Salvianolic Acid A Protects against Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting Neutrophil NETosis

Abstract

Salvianolic acid A (SAA) is one of bioactive polyphenol extracted from a Salvia miltiorrhiza (Danshen), which was widely used to treat cardiovascular disease in traditional Chinese medicine. SAA has been reported to be protective in cardiovascular disease and ischemia injury, with anti-inflammatory and antioxidative effect, but its role in acute lung injury (ALI) is still unknown. In this study, we sought to investigate the therapeutic effects of SAA in a murine model of lipopolysaccharide- (LPS-) induced ALI. The optimal dose of SAA was determined by comparing the attenuation of lung injury score after administration of SAA at three different doses (low, 5 mg/kg; medium, 10 mg/kg; and, high 15 mg/kg). Dexamethasone (DEX) was used as a positive control for SAA. Here, we showed that the therapeutic effect of SAA (10 mg/kg) against LPS-induced pathologic injury in the lungs was comparable to DEX. SAA and DEX attenuated the increased W/D ratio and the protein level, counts of total cells and neutrophils, and cytokine levels in the BALF of ALI mice similarly. The oxidative stress was also relieved by SAA and DEX according to the superoxide dismutase and malondialdehyde. NET level in the lungs was elevated in the injured lung while SAA and DEX reduced it significantly. LPS induced phosphorylation of Src, Raf, MEK, and ERK in the lungs, which was inhibited by SAA and DEX. NET level and phosphorylation level of Src/Raf/MEK/ERK pathway in the neutrophils from acute respiratory distress syndrome (ARDS) patients were also inhibited by SAA and DEX in vitro, but the YEEI peptide reversed the protective effect of SAA completely. The inhibition of NET release by SAA was also reversed by YEEI peptide in LPS-challenged neutrophils from healthy volunteers. Our data demonstrated that SAA ameliorated ALI via attenuating inflammation, oxidative stress, and neutrophil NETosis. The mechanism of such protective effect might involve the inhibition of Src activation.

Figure 1

Structural formula of salvianolic acid A.

Figure 2

Effects of SAA at 10 mg/kg on LPS-induced histopathologic changes in the lungs. (a) Representative HE staining in histological sections of the lung at 24 h after ALI modeling established by LPS (10 mg/kg) and administration of SAA (10 mg/kg) or DEX (5 mg/kg) (Scale bar, 50 μm). (b) The semiquantitative scores of the histopathologic changes (mean ± SD, ^∗∗∗p < 0.001, n = 6, each group).

Figure 3

Effects of SAA on the lung edema and leukocyte infiltration in LPS-induced ALI mice. (a) Lung tissues were weighed to calculate the W/D ratio. (b) The total protein concentration in BALF was quantified via BCA assay at wavelength 540 nm. (c) The neutrophils proportion was detected by flow cytometry in BALF. (d) The total cells count was assayed by flow cytometry in BALF. (e) The neutrophils count was measured by flow cytometry in BALF (mean ± SD, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, n = 4 or 6 for each group).

Figure 4

Effects of SAA on the production of inflammatory cytokines and oxidative stress in LPS-induced ALI mice. (a) TNF-α in BALF after ALI 24 h. (b) IL-6 in BALF after ALI 24 h. (c) IL-1β in BALF after ALI 24 h. (d) The activity of SOD in lung tissues challenged with LPS and treated with SAA or DEX. (e) MDA content in lung tissues challenged with LPS and treated with SAA or DEX. The values presented are mean ± SD (^∗∗p < 0.01, ^∗∗∗p < 0.001, n = 6 for each group).

Figure 5

Effects of SAA on NETosis and Src/Raf/MEK/ERK signaling pathway in lung tissues and plasma of ALI mice. (a) Immunofluorescence assay of MPO and citrullinated histones (Cit-H3) in lung tissue at 24 h after ALI modeling and administration of SAA (10 mg/kg) or DEX (5 mg/kg) (scale bar, 50 μm). (b) The levels of MPO-DNA complexes in the plasma of mice. (c) Protein expression in lung homogenate and the relative quantification of p-Src/Src, p-Raf/Raf, p-MEK/MEK, and p-ERK/ERK (mean ± SD, ^∗∗p < 0.01, ^∗∗∗p < 0.001, n = 4 or 6 for each group).

Figure 6

Effects of SAA on NETosis and Src/Raf/MEK/ERK signaling pathway in ARDS neutrophils. (a) Immunofluorescence assay of MPO and Cit-H3 in ARDS neutrophils at 21 h after treated with SAA (50 μM) or DEX (1 μM) and stimulated with Src family agonist (10 μM) or not (scale bar, 50 μm). (b) The levels of MPO-DNA complexes in the ARDS cells supernatant. (c) Protein expression in neutrophils lysate and the relative quantification of p-Src/Src, p-Raf/Raf, p-MEK/MEK, and p-ERK/ERK (mean ± SD, ^∗∗∗p < 0.001; n = 3 for normal volunteers and 5 for ARDS patients).

Figure 7

Effects of SAA on NETosis in normal healthy individual neutrophils. (a) Immunofluorescence assay of MPO and Cit-H3 in normal healthy individual neutrophils at 21 h after challenged with LPS (1 μg/ml) and subsequent treatment. In the SAA treatment group, interference was performed or not with agonist (scale bar, 50 μm). (b) The levels of MPO-DNA complexes in the normal healthy individual cells supernatant (mean ± SD, ^∗∗p < 0.01; ^∗∗∗p < 0.001; n = 6 for each group).