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.

Keywords: acute lung injury; lipopolysaccharide; nuclear factor-κB; peroxisome proliferator-activated receptor γ; wogonin.

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.

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.

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.

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.

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.

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.

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.

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.

Figure 9

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