Ginsenoside Rh2 Ameliorates Lipopolysaccharide-Induced Acute Lung Injury by Regulating the TLR4/PI3K/Akt/mTOR, Raf-1/MEK/ERK, and Keap1/Nrf2/HO-1 Signaling Pathways in Mice

Abstract

The anti-inflammatory effect of ginsenoside Rh2 (GRh2) has labeled it as one of the most important ginsenosides. The purpose of this study was to identify the anti-inflammatory and antioxidant effects of GRh2 using a lipopolysaccharide (LPS) challenge lung-injury animal model. GRh2 reduced LPS-induced proinflammatory mediator nitric oxide (NO), tumor necrosis factor-alpha, interleukin (IL)-1β, and anti-inflammatory cytokines (IL-4, IL-6, and IL-10) production in lung tissues. GRh2 treatment decreased the histological alterations in the lung tissues and bronchoalveolar lavage fluid (BALF) protein content; total cell number also reduced in LPS-induced lung injury in mice. Moreover, GRh2 blocked iNOS, COX-2, the phosphorylation of IκB-α, ERK, JNK, p38, Raf-1, and MEK protein expression, which corresponds with the growth of HO-1, Nrf-2, catalase, SOD, and GPx expression in LPS-induced lung injury. An in vivo experimental study suggested that GRh2 has anti-inflammatory effects, and has potential therapeutic efficacy in major anterior segment lung diseases.

Keywords: MEK; Nrf-2; acute lung injury; ginsenoside Rh2; lipopolysaccharide.

Figure 1

(A) The chemical structure of ginsenoside Rh2 (GRh2); (B) the lung-injury scores were determined; and (C) the effects of GRh2 (5, 10, and 20 mg/mL) on lipopolysaccharide (LPS)-induced lung histopathologic changes in mice. At 6 h after LPS challenge, lungs in each group were prepared for histological evaluation. Representative histological section of the lungs was stained by hematoxylin and eosin (H and E) staining, magnification (400×). The data are presented as the means ± S.E.M (n = 5). ### denotes p < 0.001 compared with sample of control group. ** p < 0.01 compared with the LPS-alone group. The red arrow indicates the symptoms of bleeding and inflammatory cell infiltration. Dexamethasone (Dex).

Figure 2

GRh2 improved (A) pulmonary edema (wet-dry (W/D) ratio); (B) myeloperoxidase (MPO) activity in vivo; and reduced (C) cellular counts and (D) total protein in bronchoalveolar lavage fluid (BALF). Lung tissues were weighed and calculated the W/D ratio. BALF was harvested to investigate at 6 h after LPS treatment. Total cells and total proteins in BALF were analyzed. Data are presented as mean ± S.E.M. (n = 5). ### denotes p < 0.001 compared with sample of control group. *** denotes p < 0.001 compared with the LPS-only group.

Figure 3

GRh2 downregulated (A) nitric oxide (NO); (B) tumor necrosis factor-alpha (TNF-α); (C) IL-1β; (D) IL-4; and (E) IL-6; and (F) increased IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-4, IL-6, and IL-10 were detected at 6 h after LPS challenge by enzyme-linked immunosorbent assay (ELISA). Data are represented as mean ± S.E.M. (n = 5). ### denotes p < 0.001 compared with sample of control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.

Figure 4

Effects of GRh2 on LPS-induced (A) iNOs, COX-2; (B) IκB-α, NF-κB; and (C) mitogen-activated protein kinase (MAPK) phosphorylation signaling expression in lungs. Protein levels of iNOs, COX-2, IκB-α, NF-κB, and MAPK phosphorylation protein expression in lung homogenates were evaluated by western blot analysis after LPS challenge 6 h later. Densitometric analysis of the relevant bands was performed. Data are represented as mean ± S.E.M. (n = 2). ## p < 0.01 and ### p < 0.001 compared with the control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.

Figure 5

Effects of GRh2 on (A) LPS-induced antioxidative enzymes (catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx)); heme oxygenase-1 (HO-1), Trx-1, NF-E2-related factor 2 (Nrf2)/Keap1 and KAP1; (B) TLR4, PI3K, Akt, and mTOR; and (C) Raf-1, p-Raf-1, Mek and p-Mek protein expression in the lungs. Protein levels of catalase, SOD, GPx, HO-1, Trx-1, Nrf2/Keap1, KAP1, TLR4, PI3K, Akt, mTOR, Raf-1, p-Raf-1, Mek and p-Mek protein expression in lung homogenates were evaluated by western blot analysis after LPS challenge 6 h hours later. Densitometric analysis of the relevant bands was performed. Data are represented as mean ± S.E.M. (n = 2). ## p < 0.05 and ### p < 0.001 compared with sample of control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.

Figure 5

Effects of GRh2 on (A) LPS-induced antioxidative enzymes (catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx)); heme oxygenase-1 (HO-1), Trx-1, NF-E2-related factor 2 (Nrf2)/Keap1 and KAP1; (B) TLR4, PI3K, Akt, and mTOR; and (C) Raf-1, p-Raf-1, Mek and p-Mek protein expression in the lungs. Protein levels of catalase, SOD, GPx, HO-1, Trx-1, Nrf2/Keap1, KAP1, TLR4, PI3K, Akt, mTOR, Raf-1, p-Raf-1, Mek and p-Mek protein expression in lung homogenates were evaluated by western blot analysis after LPS challenge 6 h hours later. Densitometric analysis of the relevant bands was performed. Data are represented as mean ± S.E.M. (n = 2). ## p < 0.05 and ### p < 0.001 compared with sample of control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.

Figure 6

(A) GRh2 and Raf-1 inhibitor (GW-5074) reduced NO; (B) TNF-α; (C) IL-1β; (D) IL-6; and (E) IL-10 in BALF. BALF was collected. NO, TNF-α, IL-1β, IL-6, and IL-10 were detected at 6 h after LPS challenge by ELISA. Data are represented as mean ± S.E.M. (n = 5). ### p < 0.001 compared with sample of control group. ** p < 0.01 and *** p < 0.001 compared with LPS-only group.

Figure 7

The schemes of the mechanism for the protective effect of GRh2 on LPS-induced inflammation.