Protective Effects of Dioscin against Lipopolysaccharide-Induced Acute Lung Injury through Inhibition of Oxidative Stress and Inflammation

Abstract

The protective effects of dioscin, a natural steroidal saponin from some medicinal plants including Dioscorea nipponica Makino, against lipopolysaccharide (LPS)- induced acute liver and renal damages have been reported in our previous works. However, the actions of dioscin against LPS-induced acute lung injury (ALI) is still unknown. In the present study, we investigated the effects and mechanisms of dioscin against LPS-induced ALI in vitro and in vivo. The results showed that dioscin obviously inhibited cell proliferation and markedly decreased reactive oxidative species level in 16HBE cells treated by LPS. In addition, dioscin significantly protected LPS-induced histological changes, inhibited the infiltration of inflammatory cells, as well as decreased the levels of MDA, SOD, NO and iNOS in mice and rats (p < 0.05). Mechanistically, dioscin significantly decreased the protein levels of TLR4, MyD88, TRAF6, TKB1, TRAF3, phosphorylation levels of PI3K, Akt, IκBα, NF-κB, and the mRNA levels of IL-1β, IL-6, and TNF-α against oxidative stress and inflammation (p < 0.05). Dioscin significantly reduced the overexpression of TLR4, and obviously down-regulated the levels of MyD88, TRAF6, TKB1, TRAF3, p-PI3K, p-Akt, p-IκBα, and p-NF-κB. These findings provide new perspectives for the study of ALI. Dioscin has protective effects on LPS-induced ALI via adjusting TLR4/MyD88- mediated oxidative stress and inflammation, which should be a potent drug in the treatment of ALI.

Keywords: TLR4 signal pathway; acute lung injury; dioscin; inflammation; lipopolysaccharide; oxidative stress.

FIGURE 1

Dioscin inhibits proliferation of the cells treated with lipopolysaccharide (LPS) in vitro. (A) The chemical structure of dioscin. (B) Effect of dioscin (75, 150, 300, 600, 1200, and 2400 ng/ml) on the viability of 16HBE cells for 24 h, and the effect of dioscin (150, 300, and 600 ng/ml) on the proliferation of 16HBE cells treated with LPS (100 ng/ml) for 6, 12, and 24 h. Values are expressed as the mean ± SEM (n = 5). *p < 0.05 and **p < 0.01 compared with model group. ^##p < 0.01 compared with control group.

FIGURE 2

Dioscin rehabilitates LPS-induced injury in vitro and in vivo. (A) Effect of dioscin (150, 300, and 600 ng/ml for 24 h pretreatment) on the cellular morphology and structure of 16HBE by bright image (200 × magnification) investigation. (B) Effects of dioscin on LPS-induced lung injury in mice and rats based on H&E staining (200 × original magnification).

FIGURE 3

Dioscin attenuates oxidative stress in vitro and in vivo. (A) Effect of dioscin (150, 300, and 600 ng/ml for 24 h pretreatment) on the ROS level in 16HBE cells by immunofluorescence assay (200 × magnification). (B) Effect of dioscin on the levels of MDA, SOD, NO, and iNOS in mice and rats. Values are expressed as the mean ± SEM (n = 8). ^##p < 0.01 compared with control group; *p < 0.05 and **p < 0.01 compared with model group.

FIGURE 4

Dioscin adjusts TLR4/MyD88 signal in vitro. (A) Effect of dioscin on the protein levels of TLR4 and MyD88 in 16HBE cells. (B) Effect of dioscin on the expression levels of TLR4 and MyD88 in 16HBE cells based on immunofluorescence assay (400 × original magnification). (C) Effect of dioscin on the expression levels of TRAF6, TKB1, TRAF3, p-PI3K, p-Akt, p-IκBα, and p-NF-κB in 16HBE cells. (D) Effect of dioscin on the mRNA levels of TNF-α, IL-1β, and IL-6 in 16HBE cells. Values are expressed as the mean ± SEM (n = 3). ^##p < 0.01 compared with control group; **p < 0.01 compared with model group.

FIGURE 5

Dioscin adjusts TLR4/MyD88 signal in vivo. (A) Effect of dioscin on the protein levels of TLR4 and MyD88 in mice and rats. (B) Effect of dioscin on the expression levels of TLR4 and MyD88 based on immunofluorescence assay in mice and rats (200 × original magnification). (C) Effect of dioscin on the expression levels of TRAF6, TKB1, TRAF3, p-PI3K, p-Akt, p-IκBα, and p-NF-κB in mice and rats. (D) Effect of dioscin on the mRNA levels of TNF-α, IL-1β, and IL-6 in mice and rats. Values are expressed as the mean ± SEM (n = 3). ^##p < 0.01 compared with control group; *p < 0.05 and **p < 0.01 compared with model group.

FIGURE 6

TLR4 DNA abrogates the protective effect of dioscin. (A,B) Effect of dioscin on the level of ROS, and the expression levels of TLR4 and MyD88 based on immunofluorescence assay (400 × original magnification). (C) Effect of dioscin on the protein levels of TLR4, MyD88, TRAF6, TKB1, TRAF3, p-PI3K, p-Akt, p-IκBα, and p-NF- κB in 16HBE cells after transfection.