Gambogic acid protects LPS-induced apoptosis and inflammation in a cell model of neonatal pneumonia through the regulation of TrkA/Akt signaling pathway

Abstract

Objective: In this work, we investigated the effects of gambogic acid (GA) on lipopolysaccharide (LPS)-induced apoptosis and inflammation in a cell model of neonatal pneumonia.

Method: Human WI-38 cells were maintained in vitro and incubated with various concentrations of GA to examine WI-38 survival. GA-preincubated WI-38 cells were then treated with LPS to investigate the protective effects of GA on LPS-induced death, apoptosis and inflammation. Western blot assay was utilized to analyze the effect of GA on tropomyosin receptor kinase A (TrkA) signaling pathway in LPS-treated WI-38 cells. In addition, human AKT serine/threonine kinase 1 (Akt) gene was knocked down in WI-38 cells to further investigate the associated genetic mechanisms of GA in protecting LPS-induced inflammation and apoptosis.

Results: Pre-incubating WI-38 cells with low and medium concentrations GA protected LPS-induced cell death, apoptosis and inflammatory protein productions of IL-6 and MCP-1. Using western blot assay, it was demonstrated that GA promoted TrkA phosphorylation and Akt activation in LPS-treated WI-38 cells. Knocking down Akt gene in WI-38 cells showed that GA-associated protections against LPS-induced apoptosis and inflammation were significantly reduced.

Conclusions: GA protected LPS-induced apoptosis and inflammation, possibly through the activations of TrkA and Akt signaling pathway. This work may broaden our understanding on the molecular mechanisms of human neonatal pneumonia.

Keywords: Neonatal pneumonia; apoptosis; gambogic acid; inflammation; lipopolysaccharide.

Fig. 1

The effect of GA on LPS-induced WI-38 cell death. aWI-38 cells were cultured in vitro, and incubated with Gambogic acid (GA) at concentrations of 0, 1, 2, 5, 10, 20, 50, 100, 200 and 500 nM for 24h. Then, potential cell death caused by GA was characterized using a viability assay (* P < 0.05). bAfter GA pre-incubation, WI-38 cells were treated with 10mg/ml lipopolysaccharide (LPS) for 24h. Again, potential cell death was characterized using a viability assay (* P < 0.05)

Fig. 2

The effect of GA on LPS-induced WI-38 apoptosis and inflammation.aWI-38 cells were cultured in vitro (Ctrl), or treated with 10mg/ml LPS for 24h (LPS), or pre-incubated with 50 nM GA for 24h prior to LPS treatment (LPS + GA). A TUNEL assay was applied to identify apoptotic cells with TUNEL (Red) immunoreaction. During the meantime, DAPI (Blue) immunoreaction was applied to identify WI-38 cell nuclei. bFor images acquired in TUNEL assay, the percentages of apoptotic WI-38 cells were compared among Ctrl, LPS and LPS + GA conditions (* P < 0.05). cFor WI-38 cells under Ctrl, LPS and LPS + GA conditions, western blot analysis were conducted to compare IL-6 and MCP-1 protein expressions. dFor western blot data in (c), relative band intensities of IL-6 and MCP-1 were compared between LPS and LPS + GA conditions (* P < 0.05)

Fig. 3

The effect of GA on TrkA/Akt in LPS-treated WI-38 cells. aWI-38 cells were cultured in vitro (Ctrl), or treated with 10mg/ml LPS for 24h (LPS), or pre-incubated with 50 nM GA for 24h prior to LPS treatment (LPS + GA). Western blot analysis was conducted to examine protein productions of TrkA, p-TrkA and Akt. bFor blot result in (A), TrkA protein expressions were quantitatively compared between WI-38 cells under Ctrl, LPS and LPS + GA conditions (∆ P > 0.05). cFor blot result in (A), phosphor-TrkA (p-TrkA) protein expressions were quantitatively compared between WI-38 cells under Ctrl, LPS and LPS + GA conditions (* P < 0.05). dFor blot result in (A), Akt protein expressions were quantitatively compared between WI-38 cells under Ctrl, LPS and LPS + GA conditions (* P < 0.05)

Fig. 4

The effect of Akt knockdown in WI-38 cells double-treated with GA and LPS. aWI-38 cells were transfected with a siRNA specifically targeting human Akt gene (si_Akt), and a non-specific control siRNA (si_C). Then, a qRT-PCR assay was performed to compare WI-38 endogenous Akt expressions (* P < 0.05). bCell viabilities was compared among WI-38 cells under control (ctrl) condition (not transfected by siRNA, nor treated with LPS), WI-38 cells transfected with si_C or si_Akt, and siRNA-transfected WI-38 cells treated with 10mg/ml LPS for 24h (* P < 0.05; ∆ P > 0.05). cSiRNA-transfected WI-38 cells were pre-incubated with 50 nM GA for 24h and then treated with 10mg/ml LPS for another 24h (LPS + GA). A TUNEL assay was applied to identify apoptotic cells with TUNEL (Red) immunoreaction. Also, DAPI (Blue) immunoreaction was applied to identify WI-38 cell nuclei. dFor siRNA-transfected WI-38 cells which were treated with LPS + GA, the percentages of apoptotic WI-38 cells were compared (* P < 0.05). eFor siRNA-transfected WI-38 cells which were treated with LPS + GA, western blot analysis was conducted to examine IL-6 and MCP-1 protein expressions. fFor western blot data in (E), quantitative assessment on relative band intensities of IL-6 and MCP-1 were performed (* P < 0.05)