Ginkgolide A Ameliorates LPS-Induced Inflammatory Responses In Vitro and In Vivo

Abstract

Ginkgolide A (GA) is a natural compound isolated from Ginkgo biloba and has been used to treat cardiovascular diseases and diabetic vascular complications. However, only a few studies have been conducted on the anti-inflammatory effects of GA. In particular, no related reports have been published in a common inflammation model of lipopolysaccharide (LPS)-stimulated macrophages, and the anti-inflammatory mechanisms of GA have not been fully elucidated. In the present study, we extensively investigated the anti-inflammatory potential of GA in vitro and in vivo. We showed that GA could suppress the expression of pro-inflammatory mediators (cyclooxygenase-2 (COX-2) and nitric oxide (NO) and pro-inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β) in LPS-treated mouse peritoneal macrophages, mouse macrophage RAW264.7 cells, and differentiated human monocytes (dTHP-1) in vitro. These effects were partially carried out via downregulating Nuclear factor kappa-B (NF-κB), Mitogen-activated protein kinases (MAPKs) (p38 mitogen-activated protein kinase and extracellular signal-regulated kinase (ERK), but not c-Jun N-terminal kinase (JNK), and activating the AMP-activated protein kinase (AMPK) signaling pathway also seems to be important. Consistently, GA was also shown to inhibit the LPS-stimulated release of TNF-α and IL-6 in mice. Taken together, these findings suggest that GA can serve as an effective inflammatory inhibitor in vitro and in vivo.

Keywords: AMPK; Ginkgolide A; MAPKs; NF-κB; inflammation.

Figure 1

GA inhibits LPS-stimulated COX-2 expression and NO production. (A) Chemical structure of GA; (B) The effects of GA on cell viability. Macrophages were treated with different concentrations of GA (5, 10, 20, and 40 μg/mL) in the absence or presence of 500 ng/mL LPS for 24 h. Cell viability was quantified with the MTT assay; (C) The effects of GA on the expression of COX-2. Following pre-treatment with GA for 1 h, macrophages were co-incubated with or without 500 ng/mL LPS for 24 h. The protein expression of COX-2 was determined by Western blotting; and (D) the effects of GA on NO production. Macrophages were treated as in (B). Production of NO in the culture medium was measured by the Griess reaction assay. GA, Ginkgolide A; LPS, lipopolysaccharide; dTHP-1, differentiated THP-1; COX-2, cyclooxygenase-2; NO, nitric oxide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Experiments were repeated three times. ^### p < 0.001 versus the control group; * p < 0.05; *** p < 0.05 versus the LPS-treated group.

Figure 2

GA inhibits LPS-stimulate expression and production of pro-inflammatory cytokines in macrophages. (A–C) Effects of GA on LPS-stimulated mRNA expression of TNF-α, IL-6, and IL-1β. Following pre-treatment with GA for 1 h, mouse peritoneal macrophages (A), RAW264.7 cells (B), and dTHP-1 cells (C) were co-incubated with or without 500 ng/mL LPS for 10 h. The total RNA was prepared, and the mRNA expression levels of IL-6, TNF-α, and IL-1β were determined by RT-qPCR. (D,E) Effects of GA on LPS-stimulated production of TNF-α, IL-6, and IL-1β. Following pre-treatment with GA for 1 h, mouse peritoneal macrophages (C) and RAW264.7 cells (D) were co-incubated with or without 500 ng/mL LPS for 24 h. Levels of IL-6, TNF-α, and IL-1β in culture media were quantified by ELISA. TNF, tumor necrosis factor; IL, interleukin; RT-qPCR, quantitative Real-Time RT-PCR; ELISA, enzyme-linked immunosorbent assay. Experiments were repeated three times. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus the control group; * p < 0.05; ** p < 0.01; *** p < 0.05 versus the LPS-treated group.

Figure 2

GA inhibits LPS-stimulate expression and production of pro-inflammatory cytokines in macrophages. (A–C) Effects of GA on LPS-stimulated mRNA expression of TNF-α, IL-6, and IL-1β. Following pre-treatment with GA for 1 h, mouse peritoneal macrophages (A), RAW264.7 cells (B), and dTHP-1 cells (C) were co-incubated with or without 500 ng/mL LPS for 10 h. The total RNA was prepared, and the mRNA expression levels of IL-6, TNF-α, and IL-1β were determined by RT-qPCR. (D,E) Effects of GA on LPS-stimulated production of TNF-α, IL-6, and IL-1β. Following pre-treatment with GA for 1 h, mouse peritoneal macrophages (C) and RAW264.7 cells (D) were co-incubated with or without 500 ng/mL LPS for 24 h. Levels of IL-6, TNF-α, and IL-1β in culture media were quantified by ELISA. TNF, tumor necrosis factor; IL, interleukin; RT-qPCR, quantitative Real-Time RT-PCR; ELISA, enzyme-linked immunosorbent assay. Experiments were repeated three times. ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 versus the control group; * p < 0.05; ** p < 0.01; *** p < 0.05 versus the LPS-treated group.

Figure 3

GA suppresses LPS-induced activation of NF-κB signaling. (A) Effects of GA on degradation of IκB. Mouse peritoneal macrophages (left), RAW264.7 cells (middle), and dTHP-1 cells (right) were pre-treated with 20 μg/mL GA for 1 h and then stimulated with LPS (500 ng/mL) for 0, 15, and 30 min. Total cellular lysates were prepared and analyzed for IκB by Western blotting. The histograms below the blots show the relative density ratios (fold changes) measured by Image J. Values are presented as means ± SD of three independent experiments. ** p < 0.01 versus the LPS group at 15 min; ^# p < 0.05, ^## p < 0.01 versus the LPS group at 30 min. (B,C) Effects of GA on nuclear translocation of p65; (B) mouse peritoneal macrophages (left), RAW264.7 cells (middle), and dTHP-1 cells (right) were pre-treated with 20 μg/mL GA for 1 h and then stimulated with LPS (500 ng/mL) for 1 h. Nuclear and cytosolic extracts were isolated, and the levels of p65 in each fraction were determined by Western blotting. Histone 1 was detected as a nuclear internal control, and GAPDH was used as a cytosolic internal control. The histogram shows relative density ratios (fold changes) measured by Image J. Values are presented as means ± SD of three independent experiments * p < 0.01 versus the LPS group. Mpm, mouse peritoneal macrophages; (C) Mouse peritoneal macrophages (left), RAW264.7 cells (middle), and dTHP-1 cells (right) were treated as in (B). The nuclear translocation of p65 was determined by immunofluorescence staining. DAPI-stained nuclei are indicated by blue fluorescence. Nuclear and cytosolic p65 are indicated by red fluorescence. Nuclei appear purple in merged images. Bar, 50 μm. NF-κB, nuclear factor kappa-B; IκB, inhibitor of NF-κB; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.

Figure 4

GA suppresses activation of MAPKs. (A) Mouse peritoneal macrophages; (B) RAW264.7 cells; and (C) dTHP-1 cells were pre-treated with 20 μg/mL GA for 1 h and then stimulated with LPS (500 ng/mL) for 0, 15, and 30 min. Total cellular proteins were obtained from the cells and analyzed for phosphorylation of ERK, JNK, and p38 by Western blotting. The histograms show relative density ratios (fold changes) measured by Image J. Values are presented as means ± SD of three independent experiments. * p < 0.05 versus LPS group at 15 min; ^# p < 0.05, ^## p < 0.01 versus LPS group at 30 min. p, phospho; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase.

Figure 5

GA upregulates AMPK signaling in LPS-stimulated macrophages. (A) Mouse peritoneal macrophages; (B) RAW264.7 cells; and (C) dTHP-1 cells were treated with the same method as in Figure 4. Cell lysates were prepared and analyzed for phosphorylation of AMPK by Western blotting. The histograms below the blots show relative density ratios (fold changes) measured by Image J. Values are presented as means ± SD of three independent experiments. ** p < 0.01; *** p < 0.001 versus LPS group at 15 min; ^# p < 0.05, ^## p < 0.01 versus LPS group at 30 min. AMPK, AMP-activated protein kinase.

Figure 6

GA inhibits NF-κB and MAPK pathways through AMPK activation in LPS-stimulated mouse peritoneal macrophages. Mouse peritoneal macrophages were pre-treated with 12.5 μM CC for 1 h, followed by GA (20 μg/mL) treatment for 1 h, and LPS (500 ng/mL) stimulation for 0 min (A) or 30 min (B) subsequently. Total cellular proteins were obtained and subjected to Western blotting to detect the phosphorylation of AMPK, ERK, and p38 and the degradation of IκB. CC, compound C.

Figure 7

GA attenuates inflammatory response in LPS-treated BALB/c mice. (A) Effects of GA on production of TNF-α, IL-6, and IL-1β in LPS-induced BALB/c mice. BALB/c mice were randomly divided into three groups. The control group was administered the same amount of solvent i.p. (n = 5). The LPS-induced group was administered LPS (250 μg/kg) (n = 6). The treatment group was administered LPS (250 μg/kg) and GA (20 mg/kg) at the same time (n = 5). Two hours after the injection, blood was harvested, and serum was separated to measure IL-6 (left), TNF-α (middle) and IL-1β (right) levels by ELISA. ^### p < 0.001 versus control group; *** p < 0.05 versus LPS-treated group. (B) The effects of GA on the AMPK pathways in LPS-treated BALB/c mice. BALB/c mice were treated with the same method as in (A). Blood samples were treated with red blood cell lysis buffer to harvest immune cells for analysis of AMPK phosphorylation by Western blotting.