The Impact Of Tmem16a Chloride Channel In At â…¡ By LPS

Objection: Acute lung injury (ALI) or acute respiratory distresssyndrome (ARDS) refers to acute progressive anoxic respiratory insufficiencyor failure induced by different factors except cardiogenic causes. Its mainclinical manifestation is stubborn hypoxemia, non-cardiogenic pulmonaryedema, pulmonary hypertension and systemic circulation hypotension. Itsmortality is50%.One of its main pathophysiology characteristics is permeablepulmonary edema. We have found that alveolar epithelial cell performs animportant role in the process of alveolar fluid clearance. Now we consider thatthe alveolar epithelial cells typeⅠ (ATⅠ) and alveolar epithelial cells typeⅡ(AT Ⅱ) play complementary roles in alveolar fluid clearance,with ATI cellsmediating basal salt and fluid clearance and ATII cells being responsible foraugmented transport. And the ion channels on the surface of alveolarplasmalemma have a close relationship with the alveolar fluid clearance.Although it is clear that the active transport of sodium drives a significantfraction of overall fluid clearance, lots of studies show that alveolar fluidclearance is a complex process that requires the coordinated transport ofsodium, chloride, water, and other molecules across the tight epithelium barrierinto the interstitial space. Chloride channel, as it is abound in organism, hasbeen ignored for a long time until recently. It is reported that cyctic fibrosistransmembrane regulator (CFTR) may pay a role in alveolar fluid clearance,whose deficiency or dysfunction could block the fluid transportation ofalveolar plasmalemma. It is proved in both experimental models and in clinicalstudies that CFTR is essential for cAMP-mediated upregulation of alveolarfluid clearance.TMEM16A is chosen to be our object of this study.TMEM16A, a memberof the TMEM family, has been proved to be expressed in the sensory neuron,the epithelial cell, the carcinoma of esophagus, bladder and breast. In2008 Caputo A et al have proved that TMEM16A is the protein associated withcalcium-dependent chloride channel.Recent studies indicate that TMEM16A isclosely related to the secretion of mucus gland in airway. At present we do notpay enough attention to the relationship between TMEM16A and alveolar fluidclearance in ALI.The further study can help us to make deeper discussionabout the mechanism of ALI. We made the LPS-induced ALI model of ATⅡin vitro, under this unique microenvironment we will investigate the expressionof TMEM16A and the relationship between TMEM16A and alveolar fluidclearance in ALI.Methods: Twelve healthy male SD rats (weight220±10g) were selected.After anaesthetizing by intraperitoneal injection, inserting a tube through thetracheotomy and puncturing into the right atrium,the lungs were lavaged viapulmonary artery and trachea. After the lung and heart were removed from thethoracic cavity, the lungs were instiled with trypsinase and collagenase anddigested in water bath at37℃for20minutes. The lungs were then minced,and the resulting suspension was filtered by cell strainer. We cultured ATⅡafter adjusting the cell concentration. Alveolar cells were purified by adhere toIgG coated dishes. The cell viability was determined by trypan blue dyeexclusion. The cell was identified by the electron microscope and AKP staining.Meanwhile, AKP staining was served to test the purity of AT Ⅱ.To make the LPS-induced ALI model of ATⅡ in vitro, we added LPS bygroup. To make sure that we have replicated the ALI model in vitrosuccessfully. We took picture by electron microscope and compared theultramicro structure of AT Ⅱ.The concentration of IL–1β in the culture mediawas determined based on ELISA. The expression of TMEM16A protein wasmeasured by Western blotting.Results:1Counting and indentifying ATⅡ: We get (4.50±1.12)×107cells per rat beforepurification and (1.55±0.41)×107cells after it. The trypan blue stain shows thatthe cells＇activity is (90.17±5.10)％before purification and (79.17±3.07)％after it. Identified with AKP stain, the purity is (79.17±3.07)％.The electron microscope can identify the ATⅡ and can also identify that we have made theALI model of AT Ⅱin vitro.2The result of ELISA: the concentration of IL–1β in the control group is(14.50±2.24)pg/mL; the concentration of IL–1β in the ALI group is(38.55±15.07)pg/mL. The concentration of IL–1β in the culture media wassignificantly increased as compared with control group(P＜0.05).3The result of Western blotting: the expression of TMEM16A in the controlgroup is (0.99±0.26); the result in the ALI group is (1.00±0.11). Theexpression of TMEM16A dose not have a significantly difference (P＞0.05).We can not consider that there is difference between the two groups in thecontent of this protein.Conclusions:1Our experiment can get enough number and purification of primy 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 type Ⅱ cells directly in vitro. It could make thesecretion of IL–1β increased significantly.3The expression of TMEM16A dose not have a significantly differencebetween the control group and ALI group in the content of TMEM16A protein.The result may owe to that the ALI of ATⅡ in vitro induced by LPS can notimpact the expression of TMEM16A significantly;or the time phase of proteinexpression level needs to be further discussed; also we can not get rid of therelationship with the immature experiment condition.