| Objective To observe the effects of insulin on the expression of Epithelial Sodium Channelαsubunit in A549 cell(sadenocarcinomic human alveolar basal epithelial cells), and impact of apoptosis. Investigate the effects of insulin on mediators of inflammation in acute lung injury induced by lipopolysaccharide in rats,also the expression of the subunit of ENaC were detected, and the mechanism was investigated.Method A549 cells were cultured with insulin with a concentration of 30μiU/ml for 30 or 60 minutes. Then ENaC-α,SGK-1(serum and glucocorticoid-inducible kinase-1) protein and mRNA were detected by western-blot and RT-PCR. Then chelidonii a blocking agent of PKC (protein kinase C), which can inhibit the effect of SGK-1, was used in the cells culture for 60 minutes, also ENaC-αprotein was detected. Cells were cultured in a hypoxia circumstance for 30 minutes, to induce apoptosis, the rate of observed by TUNEL between cells cultured with or without insulin. 72 male SD rats weighing 180·250g were randomly divided into 3 groups with 24 animals in each group after be anesthetized, jugular vein was exposed: (1) control group received only normal saline, at first 1ml saline were iv. pushed, 2 hours later saline was pumped at 1ml/h; (2) LPS group received LPS 5 mg/kg iv. then 2 hours later saline was pumped at 1ml/h; (3) treatment group received LPS 5 mg/kg iv. 2 hours later, insulin was pumped at concentration of 0.5U/ml with speed of 1ml/h, blood glucose was controled at 2.8-3.2mmol/l. Take the 2h later as first time point, then at 0, 30min, 1h, 2h, blood samples were obtained, animals were then killed by exsanguination. The lungs were removed. TNF-α, IL-1β, IL-8, PCT were detected by ELISA. Lungs were used to measure its wet/dry lung weight radio, and pathology was also observed. A part of lung was homogenated, and the expression of the subunits of ENaC were detected by RT-qPCR.Results Insulin can up-regulate the expression of ENaC-αin A549 cells, which was via the way of PKC, SGK-1, and when at 30 minutes there is a significant difference ( p<0.05). And insulin also can inhibit apoptosis which was induced by hypoxia. In LPS group the wet/dry weight radio, the mediator of inflammation TNF-α, IL-1βin serum were significantly increased compared with control group(p<0.05); after insulin treatment 1 hour, the wet/dry weight radio, the mediator of inflammation TNF-α, IL-1β, Il-8, PCT in serum were significantly decreased compared with LPS group(p<0.05). Also, in pathology, LPS group have more severe inflammation, at the same time point, compared with treatment group(p<0.05). Then come to the expression of the subunits of ENaC, when LPS was injected the ALI models were builded, the exression of subunits of ENaC were decreased significantly, compare to control group, after treated by insulin for 30min the subunits of ENaC expression were upregulated, 1h later the difference became significantly.Conclusions Insulin can up-regulate rxpression of the ENaC-αin A549 cells via PKC SGK-1 pathway, also insulin can inhibit apoptosis which was induced by hypoxia. In the pathology of ALI/ARDS, insulin can decrease mediator of inflammation, inhibit inflammatory reaction, mabe this was affected by the effect of anti-inflammation and up-regulation of ENaC, by insulin. So insulin may do some good in acute lung injury (ALI) and acute respiratory distress syndrome (ARDS). |
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