ siRNA or treated with GW9662, and then given wogonin were examined. The results are representative of three independent experiments and expressed as means ± SD. (b) The expression of PPARγ in the nucleus of RAW 264.7 and Ana-1 cells transfected with PPARγ siRNA were examined by Western blots. (c) Densitometric analysis and the relative ratios of PPARγ. The results are representative of three independent experiments and expressed as means ± SD. *P < 0·05 compared with the LPS alone group; ^#P < 0·05 compared with the LPS+wogonin group. The secretion of tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β in Ana-1 cells (d, e) and RAW 264.7 cells (f, g) after administration of GW9662 and transfection of PPARγ siRNA were detected by ELISA. The results are representative of three independent experiments and expressed as means ± SD. *P < 0·05, **P < 0·01 compared with the LPS alone group; ^#P < 0·05 compared with the LPS+wogonin group.

Wogonin represses NF-κB signalling dependent on PPARγ activation in vitro

To explore the potential role of PPARγ in NF-κB suppression induced by wogonin, the nuclear translocation of NF-κB p65 in Ana-1 and RAW 264.7 cells transfected with PPARγ siRNA or administered with GW9662 was examined. Both GW9662 and PPARγ siRNA pre-treatment reversed, at least partially, the inhibitory effect of wogonin on the LPS-induced nuclear translocation of NF-κB in the two cell lines (Fig. 8a-h).

Figure 8.

GW9662 and peroxisome proliferator-activated receptor γ (PPARγ) small interfering (siRNA) reverse wogonin-induced decreases in nuclear factor-κB (NF-κB) nuclear localization and DNA-binding ability in vitro. (a, d) Ana-1 and RAW 264.7 cells were treated with wogonin after administration with GW9662 or transfection with PPARγ siRNA. Cell lysates were prepared to detect the nuclear and cytoplasmic expression of NF-κB using Western blot analysis. (b, c and e, f) Densitometric analyses of Western blots and the relative ratios of NF-κB protein in the cytoplasm and nucleus. The results are representative of three independent experiments and expressed as means ± SD. *P < 0·05 compared with the lipopolysaccharide (LPS) alone group; ^#P < 0·05, ^##P < 0·01 compared with the LPS+wogonin group. (g and h) The nuclear localization of NF-κB was also detected using immunofluorescence. (i, j) Effect of wogonin on the LPS-activated NF-κB DNA-binding ability in Ana-1 and RAW 264.7 cells was assessed by EMSA.

Furthermore, wogonin inhibited LPS-induced DNA binding activity of NF-κB on Ana-1 and RAW 264.7 cells, and the transfection of PPARγ siRNA or administion of GW9662 reversed the effects of wogonin as observed by EMSA (Fig. 8i,j). These findings indicated that PPARγ is a promising target of wogonin in the suppression of NF-κB activation and the signal transduction that promotes the development of inflammation.

Discussion

Acute lung injury occurs in the setting of an acute severe illness complicated by systemic inflammation. This is characterized by an increased permeability of the alveolar–capillary barrier represented by the excess production of inflammatory mediators from inflammatory cells, especially cytokines, chemokines and adhesion molecules.^38–40 When LPS is administered to experimental animals, it mimics acute lung injury, leading to the recruitment of neutrophils and finally the impairment of gas exchange.^3,8,41 The results in this study showed that wogonin induced a significant decrease in thickened intra-alveolar septa, a decrease in the infiltration of prominent inflammatory cells (including alveolar macrophages and neutrophils), and a decrease in occasional alveolar haemorrhage (Fig. 2). Overall these results suggest that wogonin has potential clinical applications for ALI.

The cardinal features of inflammation in ALI animals include elevations in relevant inflammatory mediators (such as cytokines, chemokines and adhesion molecules) and acute-phase proteins in BALF and lung, which aggravate the degree of lung tissue injury.^41–43 Furthermore, cytokines, including TNF-α, IL-6, IL-1β and MIP-2, play important roles in mobilizing neutrophils and promoting LPS-induced ALI.^44–47 In this study, wogonin was concluded to successfully attenuate acute inflammation by inhibiting the release of cytokines (such as TNF-α, IL-6 and IL-1β) in BALF (Fig. 4a–c) and lung tissue (Fig. 4d–f). NO production and iNOS over-expression in the lung, known to lead to the recruitment and activation of inflammatory cells, were reduced by wogonin (Fig. 4g–j). Additionally, in vivo and in vitro experimental results showed that wogonin inhibited the nuclear localization as well as the DNA-binding activity of NF-κB, and that treatment with GW9662 partially blocked these effects (Figs 6e–i and 8). These results suggested that wogonin reduced lung inflammation in ALI mice by inducing PPARγ-mediated attentuation of NF-κB activation (Fig. 9).

Figure 9.

Possible mechanisms of wogonin. Possible mechanisms underlying the inhibitory effects of wogonin on lipopolysaccharide (LPS) -induced acute lung injury.

Lipopolysaccharide stimulates the migration of circulating leucocytes and neutrophils into sites of infection, promoting a rapid regulation of the immune response.^21,48,49 Our present results indicated that circulating leucocytes and neutrophils significantly decreased initially after LPS challenge and that this effect was reversed by wogonin administration (Fig. 1). These results were consistent with the previous studies.²¹ In response to the challenge from endotoxin or bacteria, alveolar macrophages and especially neutrophils mobilize from the peripheral blood and are recruited into the airspaces of the lung leading to acute lung inflammation and injury.^7,8,48,50 Infiltrating peripheral blood monocytes play major roles in regulating neutrophil emigration into tissues^51,52,53 and consequent vascular leak, and the depletion of the peripheral blood monocyte pool is a valuable interventional strategy. This study investigated wogonin inhibition of the recruitment of macrophages and neutrophils in a murine ALI model.

An initial infiltration of CD11b⁺ F4/80⁺ macrophages and CD11b⁺ Gr-1⁺ neutrophils into lung tissues in ALI mice was found, and these pathological changes were reversed by the administration of wogonin (Fig. 2b,c). Decreases of MPO activity and MIP-2 expression in lung tissues could also reflect wogonin-reduced inflammatory cell infiltration (Fig. 2d,e). All these observations suggested that wogonin prevented neutrophils and macrophages trafficking into the lung tissues.

It is generally accepted that M1 macrophages are polarized in an inflammatory environment to produce pro-inflammatory cytokines (e.g. TNF-α, IL-1, IL-6, IL-12 and IL-23) and iNOS (NOS2), while M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Moreover, M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation.^54,55 Therefore, an in vitro approach with RAW 264.7 cells as M1-like macrophages was used to evaluate the inflammatory response.

Wogonin reportedly decreased palmitate-induced reactive oxygen species generation to ameliorate the lipotoxicity-induced apoptosis of cultured vascular smooth muscle cells.⁵⁶ As we know, NF-κB activation with inflammation occurs in a wide array of diseases and animal models,⁵⁷ and there is a staggering amount of literature that correlates wogonin with the inhibition of NF-κB expression levels and activation in vivo and in vitro.^58,59 Therefore, wogonin potentially acts as a reactive oxygen species scavenger that reduces NF-κB activation and the general inflammatory response. However, whether wogonin inhibits inflammatory stimulators induced by NF-κB activation via decreasing ROS accumulation needs to be investigated in a future study.

PPARγ agonists are known for their strong inhibitory effect on inflammatory mediator-induced NF-κB transcriptional activity.⁶⁰ Our results demonstrated that wogonin suppressed the degradation of IκBα and NF-κB DNA binding activity in vivo and in vitro (Figs 6 and 8). However, wogonin had less effect on the LPS-induced nuclear localization and DNA-binding activity of NF-κB after administration of the PPARγ antagonist GW9662, which suggests that PPARγ is a promising target of wogonin in the suppression of NF-κB activation and the signal transduction. Previous studies have also shown that ligand-activated PPARγ can down-regulate the LPS-induced activation of NF-κB through direct interactions with the p65 subunit of NF-κB or through increasing the synthesis of IκB, a known NF-κB inhibitor.^61,62 Therefore, future studies need to determine whether wogonin affects the direct binding of p65/p50 and PPARγ, the activity of c-Jun N-terminal kinase and IκB kinase, or other unidentified pathways in inflammatory cells.

Our results show for the first time that wogonin ameliorates LPS-induced ALI through a mechanism that depends on PPARγ-regulated NF-κB signalling in the inflammatory cells. In previous studies, the blockade of IL-6 and TNF-α production by wogonin was mainly attributed to interference of the Jak-1/-3-STAT1/3 signalling pathway.⁶³ It was also reported that wogonin could alleviate the inflammation through inhibiting the expression and activity of cyclooxygenase-2.⁶⁴ Moreover, wogonin demonstrated potential as a neutrophil apoptosis-inducing anti-inflammatory agent.⁶⁵ Therefore, other pathways may be involved in the anti-inflammatory response and will be studied in future work.

Several flavonoid compounds reportedly activate and up-regulate PPARγ in various states. A recent study investigated the anti-inflammatory action of baicalin, which depended on its ability to activate PPARγ, subsequently suppressed NF-κB-mediated inflammation in vivo and in vitro.⁶⁶ In addition, luteolin influenced the production of cytokines by activating the PPARγ pathway.⁶⁷ The present study also demonstrated that wogonin exerted protection against ALI through PPARγ; however, the possibility that wogonin could be a potent PPARγ activator requires further assessment.

In this study, wogonin alleviated LPS-induced inflammation via the augmention of PPARγ expression and activity, leading to the abrogation of NF-κB activation in vivo and in vitro. These results extend our understanding of the molecular mechanisms underlying the clinical applications of wogonin for ALI.

Glossary

ALI

acute lung injury

BALF

bronchoalveolar lavage fluid

EMSA

electrophoretic mobility-shift assay

IL-1β

interleukin-1β

iNOS

inducible nitric oxide synthase

LPS

lipopolysaccharides

MIP-2

macrophage inflammatory protein 2

MPO

myeloperoxidase

NF-κB

nuclear factor-κB

NO

nitric oxide

PPARγ

peroxisome proliferator-activated receptor γ

siRNA

small interfering RNA

TNF-α

tumor necrosis factor alpha

Disclosures

There is no conflict of interest with this article. All authors have been involved with the work, have approved the manuscript, and agree to its submission.