Effect Of1,25-（OH）₂D₃on The Expression Of HMGB1and TLR4in Lung Of Asthmatic Mice

Bronchial asthma is a higher incidence of childhood a chronic inflammatorydisease of respiratory system, It is a respiratory tract of chronic immune allergicdisease which a variety of cells (airway inflammatory cells, structure cell) and cellcomponents involved in it. Chronic inflammation and airway hyperreactivity、airway remodeling are It’s basic pathological feature. The airway inflammation isthe main pathological changes, It determine the degree of airwayhyperresponsiveness and airway remodeling. The pathogenesis of asthma are stillnot entirely clear, A large amount of test research shows that: the body’s ownimmune disorders are closely associated with the occurrence of asthma,At presentstudy mainly associated with the body’s own immune abnormal response, Focusedon Th1/Th2cells differentiation imbalance caused by a series of abnormalimmune response, At present about Th1/Th2cells’ secretion of cytokines andTh1/Th2cells differentiation direction has gradually been clear,Stimulation ofthe molecular mechanism of Th1/Th2differentiation is not clear. In recent years,the incidence of childhood asthma, and particularly fatal cases of this disease,have increased on a yearly basis, and seriously affects the quality of life for manychildren. HMGB1is a widespread and highly conserved nucleoprotein which ismainly released upon stimulation by inflammatory factors by pituitary cells,monocytes, macrophages, and can also be released by dead cells. HMGB1can stimulate the immune system to produce inflammatory responses to certain typesof stress. HMGB1is not only involved in signal transduction, but also participatesin inflammatory responses caused by a variety of cytokines, and chemotaxis ofproinflammatory cells. Recent studies on signal transduction mechanisms haveconfirmed that HMGB1can interact with its receptor (TLR4) by activatingNF-κB, and then inducing release of downstream inflammatory mediators.The molecule1，25-(OH)2D3is the active form of vitamin D3.1,25-(OH)2D3combine with intracellular vitamin D receptors play a biological effection,1,25(OH)2d3is a kind of immune modulators, its function mainly by changing theactivity of dendritic cells and mononuclear cell.It has extensive influence onlymphocyte development, the process of cell cycle, cell differentiation.About1,25-(OH)2d3protective effects on asthma, It has been confirmed inepidemiological investigation. But the specific mechanism is not clear.weestablished asthma mouse’s airway remodling model, through1,25-(OH)2D3intervene, get the change of HMGB1/TLR4protein and it’s mRNA、the mouseairway remodling condition, To explore the pathogenesis of asthma mechanism ofmolecular biology, may provide a new target for treatment of asthma.ObjectiveAfter the airway remodeling of asthma mice model is set up,Use1,25-(OH)2D3by intraperitoneal injection asthma mice to intervene,Then determined theairway thickness of the three group mouses and the changes of expression ofHMGB1and TLR4in the lungs of asthmatic mouse, To further understand themolecular biology mechanism of the onset of asthma, offers a new thought andmethod for the treatment of asthma.Materials and Methods1Animal groups and asthma model preparationAccording to the principle of random grouping30SPF mouse, By intraperitoneal injection and atomization stimulating made asthma model in mice,And given intervention treatment at0.5hour before each atomization excitation.2Preparation of lung tissue specimensAll groups of mouse were anesthetized by inhale ether within24hour ofthe last treatment of atomization stimulation. Following inhalation of ether, madeLung tissue samples, Used in the immunohistochemical staining and HE staining.3HE stainingAfter the paraffin section, and then HE dyeing operation， In opticalmicroscopy at high magnification vision (10x40) under the application ofcomputer pathological image analysis system to observe the type bronchial airwaywall structure shape change, Determination of different groups bronchial wallthickness, at the same level in mice and observe each group mice inflammationcells infiltration around the bronchi.4immunohistochemical stainingUsing serial section，Choose one slice from Every3slices，Each mouserandomly selected from three section, Made Immunohistochemical stainingoperation in tissue section operation. In optical microscopy at high magnificationvision (10x40) under the application of computer determination of pathologicalimage analysis system to observe the expression of HMGB1/TLR4proteinpositive cells. Randomly selected more than5at high magnification vision fromeach section, take the average values of IOD as protein semi-quantitative result ofthe slice.5RT-PCR analysis of HMGB1and TLR4mRNA expression,Use-80℃keep fresh lung tissue of mice for rt-pcr operation, Geneexpression of HMGB1/TLR4mRNA amount is the ratio of HMGB1/TLR4DNA grey banding and GAPDH DNA grey banding. 6Statistic alanalysisStatistical analyses were conducted using SPSS17.0statistical software.Data are expressed as the mean value±standard deviation (x±s). Comparisonsof were done using single factor analysis of variance. Correlation analysis of twovariables was done using used Pearson’s correlation analysis. As P value <0.05indicates the difference has statistical significance.Results1The pathomorphology of each group of miceMouse lung tissue pathology Observations by optical microscopy (10x40))Bronchial walls of control mice are smooth and show alignment of epithelial cells.Airway wall thickness is moderate, with little evidence of inflammatory cellinfiltration.No metaplastic goblet cells are observed.Bronchial walls in OVA-induced asthma mice show thickening and damage. Airway lumen shows stenosis.Epithelial cells are disordered and detached; numbers of metaplastic goblet cellsare increased. Airway smooth muscle is thickened, and inflammatory cells haveinfiltrated around the bronchi.1,25-(OH)2D3intervention group show lessairway wall thickening, less lumen stenosis, less inflammatory cell infiltration,and fewer metaplastic goblet cells. Comparison of these three bronchial wallthickness among the groups’difference, It has statistically significance.2Immunohistochemical results of mice in each groupHMGB1protein is primarily located in the nucleus and cytoplasm ofinflammatory and epithelial cells. TLR4is primarily located in the cell membraneand cytoplasm of inflammatory cells and epithelial cells. Mice in the controlgroup showed only sight expression of HMGB1and TLR4protein, while mice inthe OVA-induced asthma groups showed strong expression of HMGB1andTLR4, and the difference was statistically significant (P <0.05). Expression ofHMGB1and TLR4protein in the1，25-(OH)2D3intervention group wassignificantly lower than expression in the asthma group (P <0.05). Comparison among the three groups’difference,It has statistically significant.3HMGB1mRNA and TLR4mRNA expression:RT-PCR detection results showed that expression of both HMGB1mRNAand TLR4mRNA in the lungs of asthma mice was significantly greater thanexpression in control mice (P <0.05). Expression of HMGB1mRNA and TLR4mRNA in1,25-(OH)2D3intervention group was significantly lower thanexpression in the asthma group (P <0.05). Comparison among the threegroups’difference, It has statistically significant.Conclusions1．The1,25-(OH)2D3intervention therapy of asthma mice can obviouslyrelieve asthma airway inflammation, and delay the onset of airway remodeling.2．1,25-(OH)2D3may be by blocking HMGB1-TLR4downstreamsignaling pathways to suppress the release of inflammatory factors, so as torelieve asthma airway inflammation and airway remodeling in mice.