Effect of the BMP-2 on Pulmonary Vascular Remodelling in Rats with Acute Lung Injury by Endotoxin and Interventional Role of Intravenous Anesthetic
|School||China Medical University|
|Keywords||bone morphogenetic protein-2 lung injury intravenous anesthetics pulmonary vascular remodelling lipopolysaccharide gene|
IntroductionVascular remodelling refers to smooth muscle cell of the vascular medial layer migrate to internal membrane and the proliferation of vascular smooth muscle cell which induced by damage of blood vessel endothelium and exceptional agglomerates of the extracellular matrix, then causes endomembrane and the medial layer thicken and being narrow of the vascular lumen, these result creates blood vessel wall disease. Abnonnal proliferation and apoptosis of pulmonary artery smooth muscle cell（PASMC） is the common pathological change of this kind of disease. Disease cause of the pulmonary artery hypertension is quite complex, but two final ways is essential: First, lung vascular constriction enhance.Second, the lung vascular configuration remodel. The typical manifestation is disfunction of endothelial cell induced by all kinds of etiological factor, and furthermore causes this result that metabolism, the release, the production, the transportation and the secretion process of body fluid factor which adjust the relaxation and proliferation of vascular smooth muscle cell is at the disorder condition.Meanwhile, activeness changes of some transcription factors cause some protein expression which regulate apoptosis and proliferation of vascular smooth muscle cell by signal transduction pathway.The pulmonary artery wall are made of the endomembrane and the tunica media vasorum （non-muscle artery does not have tunica media vasorum） and adventitia.The internal membrane is made of continual flat endothelial cell and the basal membrane as well as the cavity under endothelium; The blood vessel tunica media vasorum mainly is constituted by the vascular smooth muscle cell（VASMC）, VASMC is one of most plastic cells which can carry on the response to the different growth factor. The vascular smooth muscle cell may multiply （proliferation which concomitance cell quantity increases）,hypertrophy （cell volume gain, the DNA content invariable）, intranuclear duplicates （increasing of the DNA content and being invariable of cell volume） and apopotosis.Each kind of stimulation factor regulate these reaction through the growth way of autocrine and paracrine secretes. The vascular smooth muscle cell has the plasticity ,which refers to VASMC can carries on some response to the haemodynamics, the growth and the damage stimulation （growth mechanism of autocrine and paracrine secretes） and causes the vasclar smooth muscle cell adapt the important biology function of blood vessel: For example, blood vessel growth during the physiology process, the response of the blood vessel to damages and the organization remodelling of the blood vessel occur to the organization needs, pathological arteriosclerosis, hypertension, renarrow and vasculitis of blood vessel formation operation. Obviously, during each kind of situation, endothelial cell and vascular smooth muscle cell of the blood vessel wall as well as interaction between the vascular smooth muscle cell and other cells （becomes ciliary cell, tree cell, phlogocyte） decide nature of growth response. Many gene transcription and event about the growth factor participate process which blood vessel smooth muscle cell create tunica media vasorum, these processes may reappear in the grown-up artery and the blood vessel formation. In the adult blood vessel, growth of the smooth muscle cell appears after the blood vessel damage. Repair of the blood vessel and proliferation process of the internal membrane is similar among the different species and the different artery. At present, mechanism of terminating vascular smooth muscle cell growth and adjusting vascular smooth muscle cell quantity are still not clear, although the mechanism research about apopotosis of the vascular smooth muscle cell make the very dramatical progress, but adjustment mechanism of the related blood vessel size and medial layer thickness still need to study, but it might affirm that the growth mechanism of autocrine and paracrine secretes is necessary to form the newborn internal membrane.The acute lung injury induced by the endotoxin is the clinical common disease, which is contributed by infection and further lead system inflammation response sydrome （SIRS）, its final result is multi-organ system disfunction （MOSD） and multi-organ function failure （MOSF）, in which lung is even the first occurred organ, because the disease condition is complex and not easy under control, the mortality rate is extremely high, at present ,is one of difficult treat illness, also is hot spot which is studied at present. The endotoxin is lipopolysaccharide（LPS） ingredient into cell wallof Gram-negative bacteria, TNF-αis causative agent that leads the acute lunginjury（ALI） induced by LPS. Our preliminary animal experimentation also indicated that when lipopolysaccharide （LPS） causes the acute lung injury（ALI）, the lung vascular endothelial cell meet with the attack firstly and lead to necrosis or fall off of endothelial cell and vanishing of barrier function, causes secondary proliferation and the migration of the PASMC, meanwhile appears obvious worsening of thehaemodynamics: For example the lung arterial pressure ascend, the average arterialpressure drop, increasing of the airway pressure, the artery blood oxygen partial pressure obvious drop, water content of the lung rise and so on. Moreover these sympotm can gradually aggravate along with the condition development, finally causes the pulmonary artery hypertension and system function of respiration and circulation are failure which controls with difficulty, system function failure of respiration and circulation can create insufficiency of other organ blood and oxygen supply, which further induces multi-organ disfunction of all over the body, finally causes MOSF.Therefore, many domestic and foreign scholars devote to preventing and controlling research of the pulmonary artery hypertension which causes by the lung artery configuration remodelling, has basically been clear about molecular and genetics mechanism of signal transduction process within the lung vascular configuration remodelling. Research about molecular mechanisms of the pulmonary artery hypertension have yielded the following information. （1） BMP/TGF-βsignal transduction pathway（includingSmad-dependent signaling and Smad-independent signaling）; （2） The serotonin signalling pathway; （3） Extracellular matrix signalling pathway; （4） Role of ion channels; （5） Inflammatory components. Sometimes these pathways mutually affect and overlap and play their regulative role together, in which BMP signal transduction pathway has been emphasized by more and more peoples.Bone morphogenetic proteins （BMPs） become one member of TGF-βsuperfamily’s, not only has the vital role during production and differentiation and growth of the bone and the cartilage, moreover has the expression in formation process of other many organization and organ （heart,lung, brain and so on）, and regulate proliferation and differentiation and migration and apoptosis of many kinds of cells （mesenchymic cell, becomes textile fiber metrocyte, keratinizing cell, star cell, kidney cortical cell, endothelial cell as well as tumor thin and so on）, and is advantageous to the adult organization’s maintenance and repair. Among these factors, Bonemorphogenetic proteins type II receptor （BMPR- II） is important ingredient on theBMPs signal transduction pathway, play an important role during process which accomplish information transmission inside and outside of the cell. However, causesnormal expression drops or the function flaw of BMPR- II by stimulation of certaindiseases （for example primary pulmonary hypertension） and environmental factors, which causes change of the lung vascular cell growth condition and induce restructuring of lung blood vessel and dysfunction of the signal transduction. The bonemorphogenetic protein - 2 （BMP-2） is the important active ligand of BMPR- II andplay the decisive role on this pathway. In recent years studies,some scholars discovers BMP-2 not only regulate occurrence and formation of the bone or the cartilage and the embryo, but also regulate growth and proliferation and apopotosis and migration and cell division of pulmonary artery smooth muscle cell （PASMC） and has the vital role during the matrix synthesis, then regulate lung vascular configuration reconstruction. This research focus on the lung blood vessel configuration reconstruction after the acute lung injury induced by the endotoxin, discuss function and the possible mechanism of exogenetic BMP-2 in this process, furthermore ,discuss the effect of the protective function intravenous anaesthetic during acute lung injury induced by the endotoxin on the expression of BMP-2 gene. In order to discuss the question which must solve by different levels, this experiment divides into three parts: The first part adopt to injects rhBMP-2 directly, inspects BMP-2 to send the function and the mechanism during the lung vascular remodelling after acute lung injury induced by the endotoxin in rats, provides the research foundation for following experiment; The second part focus on the level of pulmonary artery smooth muscle cell （PASMC） in vitro, simulates damage environment of toxin-endothelial cell in vivo, and eliminate interventional factors in the body environment, detect the influence and the mechanism of exogenetic rhBMP-2 on proliferation/apoptosis of the pulmonary artery smooth muscle cell （PASMC）, discusses the inhibitory action and the possible mechanism of BMP-2 which restructures to the lung blood vessel in the cell level; The third part mainly inspect that intravenous anaesthetic sends influence of BMP-2 gene expression and the lung arterial lumen after acute lung injury induced by endotoxin in rats, provides the theorial basis for the clinical reasonable medication of anaesthesia after acute lung injury induced by the endotoxin. It is worth discussing that the process of lung vascular remodelling is contributed whether the pathway about the BMP-2 signal transduction simultaneously regulates many kinds of gene expression or regulates many kinds of gene cascade response successively the effect way affects lung blood vessel restructuring, waits for in further studies and confirmation.Materials and MethodsExperimental animal and reagent drugs1、Experimental animal: Wistar rats weighing 180-260g are provided by the China Medical University experimental animal center. 2、Important reagent and drugs:endotoxin （Escherichina coli LPS,055:B5,America Sigma company） ; pentobarbitol sodium; rhBMP-2; Bc1-2、Bax、PCNAantibody; TUNEL kit; fetal bovine serum; Dulbecco’s Modified Eagle Medium; AnnexinV-FITC kit; propidium iodide; SM-α-actin antibody;propofol;ketamine;PBS; 4%paraform;2.5%glutaral etal.3、Mainly Experimental instrument:RM-6000multiplying channel physio-monitor;PTC-100PCR amplification meter; KADAKID gel image analysis system; GIS-700D figure gel scanning analysis system;visible light contimuous spectrum meter; DYY-III31A electrophoresis apparatus; JUNG-CM1800 section cutter; Olympus microscope; Metamorph/Olympus DP10/BX 51 microgram analysis system; HITACH transmission electron microscope; carbon dioxide incubator; centrifugal machine; flow cytometry; inverted phase contrast microscope; fluorescence microscope; SW-CJ-1FDsuperclean bench; patch clamp.Methods1、Model preparationWistar rats were basal anesthesia by intraperitoneal injection 1% embutal with 50 mg/kg. Uses constant speed input method of small dosage endotoxin: In 30min inputs LPS 1mg/kg from the femoral vein, establishes rat model of acute lung injury.2、Cell culturePulmonary artery smooth muscle cell of the rat culture（pasting block method）, pulmonary artery of scraping off the internal membrane were flushed by using PBS and were cut about 1mm3 scrap, organization was placed in the cell culture dish by 3-5 /cm2density, will paste wall 2h 37°C condition, will culture staticly for 5 day under thenutrient fluid 2ml including 20% FBS DMEM,37°C 5% CO2.When cells grow into themonolayer, takes out the organization block, renoveatur the fluid and continue to culture, until the cytomixis,then digests the cell with 0.125% trypsin, serial subcultivation.This experimental use 35th generation of cell.3、experimental groups（1） The first part of experiment divides into three groups:30 wistar rats were divided randomly into three groups with 10 each group.control: Through femoral vein infusion physiological saline53ml within 1.5h; Group L:Through femoral vein infused physiological saline 3ml, then infusion LPS1mg/kg （dissolves in 2ml 0.9% physiological saline）, within 30min the infusion finished; Group LT: Through femoral vein infused LPS1mg/kg （dissolves in 2ml 0.9% physiological saline） within 30min,then infused rhBMP-2 10μg/ kg（dissolves in 3ml 0.9% physiological saline） within 1h and injects separately rhBMP-2 4μg/kg throug the detaining ductus venosus at 24h and 48h.（2） The second part of experiment divides into five groups:The PASMCs were cultured by EC-CM1 （Group I）; The PASMCs were cultured by EC-CM2（Group II）; The PASMCs were cultured by EC-CM2 and BMP-2 1ng/ml （Group III）; ThePASMCs were cultured by EC-CM2 and BMP-2 10ng/ml （Group IV）; The PASMCswere cultured by EC-CM2 and BMP-2 100ng/ml （Group V）.After 24h collected cells to detect index.（3） The third part of experiment divides into six groups: 60 wistar rats were divided randomly into six groups with 10 each group.Through femoral vein infusion physiological saline 5ml within 1.5h（Group C）; Through femoral vein infused physiological saline 3ml, then infusion LPS 1mg/kg （dissolves in 2ml 0.9% physiological saline） within 30min （Group L）; Through femoral vein infused propofol 20mg/kg/h（dissolves in 3ml 0.9% physiological saline） within 1h,then infused LPS 1mg/kg （dissolves in 2ml 0.9% physiological saline） within 30min (Group P20L); Through femoral vein infused propofol 50mg/kg/h（dissolves in 3ml 0.9% physiological saline） within 1h,then infused LPS 1mg/kg （dissolves in 2ml 0.9% physiological saline） within 30min (Group P50L); Through femoral vein infused ketamine 20mg/kg/h（dissolves in 3ml 0.9% physiological saline） within lh,then infused LPS 1mg/kg （dissolves in 2ml 0.9% physiological saline） within 30min (Group K20L); Through femoral vein infused ketamine 50mg/kg/h（dissolves in 3ml 0.9% physiological saline） within 1h,then infused LPS1mg/kg （dissolves in 2ml 0.9% physiological saline） within 30min (Group K50L).4、Examination target and examination method（1） The first part of experiment: Through transmission electron microscope to observe synthesis situation of the cell organ and the phenotype differentiation of pulmonary artery smooth muscle cell in each group rats;Measured medial layer thickness of pulmonary arteriole by microimage analysis system;Using immumofluorescence method localized the expression of Bcl-2 and Bax gene at pulmonary arteriole;Detected proliferation and apoptosis of pulmonary artery smooth muscle cells by PCNA and TUNEL methods;Quantitated mRNA and protein expression of Bcl-2 and Bax gene by RT-PCR and western-blot methods.（2） The second part of experiment: Through MTT chromatometry observed proliferation ratio of pulmonary artery smooth muscle cells;Analysised cell life cycle by flow cytometry with PI staining method;Detected earlier apoptosis of pulmonary artery smooth muscle cells cultured in vitro by the way of Annexin V-FITC/PI; Quantitated mRNA and protein expression of cyclinD1 and p21 gene by RT-PCR and western-blot methods;using the patch clamp technique proved the activity of delayed rectification K+ channel.（3） The second part of experiment:localization of BMP-2 gene expression onpulmonary artery through immunohistochemical method; Quantitated mRNA and protein expression of BMP-2 gene by RT-PCR and western-blot.statistical treatment: This experiment adopted completely random design, all datasby （x|-）±s expression, statistical analysis system（SAS8.1） package carry out statistics analysis. During the multi-groups compares uses the single factor variance analysis, Between two groups comparison applies student newman-keuls the t-test. ResultsPart 1:Effect of rhBMP-2 on pulmonary vascular remoding in rats with acute lung injury caused by endotoxin and mechanism therefor1、Through transmission electron microscope to observe synthesis situation of thecell organ and the phenotype differentiation of pulmonary artery smooth muscle cell in each group rats. In C group ,normal blood vessel wall tunica media vasorum PASMC includes the massive myofilament ingredient, its structure is compact, display fasciculation arrangement, quantity of cell organ are few, enrichment of the nucleus chromatin, has typical characteristic of the contraction smooth muscle cell, has not discovered characteristic of the lung vascular configurationremodelling. In group L, the myofilament ingredient reduces in medial layer of the smooth muscle cell, the golgiosome and the chondriosome and the ribosome increase, these symbolized that the majority of pulmonary artery smooth muscle cells already changed from the differentiated contraction phenotype to synthesis phenotype, has the characteristic which the lung blood vessel restructured. In group Lt, increasing tendency of the golgiosome and the chondriosome and the ribosome of vascular smooth muscle cell were weaken obviously, indicated the characteristic of the lung vascular restructures isweaken.2、Compared with C group and Lt group, group L of lung arterial mediathickness percentage obviously increases （P＜0.01）, compared with group C, the lung arterial media thickness percentage of group Lt does not have the difference （P>0.05）.3、On pulmonary arterial media of group C , of group L,of group Lt Bcl-2 and Bax gene has the expression, the green fluorescence means positive expression.4、Compared with C group and LT group, proliferating cell nuclear antigen expression of pulmonary arterial smooth muscle cells increased in group L （P< 0.01） ;contrast to this, apoptosis ratio of pulmonary arterial smooth muscle cells attenuated in group L （P<0.01） .5、Bax、Bcl-2 gene all expressed in pulmonary artery.Expression of Bax mRNA significantly decreased in L group comparison with C group （P <0. 01）. Expression of Bax mRNA decreased in LT group compared with C group （P <0. 05）, but increased in comparison with L group （P <0. 05） ; Level of Bax protein was dramaticly lower in L group than C group,contrast to that Bcl-2 was higher （P <0. 01） .Part 2:Effect of recombinant human bone morphogenetic protein-2 on proliferation/apoptosis of pulmonary artery smooth muscle cells induced by endothelial cell-conditioned medium liquid in vitro and mechanism therefor1、The result of MTT did not show obviously pulmonary arterial smooth muscle cells proliferation in group I.compared with group I, EC-CM2 trigered dramaticaly proliferation of PASMC in group II （P < 0.01） . compared with group II,proliferation ratio of PASMCs did not appear significant changes in group III （P>0.05） .but the proliferation of PASMCs has been inhibited in group IV and V.2、Cell life cycle analysis showed amount of phase S and G2/M were less.meanwhile, amount of phase S and G2/M increased obviously（P < 0.01）.compared with group II, amount of phase S and G2/M dramatically attenuated in group IV and V.3、Result of Annexin V-FITC/PI showed apoptosis ratio of PASMC in group II. apoptosis ratio of PASMC decreased markly in group III and IV and V with increasing rhBMP-2 concentration.4、Compared with group I, Cyclin-D1 mRNA and protein expression of Group II obviously increases （P < 0.01）. In comparison to Group II, Cyclin-D1 mRNA and protein expression of Group III did not appear the difference （P>0.05）; the protein and mRNA expression of cyclin-D1 striking droped Group IV and V; the protein and mRNA expression of p21 did not show notable difference in group I and II and III.compared with group II, the protein and mRNA expression of p21 increased significantly in group IV and V.5、Electrophysiology experiment approved intensity of delayed rectification K+ current of PASMCs weakened dramatically in group II in comparision to group III and IV and V.Part 3:Effect of intravenous anesthetics on BMP-2 gene expression in rats with acute lung injury by endotoxin 1、Immunohistochemistry result showed BMP-2 gene expressed on medial layer of pulmonary artery,and compared with group C,positive expression index dropped in group II （p<0.01） . and compared with group L, positive expression index dropped in group P20L and P50L and K20L and K50L （p＜0.01） .2、With coincidence to Immunohistochemistry result, compared with group C,mRNA and protein expression of BMP-2 dropped dramatically in group II （p<0.01）. and compared with group L, mRNA and protein expression of BMP-2 soared in group P20L and P50L and K20L and K50L （p<0.01） .Conclusions1、Upregulating the Bax gene expression and declining the Bcl-2 gene expression by giving rhBMP-2 in vivo promoted apoptosis of PASMC on the pulmonary artery,further suppressed the pulmonary artery configuration remodelling induced by exceptionally proliferation of PASMC after the acute lung damage by the endotoxin in rats.2、The endotoxin-endothelial cell-conditioned medium liquid can enhanceproliferation of PASMC cultured in vitro,certain concentration of recombinant human bone morphogenetic protein inhibited the expression of cyclinD1 and promoted the expression of p21 in PASMCs cultured by endotoxin-endothelial cell-conditioned medium liquid,meanwhile enhanced the activity of delayed rectification K+ channel, those factor suppressed proliferation and triggered apoptosis in PASMCs cultured by endotoxin-endothelial cell-conditioned medium liquid.3、Intravenous anesthetic including propofol and ketamine played protective roleduring acute lung injury by endotoxin in rats,caused expression of BMP-2 on pulmonary arterial after acute lung injury by endotoxin in rats.