Protective Effects Of Perfluorocarbon On Lipopolysaccharide-induced Down-regulation Of Alveolar Fluid Clearance Related Ion Channels

ObjectivesMany causes of ALI/ARDS can damage the ability of alveolar fluid clearancethrough down-regulating the expression of the related ion channels and increasingalveolar space fluid accumulation which can directly lead to respiratory distress andbad prognosis of ALI/ARDS. Improving the ability of AFC may be a new approachfor the future of ALI/ARDS. This study investigated protective effects ofperfluorocarbon on lipopolysaccharide-induced down-regulation of alveolar fluidclearance related ion channels.Methods(1) Four different ratio (0%,10%,30%,50%) of PFC obtained by the ultrasoniccell crusher were respectively used on A549cells for0.5h,1h,2h,4h.Then we selectedthe most appropriate concentration and time;(2) The cell model of lung injury was setup by using the LPS(10ug/ml);(3) To explore the effects of PFC on the AFC relatedion channels the A549cells were treated with either DMEM, LPS(10ug/ml),10%PFC-in-DMEM,and10%PFC-in-DMEM~+LPS(10ug/ml). And cells treated withDMEM served as control group. After4hrs reaction, each group was detected by①real time quantitative PCR(q PCR) to identify the mRNA expression of ENaC-α,β, γ, Na~+-K~+-ATPase-α1, β1, CNG1-αand AQP3;②western-blot to detect thechanges of ENaC-α, Na~+-K~+-ATPase-α1protein abundance;③Na~+-K~+-ATPaseactivity kit to analyse the Na~+-K~+-ATPase activity in A549cells.Results(1) When10%PFC used in A549cell for4hrs, the mRNA expression ofENaC-α, Na~+-K~+-ATPase-α1up-regulated most obviously;(2) The mRNA expression of IL-1β, IL-6and TNF-α up-regulated most in the cells with LPS(10ug/ml), which got a peak in4hrs reaction and descend after6hrs, but still higherthan control group; the expression of ENaC-α and Na~+-K~+-ATPase-α1in theLPS(10ug/ml) group also showed a gradual upward trend in half an hour anddescend mostly after4hrs(P=0.037，P=0.046，respectively);(3) Compare with thecontrol group, the mRNA expression of ENaC-α、β, Na~+-K~+-ATPase-α1、β1and theexpression of ENaC-αand Na~+-K~+-ATPase-α1protein in LPS group decreasedsignificantly, while no statistical difference was observed in the mRNA expression ofENaC-γ、CNG1-αand AQP3.(4) Compared with LPS group, the mRNA expression ofENaC-α、γand Na~+-K~+-ATPase-α1、β1and CNG1-αand the protein level of ENaC-αand Na~+-K~+-ATPase-α1in PFC~+LPS group was significantly higher;(5) Comparewith the LPS group, PFC~+LPS4hrs group can also increase the activity of Na~+-K~+-ATPase.Conclusions1.10ug/ml LPS can lead to a large number of IL-1β, IL-6and TNF-α produced bythe A549cells which means lung injury model established successfully. And it alsocan down-regulate the gene expression of ENaC-α、β，Na~+-K~+-ATPase-α1、β1andthe protein levels of ENaC-αand Na~+-K~+-ATPase-α1which means this can be used inthe cell research of the alveolar fluid clearance in ALI.2. PFC can improve the gene expression of ENaC-γ and CNG1-αand protein level ofENaC-α and Na~+-K~+-ATPase-α1in normal A549cells.3. PFC may enhance the mRNA expression of ENaC-α、γ，Na~+-K~+-ATPase-α1、β1and CNG1-α and the protein level of ENaC-α and Na~+-K~+-ATPase-α to reduce theLPS’injuries.