The Impact Of TMEM16A Chloride Ion Channels On Acute Lung Injury

Objective: Acute lung injury(ALI) or acute respiratory distress syndrome(ARDS) refers to acute progressive respiratory failure induced by different factors except cardiogenic causes. It is a serious and acute disease with high mortality. In clinic, its main appearance is stubborn hypoxemia, non-cardiogenic pulmonary edema, pulmonarty hypertension and systemic circulation hypotension. One of its main pathophysiology characteristic is acute pulmonary edema(APE). The phenomenon refers to excessive fluid accumulates at intersistial tissue and/or alveolus space which caused by different factors. It will form to stroma and/or alveolus space edema. Alveolar epithelial cell performs a key function in the process which against with pneumonedema generation. When alveolar epithelial cells are injuried by kinds of direct or indirect factors, they will lose their activity. Inaddition, reduction of surfactant protein(SP), the normal volume cell will change, fluid transportation of alveolar plasmalemma will be blocked. Finally, more and more fluid will be deposited, which will lead to more serious pulmonary edema. And all of these phenomenon have a close relationship with ion channels on the surface of alveolar plasmalemma.Among all of the ion channels, the function of sodium channel towards fluid clearance has been proved. Lin liu and her team have proved that there exists aquaporin1 (AQP1) in alveolar epithelial cells. And its level will drop obviously in the state of ARDS[1]. Chloride channel, as it is abound in organism, has been ignored for a long time until recently. Scientists gradually find out many kinds of chloride channels, such as currents mediated by calcium-activated chloride channels(CaCCs),volume-regulated anion channels (VRAC, ICl. swell), votage-gated chloride channels ClC family, cAMP/protease-activated chloride channels, ligand-activated chloride channels etc. They have kinds of functions. There are a few reports about cyctic fibrosis transmembrane conductanceregulator (CFTR), they says that CFTR has function of alveolus fluid clearance. There are also some reports say that VRAC can keep cell normal physiological function. But there is still not enough people pay much attention to the function of chloride channels in acute lung injury.TMEM16A was originally reported in 2008, it has many characteristic, such as 1 Its function is multitude. It is important for the development of mice trachea. It can reduce the mucous secretion in trachea. It also plays a role in signal conduction. What is more, it has volume-regulation fuction. 2 It is abundant in mankind, and expressed in many organism. 3 It has specific blocking agent, such Niflumic acid (NFA), it is necessary in most experiments. 4 There is a high range of homology (91%) between mice and human being. 5 Its molecule characteristic is distinctive. 6 There are already some other researches of chloride channels, they can be the basis for other experiment. 7 It may be the molecular basis of calcium activated chloride channels activated.It is reported that chloride channels may play a role in alveolar fluid clearance in the state of ALI. And in our pre-experiment, we have already found that TMEM16A levels are different between different kinds of ALI. So we duplicated two kinds of ALI model:Escherichia coli (E.coli) group and lipopolyaccharide (LPS) group, to simulate endogenous and exogenous ALI. For the purpose to find out the difference of TMEM16A level between the two groups.Methods: Select 18 healthy male SD rats (weight 250±10g), and divide them into 3 groups at random with 6 animals for each group: control group, LPS group and Ecoli group. LPS group:injecting LPS(8mg/kg) in their caudal vein,take the materials 4 hours later. Ecoli group:instill Ecoli suspension(concentration:109) in rats trachea, take the materials 24 hours later。Anaesthetize the rats by intraperitoneal injection with 10% chloral hydrate (3ml/kg) and hold them on the operating table after making models successfully. Fiat venaesection from femoral artery to kill the rats. And then take the fresh lung tissues immediately. Take the inferior lobe of right lung on ice, keep it in marked tube, and store it at -80℃for western blot(WB). Take superior lobe of right lung for the test of wet/dry(W/D). Take middle lobe of right lung, fix it with 10% neutrophilia formalin fluid for pathology and immunohistochemistry. Separate the trachea, operate trachea intubation. Use 4ml sterile normal saline (NS) for bronchoalveolar lavage,keep the lavage inside the lung tissues for at least 20 seconds, and wave the operating table repeatedly during the period to make sure that the lavage can permeate into the alveolar space. Collect the lavage after repeating the process for 3 times, and centrifuge it (1200rpm, 10min). Keep the supernatant for measuring protein concentration.Results: 1 result of WB there is no difference between the two model groups: control group (1.599±0.142) and LPS group (1.652±0.036) (P>0.05). Compared with the other two groups, the level of E.coli group (1.149±0.125) decreases obviously (P<0.01).2 result of pathology the pathologic grade of the two groups are much higher than control group: LPS group (11.33±1.37), E.coli group (11.83±0.75).3 result of immunohistochemistry Alveolar interstitial and alveolar epithelial cell are specific stained.4 result of protein concentration in BALF and W/D the protein concentration of LPS group (1.15±0.12) and E.coli group (0.91±0.29) are much higher than control group (0.17±0.05) (P<0.01);the W/D level of LPS group (5.35±0.45) and E.coli group (4.61±0.19) are also much higher than control group (3.79±0.42) (P<0.01)。Conclusions: 1 We duplicated the two model groups successfully. They are stable and easy to be operated. Their prediction and achievement tatio are also perfect. So they are good models for research of ALI.2 The immunohistochemistry manifestation of lung shows pulmonary vascular smooth muscle(PVSM) cell and alveolar epithelial cell are specific stained in LPS group and control group. but the specific stain of alveolar epithelial cell can hardly be seen in E.coli group.3 The level of protein concentration in BALF (bronchoalveloar lavage fluid) and W/D are increased in LPS group and E.coli group. Shows that the AFC is decreased in both two model groups. It is in accordance with the change of TMEM16A level of E.coli group.4 There is no difference between control group and LPS group. Compared with the other two group, the level of E.coli group decreases obviously. The level of TMEM16A in different groups caused by different factors is different. It implies that ALI/ARDS induced by different factors can cause different influence to TMEM16A, so it should be treated differently. And the compare between LPS group and E.coli group should be strengthened.