The Effects And Mechanisms Of Bone Morphogenetic Protein-4 On The Rats With Acute Lung Injury Induced By Acid Inhalation

IntroductionThe acute lung injury (ALI) induced by aspiration of gastric contents is one of primary causes of acute respiratory distress syndrome( ARDS) , and the mortality of ARDS reach up to 40% -60% .At the morning stage of ALI, neutrophil and many cytokines, which mediate cascade reactions during inflammation, are principal account for it, they are both in charge of the damage of capillary endothelium cell and alveolar epithelium. It could lessen the lung injury induced by acid - inhalation by blocking the products of neutrophil, consuming neutrophil or inhibiting the immigration of it to alveolar space, but for the multiplicity and complexity of the cells and cytokines which participate in the course of lung injury, the effect and mechanism of some of them are not known clearly, the injury can not been blocked completely;At the stage of fabric hyperplasia, fibroblast play a key role and become target cell of regulatory factor of matrix apposition, the balance of two aspects which promote or inhibit fibroblast to proliferate and differentiate determines the results of repair and then, affect prognosis. Two phases of ALI intercross, intervention about any links of them may influence the course of ALI.BMPs is secreted functional protein, one of the superfamily of TGF - β. By regulating gene activity, they control many function such as formation of meso-blastema, bilateral symmetry, myelogenesis, development of somite and ossa-ture, formation of limbs and the development of kidney, stomach intestine, liver, lung, tooth and so on, and the maintenance of various tissue s morphous. The studies of recently showed that BMPs play a crucial part not only in develop-ment of tissue and maintenance of morphous, but also in nosogenesis and development of diseases or injury and repair of tissue, the empirical studies about treatment of acute or chronic nephropathy had gained positive curative effect. BMPs ( principal of BMP4) has not only crucial contribution to the graphic development of embryo lung, but also induction to fibroblast, myocyte and inflammatory cell. But so far, there are not internal or overseas reports on the effects of BMPs during ALI. A lot of records had confirmed that the model of rats induced by acid - inhalation was analogue with human ALI induced by aspiration of gastric contents, so, we designed series of empirical studies: making use of the rats model of ALI induced by acid - inhalation to simulate human ALI induced by aspiration of gastric contents ( aspiration) , we investigate the effect and mechanism of BMP - 4 during ALI at organ, tissue and molecular level, to provide experimental foundation for clinical treatment of ALI, preventing the development to ARDS, and improving prognosis.METHODS1. Experimental animal:110 Wistar rats writh body weight 250 - 300g were provided by the experimental animal center of China Medical University.2. Main Reagent and Apparatus;Pentobarbital sodium;HCL;BcaBEST RNA RCR assay kit;Multiclone goat - anti - rat BMP - 4 antibodies;Goat - anti - rabbit IgG antibodies;Primers of BMP - 4 internal reference GAPDH;Recombinate BMP - 4 ( Human);Recombinate Noggin (rat);Kits of TNF-a, NO, MDA, SOD, HYP;Various kinds of enzyme;Stain;Pure agent of analysis.Model 683 animal breathing machine;Monitor;PERKIN ELMER PE9600 PCR amplification device;A -200 Ds Electric balance;Dupont ST-21 hypothermia high speed centrifuger;Kodak ID gel imaging analytical system;GIS -700D digital gel scouding analytic system;EPS - 300 electrophoresis apparatus;HEIDOLPH DIAX900 homogenate apparatus;JUNG - CM1800 constant fridge microtome;Deep freeze refrigerator;DG3022A enzyme -linked immunodetec-tion apparatus;HP1100 high performance liquid chromatogram apparatus;721A spectrophotometer;Olympus light microscope;Animal operation instrument.3. Experimental protocols-.Rats were randomly divided into five groups: 1 ) control group ( A, n = 35 ) , only operating as ALI group, not HCL - dropped, then divided into 4 subsections according to the time of obtaining sample;2) ALI group ( B, n = 35 ) , according the methods of records, rats were anesthetized, intubated, HCL (PH = 1. 25) was dropped through trachea cannula at inhalation phase, and then divided into 4 subsections according to different dummy intervention;ALI + BMP group (C, n = 10) , rBMP -4 ( 15jxg/kg) was injected through vena caudalias at once after HCL - inhalation and duplicate injected at 3d, 5d;ALI + Noggin group (D, n=20) was divede into 2 subsections, Noggin (30(xg/kg) was injected through vena caudalias at once after HCL - inhalation and duplicate injected at 3d, 5d or just after HCL - inhalation;ALI + Lye group ( E, n = 10) , a week before modeling of ALI, rats were intragastric administrated with lycopene (5mg/kg) once a day.Rats were re - anesthetized at corresponding time points, 5ml of blood was drawn from abdomen cardinal vein before executing, centrifuged at 3000 rpm for 10 min at 4°C , the supernatant was preserved below - 20°C for measurement, then rats were executed by exsanguinating, lung was obtained and left fixed with 10% formalin, routine embedded, sliced and stained, right lung was preserved below -10X, after washing and quick freezing for measurement.4. Indexes and Methods of Observation:1) Observing the morphologic changes of the lung ( hematoxylin and eosin or Masson Staining).2) Assaying the expression of BMP -4 mRNA in the lung ( RT - PCR).3) Detecting the protein expression of BMP - 4 in the lung ( Western -blot).4) Detecting the content of TNF - o^NO^MDA in plasma ,the content of HYP and the activity of SOD in lung ( enzyme - linked immunosorbent and radio- immunity analytical assay)5) Observing the state of AECs of rats (in situ end labeling assay).5. Statistics:Values are expressed as means ± SD, group comparison was analysed by t test, statistical significance was set at P <0. 05.Results1) The changes of morphology in lung: Stained with HE, we could see e-dema, inflammatory cell infiltrate, telangiectasis and haemorrhage, interval widen, structure of alveolus destroyed in the lung at 6h, there were ponderosus inflammatory cell infiltrated, a large area alveolus collapsed, connective tissue and micrangium proliferated in interstitial at 7d, alveolar space disappeared and been replaced of collagen fibers, collagenoblast and lymphocyte at 14d;Stained with Masson, a great quantity of collagen fibers could be seen in lung interstitial at 7d, a great quantity of collagen fibers connected into lamellar in lung interstitial at 14d in group ALI;The changes described aforesaid were aggravated at 7d and 14d in group ALI + BMP, but lessened obviously in group ALI + Noggin and ALI + Lye.2) The expression of BMP -4 mRNA;there is some degree expression of BMP - 4 mRNA in lung of Group Control, but in Group ALI, the expression was up - regulated significantly at 6h (compared with Group Control, P <0. 05);In Group ALI + Lye, the expression was degraded significantly ( compared with Group ALI).3 ) The protein expression of BMP - 4;In Group ALI, the expression of precursor protein of BMP - 4 was up - regulated obviously, values was doubled of that in Group ALI, but the expression of mature protein of BMP - 4 was degraded significantly, values was half of that in Group ALI.4) The changes of biochemical indicator;Compared with Group control, the content of TNF - o^NC\MDA raised up and the activity of SOD decreased obviously (P < 0. 01) in Group ALI at 7d, 14d, the content of HYP in lung raised up obviously ( P < 0. 01) in Group ALI at 14d;Compared with Group ALI, the content of HYP in lung raised up obviously (P<0.01)in Group ALI + BMP at 7d, 14d, but decreased obviously (P <0. 01) in Group ALI + Noggin at17d, others remained no significant changes;5) Apoptosis of AECs;The cells that nucelus marked positive could be seen occasionally in Group Control at 6h and 20h, but the number of that increased in Group ALI, particularly in areas of the corner of alveolar wall, connected with ductuli alveolares, it is consistent with the distribution of type II AECs;The cells that nucelus marked positive emerged in alveolar space at 2Qh. Quantitative analysis, The number of the cells that nucelus marked positive increased obviously in Group ALI at 6h and 20h ( compared with Group Control, (P<0.01 or0.05 ), and it decreased obviously in Group ALI + Noggin at 6h (compared with Group ALI, P < 0.05).Conclusions1. The expression of BMP - 4 mRNA and preproprotein up - regulated but the expression of BMP -4 mature protein decreased during ALI, which illustrated that BMP -4 participated the course of ALI.2. During ALI, intervention with recombinated BMP -4 aggravated pulmonary fibrosis, and its antagon (recombinated Noggin) lessened pulmonary fibrosis, which showed that BMP -4 had the effect of pro - fibrosis.3. Intervention of anti - oxidative damage lessoned pulmonary fibrosis and decreased the expression of BMP - 4 mRNA during ALI, so the signal pathway of BMP -4 may involved in the lung protection of anti - oxidative damage.4. Recombinated Noggin lessened the apoptosis of AECs during ALI, and the apoptosis of AECs is an important mechanism of pulmonary fibrosis, which demonstated that the apoptosis of AECs may be one of the pro - fibrotic mechanisms of BMP -4.