Experimental Study On The Disequilibrium Of The Cytokine Network Balance With The Mechanism Of External Dryness Injurying Lung

Objective:The classical Chinese medicine theory that pulmonary inhaled the fresh air of nature and scattered,moderately,airway smooth was in circulation. Small blood vessels and capillaries in the modern medicine including microcirculation with highly relevant rich lung collaterals was the material basis for the"department, breathing" function.Gas exchange between gas and lung capillary blood through six layer thickness was about 0.2 μmm-1μm structure called the respiratory membrane(RM),the diffusion rate was inversely proportional with the thickness of the RM.Fine knowledge of anatomy and physiology mechanism for the film and the theory of division,breathing breathing at the micro level structure,gas exchange function in vivo and in vitro and in asthmatic cough more primary symptom similarity to provide physiological, biochemical,pathological objective standard and frame of reference,such as communication become human syndrome and syndrome of animal models with Bridges.Externess dry injurying lung involved in the pathogenesis essence of invaded and microscopic material body,such as proinflammatory cytokines/anti-inflammatory cytokine balance disorders, liquid water metabolism related gene expression.Novelty search data shows nearly 20 years did not see the same research report.This dissertation topic in neijing outside the six evils of the dry lung injury theory as the instruction,on the basis of previous studies, combined with micro super pathology,immunology,molecular biology theory and technology,to airway cytokine network balance as the breakthrough point,study of proinflammatory cytokines/ anti-inflammatory cytokine balance disorders, abnormal AQP5 gene expression, confirmed"airway cytokine network cause lung inflammatory pathological changes was the material base injured lung asthmatic cough syndrome"hypothesis. According to the results of the study expatiates on airway cytokine network imbalance causes dry lung injury asthmatic cough the material basis for the pathogenesis and pathophysiology mechanisms,to promote the six evils of the lung injury asthmatic cough common material provides the experimental basis for basic research,theoretical innovation and the guidance of clinical significance.material:The first.Experimental animals:the 210 kunming mice (SPF,17±1 g,male and female half) were provided experimental animal center of Hubei province,(license: 2008-2005).Second.the main instruments:LRH- 250 climate box, artificial intelligence temperature (6-65 ℃)±-1 ℃, relative humidity (30%-90%)±3%(guangdong medical instrument factory),the MASTER-K30H fan program-controlled instrument (South Korea’s Best mechanical and electrical co.,LTD.),HITACHI-7500 transmission electron microscopy (Japan),enzyme mark BioTek instrument(USA),fluorescence quantitative PCR system(SLAN,HONGSHI,U.S.A),scanner(V300,EPSON),leica ordinary and ultra-thin slicing machine (Germany),refrigerated centrifuge (15R,heal force,Hong Kong,China),DYY-6celectrophoresis apparatus(Beijing instrument factory),ultraviolet spectrophotometer (752-P,Shanghai now instrument co.,LTD.).Third.the mainagent:rabbit polyclonal antibody against mice AQP5 kits(DA0648, 200902,wuhan boster biotechnology co.,LTD.),biotin sheep rabbit 2 resisting and SABC kit,DAB,L-poly lysine and positive for more photos for wuhan boster biological technology co.,LTD.;Superscript II retroviras kit(FSK-100) and SYBR Green realtime PCR master mix-plus(QPK212 plus,TOYOBO company);Antibody Actin (Sc-1616-r) for Pierce companies,U.S.A);Formaldehyde (20090325,Shanghai).Methods:The 132 mice were randomized assigned into the normal temperature and humidity group(group A),warm dryness group(group B) and cool dryness group(group C),with 44 in each.The models were established with the simulation of temperature, rative humidity and wind.On the 6th and 12th day of moderling, According to the document,we dealed with each group after the 6th and 12th to detect the following experimental items:A.lung tissue pathology observation1.The common pathology observation:broken neck death mice,take right lung tissue in mice (40±10 mg/only),conventional paraffin section,HE staining and light microscope observation.2.The ultrastructure observation:each group randomly take 4 mice by ether anesthesia,injections into the lungs by endotracheal intubation 2% glutaraldehyde 1.0 ml fixed after 1 h,in the middle of the left lung tissue into 1cmx1cmx2cm,conventional shoot slices,Uranium acetate staining and transmission electron microscopy;Observeed ultrastructure,pulmonary interstitial of respiratory membrane,analysis RM membrane average thickness with JEDA 80ID image software.3.proinflammatory/anti-inflammatory cytokine level detection:each group through regular tracheal intubation in bronchial lung lavage 1.5 ml,place-82℃,unified inspection within 7 days.Using monoclonal antibody ELISA kit(R&D in U.S.A),according to the manual operation,CK,PBS negative contrast;BioTek enzyme immunoassay analyzer 495 nm OD value of standard curve to calculate inspected samples content.B.molecular biological detection(1) AQP5 mRNA measurementReal-time fluorescent PCR detection.Retrieve NCBI (the national biological information center) query gene sequences, Oligo 6 software design template primer amplification (Shanghai sangon biotech companies),upstream primer:5 ’-GGCTGCAATCCTCTACTTCTAC-3’,downstream primers:5’-TTCTTCCGCTCCTCT-CTATGA-3’(127 bp);Reference gene in beta actin (beta actin) primer sequence: upstream primer:5’-CCGTGAAAAGATGACCCAG-3’,downstream primers:5’-TA-GCCACGCTCGGTCAGG-3’(191 bp).RT-PCR reaction system for 50 ul;as 95℃ degeneration,55,72℃ extends 40℃ annealing cycle.In 2 -△△ Ct method to calculate AQP5 mRNA expression level.(2) AQP5 protein expression levelWestern blotting methods.The main steps:extraction of total protein,BCA (bicinchoninic acid) method determination of protein content in supernatant,SDS-page electrophoresis, transfer film5immune response,and chemiluminescence, enhancement, fixing; Scan film gel image analysis was carried on.C.statistical methods:Experimental data to mean±standard deviation (x±s),analyzed with SPSS 13.0 software,the significance test using multiple between the sample mean q test (Newman-Keuls method);Difference was statistically significant (P<0.05),inspection level for a= 0.05 on both sides.Results:A:histopathological observation1.Common lung histopathological observation shows again that group A of 6th and 12th days without abnormal,the alveolar of group B and C flake blood stasis,edema and fibrinous exudate,pulmonary interstitial thickening and inflammatory cells invasion,with emphysema phenomenon, in 12th days.2.The ultrastructure observation(1) In 6th and 12th days Respiratory membrane(RM) of group A was clear and the blood capillary endothelial cell structure was abnormal;type Ⅱ alveolar epithelial cell(AT Ⅱ) and type Ⅱ alveolar cell(AT Ⅰ) connection were cleared,riched microvilli and regularly arranged on side of alveolar cell membrane of AT Ⅱ and Osmiophilic Multilamellar Body (OMB);the mitochondria was presents typical membrane structure and the abundance.The alveolar septum broadening with collagen fiber of group B and Cwere hyperplasia and reduce of AT Ⅱ microvilli, obviously broadening swelling of the respiratory membrane and the capillary endothelial cells;swelled the joint line between AT Ⅰ and AT Ⅱ,and fewered of AT Ⅱ microvilli and arrangement planning,the cell cytoplasm was rich but decreased the number of intracellular mitochondria and swelling to the loaf of bread for the more mitochondria.(3)In the 6th and 12th days of group A,the alveolar septum and the structures of AT I and AT II and the alveolar capillary membrane structure and capillary structure were cleared;in group B and C of the alveolar septum broadening with collagen fiber hyperplasia,swelling of the alveolar capillary endothelial cells and the basilemma of AT I as more irregular and more dot fracture,and widened obviously of AT I basal membrane and blood capillary basilar membrane gap.With the image analysis, comparied with group A,the average thickness of group B and Cwere obvious thickening (p< 0.05).B.the levels of proinflammatory cytokines/anti-inflammatory cytokine in the bronchoalveolar lavagefluid1.Comparing with group A,the levels of NE and a1-AT of group B and C in the 6th and 12th days were elevated as obvious of cool-dryness(p< 0.05);the level of al-AT in the two groups were down,as obviousof cool-dryness group (p< 0.05).2.Comparing with group A of the level of TNF-a and IL-10 in the 6th and 12th days,group B and C were declined but without statistical significance;the level of IL-10 in the two groups were decreased (p< 0.01).3.Comparing with group A,the level ET of group Band C were increased but without statistical significance,but the level of PAF were increased (p< 0.05).C.Detection to the AQP5 mRNA and AQP5 protein expression levelComparing with group A, AQP5 mRNA expression of group B and C in the 6th and 12th days were cuted (p< 0.01);the level of AQP5 protein expression in the 6th and 12th days were continuesly declined,and the cool dryness group was elevated in the 6th but lowered in the 12th (p< 0.01).Discourses:1.The pathological characteristic of respiratory membrane ultras-tructure of group Band C in extern dryness injury lung showed that the AT Ⅰ membrane in was swelling and irregular breathing lung membrane thickening and fibrous tissue hyperplasia,thickening and swelling of the endothelial cells nad the capillary basement membrane as partly been stripped shape and obvious exudation stasis.The first image analysis was that RM of group B and C were markedly thickened as compared with group A in the 6th and 12th days(p< 0.05).Combined with literature reports that energy metabolic disorders of AT Ⅱ with swollened the mitochondria and hint the synthesis impaired function of surfactant AT Ⅱ as reduced the eaverage of OMB;the layer water molecules to reduce would influenced the formation of lung surfacant on the stability of single molecule lecithin membrane and lung moisten,was less regulated the surface tension of the alveolar surface,thickening and alveolar inflammatory lung RM,and damage the elasticity of the alveoli and alveolar ventilation dysfunction,which was put forward that RM thickening as one of the histopathological basis and the specificity ultrastructure pathological indicators in extern dryness injurying lung.2.It was put forward that the disordered the synthesis and secretion of alveolar surfactant of AT II and caused the lung-lose moisting and damaged of the alveolar ultrastructure,which was that one of the"Damage associated molecular patern" (DAMP) to activated the monocyte/macrophage lungs,and increased of the level of proinflammatory cytokines and release of proinflammatory cytokines as reduce the anti-inflammatory cytokine IL-10 and NE resistance mediated al-AT lower levels of the role of lung injury,cause cytokines network disordered of the proinflammatories /anti-inflammatories,so that started the center inflammatory cytokine cascade out-controling as the neutrophils release protease (NE) and PAF,which would one of the molecular biological basis of extern dryness.3.That the imbalance of the pro-inflammatory/anti-inflammatory cytokine network was caused cut AQP5 gene expression and drop the protein levels were prompted disordered the alveolar’s liquid transshipment;the out-controled of the inflammatory response caused stasis the inflammatory medium,and the swelled and increased the vascular permeability of pulmonary capillary endothelial cells with liquid leakage cause edema of the membrane tissue interstitial and thickened the RM and stasis and edema of the alveolar blood,leading to decreased the diffusion ratiothe O2 and CO2/blood loss and damage the ventilation of lungs through the weather",which was the based of pathophysiology extern dryness injurying lung.4.The results show that the extern dryness injurying lung was different with the "give priority to with TNF-a inflammatory cytokines’waterfall effect damage all asthmatic cough due to lung collaterals disease pathology basis",that was associated with the pathogenic characteristics with extern dryness.The innovation thesis of the dissertation:1.It was frist system observated that the characterized of damage ultrastructure in extern dryness was thickened of RM,swelled the AT I and the capillary basement membrane thickening and the endothelial cells,which was the the superfine histopathological basis of the cough and asthma in extern dryness.2.It was frist put forwarded that the quantitative measurement method of the RM average thickness was measuremented the RM thin sides as centering the pulmonary capillaries.3.It was put forward that the one type of "damage associated molecular patern" (DAMP) as injuring lung-fluids and the lung-lose moisting and damaged of the alveolar ultrastructure casued the balance disordered of the proinflammatories /anti-inflammatories network and cut AQP5 gene expression and protein levels,started the out-controling inflammatory cascade as centering of the neutrophils release protease (NE) and PAF,and cause swelled of pulmonary capillary endothelial cells and thickening of the basement membrane of interstitial edema and RM and damage the function of’lungs through the weather".It was frist proposed that the imbalance of proinflammatory/anti-inflammatory cytokine and disorder AQP5 gene expression would be the molecular pathophysiological basis and one of the characteristic index in extern dryness injurying lung and innovated the theoretical and clinical significance.