Expression Of Tmem16a Protein In Alveolar Type Epithelia Cells Of ALI Rats

Objective: Acute lung injury(ALI),a syndrome of pulmonaryinflammation characterized by alveolar-capillary increased permeability,is aserious complication in the patients with critical illnesses,resulted from avariety of clinical reasons.All often leads to acute respiratory distresssyndrome (ARDS)without effective treatment.In clinical,its main appearanceis stubborn hypoxemia, non–cardiogenic pulmonary edema,pulmonaryhypertension systemic circulation hypotension.Despite improvement insupportive care and multiple therapeutic efforts directed at modifying thecourse of this condition,mortality still remain about50%. Acute pulmonaryedema(APE)is one of the main pathophysiology characteristic of ALI／ARDS.A large of clinical research indicated that alveolar fluid clearance early is akey to clinical treatment.Alveolar fluid clearance(AFC)is closely related topulmonary edema.It is shown in experimental studies that intact alveolarepithelial fluid transport function is critical for resolution of pulmonary edemaand acute lung injury.The increase in alveolar fluid clearance can acceleratethe resolution.AFC is one of the important effects of alveolar epithelium.Alveolar epithelia cells, consisted of flat type Ⅰcells and caboidaltypeⅡ cells,are considered to be not only an important barrier to alveolarflooding but also the most likely site of fluid reabsorption after pulmonaryedema.Meanwhile AT Ⅱmaintain normal gas exchange.There are various ofion pumps and channels on the alveolar epithelium,which in turn can drivethe movement of lung edema fluid out of the airspaces.Among all of the ionchannels,the function of sodium channel towards fluid clearance has beenproved.AQP1have been proved to exit in alveolar epithelial cells,and itslevel will drop obviously in the state of ARDS.Lihua zhu and her team haveproved that there exits AQP4in alveolar epithelial cells,and its level will increase in the state of ARDS.Chloride channel is abound in organism,butthere are few reports about the function of chloride channel in alveolar fluidclearance.The CFTR is a cAMP-dependent Protein kinase and ATP-regulatedCl-channel.CFTR has been proved to exit in alveolar epithelial cells.Xiu Guand her team have proved that both denopamine (β-1agonist) and NS004(aCFTR opener)can increase alveolar fluid clearance significantly in theisolated rat lungs.The efforts of denopamine maybe mediated by CFTR inpart.Several studies show that CFTR plays role in alveolar fluidclearance.Recently,there is no report shown that the effect of β-2agonisttreatment can improve the prognosis of ARDS, indicating that the influencingfactors are complicated.We selected TMEM16A as the object of this study.TMEM16A wasoriginally reported in2008. It is the calcium-activated chloride ion channel.There is still not enough people pay much attention to the function ofTMEM16A in ALI.We detected the level of TMEM16A to find out thedifference between the control group and ALI group.Methods：Select24healthy male SD rats (weight180-220g), dividedinto2groups randomly with12animals in each:control group and LPSgroup.In each group,6rats were selected to observe histopathology,6ratswere measured the expression of TMEM16A protein.By injecting LPS intheir caudal vein LPS (8mg／kg), the material was taken2hours later in LPSgroup. By fiatting venesection from artery to kill the rats after anesthesia,thenremoved the fresh lung tissues immediately,took the left lobe,fixed it with10%neutrophilia formalin fluid for pathology.Alveolar type Ⅱcells wereisolated by classic methods that had been proved.Briefly,afteranesthesia,tracheotomy,and cannulation of the pulmonary artery,the lungswere lavaged and inflated simultaneously with air.After removal from thethoracic cavity.They were digested by trypsin and collagenase.The lung werethen minced and the resulting suspension was filtered.We cultured ATⅡ aftercentrifugation of cell suspension and adjust the cell concentration.The cellspurified by adhered to IgG coated dished and identified with AKP stain and electron microscope.The concentrations of IL–β in the culture media weredetermined based on ELISA.The expression of TMEM16A protein wasmeasured by Western blotting.Results:1result of pathology:HE coloration of LPS group shown thatthe alveoli and interstitial became dropsical and hemorrhagic,capillary vesselbeen overspread,engorgement and leukocyte infiltration.The pathologicgrade of LPS group(9.42±0.72) is much higher than controlgroup(0.70±0.52)(P <0.01).2Isolating,purifying and identifying ATⅡ of rat: AT Ⅱcells wereidentified with AKP stain and electron microscopic.The AT Ⅱcells wasobserved by electron microscopic that they had regular shape,cube-shaped orround.The isolated ATⅡ had typical features including lamellar bodies andmicrovilli. We get(1.35±0.04)×107cells of LPS group after purification.Thetrypan blue staining shows that the cellˊactivity of LPS group over the(83±2.32)%.3result of ELISA: The level of IL-1β in the culture media of LPSgroup is much higher than control group (P <0.01), respectively control groupwith14.68±0.95and LPS group with33.33±2.484result of Western blotting: The expression of TMEM16A protein isno difference between the two groups (P>0.05),in which,control group with1.01±0.10,LPS group1.00±0.07Conclusions:1The rats in the model group by intravenous injection ofLPS were characterized by diffuse alveolar damage and severe systemicinflammatory response,which indicated we succeed in replicating ALImodel.2Fitting the concentration of digestive enzyme and digestion time arecritical for isolation of ATⅡ cells.Adhered to IgG coated dishes afterisolating provided more high cell purity than before.Lamellar bodies could befound by electron microscope.3The secretion level of IL-1β in ATⅡ cells increased significantly inLPS group. The expression of TMEM16A protein does not have a significantly change in LPS group.