Emodin Protects Against Lipopolysaccharide-Induced Acute Lung Injury via the JNK/Nur77/c-Jun Signaling Pathway

Abstract

Background: Acute lung injury (ALI) is a serious inflammatory disease with clinical manifestations of hypoxemia and respiratory failure. Presently, there is no effective treatment of ALI. Although emodin from Rheum palmatum L. exerts anti-ALI properties, the underlying mechanisms have not been fully explored. Purpose: This study aimed to investigate the therapeutic effect and mechanism of emodin on LPS-induced ALI in mice. Methods: RAW264.7 cells and zebrafish larvae were stimulated by LPS to establish inflammatory models. The anti-inflammatory effect of emodin was assessed by ELISA, flow cytometric analysis, and survival analysis. In vitro mechanisms were explored by using Western blotting, luciferase assay, electrophoretic mobility shift assay (EMSA), and small interfering RNA (siRNA) approach. The acute lung injury model in mice was established by the intratracheal administration of LPS, and the underlying mechanisms were assessed by detecting changes in histopathological and inflammatory markers and Western blotting in lung tissues. Results: Emodin inhibited the inflammatory factor production and oxidative stress in RAW264.7 cells, and prolonged the survival of zebrafish larvae after LPS stimulation. Emodin suppressed the expression levels of phosphorylated JNK at Thr183/tyr182 and phosphorylated Nur77 at Ser351 and c-Jun, and increased the expression level of Nur77 in LPS-stimulated RAW264.7 cells, while these regulatory effects of emodin on Nur77/c-Jun were counteracted by JNK activators. The overexpression of JNK dampened the emodin-mediated increase in Nur77 luciferase activity and Nur77 expression. Moreover, the inhibitory effect of emodin on c-Jun can be attenuated by Nur77 siRNA. Furthermore, emodin alleviated LPS-induced ALI in mice through the regulation of the JNK/Nur77/c-Jun pathway. Conclusions: Emodin protects against LPS-induced ALI through regulation on JNK/Nur77/c-Jun signaling. Our results indicate the potential of emodin in the treatment of ALI.

Keywords: JNK/Nur77/c-Jun; acute lung injury; emodin; inflammation; lipopolysaccharide.

FIGURE 1

Emodin suppresses inflammatory response and oxidative stress in vitro and in vivo. (A) The chemical structure of emodin. (B) Emodin inhibits the production of inflammatory factors TNF-α, IL-6, MCP-1, and MIP-2 in LPS-induced RAW264.7 cells. RAW264.7 cells were pretreated with emodin (30, 35, or 40 μM) for 2 h and then treated with LPS (100 ng/ml) for 18 h. The levels of TNF-α, IL-6, MCP-1, and MIP-2 in supernatant were measured by ELISA. (C) Emodin inhibits oxidative stress in LPS-induced RAW264.7 cells. RAW264.7 cells were pretreated with emodin (30, 35, or 40 μM) for 2 h and then treated with LPS (100 ng/ml) for 18 h. The cells were collected and the ROS production was determined by flow cytometric analysis. Data are presented as mean ± SEM, n = 3. ^*** p < 0.001 compared with the control group, ^### p < 0.001 compared with the LPS group. (D) Protective effects of emodin on zebrafish challenged by LPS. 3 dpf zebrafish larvae were yolk-microinjected with LPS (0.5 mg/ml), and then treated with emodin (0.05 μM) or DEX (5 μg/ml). Survival of zebrafish larvae was assessed within 72 h. PBS-microinjected zebrafish larvae were served as the negative control. Data are presented as mean ± SEM. ^*** p < 0.001 compared with the PBS group, ^### p < 0.001 compared with the LPS group.

FIGURE 2

Emodin inhibits LPS-induced inflammation via the JNK pathway in RAW264.7 cells. (A) Emodin suppresses activation of the JNK pathway in LPS-stimulated RAW264.7 cells. RAW264.7 cells were pretreated with emodin (40 μM) for 2 h followed by exposure to LPS (100 ng/ml) for 18 h. Then the expression levels of p-p38 (Thr180/Tyr182), p38, p-JNK (Thr183/Tyr185), JNK, p-ERK (Thr202/Tyr204), and ERK were assessed using Western blotting. (B,C) Anisomycin counteracts the inhibitory effects of emodin on the secretion of inflammatory cytokines. RAW264.7 cells were pretreated with SP600125 (8 μM) for 2 h or anisomycin (18 nM) for 4 h, and then treated with emodin (40 μM) for 2 h followed by exposure to LPS (100 ng/ml) for another 18 h. The levels of TNF-α (B) and IL-6 (C) in supernatant were measured by ELISA. Data are presented as mean ± SEM, n = 3. ^*** p < 0.001 compared with the control group; ^## p < 0.01, ^### p < 0.001 compared with the emodin group; ^† p < 0.05, ^†† p < 0.01, ^††† p < 0.001 compared with the emodin group.

FIGURE 3

Emodin regulates the Nur77/c-Jun pathway in LPS-stimulated RAW264.7 cells by inhibiting the JNK pathway. (A) Emodin regulates the Nur77/c-Jun pathway. RAW264.7 cells were pretreated with emodin (40 μM) for 2 h followed by exposure to LPS (100 ng/ml) for 18 h. The expression levels of p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun were measured by Western blotting. (B) The functional verification of SP600125 and anisomycin. RAW264.7 cells were treated with SP600125 (10 μM) for 22 h or anisomycin (18 nM) for 24 h. The expression levels of p-JNK (Thr183/Tyr185), JNK, p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total cell lysates were detected by Western blotting. (C) Anisomycin blocks the regulatory effects of emodin on the Nur77/c-Jun pathway. RAW264.7 cells were pretreated with SP600125 (8 μM) for 2 h or anisomycin (18 nM) for 4 h, and then treated with emodin (40 μM) for 2 h, followed by exposure to LPS (100 ng/ml) for another 18 h. The expression levels of p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total cell lysates were detected by Western blotting. (D) Emodin increases the transcriptional activity of Nur77 via the JNK pathway. HEK293.7T cells were transfected with plasmid Nur77-luciferase (1.6 μg/ml) and pRL-TK (1.6 μg/ml) in the presence or absence of JNK1 plasmid (3.2 μg/ml). After 24 h transfection, cells were treated with emodin for 8 h, and then luciferase assay was performed. Relative luciferase activity was expressed as the ratio of firefly to Renilla luciferase. Data are presented as mean ± SEM, n = 3. **p < 0.01 compared with the control group; ^### p < 0.001 compared with the emodin group. (E) Emodin increases the protein expression of Nur77 via the JNK pathway. HEK293.7T cells were transfected with JNK1 plasmid (3.2 μg/ml). After 24 h transfection, cells were treated with emodin for 8 h, and then the expression levels of p-JNK (Thr183/Tyr185), JNK, p-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total cell lysates were assessed using Western blotting.

FIGURE 4

Emodin suppresses LPS-induced activation of c-Jun through regulation of Nur77. (A) Emodin decreases the expression levels of p-c-Jun (Ser73) and c-Jun through regulation of Nur77. RAW264.7 cells were transfected with Nur77 siRNA (120 nM) for 24 h, and then treated with emodin (40 μM) for 2 h, followed by exposure to LPS (100 ng/ml) for another 18 h, p-c-Jun (Ser73), and c-Jun were detected by Western blotting. (B) Emodin inhibits the DNA-binding activity of c-Jun through regulation of Nur77. The nuclear lysates were harvested and the DNA-binding activity were measured using EMSA.

FIGURE 5

Emodin inhibits LPS-induced acute lung injury (ALI) in mice via the JNK pathway. (A) SP600125 alleviates the protective effects of emodin on lung histopathological changes in ALI mice. The lung tissues were stained with hematoxylin and eosin (H&E) (40 ×). (B–E) Anisomycin counteracts the inhibitory effects of emodin on inflammation and edema in ALI mice. At 8 h after stimulation by LPS, lung tissues were separated and then weighted to obtain the W/D ratio (B). BALF was collected to detect the total protein concentration (C) using the BCA protein assay kit, and the levels of TNF-α (D) and IL-6 (E) in the supernatant were measured by ELISA. Data are presented as mean ± SEM, n = 8. ^*** p < 0.001 compared with the control group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001 compared with the LPS group; ^† p < 0.05, ^††† p < 0.001 compared with the emodin-40 group.

FIGURE 6

(A) Emodin regulates the Nur77/c-Jun pathway through inhibition of the JNK pathway in LPS-induced acute lung injury mice. The lung tissues were harvested and homogenized. The expression levels of P-Nur77 (Ser351), Nur77, p-c-Jun (Ser73), and c-Jun in total lysates were examined using Western blotting. (B) Illustration of protection provided by emodin on LPS-induced inflammation.