The Experimental Study About The Effect Of Propofol On The Expression Of BMP-4 In The Lung Of Rats Asthma Remodel

IntroductionInduction of anesthesia and intubation of the trachea causes airway constriction. In patients with asthma, tracheal intubation can increase the risk for development of severe bronchospasm. Propofol is a short - acting intravenous anesthetic largely used, both in anesthesia and intensive care units, for sedation during mechanical ventilation. Several clinical studies have suggusted that propofol may be more protective against tracheal intubation induced brochoconstriction compared with thiopental in patients with or without asthma. Moreover, propofol has also been reported to produce brochodilation in patients with chronic obstructive pulmonary disease undergoing mechanical ventilation. Jn contrast, in vitro studies have suggested that clinically achievable concentration of propofol may not inhibit airway smooth muscle contraction induced by histamine, acetylcholine , or high potassium level. All these suggested us to study airway protectivies of propofol further more.Members of the transforming growth factor ( TGF) - β family play central roles in controlling cellular proliferation, differentiation, and survival. The TGF -β family can be divided into three subgroups: TGF - βs, the activins/inhib-ins, and BMPs, of which the latter constitutes the largest subfamily. BMPs are 30 - 35 KD hetero - or homodimeric proteins originally identified by their ability to induce ectopic cartilage and bone formation when injected subdermally in rats, and later studies demonstrated the importance of BMP - 4 for normal lung development. Overexpression of BMP -4 in transgenic animals under the surfactant protein C enhancer reverled a phenotype with abnormal morphogenesis asso-ciated with cystic terminal sacs and markedly inhibited overall proliferation. In addtion, the epithelium was dominated by type Ⅰ epithelial cells and the member of type Ⅱ cells was markedly reduced. The importance of controlled BMP signaling during lung development was further delineated in transgenic animals expressing endogenous BMP antagonist, XNoggin, in the lung. Epithelial cell proliferation was significantly inhibited in these animals. This was accompanied by severely reduced distal epithelial cell formation with a concurrent increase in proximal cell phenotype. Thus, these BMP -4 mechanisms may contribute to the disease pathphysiology.A rat asthma remodel was established by sensitization and challenge with ovalbumin in our study. The change of airway and the expression of BMP - 4 were determined by image analysis system, immunohistochemistry and Westen Blotting. At the same time, we apply intravenous anesthetics, propofol, to observe the effect of propofol on the expression of BMP - 4 in the lung of rats asthma remodel, and try to get one of the mechanisms for airway protects of propofol.Materials1. Animals; Forty -five SD rats (body weight: 160 -210g) were provided by Animal Center, China Medical University.2. Main reagents and apparatus: Ovalbumin (Sigma) ; Rabbit - anti -BMP -4 ( Boster, Wuhan) ; Gaot - anti - Rabbit - IgG ( Boster, Wuhan) ; Immunohistochemistry kit ( Maixin,Fuzhou) ; Western Blotting kit (Boster, Wuhan) ; ECL plus reagents ( Amershem, Sweden) ; SDS - PAGE protein Mark (Shanghai) ; Eppendorf Centrifuge 5810R (Germany) ; BIO - RAD's Model 200/2.0 (Canada); Chemilmager 5500 (America)Methods1. Rats asthma remodel and Groups: Forty - five SD rats were randomly divided into three groups of 15 each; Group Ⅰ , healthy rats as control group;Group Ⅱ , asthma group ,a rat asthma remodel was established by sensitization and challenge with ovalbumin (OVA) ; Group Ⅲ, propofol group, propofol 50 mg · kg-1 · h-1 was continuously infused iv for 2 hours after the same treatment as Group Ⅱ .2. Immunohistochemistry: Brieftly, after blocking, sections were Incubated with primary antibody followed by biotinylated secondary antibody. Slides were developed in DAB and couterstained with hemotoxylin.3. Western Blotting: The proteins were extracted in protease inhibitor. An equal amount of lung protein was run under conditions on a 12% SDS - PAGE Bis/tris gel and thereafter transferred to PVDF membranes by elecblotting using transfer buffer. Blots were blocked in 5% milk - PBS -0. 1% Tween 20 and then incubated with 1:400 of primary antibody for 1h, at room temperature. Thereafter, the membranes were washed 3 × 15min, with blocking buffer, incubated for 1 h with the secondary HRP - linked Goat anti - rabbit antibodies at 1: 400, washed 3 × 15min, incubated with ECL - substrate, and finally exposed to radiographic films.Results1. BMP -4 was found on the bronchial epithelium, in the vascular endothelium, and on a few scattered alveolar cells of all groups.2. There were decline in A1/D and increase in A2/D in the airways of Group Ⅱ (153.4 ± 10.6 and 72.2 ±5.7) as compared to that of Group Ⅰ (186. 9 ±13.6 and 60.9 ±7.1)P<0.05; The BMP -4 level in Group Ⅱ(11.5% ± 3.5%) was lower than that of Group Ⅰ ( 19. 4% ±4.4% ) P <0. 05. Group Ⅲ has a higher A,/D( 166. 8 ± 10.9) compared to that of Group Ⅱ (153.4 ± 10.6 ) and the level of BMP -4 also increased 4. 1% ,P <0.05.DiscussionMorphogenesis is responsible for bringing cell populations together for new inductive interactions and for building complex structures such as hearts, lungs,and eyes out of simple epithelial sheets and mesenchymal cell masses. Research over the past two decades have elucidated many of the genetic pathways underlying cell division, and differention, and has shown them to be evolutionally conserved. Evidence is beginning to accumulate that the majority of the signaling factors are proteins encoded by a relatively small number of conserved multi-genefamilies, including BMPs. BMP signaling is mediated by specific receptors. In the mouse, a single type Ⅱ subunit has been identified, while at least three type Ⅰ subunits are known. Upon binding of the ligand, the type Ⅱ receptor phosphorylates cytoplasmic targets, including members of the Smad family of proteins. These phosphorylated Smads then form complexes with a common effector, Smad4, and translocate into the nucleus to regulate gene expression.It is now clear that the name BMP is misleading because there is strong genetic and experimental evidence that these molecules regulate biological processes as diverse as cell proliferation, differentiation, and morphogenesis. Moreover, the vertebrate BMPs are involved in the development of nearly all organs and tissues, including the nervous system, lung, kidney and skin, as well as in critical steps in the establishment of the basic embryonic body play.There is evidence that BMPs can either stimulate cell proliferation or promote cell differention and exit from the cell cycle, depending on the target cell type. This raises the possibility that the same BMP could affect cells in different ways, depending on the concentration of ligand to which the cell are exposed; for example, low concentrations might stimulate cell proliferation, whereas higher concentrations might promote differentiation. BMP - 4 expressed ectopically has different effects on epithelial versus mesenchymal cells, inhibiting the proliferation and differentiation of the former, and stimulating either proliferation or cell death in the latter.Even though BMP - 4 is involved in lung development, its role during inflammatory responses in adult lung has not been investigated so far. Allergic asthma is a chronic disease representing the final stage of repeated airway antigen challenges. The disease is associated with widespread narrowing of the bronchial airways, pulmonary eosinophilia, mast cell infiltration, mucus hypersecretion, excessive IL -4 and IgE production, and finally remodeling of airways.