Effect And The Mechanism Of Sodium Tanshinone IIA Sulphonate On Pulmonary Edema Of Seawater Drowning

Background:Drowning is one of the most important public health problems , ranking the second death-causing factor in fatal accidents. Seawater drowning is not only the main death cause of offshore operation and maritme accident, but also an important reason for member depletion of army sea war, training and non-combat affairs. Besides the acute asphyxia caused by reflex laryngismus and tracheospasm, pulmonary edema of seawater drowning, PE-SWD is the major death cause drown by the sea. If the patient was not treated in time, seawater respiratory distress syndrome,SW-RDS will occur which enhances the death rate. Therefore, relieving pulmonary edema and injury caused by pulmonary edema is the key of a successful treatment to seawater drowning patients.It is known that basolateral Na,K-adenosine triphosphatase (Na-K-ATPase) located in the alveolar epithelial cells(AEC) is critical for clearance of edema fluid from the pulmonary alveoli. So up-regulating the activity of Na-K-ATPase accelerates clearance of edema fluid of alveolar and improves PE-SWD.Sodium tanshinone IIA sulphonate (STS),a derivative of phenanthrenequinone, is a major compound extracted from Danshen. It exhibits multiple pharmacological actions and has been commonly used in traditional oriental herbal medicine to prevent or manage cardiovascular diseases and treat inflammatory diseases.Recent studies have reported that it can reduce lung edema and protect lung from LPS-induced acute injury.However, little is known about its effect and mechanism on seawater aspiration-induced acute pulmonary edema. In the present study, we explored the effect of STS on PE-SWD both in vivo and in vitro,and examine the effects of STS on Na-K-ATPase in PE-SWD and its underlying mechanisms.Aim of the study:To investigate whether STS treatment attenuates PE-SWD,and examine the effects of STS on Na-K-ATPase in vivo and in vitro and its underlying mechanisms by ERK signaling pathway.Methods:Animal experiment1.Group of experiments and model making:48 rats were randomly divided into four groups: normal group(NG), STS group, seawater(SW) group, and seawater (SW)+STS group . The model of PE-SWD in rats were established by seawater (4 ml/kg body weight) instillation.STS(25mg/kg) was injected through peritoneal cavity at once after seawater aspiration.2. Index of experiments: 1).PaO₂: Arterial blood gas of rats were observed before seawater aspiration, and 30 min, 1 h, 2 h, 4 h and 6h after seawater aspiration. 2).Lung histology: At 4h after seawater aspiration, lung histology was observed. 3).Index of Pulmonary edema : At 4h after seawater aspiration, lung wet/dry (W/D) ratio and pulmonary microvascular permeability (PMVP )were observed. 4).Measurement of Na-K-ATPase activity and expression: The activity of Na-K-ATPase was measured by a Na-K-ATPase activity assay kit. Western blot,RT-PCR were used to test the effects of STS on Na-K-ATPaseα1,β1 subunit plasma membrane protein expression and mRNA level in PE-SWD.Cell experiment1.Group and preparation of the cells: The A549 cells were randomly divided into five groups: normal group(NG), STS group, seawater(SW) group, seawater(SW) +STS group, seawater(SW)+ STS+ ERK inhibitor(U0126) group. The A549 cells were incubated for 4 h with seawater. STS (25 mg/L) was added immediately after seawater treatment. The A549 cells were pretreated with U0126 as inhibitor of the ERK1/2 activity for 30 min before STS administration.2. Index of experiments: 1).Na-K-ATPase activity and expression : We detected Na-K-ATPase activity and Na-K-ATPaseα1,β1 subunit protein expression by western blot. 2).ERK1/2 expression: Western blot was used to test the protein expression of ERK1/2.Results:Animal experiment1.PaO₂:The oxygen partial pressure reached the lowest in 30 minutes after seawater aspiration,then increased slowly,but manifested obvious hypoxemia. Treatment with STS, PaO₂ was significantly increased compared with that of seawater group from 2 hours(P<0.05),and increased to peak at 4h(P<0.01) post seawater instillation. It suggested that STS improved hypoxemia.2.Lung histopathology: After seawater aspiration, there were serious lung injuries such as hemorrhage, the markedly thickened alveolar wall, and the infiltration of many inflammatory cells in the alveolar spaces. However, treatment with STS the destruction of lung structure was reduced significantly.3.Index of Pulmonary edema: The lung W/D ratio and PMVP in rats were significantly increased following seawater aspiration. STS treatment reversed such effect in part (P<0.05). It suggested that lung edema induced by seawater was attenuated by STS.4.The activity and expression of Na-K-ATPase in lung tissue: Seawater aspiration decreased the Na-K-ATPase activity,protein expression and mRNA level. STS reversed seawater-induced reduction of Na-K-ATPase activity and the western blot results showed that both Na-K-ATPaseα1 andβ1 subunit plasma membrane protein expressions following seawater aspiration were increased by STS treatment compared with seawater group(P<0.05).However, administrated with STS, the Na-K-ATPaseβ1 mRNA level following seawater aspiration was increased compared with seawater group (P<0.05). There was no significant difference in the Na-K-ATPaseα1 mRNA level between seawater group and seawater with STS treatment group.Cell experiment1.The activity and expression of Na-K-ATPase in A549 cells:The cell experiment indicted that the change of Na-K-ATPase protein expression and activity in A549 cells were similar to the lung tissues. Moreover, pretreated with ERK1/2 inhibitor U-0126,Na-K-ATPase protein expression and activity were decreased compared with STS treatment following seawater incubation (P<0.05).2.The protein expression of ERK1/2: Seawater induced the decrease in ERK1/2 protein expression. However, STS treatment increased ERK1/2 phosphorylation following seawater incubation, which manifested STS can activat ERK1/2 in A549 cells.Conclusion1.Hypoxemia, acute pulmonary edema and lung injury induced by seawater aspiration were improved by STS administration.2.STS treatment could improve PE-SWD by up-regulating Na-K-ATPase activity and expression both in vivo and in vitro.3.STS activated ERK1/2 in A549 cells following seawater incubation. Moreover, the ERK1/2 inhibitor partially blocked the improved effect of STS on the Na-K-ATPase expression and activity,which indicated that activation of ERK1/2 mediated STS up-regulating the Na-K-ATPase protein expression and activity.