Rosavin Alleviates LPS-Induced Acute Lung Injure by Modulating the TLR-4/NF-κB/MAPK Singnaling Pathways

Abstract

Acute lung injury (ALI) is a serious inflammatory disease with high morbidity and mortality. Rosavin is an anti-inflammatory and antioxidant phenylpropanoid and glucoside, which is isolated from Rhodiola rosea L. However, its potential molecular mechanisms and whether it has protective effects against lipopolysaccharide (LPS)-induced ALI remain to be elucidated. To assess the in vitro anti-inflammatory effects and anti-lung injury activity of rosavin, RAW264.7 and A549 cells were stimulated using 1 μg/mL LPS. Rosavin attenuated LPS-induced activation of the TLR-4/NF-κB signaling pathway in RAW264.7 cells and inhibited LPS-induced release of inflammatory factors in A549 cells. A mouse model of acute lung injury was constructed by intraperitoneal injection of 5 mg/kg LPS to observe the therapeutic effect of rosavin. Transcriptomics analysis and Western blot assays were utilized to verify the molecular mechanism, rosavin (20, 40, and 80 mg/kg) dose-dependently ameliorated histopathological alterations, reduced the levels of inflammatory factors, and inhibited the TLR-4/NF-κB/MAPK signaling pathway and apoptosis activation. Rosavin is a promising therapeutic candidate for acute lung injury by inhibiting the TLR-4/NF-κB/MAPK pathway.

Keywords: TLR-4/NF-κB/MAPK pathway; acute lung injury; anti-inflammatory; network pharmacology; rosavin; transcriptome sequencing.

Figure 1

Network pharmacological analysis of rosavin and ALI. (A) Venn analysis of rosavin and ALI targets. (B) Protein network interactions. (C) Histogram of top 20 genes acted on by PPI. (D) KEGG enrichment bubble diagram. (E) Target-pathway diagram.

Figure 2

Rosavin inhibits pro-inflammatory mediator release and oxidative stress generated by LPS in RAW264.7 cells. RAW264.7 cells were treated with 64 μM rosavin and LPS (1 μg/mL) for 24 h. (A) Chemical structure of rosavin. (B) Effect of rosavin on the viability of RAW264.7 cells. (C) Measurement of NO content. (D) ROS measurements by microplate reader. (E) ROS measurements by fluorescence microscope. Scale bar = 50 μm. Data are expressed as the mean ± standard deviation (n = 3); ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 3

Rosavin inhibits the TLR-4/NF-κB signaling cascade generated by LPS in RAW264.7 cells. RAW264.7 cells were treated with 0, 16, 32, 64 μM rosavin and LPS (1 μg/mL) for 24 h. (A) Relative protein levels of TLR-4 and MyD88 in RAW264.7 cells. (B) Subcellular localization of NF-κB p65 in LPS-stimulated RAW264.7 cells. Scale bar = 50 μm. (C) Relative protein levels of COX-2 and TNF-α in RAW264.7 cells. Data are expressed as the mean ± standard deviation (n = 3); ^## p < 0.01 vs. control group; ** p < 0.01 vs. LPS group.

Figure 4

Rosavin attenuated the inflammation response cascade of LPS-induced in A549 cells. A549 cells were treated with 0, 16, 32, 64 μM rosavin and LPS (1 μg/mL) for 24 h. (A,B) Effect of rosavin on the viability. (C) Measurement of TNF-α content. (D) Measurement of IL-6 content. (E) Effect of rosavin on MCP-1, CXCL-2, MIP-3α protein expression. Data are shown as the mean ± standard deviation (n = 3); ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 5

Effect of rosavin on p-ERK and p-p38 protein expression in A549 cells. A549 cells were treated with 0, 16, 32, 64 μM rosavin and LPS (1 μg/mL) for 24 h. Data are shown as the mean ± standard deviation (n = 3); ^# p < 0.05 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 6

Effect of rosavin on Bax and Bcl-2 protein expression in A549 cells. A549 cells were treated with 0, 16, 32, 64 μM rosavin and LPS (1 μg/mL) for 24 h. Data are shown as the mean ± standard deviation (n = 3); ^# p < 0.05, ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 7

Rosavin protects mice from LPS-induced acute lung injury. (A) HE staining results of lung tissue (scale bar = 50 μm, black arrows indicate inflammatory infiltration). (B) Lung injury score. (C) W/D of lung tissue. (D) Myeloperoxidase activity of lung tissue. Data are expressed as the mean ± standard deviation (n = 6); ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 8

Rosavin protects mice from LPS-induced acute lung injury. (A) Micrographs of Giemsa staining of lung tissue (scale bar = 100 μm, blue arrows indicate monocytes, yellow arrows indicate neutrophils, red arrows indicate lymphocytes). (B) Total cell count results in alveolar lavage fluid from each group of mice. (C) Monocyte count in alveolar lavage fluid. (D) Neutrophil count in alveolar lavage fluid. (E) Lymphocyte count in alveolar lavage fluid. Data are expressed as the mean ± standard deviation (n = 6); ^## p < 0.01 vs. control group; ** p < 0.01 vs. LPS group.

Figure 9

Effect of rosavin on BALF liberation of IL-6, IL-1β and TNF-α and the effect of rosavin on MDA, T-SOD, and GSH activity in lung tissues. (A) IL-6 concentration. (B) IL-1β concentration. (C) TNF-α concentration. (D) MDA content. (E) T-SOD activity. (F) GSH activity. Data are shown as the mean ± standard deviation (n = 6); ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 10

Transcriptomics analysis results of the control group and LPS group. (A) Volcano graph. Black dots represent genes, red dots represent up-regulated genes, green dots represent down-regulated genes, and the red line represents the screening screening condition of |log₂FC| > 1, and the blue line represents the screening condition of p < 0.05. (B) GO biological process enrichment graph. (C) KEGG enrichment bubble graph, red boxed options are key signaling pathways. (D) Heat map.

Figure 11

Transcriptomics analysis results of the LPS group and rosavin high dose group. (A) Volcano graph. Black dots represent genes, red dots represent up-regulated genes, green dots represent down-regulated genes, and the red line represents the screening screening condition of |log₂FC| > 1, and the blue line represents the screening condition of p < 0.05. (B) GO biological process enrichment graph. (C) KEGG enrichment bubble graph, red boxed options are key signaling pathways. (D) Heat map.

Figure 12

Effect of rosavin on TLR-4, MyD88, NF-κB p65, and iNOS protein expression in lung tissue. Data are shown as the mean ± standard deviation (n = 3); ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 13

Effect of rosavin on p-ERK, p-p38, and p-JNK protein expression in lung tissue. Data are shown as the mean ± standard deviation (n = 3); ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 14

Effect of rosavin on Bax and Bcl-2 protein expression in lung tissue. Data are shown as the mean ± standard deviation (n = 3); ^# p < 0.05, ^## p < 0.01 vs. control group; * p < 0.05, ** p < 0.01 vs. LPS group.

Figure 15

Schematic diagram of the molecular mechanism of rosavin alleviated LPS-induced ALI.

Figure 16

Laboratory flow diagram.