HMGB1/TLR4/NF-kappa B Signaling Pathway In The Role Of Asthma Model In Mice And The Regulatory Mechanism Of Vitamin D

Introduction Asthma is a common and complex chronic respiratory disease, caused by the interaction between genes and environmental factors. pathogenesis, treatment, and many other link still has not yet fully understood, thus exploring the pathogenesis of asthma, looking for a new control target is still an important part of the study of asthma. Lung tissue high mobility group protein B1（HMGB1） is a kind of highly conservative widespread nucleoprotein, can be used as an immune regulating factor and inflammatory factor involved in the inflammatory response; Toll-like receptor 4（TLR4） is a kind of important pattern recognition receptors, connect innate and acquired immune which play an important role in the initiation and regulation of airway inflammation. HMGB1 can combine with TLR4 and interaction, by activating the nuclear transcription factors（NF-KB）, cause downstream release of inflammatory mediators. 1,25-（OH）₂D₃ is the active form of vitamin D3, in addition to regulating calcium phosphorus metabolism, also have immunomodulatory effect, play a broadspectrum anti-inflammatory effects through influencing the functions of monocytes, dendritic cells（DC）, lymphocytes and other immune cells, and adjust the airway structure by influencing a variety of cell growth and differentiation. at present there are some reports about vitamin D and asthma research at home and abroad, but the specific mechanism is still not clear. there are less asthma animals study about the expression of HMGB1, TLR4 in lung tissue and the intervention of vitamin D. and the study about the expression of HMGB1, TLR4 in asthma animal peripheral blood and bronchoalveolar lavage fluid（BALF） are much less, the study about HMGB1/ TLR4 / NF-KB signaling pathway and the regulation of vitamin D was not reported at home and abroad. This topic proposed by establishing the asthma model in mice and observe the lung tissue pathology change, the cell count and classification change in BALF, the content changes of HMGB1, TLR4, IL-4 and INF-γ in peripheral blood and BALF; and the expression changes of HMGB1, TLR4 and NF-KB m RNA and protein in lung tissue, to further explore the pathogenesis of asthma. Vitamin D is a kind of biological agents, and cheap, it’s side effect is small, it has a good drug compliance and similar role with steroid hormones; This experiment used vitamin D for interventional trials, observe the influence of HMGB1 / TLR4 / NF-KB signaling pathway and the effect of airway inflammation and airway remodeling, interpret its role in the prevention and control of asthma from a new angle, to explore new uses of older drugs, and provides the basic experiment basis for the clinical research and application.PartⅠ Effect of different 1,25-（OH）2 D3 doses on HMGB1 and TLR4 expression in lung tissue of asthmatic miceObjective: To establish a mice model of asthmatic airway remodeling and observe the expression of HMGB1 and TLR4 in lung tissue of asthmatic mice; investigate the effects of different doses of 1,25-（OH）2 D3 on airway remodeling and expression of HMGB1 and TLR4 in asthmatic mice.Methods: A total of fifty female mice（BALB/c strain） were randomly assigned to one of 5 groups, each containing 10 mice. These groups consisted of a control group, asthma group, 1,25-（OH）2 D3 low dose group, 1,25-（OH）₂D₃ medium dose group, and a 1,25-（OH）₂D₃ high dose group. An asthmatic mice model was induced using ovalbumin, The control group use physiological saline instead. And then 1,25-（OH）2 D3 low、medium and high dose group was given different doses of 1,25-（OH）₂D₃ to intervene, The asthma group use physiological saline instead. The pathology changes of lung tissue in mice were observed under optical microscopy at high magnification vision（10 x 40） by staining with hematoxylin-eosin（HE）. Application of the Image Pro Plus 6.0 computer pathological image analysis system software to determine the same level of bronchial airway wall thickness changes in cross-section. Using the immunohistochemical method detected the HMGB1 and TLR4 expression from the level of protein, application of computer pathological image analysis system to determine the expression of the protein positive cells. Changes in m RNA levels expression of HMGB1 and TLR4 were monitored by RT- PCR analysis, According to the grey value ratio formula to calculate the relative gene expression.Results: 1 Asthma mice appear a runny nose, sneezing, shortness of breath, coughing, nodding breathing such as asthma, severe case followed by cyanosis and limbs collapse; The above prove that model making is successful. 2 Compared to the control group, obvious changes including luminal stenosis, epithelial cells arrange disorder, fall off, more inflammatory cells infiltration around the bronchi were noted in asthma group, These changes were less pronounced in the low and medium doses of 1,25-（OH）₂D₃ group, but were more pronounced in the high dose of 1,25-（OH）₂D₃ group. 3 Compared with control group, the airway wall thickness was significantly increased in the asthma group; compared with asthma group, the airway wall thickness was significantly decreased in the low and medium doses of 1,25-（OH）₂D₃ group（P<0.05）, but significantly increased in high dose of 1,25-（OH）₂D₃ group（P < 0.05）. 4 Immunohistochemistry studies showed that HMGB1 and TLR4 proteins are mainly located in the nucleus and cytoplasm of inflammatory cells and epithelial cells. the expression of HMGB1 and TLR4 in the asthma groups was higher than in the control group（P<0.05）, low and medium dose group compared with asthma group was significantly decreased（P<0.05）, while higher than control group; high dose group was significantly increased in high dose group compared with asthma group（P<0.05）. 5 RT-PCR results showed that the expression of HMGB1 m RNA and TLR4 m RNA in the asthma group was higher than in the control group（P<0.05）; the expression of HMGB1 m RNA and TLR4 m RNA in low and medium dose groups was lower than in the asthma group（P<0.05）, but significantly increased compared to asthma group（P<0.05）.Conclusion: 1 In the OVA sensitization asthmatic airway remodeling model, the expression of HMGB1 and TLR4 significantly increased in lung tissue; 2 The appropriate amount of 1,25-（OH）₂D₃ can decreased the expression of HMGB1 and TLR4 in the lung tissue of asthmatic mice and reduce airway remodeling; overdose may increase HMGB1 and TLR4 expression, aggravate airway remodeling; 3 1,25-（OH）₂D₃ may inhibit downstream inflammatory cytokines release by blocking HMGB1/TLR4 signaling pathway, thereby relieve asthma airway remodeling in mice.Part ⅡThe role of HMGB1 / TLR4 / NF-ΚB signaling pathway and vitamin D in asthmatic miceObjective: From different time points of asthma mice, observe the pathology change, the total number and classification of inflammatory cells in BALF, the content change of HMGB1, TLR4, IL-4 and IFN–gamma in peripheral blood and BALF, the expression of HMGB1, TLR4 and NF-KB m RNA and protein in lung tissue of asthma mice; and investigate the intervention effect of vitamin D.Methods: A total of forty-eight female mice（BALB/c strain） were randomly assigned to one of 3 groups, each containing 16 mice. These groups consisted of a control group,asthma group and 1,25-（OH）₂D₃ group. After atomization inhalation process, specimens were taken according to the two observation time points（1 and 2 weeks） in each group, each time point 8 mice. Two asthmatic mice model of asthma trigger 1 and 2 weeks were established, the same period, the moderate doses of 1,25-（OH）₂D₃ was used to do intervention trial. According to different time points, the middle of the right lung in each group mice was done with HE and immunohistochemical staining, observing the pathological change and the expression of HMGB1, TLR4 and NF-KB in the lung tissue. and determinating of the airway wall thickness. At the same time, collecting BALF and peripheral blood, BALF was used cytological examination, determinating the content of HMGB1、TLR4, IL-4 and IFN-gamma in BALF and peripheral blood by using enzyme-linked immuno sorbent assay（ELISA）. Changes in m RNA levels expression of HMGB1、TLR4 and NF-KB were monitored by Real-time PCR analysis;Using Western blot method detected the HMGB1、TLR4 and NF-KB expression from the level of protein, Relative expression of genes is calculated by 2-△△Ct,According to the grey value ratio formula to calculate the relative protein expression.Results: 1 The airway wall thickness and airway damage of the asthma group 1, 2 weeks significantly increased than control group 1, 2 weeks（all P <0.05）; Intervention group 1, 2 weeks significantly decreased than asthma group 1, 2 weeks（all P < 0.05）. 2 Asthma group1, 2 weeks compared with control group 1, 2 weeks, total number of leukocytes, the percentage of eosinophils, neutrophils and lymphocytes in BALF increased significantly, monocyte/macrophage percentage significantly decreased（all P< 0.05）; Intervention group 1, 2 weeks compared with asthma groups1, 2 weeks, the total number of leukocytes, the percentage of eosinophil, neutrophils and lymphocytes in BALF decreased obviously, monocyte/macrophage percentage increased significantly（all P < 0.05）. 3 The content of HMGB1, TLR4 and IL–4 in BALF of Asthma group 1, 2 weeks were significantly higher than that of control group 1, 2 weeks, IFN-gamma levels were significantly lower than the control group 1, 2 weeks（all P < 0.05）; The content of TLR4 and IL–4 in BALF of intervention group 1, 2 weeks were significantly lower than asthma group 1, 2 weeks, IFN-gamma levels were significantly higher than in asthma group 1, 2 weeks（all P < 0.05）;At the first week of intervention group, The content of HMGB1 in the BALF had no statistical differences with the asthma group（P > 0.05）, but in the second week, intervention group was lower than that in asthma group（P < 0.05）. 4 The content of HMGB1 and IL–4 in peripheral blood of asthma group 1, 2 weeks were significantly higher than that of control group 1, 2 weeks, IFN-gamma levels were significantly lower than the control group 1, 2 weeks（all P < 0.05）; The content of HMGB1 and IL–4 in peripheral blood of intervention group 1, 2 weeks were significantly lower than asthma group 1, 2 weeks, IFN-gamma levels were significantly higher than in asthma group 1, 2 weeks（all P < 0.05）; At the first week of asthma group, The content of TLR4 in the peripheral blood had no statistical differences with the control group（P > 0.05）, but in the second week, asthma group was higher than that in asthma group（P <0.05）. At the first week of intervention group, The content of TLR4 in the peripheral blood had no statistical differences with the asthma group（P >0.05）, but in the second week, intervention group was lower than that in asthma group（P < 0.05）. 5 The expression of HMGB1, TLR4 and NF-KB m RNA in lung tissue of asthma group 1, 2 weeks were significantly higher than that of control group 1, 2 weeks; the expression of HMGB1, TLR4 and NF-KB m RNA in intervention group 1, 2 weeks significantly lower than that of asthma group 1, 2 weeks（all P < 0.05）. At the first week of intervention group, The expression of HMGB1 m RNA in lung tissue had no statistical differences with the asthma group（P >0.05）, but in the second week when intervention group were significantly decreased（P < 0.05）.6 The expression of HMGB1, TLR4 and NF-KB protein in lung tissue of asthma group 1, 2 weeks were significantly higher than that of control group 1, 2 weeks; the expression of HMGB1, TLR4 and NF- KB protein in intervention group 1, 2 weeks were significantly lower than that of asthma group 1, 2 weeks（all P < 0.05）. 7 There were positive correlation between airway wall thickness and HMGB1, TLR4 and NF- KB m RNA expression of lung tissue in mice（ r= 0.802, 0.895, 0.834; all P <0.05）; There were positive correlation between m RNA expression and protein expression of HMGB1, TLR4 and NF-KB（r= 0.871, 0.841, 0.823; all P < 0.05）. 8 There were positive correlation between IL-4, IFN- gamma concentration in peripheral blood and BALF（r = 0.600, 0.743; all P < 0.05）; In the peripheral blood and BALF, There were positively correlation between IL-4 concentration and HMGB1, TLR4 concentration（r1 = 0.696, 0.331, r2 =0.695, 0.648; all P < 0.05）. 9 There were positive correlation between HMGB1, TLR4 content in BALF and HMGB1 m RNA, TLR4 m RNA expression in lung tissue; There were positive correlation between HMGB1, TLR4 content in peripheral blood and HMGB1 m RNA, TLR4 m RNA expression in lung tissue（r1 = 0.756, 0.762, r2 =0.762, 0.327; all P < 0.05）; and there was a positive correlation between the HMGB1 and TLR4 content in BALF and peripheral blood（r1= 0.617, r2= 0.325; all P < 0.05）. 10 There were positive correlation between HMGB1 and TLR4 content in BALF and the total number of cells, the percentage of neutrophils, eosinophils and lymphocyte（r1=0.796,0.787,0.754,0.749; r2=0.730,0.698,0.558,0.480; all P<0.05）.Conclusion: 1 In the early stage of airway remodeling, the expression of HMGB1, TLR4 and NF– KB were obvious in lung tissue of mice; 2 HMGB1 and TLR4 were associated with airway inflammation and immune disorders; the appropriate amount of 1,25-（OH）₂D₃ can relieve airway inflammation, may be associated with regulating Th1 / Th2 cells balance. 3 HMGB1 and TLR4 protein were highly expressed in peripheral blood and BALF of asthmatic mice; the appropriate amount of 1,25-（OH）₂D₃ can down-regulate the expression of HMGB1 and TLR4 protein, and relieve airway inflammation.4 HMGB1, TLR4 and NF-KB m RNA and proteins were highly expressed in asthmatic mice lung tissue, which were associated with airway remodeling. The appropriate amount of 1,25-（OH）₂D₃ can down-regulate the expression of the HMGB1, TLR4 and NF-KB m RNA and proteins, and improve airway remodeling. 5 HMGB1/TLR4/NF-KB signal transduction pathway plays an important role in the pathogenesis of asthma, The appropriate amount of 1,25-（OH）₂D₃ may prevent the progress of asthma by regulating this signaling pathway, which is expected to become the new choice for treatment of asthma.