Between The Expression Of HMGB1and COPD Rat Pulmonary Artery Smoothmuscle Cells Synthesis And Secretion Of Correlation

Chronic obstructive pulmonary disease is a major chronic respiratorydisease, the number of patients, mortality is high, has become theworld’s fourth largest killer diseases, while the total number of COPDpatients up to43million, an average of2.5per minute people died inCOPD. Because of its slow sexual development, seriously affecting theability to work and quality of life of patients, people emphasis on diseasehas become increasingly high. COPD patients after acute exacerbation ofclinical symptoms despite the relief, but lung function continues todeteriorate, and by reducing the harmful impact of external factors aswell as self defense and immune function, often recurrent, easysecondary pulmonary pressure, and gradually produce a variety of heartand lung complications. Complex pathogenesis of COPD, and personalsmoking, environmental destruction, closely related air pollution, smallairway infections, pneumoconiosis, etc., especially PM2.5concentrationsclosely with COPD. A six-month longitudinal study showed that indoorconcentrations of PM2.5and NO2elevated in COPD patients with nocturnal symptoms aggravate and increase in acute attacks. Therefore,the main measures to prevent COPD is to avoid the incidence of riskfactors, predisposing factors of acute exacerbations and enhancedimmunity. Quitting smoking is easy and effective measures to preventCOPD. Control of occupational and environmental pollution, reduceinhalation of harmful gases or harmful particles, reduce airwayinflammation and lung abnormalities; another, early detection and earlyintervention in favor of prevention and treatment of COPD.Studies confirm, COPD is a chronic systemic inflammatory disease.Various cytokines and inflammatory mediators such as HMGB1, IFN-γ,IL-6and other processes involved in inflammation. High mobility groupprotein system in the1960s, was first discovered, a content rich highlyconserved non-histone nucleoprotein, is widely distributed inmammalian cells, with the late pro-inflammatory effects, its mobility ingel electrophoresis fast named. High mobility group protein present onthe cell nucleus, cytoplasm and cell membrane of a variety of cells, incombination with DNA by HMGB box. Contains high mobility groupprotein HMGB1, HMGB2, HMGB3three related members, and85%homology, while HMGBI under a variety of factors to active secretionand passive release of two ways to arrive outside the nucleus. HMGB1isa proinflammatory cytokine newly discovered immune stimulatingproperties, has an important role in the pathogenesis of COPD and otherdiseases. In-depth study found, HMGB1with elevated serum TNF-a,,IL a p\lL levels of inflammatory cytokines, such as a6upregulation.When cell necrosis or damaged, the role of TNF HMGBI nucleus can bereleased into the extracellular triggered monocyte macrophages secrete proinflammatory cytokines; while the pro-inflammatory cytokines can befurther promote the secretion of HMGB1to form a positive feedbackloop. In the late inflammatory response, such a positive feedback effecton the maintenance of the inflammatory response has an important role.1973Younger and Salvin found γ interferon, which comes fromlymphocytes. Its main function is an immunomodulatory biologicalinducing antigen-presenting cells MHC-I/II various molecules, activatedmonocytes, macrophages secrete IL-1,6,8, TNF-a and the like. IFN-γcan activate neutrophils, endothelial cells to stimulate and promote thedevelopment and inhibition of Th1cell activation and proliferation ofTH2cells. IFN-γ-induced macrophage inducible nitric oxide synthase(iNOS) to produce, promote the synthesis of NO, IFN-γ can increase theLPS-induced IL-1transcription and translation and secretion. IL-6ismainly composed of a variety of macrophages, T cells, B cells generatedto regulate the growth and differentiation of many cell types, such as theregulation of the immune response, acute phase response andhematopoiesis, and immune response from the anti-infection importantrole. IL-6in COPD and other diseases has changes when significant;IL-6expression imbalance can cause many diseases, its clinicalmanifestations at onset increased IL-6levels. IL-6levels and increasedactivity of the disease and the treatment are closely related, andtherefore, the detection of the patient’s body fluid levels of IL-6to reflectthe patient’s condition changes.However, whether the level of performance of HMGB1expressionand IFN-γ, IL-6levels of inflammatory cytokines such as upregulation inpatients with COPD and related pulmonary arterial smooth muscle cells whether positive feedback loop relations and IFN-γ, IL-6is inducedpulmonary arterial smooth muscle related cells to be studied, such asHMGB1release, this experiment COPD rat model for the study,pulmonary artery smooth muscle cells in primary culture, based on theuse of molecular biology techniques, and the relationship betweencytokine levels from cells described above.Part One Primary culture rat distal pulmonary arterial smoothmuscle cells and research biological characteristics or functionObjective Research using tissue adherent culturedprimary rat distal pulmonary arterial smooth muscle cells (PASMCs),chronic obstructive pulmonary disease (COPD) rats with normal rat distalPASMCs biological characteristics and functions of the similarities anddifferences. Methods SPF SD male rats12, divided into two groups:(1)control group: the first1,14days after intratracheal injection of the samedose saline, no cigarette smoke exposure, under the same conditions withthe model group, normal feeding.(2) COPD rat model groups: theexperimental section1,14days after intratracheal injection oflipopolysaccharide (LPS) solution,200μg/times, cigarette smokeexposure1h/d, a total of eight weeks. Model is established, adherenttissue extracts and primary cultures of rat distal PASMCs. Cells wereobserved under inverted phase contrast microscope morphology and growth characteristics; cell immunohistochemistry, electron microscopyidentified PASMCs; CCK8fluorescence flow cytometry and cellproliferation of cells in each group. Results PASMCs inverted phasecontrast microscope for the long spindle or "fishtail" shape, for the typical"peak-valley"-like growth, cell morphology and growth characteristicsof the two groups had no significant difference; cellimmunohistochemistry, electron microscopy identified the cultured cellsmore than98%of PASMCs; CCK8test results show: COPD model groupPASMCs absorbance (A value) than the saline control group (P <0.05);using CFDA SE shown by flow cytometry: COPD model group PASMCsproliferation was stronger than saline control group (P <0.05), namelysaline control group PASMCs fluorescence is enhanced. ConclusionCOPD model group and normal group primary cultured PASMCs cellmorphology similar, but different biological characteristics, functionalityis also different. Part Two The correlation between the expression of HMGB1andCOPD rat pulmonary artery smoothmuscle cellssynthesis and secretionIFN-γ、IL-6and clinical significanceObjective:Investigate Chronic obstructive pulmonary disease (COPD) model rat distal pulmonary arterial smooth muscle cells(PASMCs) high mobility group protein B1(HMGB1) expression levelsof its synthesis and secretion of interferon-γ(IFN-γ)、IL-6and clinicalsignificance of the correlation. Methods: Establish COPD rat model,primary cultured PASMCs and identification, detection cigarette smokeextract (CSE) and LPS-induced cytokine synthesis and secretion underPASMCs function experimental groups: Group A (control group), Bgroup (CSE group), C group (LPS group), D group (CSE+LPS group),while A, B, C, D four groups of anti-HMGB1antibodies were added0.2ml1:1000separate flask culture (i.e. A1, B1, C1, D1); were cultured12h,24h,48h,72h after each group was detected by Western blot proteinexpression of HMGB1PASMCs, ELISA detection of cell culturesupernatant HMGB1, IFN-γ、 IL-6concentration and correlationanalysis.Results:(1) light microscope fusiform cells or "peaks andvalleys" like growth, α-actin immunohistochemistry andimmunofluorescence identified as PASMCs;(2) Western blot and ELISAassay showed that cultured12h After HMGB1expression and cellsupernatant HMGB1, IFN-γ、IL-6concentration in group B than ingroup A stronger (P <0.05), C group stronger than the B group (P <0.05),D group is stronger than in group C (P <0.05); HMGB1proteinexpression level of IFN-γ concentration was positively correlated (r= 0.91,0.83); join anti-HMGB1antibodies develop after12h, A1, B1, C1,D1intervention group compared with the corresponding experimentalgroup were decreased (P <0.05); Within the same group, in cell culture12h,24h,48h,72h, Western blot and ELISA assay results were notsignificantly different (P>0.05).Conclusion: The expression level ofHMGB1of COPD model rat synthesis and secretion in its distal PASMCsIFN-γ、IL-6is positively correlated，and HMGB1for clinical treatment ofCOPD inhibiting its systemic inflammatory response provides a newintervention targets.