Effects Of Epigallocatechin-3-gallate On Seawater Aspiration-induced Acute Lung Injury

Background:Seawater aspiration-induced acute lung injury(Seawater aspiration-induced ALI) is theserious complication of seawater drowning. Uncontrollable inflammation is the mainmechanism of Seawater aspiration-induced acute lung injury. Meanwhile, extracellularfluid is hypertonic after aspirating seawater. JAK/STAT1pathway is an important signaltransduction pathway in inflammatory response, and also plays an important role in theinduction of epithelial cell apoptosis. At the same time, JAK and STAT1can be activatedby hypertonic extracellular environment. So We think JAK/STAT1pathway may play animportant role on inflammation and epithelial cells apoptosis in seawater aspiration-induced ALI.Epigallocatechin-3-gallate(EGCG), an STAT1inhibitor, is the major catechin in greentea. Previous studies showed that EGCG exhibited the potent anti-cancer and inhibitiontumor metastasise ffects, and effective on diabetes, Parkinson and Alzheimer’s disease. Astudy showed EGCG can reduce myocardial ischemia-reperfusion injury throughinhibiting the activity of STAT1. However, what effects of EGCG on Seawateraspiration-induced ALI is remain unknown. Objective(1) Investigate the mechanism of JAK/STAT1pathway in seawater aspiration-inducedALI;(2) Observe the effects of EGCG on inflammation and epithelial apoptosis in seawateraspiration-induced ALI;(3) Investigate the mechanism of EGCG to protect seawater aspiration-induced ALI.SignificanceThrough in vitro and in vivo experiments to explore the mechanism of EGCG toprotect seawater aspiration-induced ALI and the mechanism of JAK/STAT1pathway inseawater aspiration-induced ALI, provide new ideas for treating seawater aspiration-inducedALI.Methods:1. Animal experiments:(1) Male SD rats(180-220g) were randomly assigned to four groups (all n=8):control group; seawater instilled1h group; seawater instilled3h group; seawater instilled6h group; seawater instilled12h group. Rats received seawater(4ml/kg) instilled into ratlungs through trachea in4min at a steady speed with a1mL syringe except the controlgroup. The rats were sacrificed at the indicated time points after seawater treatment.Arterial partial pressure of oxygen, the inflammatory cytokines of IL-1,IL-10and TNF-α,expression of mRNA of JAK,STAT family and proteins STAT1,P-STAT1were measured.Histological section of rat lungs were observed.(2) Male SD rats(180-220g) were randomly assigned to four groups (all n=12):control group, EGCG group, seawater group and seawater+EGCG group. Rats receivedintraperitoneal injection of1ml normal saline with (for EGCG and seawater+EGCGgroup) or without (for control and seawater group) EGCG (10mg/kg)0.5h beforetrachea was exposed. In seawater and seawater+EGCG group, seawater(4ml/kg) wasinstilled into rat lungs through trachea in4min at a steady speed with a2mL syringe. Therats were sacrifced by aortic transection at6h after trachea exposed. Observed thedynamic changes of arterial partial pressure of oxygen from0hour to3hour. Lungwet-to-dry ratio(W/D), the protein in bronchoalveolar lavage fluid, the inflammatory cytokines of IL-1,IL-10and TNF-α, expression of mRNA of STAT1and proteinsSTAT1,P-STAT1were measured on6hour after instilled seawater. Measurements alsoinclude histological section, fluorescent TUNEL, immunohistochemistry and lung tissueultrastructure through electron microscopy.2. Cell experiments:(1) The rat alveolar macrophage cell line NR8383. Exponentially growing NR8383were deprived of FCS for16h, and divided into4groups: control group, EGCG group,seawater group and seawater+EGCG group. After incubated in the presence or absenceof EGCG(10uM), seawater (30%,0.3ml seawater per1ml total volume) were added tostimulate the cell for6h. The inflammatory cytokines of IL-1,IL-10and TNF-α,expression of mRNA of JAK1,JAK2,STAT1and proteins JAK1,P-JAK1,JAK2,P-JAK2,STAT1,P-STAT1were measured.(2) Human alveolar epithelial cell line A549. Exponentially growing A549weredeprived of FCS for16h. Seawater was added into medium to obtain a final concentrationof10%,20%,30%and40%for6hour to induce A549cell apoptosis. To further explorethe effect of EGCG on A549cell, we chose the30%concentration for6hour in A549cell.A549cell were divided into4groups: control group, EGCG group, seawater group andseawater+EGCG group. Cell received with (for EGCG and seawater+EGCG group) orwithout (for control and seawater group) EGCG (10цM)0.5h before seawater wereadded. The cells undergoing apoptosis were detected by annexin V-FITC and PI stainingand flow cytometry.Results:1. The most serious lung injury appeared on6hour. The expression of STAT1is elevatedfrom1hour and reached the highest on6hours after injury.The histopathology results showed lung tissue edema, inflammatory cell infiltration.Lung injury gradually increased with time depended, the most serious lung injuryappeared on6hour. After lung injury, hypoxemia is significant, pro-inflammatorycytokines(IL-1,TNF-α) are increased and suppression of inflammatory cytokine (IL-10) isdecreased(P<0.05). Expression of protein STAT1is began to increase from1hour, andreach the highest on6hour after lung injury. 2. In vivo, EGCG pretreatment can reduce inflammation and inhibit apoptosis of lung tissue.The results showed that EGCG pretreatment significantly improved hypoxemia andhistopathologic changes(P<0.05), alleviated pulmonary edema and lung vascularleak(P<0.05), reduced the production of TNF-a and IL-1, and increased the production ofIL-10in seawater aspiration-induced ALI rats(P<0.05). Moreover, EGCG pretreatmentreduced the total and the phosphorylated protein level of STAT1, and reduced protein levelof P21and caspase-3in lungs in seawater aspiration-induced ALI rats. FluorescentTUNEL shows EGCG pretreatment can reduce cell apoptosis. Histological section andelectron microscopy results shows EGCG pretreatment can reduce lung edema andinflammatory cell infiltration.3. In vitro, EGCG pretreatment can reduce inflammation of NR8383cells and suppressapoptosis of A549.The results showed that EGCG pretreatment reduced inflammation reaction, reducedthe production of TNF-a and IL-1, and increased the production of IL-10(P<0.05),reduced the total and the phosphorylated protein level of STAT1, and reduced protein levelof P-JAK1,P-JAK2. EGCG pretreatment had no effect on the total protein level of JAK1and JAK2. After intervention with different concentrations of seawater, the apoptosis rateof A549cell increased with the concentration increased(P<0.05). EGCG pretreatmentreduced the apoptosis rate of A549cell after seawater intervention(P<0.05).Conclusions:1. The expression of protein STAT1is signifficantly increased in seawater aspiration-induced ALI. The degree of lung injury is related to the activited STAT1.2. EGCG pretreatment ameliorates inflammation, lung edema and vascular permeability inseawater aspiration-induced ALI via inhibiting JAK/STAT1pathway.3. EGCG pretreatment reduced the apoptosis of alveolar epithelial cell in seawateraspiration-induced ALI via inhibiting JAK/STAT1pathway and protein caspase-3,P21.