Epigallocatechin-3-gallate ameliorates seawater aspiration-induced acute lung injury via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats

Abstract

Signal transducers and activators of transcriptions 1 (STAT1) play an important role in the inflammation process of acute lung injury (ALI). Epigallocatechin-3-gallate (EGCG) exhibits a specific and strong anti-STAT1 activity. Therefore, our study is to explore whether EGCG pretreatment can ameliorate seawater aspiration-induced ALI and its possible mechanisms. We detected the arterial partial pressure of oxygen, lung wet/dry weight ratios, protein content in bronchoalveolar lavage fluid, and the histopathologic and ultrastructure staining of the lung. The levels of IL-1, TNF-α, and IL-10 and the total and the phosphorylated protein level of STAT1, JAK1, and JAK2 were assessed in vitro and in vivo. The results showed that EGCG pretreatment significantly improved hypoxemia and histopathologic changes, alleviated pulmonary edema and lung vascular leak, reduced the production of TNF-α and IL-1, and increased the production of IL-10 in seawater aspiration-induced ALI rats. EGCG also prevented the seawater aspiration-induced increase of TNF-α and IL-1 and decrease of IL-10 in NR8383 cell line. Moreover, EGCG pretreatment reduced the total and the phosphorylated protein level of STAT1 in vivo and in vitro and reduced the phosphorylated protein level of JAK1 and JAK2. The present study demonstrates that EGCG ameliorates seawater aspiration-induced ALI via regulating inflammatory cytokines and inhibiting JAK/STAT1 pathway in rats.

Figure 1

Structures of epigallocatechin-3-gallate (EGCG).

Figure 2

Effect of EGCG on PaO₂ in seawater aspiration-induced ALI. Seawater aspiration-induced ALI caused a significant decrease of PaO₂ in a time dependent manner, which could be partly reversed by EGCG pretreatment. Data were presented as means ± SEM (n = 8). *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.

Figure 3

Effect of EGCG pretreatment on the lung edema (a) and the concentrations of total protein in BALF (b). EGCG significantly decreased lung wet/dry ratios and total protein in BALF in seawater aspiration-induced ALI rats. Data were presented as mean ± SEM (n = 12 for lung wet/dry ratios and n = 6 for total protein in BALF). *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.

Figure 4

Effect of EGCG pretreatment on the histopathologic changes in lungs in seawater aspiration-induced ALI: (a) control group: the lung structure was normal; (b) EGCG (10 mg/kg) group: there was no obvious change in lung structure compared with control group; (c) seawater group (4 mL/kg): alveolar wall thickened, edema and hemorrhage, less alveolar space and obvious inflammatory cells infiltration into interstitial and alveolar spaced; (d) seawater (4 mL/kg) + EGCG (10 mg/kg): lung injury was significantly alleviated compared with seawater group (hematoxylin and eosin staining, original magnification 200×).

Figure 5

Effect of EGCG pretreatment on the expression of STAT1 and p-STAT1 in lungs in seawater aspiration-induced ALI: (a)–(d) the expression of STAT1; (e)–(h) the expression of p-STAT1; (a) and (e) the control group; (b) and (f) the EGCG group; (c) and (g) the seawater group; (d) and (h) the seawater + EGCG group (original magnification 200×).

Figure 6

Effects of EGCG pretreatment on the expression of STAT1 and p-STAT1 in rats. (a) mRNA expression of STAT1 in rats; (b) representative western blot for the phosphorylated STAT1 and the total STAT1 protein in different groups. Samples were prepared for western blot analysis using anti-STAT1, anti-p-STAT1, and anti-β-actin; (c)-(d) summarized data of the total (c) and the phosphorylated (d) STAT1 protein in different groups. Data represent three independent experiments and are expressed as mean ± SEM; *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.

Figure 7

Effect of EGCG pretreatment on pulmonary ultrastructure. (a) Control group: the normal alveolar epithelial cell type II and thin alveolar (AL) wall. (b) EGCG group: there was no obvious change in pulmonary ultrastructure compared with control group. (c) Seawater group: neutrophil (N) infiltration in the alveolar lumen and thickened alveolar wall were observed. Meanwhile, endoplasmic reticulum expanded and lamellar bodies (LB) emptying increased. (d) Seawater + EGCG group: the change of alveolar wall and the alveolar epithelial cell type II were alleviated compared with seawater group ((a) 5000×; (b) 4000×; (c) 5000×; (d) 8000×). NU: nucleus; ER: endoplasmic reticulum.

Figure 8

Effects of EGCG pretreatment on IL-1, TNF-α, and IL-10 of lung tissue in seawater aspiration-induced ALI. IL-1 (a), TNF-α (b), and IL-10 (c) increased in seawater group. EGCG pretreatment downregulated the levels of IL-1 and TNF-α and upregulated the level of IL-10. Data were presented as mean ± SEM (n = 12). *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.

Figure 9

Effects of EGCG pretreatment on IL-1, TNF-α, and IL-10 in NR8383 cells. IL-1 (a), TNF-α (b), and IL-10 (c) increased in seawater group. EGCG pretreatment decreased IL-1 and TNF-α but increased IL-10. Data were presented as mean ± SEM (n = 12). *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.

Figure 10

Effect of EGCG pretreatment on the expression of STAT1 and p-STAT1 in the cell line NR8383. (a) mRNA expression of STAT1 in NR8383 cells. (b) Representative western blot for the phosphorylated STAT1 and the total STAT1 protein in different groups. (c)-(d) Summarized data of the total (c) and the phosphorylated (d) STAT1 protein level in different groups. Data represent three independent experiments and are expressed as mean ± SEM; *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.

Figure 11

Effect of seawater and EGCG pretreatment on the expression of JAK1, p-JAK1, JAK2 and p-JAK2 in the cell line NR8383. (a) mRNA expression of JAK1 in NR8383 cells. (b) mRNA expression of JAK2 in NR8383 cells. (c) Representative western blot for the JAK1, p-JAK1, JAK2, and p-JAK2 protein in different groups. (d)–(g) Summarized data of the JAK1 (d), p-JAK1 (e), JAK2 (f), and p-JAK2 (g) protein level in different groups. Data represent three independent experiments and are expressed as mean ± SEM; *P < 0.05 versus control group; ^# P < 0.05 versus seawater group.