Expression And Significance Of TMEM16A In The Lipopolysaccharide-induced A549Cells

Objective:Acute lung injury(ALI) refers to the acute and reprogressiveinflammatory syndrome of pulmonary with the increase in alveolar capillarymembrane permeability, induced by a variety of incentives, and withouteffective treatment can be developed into acute respiratory distress syndrome(ARDS). Despite etiology and pathogenesis in patients with ALI/ARDS arecomplicated,pulmonary interstitial and alveolar edema are commonpathological manifestations, suggestting alveolar fluid clearance barrier.Previously, the studies of ALI/ARDS has focused on the inflammatoryprocess of pulmonary, in recent years, much attention is given on the processof formation and clearance of pulmonary fluid in ALI, so the ion channelabout water transport givern more attention.Recently, TMEM16A as one of TMEM family, has been concerned on thecell secretion. The significantly enhanced expression of TMEM16A is foundin alveolar epithelial cells of a rat model of ALI and there are differencesamong models induced by different incentives. Therefore, it is necessary tofurther explore the relationship of the activity of this protein with thepulmonary fluid clearance of ALI/ARDS. The expression of TMEM16A gene,as one of TMEM family, also known as the TAOS2, DOG1, FLJ10261orORAOV2, has been proved in esophageal cancer, bladder cancer, breastcancer, sensory neurons and epithelial cells.2008, CaputoA et al. confirmed that the protein is a calcium-activatedchloride channel, relating to airway mucus glands secretion. Some researchersare exploring the characteristics of the chloride channel in Alveolar epithelialcells the type II(AT II) in the lung, which plays a key role in fluid clearance. Recently a study done by Yang, C, prompt: ATⅡis involved in fluidsecretion and urinary purine adenosine triphosphate (UTP), and other studiesRock JR done have shown that the TMEM16A is involved in fluid secretionand UTP. There are no studies about TMEM16A in ALI relating the functionon lung fluid clearance, as a chloride ion channel.The objective of the experiment:(1) Detecting the concentration of TNF-α in the supernatant of A549cellsstimulated by LPS to understand the relationship of TNF-α and the degree ofcell damage.(2) Detecting whether there is TMEM16A protein postive expression inA549alveolar epithelial cells, and watching the differences of TMEM16Aprotein expression caused by different concentrations of lipopolysaccharide(LPS) to provide new ideas and experimental evidence for further exploringthe relationship between the activity of this protein and lung water clearancein the ALI/ARDS.Methods: After A549cell have been cultivated for a long time, it hasbeen devided into the control and the experimental group. A549Cells wereincubated with lipopolysaccharide (LPS) at different concentration (5μg/ml；10μg/ml；15μg/ml) during24hours and cell proliferation was measured byMTT. We observe the organelle change of the cultured cell by transmissionelectron microscope. Western blot was performed to detect protein TMEM16Aexpression in A549Cells, and the concentration of TNF-α was determined byELISA.Results:1. The morphology of A549cell In the control group: the shapeof A549cell is polygon, abundant kytoplasm, cell process manifest. Afterlipopolysaccharide (at5μg/ml concentration) treated, A549cells were assimilar as that in the control group. After lipopolysaccharide (at10μg/ml and15μg/ml concentration) treated, A549cells were incubated with the shape ofA549cell change round, volume contract and appear inequality of sizethickness grain in the kytoplasm.Electron microscopy: The Organelles of A549cell In the control group (a),there were many microvilli on the surface of A549cells, the cytoplasmwas rich, concluding many Organelles,such as Mitochondria、lysosomes、Endoplasmic Reticulum、 Golgi complex、 lamellar body; mucusvacuole、microcapsule and so on. After lipopolysaccharide (at5μg/ml concentration)treated （.b）the change of A549cells was as similar as that in the control group.After lipopolysaccharide (at10μg/ml and15μg/ml concentration) treated.compared with control group：microvilli on the surface of A549cells reduced,a large number of vacuoles, part of the ridge and membrane fusion in themitochondria can be seen in the cells, showing t the absence of the ridge.Rough endoplasmic reticulum was dilated, with granule fusion anddegranulation.2. The percent cell growth inhibition rate (IR) was determined using theMTT method. The cell proliferation was suppressed after A549cells inducedby LPS for3，6，12and24h. However, there was no significant difference incell proliferation rate between cells treated with LPS（5μg/ml）for3and6h.there was significant difference in cell proliferation rate between any othertwo groups at any time point. Then,at the same time point, there was also nosignificant difference in cell proliferation rate between cells treated with LPS（10μg/ml and15μg/ml）for3、12and24h， there was significantdifference in cell proliferation rate between any other two groups.3. result of ELISA the concentration in experimental group of TNF-α inthe cell culture supernatant compared with the control group was significantlyincreased. There was significant difference between experimental group andcontrol group.4.result of Western-blot TMEM16A expression protein of experimentalgroup5μg/ml (0.3028±0.01898) and10μg/ml (0.2946±0.00535) was higherthan that in the control group (0.2764±0.00814), but that in15μg/ml(0.2190±0.00865) was lower than that in the control group.15μg/ml groupwith lower protein level compared with the control and other experimentalgroups was statistically significant. there is statistical difference betweenexperimental (5μg/ml) with higher protein level and the control group. Conclusion:1. LPS could successfully induce acute injury in A549cells;2. TNF-α level in the cell culture supernatant increased by cell damageincreasing gradually in different LPS-induced concentrations and differenttimes; There was significant statistical difference between experimental andcontrol group.3. Western blot proved that TMEM16A protein was postive in A549cells,and initially showed a trend of gradual increasing with the severity of celldamage, but when the injury of the cells became more serious, TMEM16Aprotein level turned decreased. TMEM16A protein expression level increasedwith less cell damage, but when the cell structure、morphology and functionhad been damaged significantly protein levels in TMEM16A was more lower.