Ginsenoside Rg3 Attenuates Lipopolysaccharide-Induced Acute Lung Injury via MerTK-Dependent Activation of the PI3K/AKT/mTOR Pathway

Abstract

Acute lung injury (ALI) is a common clinical disease with high morbidity in both humans and animals. Ginsenoside Rg3, a type of traditional Chinese medicine extracted from ginseng, is widely used to cure many inflammation-related diseases. However, the specific molecular mechanism of the effects of ginsenoside Rg3 on inflammation has rarely been reported. Thus, we established a mouse model of lipopolysaccharide (LPS)-induced ALI to investigate the immune protective effects of ginsenoside Rg3 and explore its molecular mechanism. In wild type (WT) mice, we found that ginsenoside Rg3 treatment significantly mitigated pathological damages and reduced myeloperoxidase (MPO) activity as well as the production of pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6); furthermore, the production of anti-inflammatory mediators interleukin-10 (IL-10) and transforming growth factor-β (TGF-β), polarization of M2 macrophages and expression levels of the phosphorylation of phosphatidylinositol 3-hydroxy kinase (PI3K), protein kinase B (PKB, also known as AKT), mammalian target of rapamycin (mTOR) and Mer receptor tyrosine kinase (MerTK) were promoted. However, there were no significant differences with regards to the pathological damage, MPO levels, inflammatory cytokine levels, and protein expression levels of the phosphorylation of PI3K, AKT and mTOR between the LPS treatment group and ginsenoside Rg3 group in MerTK^-/- mice. Taken together, the present study demonstrated that ginsenoside Rg3 could attenuate LPS-induced ALI by decreasing the levels of pro-inflammatory mediators and increasing the production of anti-inflammatory cytokines. These processes were mediated through MerTK-dependent activation of its downstream the PI3K/AKT/mTOR pathway. These findings identified a new site of the specific anti-inflammatory mechanism of ginsenoside Rg3.

Keywords: MerTK; acute lung injury; ginsenoside Rg3; inflammatory; lipopolysaccharide.

FIGURE 1

(A) Chemical structure of ginsenoside Rg3. (B) HPLC chromatogram of ginsenoside Rg3.

FIGURE 2

Anti-inflammatory effects of ginsenoside Rg3 in WT mice. (A) Histopathological analysis in lung tissues in WT mice. (B) The production of MPO of lung tissues in WT mice. (C) Effects of ginsenoside Rg3 on cell viability. (D) The proteins expression levels of TNF-α, IL-1β, IL-6, IL-10 and TGF-β in lung tissues were measured by ELISA. (E) The proteins expression levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β in RAW264.7 cells were measured by ELISA. (F) The mRNA expression levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β in RAW264.7 cells were measured by qPCR. β-actin was used as a control. (G) Effects of ginsenoside Rg3 on LPS-induced macrophage polarization in RAW264.7 cells. M1 macrophages were labeled with FITC Rat Anti-Mouse CD11b, PE Rat Anti-Mouse F4/80, APC-Cy7 Hamster Anti-Mouse CD11c and PE-Cy7 Rat Anti-Mouse CD16/CD32 antibodies. M2 macrophages were labeled with PE Rat Anti-Mouse F4/80, FITC Rat Anti-Mouse CD11b and Rat Anti-Mouse CD206 antibodies. The M2/M1 ratio in RAW264.7 cells was analyzed by flow cytometry. Gating was performed on F4/80⁺ cells. CG is the control group. LPS is the LPS-stimulated group. DEX is the dexamethasone group. Ginsenoside (10, 20, and 30) represent ginsenoside Rg3 (10, 20, and 30 mg/kg) + LPS in animals and Ginsenoside (25, 50, and 100) represent ginsenoside Rg3 (25, 50, and 100 μg/mL) + LPS in cells, respectively. The data are presented as the mean ± SEM of three independent experiments. ANOVA, p < 0.01, post hoc #p < 0.05 vs. CG. ^∗p < 0.05 vs. LPS group.

FIGURE 3

Effects of ginsenoside Rg3 on LPS-induced activation of the PI3K/AKT/mTOR pathway in WT groups. (A) The levels of PI3K, AKT and mTOR proteins in lung tissues were measured by western blotting. (B) The levels of PI3K, AKT and mTOR proteins in RAW264.7 cells were measured by western blotting. β-actin was used as a control. CG is the control group. LPS is the LPS-stimulated group. DEX is the dexamethasone group. The data are presented as the mean ± SEM of three independent experiments. ANOVA, p < 0.01, post hoc #p < 0.05 vs. CG. ^∗p < 0.05 vs. LPS group.

FIGURE 4

Effects of ginsenoside Rg3 on the expression of MerTK in WT groups. (A) The expression of MerTK protein in lung tissues was measured by western blotting. (B) The expression of MerTK protein in RAW264.7 cells was measured by western blotting. (C) The expression of MerTK protein in lung tissues was measured by immunofluorescence. p-MerTK protein was labeled with a red fluorophore, and the cell nucleus was labeled with a blue fluorophore. CG is the control group. LPS is the LPS-stimulated group. DEX is the dexamethasone group. Ginsenoside (10, 20, and 30) represent ginsenoside Rg3 (10, 20, and 30mg/kg) + LPS in animals and Ginsenoside (25, 50, and 100) represent ginsenoside Rg3 (25, 50, and 100 μg/mL) + LPS in cells. The data are presented as the mean ± SEM of three independent experiments. ANOVA, p < 0.05, post hoc ^#p < 0.05 vs. CG. ^∗p < 0.05 vs. LPS group.

FIGURE 5

Anti-inflammatory effects of ginsenoside Rg3 in MerTK^-/- mice. (A) Histopathological analysis of lung tissues in MerTK^-/- mice. (B) The production of MPO in lung tissues in MerTK^-/- mice. (C) The protein expression levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β in lung tissues of MerTK^-/- mice were measured by ELISA. (D) The mRNA expression levels of TNF-α, IL-1β, IL-6, IL-10, and TGF-β in lung tissues of MerTK^-/- mice were measured by qPCR. β-actin was used as a control. CG is the control group. LPS is the LPS-stimulated group. DEX is the dexamethasone group. Ginsenoside (10, 20, and 30) represent ginsenoside Rg3 (10, 20, and 30mg/kg) + LPS in animals. The data are presented as the mean ± SEM of three independent experiments. ANOVA, p < 0.01, post hoc ^#p < 0.05 vs. CG.

FIGURE 6

Effects of ginsenoside Rg3 on the LPS-induced activation of the PI3K/AKT/mTOR pathway in MerTK^-/- mice. The levels of PI3K, AKT and mTOR proteins in lung tissues were measured by western blotting. CG is the control group. LPS is the LPS-stimulated group. DEX is the dexamethasone group. Ginsenoside (10, 20, and 30) represent ginsenoside Rg3 (10, 20, and 30 mg/kg) + LPS in animals. The data are presented as the mean ± SEM of three independent experiments. ANOVA, p < 0.01, post hoc ^#p < 0.05 vs. CG.