The Impact Of TMEM16A In ATâ…¡ By LPS In Vitro

Objective: ALI/ARDS is characterized by diffuse alveolar damage withalteration of the alveolar-capillary membrane.These alteration lead toflooding of the alveolar airspaces by protein-rich edema. Clinical studiessuggest that alveolar epithelial damage and the decrease in the capacity of thealveolar epithelial barrier to remove alveolar fluid are important determinantsof poor outcome in humans. Therefore strategies aimed at decreasing theinitial alveolar epithelial damages and at accelerating the reabsorption of lungedema may be beneficial in patients with ARDS.Alveolar fluid clearance is an integrated function that depends on activeion channels transport, as well as an intact epithelial physical barrier thatmaintains relatively dry alveolar airspaces. Alveolar type Ⅱ cells (AT Ⅱ)notonly can secrete surface active substance, but also can proliferate anddifferentiate into type Ⅰ cells (AT Ⅰ)in acute lung injury. In addition, ATⅡplays an important role in alveolar fluid clearance.The ions and watertransport across the alveolar epithelium is regulated by mechanisms that havenot been fully defined. The activity of sodium channel (ENaC), Na+-k+atpase(NKA) and aquaporin (AQP) are contribute to alveolar fluid clearance andplay an important role. Chloride channel, as it is abound in organism, has beenignored for a long time until recently. It is reported that cyctic fibrosistransmembrane conductance regulator (CFTR) pay a role in alveolar fluidclearance. In2008Schroeder et al have proved that TMEM16A is the proteinassociated with calcium-dependent chloride channel. Recent studies indicatethat TMEM16A is related to the secretion of epithelial cells. Yang haveconfirmed the TMEM16A expression in alveolar epithelial cells byimmunohistochemical, there is a few studies about TMEM16A in acute lunginjury relating the effects of alveolar fluid clearance.Lipopolysaccharide (LPS) which is the main component of bacterial endotoxin, may be an important factor leading to ARDS occurrence,development when the large and sustained of LPS release. We made theLPS-induced ALI model of AT Ⅱi n vitro, we investigate the relationshipbetween TMEM16A and alveolar fluid clearance in ALI. The purposes of thisexperimental research are:①s eparation and purification of rat AT Ⅱepithelialcells，then identify by the electron microscopy and AKP alkaline phosphatasestaining.②From the change of inflammatory factors，in view of the change ofIL-8in vitro which cause by LPS stimulated AT II epithelial cells.③toobserve the change of TMEM16A mRNA under the stimulation of differenttime and different concentration of LPS.Methods:1twenty-four healthy male clean SD rats (weight220±10g) were selected.AT II cells were isolated By Dobbs’method which had been improved, thecells purified by adhered to IgG coated dishes.①The cell viability wasdetermined by trypan blue dye exclusion, total cells amount by cell countingchamber.②The cell was identified by the electron microscope andAKPstaining.③A KP staining was served to test the purity of AT Ⅱ.2Make the LPS-induced ALI model of AT Ⅱin vitro, after cultivated24h, ATⅡ cells in primary culture were divided in to three groups. In group Ⅰ,the same volume of normal medium was added to the culture medium, ingroup Ⅱ, LPS1ug/ml and in groupⅢ, LPS10ug/ml. then determined indexchanges of each group after4h,8h.①We took picture by electronmicroscope and compared the ultramicro structure of AT Ⅱ.②Theconcentrations of IL-8in the culture media were determined based on ELISA.③F luorescence-quantitative PCR technique was performed to quantitate thelevels of TMEM16A.Results:1Counting、viability and identifying ATⅡ: We get (1.84±0.14)×107cellsafter purification. The trypan blue stain shows that the cells’ activity is(93.69±1.16)％. Identified with AKP stain, the purity is (84.66±1.47)％.Theelectron microscope can identify the ATⅡ. 2Transmission electron microscopy confirmed that the model of acutelung injury was successful, we can observed cell degeneration, irregular shape,the cell surface microvilli decreased or even disappeared, the cell membranedisintegration, cytoplasmic organelles decreased significantly, lamellar bodiesemptying and shedding, cell vacuoles increased.3The result of ELISA: The concentration of IL-8was increasedsignificantly by LPS（1,10μg/ml）for4and8h.There was significant statisticaldifference between experimental（1,10μg/ml）and control group.4The result of real-time quantitative PCR：there was no significantdifference between experimental(LPS=1μg/ml)and normal group(P>0.05)after LPS stimulated AT Ⅱfor4hours, But there was significant statisticaldifference between experimental(LPS=1μ g/ml)and control group after LPSstimulated AT Ⅱ for4hours（P＜0.05）. there was also significant statisticaldifference between experimental(LPS=10μ g/ml)and control group after LPSstimulated AT Ⅱ for4hours and8hours（P＜0.05）.Conclusions:1Our experiment can get enough number and purification of primary ratAT Ⅱ, to take the next step of the experiment. And we have replicated the ALImodel of ATⅡ in vitro successfully.2LPS could injured alveolar typeⅡ cells directly in vitro. It could makethe secretion of IL-8increased significantly.3The AT Ⅱ in vitro induced by LPS may impact the expression ofTMEM16A mRNA; in a certain time and concentration, the expression level ofTMEM16A may change by the LPS concentration and stimulus duration,TMEM16A as the chloride channels may be involved in ion transport functionduring acute lung injury.