The Expression Of IL-10 And VCAM-1 Of Asthma Mice Vaccinated With BCG At Forepart Life

BackgroundIn the past decades, the prevalence of asthma over the world has arisen steadily especially in high-income countries. Though the underlying mechanism for bronchial asthma has been deeply explored, and there are more therapeutic tools than ever, the rate of morbidity and mortality still remains as ever, it even arises in many countries, so asthma has become a severe threat to people's health.Recent researches have indicated that it is the functional unbalance of Th1 and Th2 and the activation of Th2 that have important effect in the mechanism for bronchial asthma. In the body, the redundant Th2 cells, which are differentiated from Th0 cells, will secrete lots of cytokines, such as IL-4, IL-5, IL-13, and so on. Those cytokines will promote synthesis of allergen specificity IgE, mediating EOS differentiation in bone marrow, helping EOS mature and chemotaxis, accelerating mucous secretion, inducing airway hyperreactivity, which will result in asthma finally. Bacille Calmette-Guerrin(BCG) is an attenuation live vaccine of bacillus tuberculosis. It is a strong inducer of Th1 type immune response,and retrieves the disequilibrium of Th1/Th2. Up to now, the studies of animal models have revealed that BCGvaccination could suppress EOS infiltration in airway, decrease airway hyperreactivity and suppress Th2 type cytokines in animal models allergized by OVA.Interleukin-10 is an inhibitive factor of inflammation. It is secreted by Th2 cell, DC, Macrophage, Treg, and so on. Being secreted from DC, IL-10 would induce Tr1, one kind of Treg, to secrete more IL-10. The large amount of IL-10 would impress Th2 cell all the more, then impress asthma and hypersensitivity diseases. The mechanism of IL-10 of asthma and hypersensitivity disease is still unclear. There are great diversities among all kinds of data. Some reported that IL-10 and BALF in peripheral blood had been elevating, while others reported the reverse. IL-10 can inhibit the secretion of IL-1, IL-4, IL-12, TNFα, NF-κB, and other inflammatory factors. As a result, it can inhibit EOS inflammation of lung tissue and airway hyperreactivity of asthma indirectly. The secretion of IL-10 means the protectant accommodation of the body.It is an important feature of allergy inflammation that there is EOS infiltration in the lung of asthma. VCAM-1 plays an important part in asthma, as an adhesion moleculer specially mediating EOS adhesion and recruitment to lung tissue. Besides EOS infiltration, VCAM-1 may participate in body immunoloregulation as costimulatory molecules. VCAM-1 is one kind of glycoprotein on cell membrane, which mediates adhesion between cell and cell or cell and ground substance. VCAM-1 is expressed in endothelial cell, epithelium cell, Macrophage and dendritic cell. The gene expression of VCAM-1 is controlled by transcription factor NF-κB. It is induced by lots of inflammation factors such as IL-1, IL-4, IL-12, TNFα, IFN-γ, and so on. Because IL-10 impresses such inflammation factors widely and indirectly, it may inhibit VCAM-1 expression indirectly.This study is attempted to explore the underlying mechanism of BCG and asthma, and to provide evidence for empirical study of asthma therapy by observing the expression of IL-10 and VCAM-1.ObjectiveIn this study we vaccinated neonatal mice with minidose BCG many a times, andthen observed the long-term effect of asthma. The purpose of this study is to explore the underlying mechanism of BCG by observing the expression of IL-10 and VCAM-1 in BALF and lung tissue and the relationship of IL-10, VCAM-1 and BCG.MethodsC57BL/6 mice were divided into four groups, NS/NS group (control group), NS/OVA group (asthma model group), BCG10⁵/OVA group, BCG10^5-3/OVA group. Neonatal mice were vaccinated subcataneously with BCG on 0, 7 and 14 days. In the 5^th and 7^th week, mice were sensitized by ovalbumin (OVA) twice by peritoneal injection. In the 25^th week, mice were challenged three times by OVA through atomization. After the last challenge of 48h, BALF and lung tissue were collected and preserved at low temperature. Lung tissue was HE stained and observed if it were asthma model. The expression of VCAM-1 mRNA and IL-10 mRNA in the lung were measured by reverse trancription-polymerase chain reaction (RT-PCR). IL-10 of BALF was measured by ELISA. The expression of VCAM-1 was observed by immunohistochemistry.Results1. The changes of pulmonary pathology and inflammationPulmonary pathology was HE stained. Compared with the control group, there was more eosinophils infiltration around bronchi and pulmonary vessels after 48 h challenged in asthma model group(n=6,P<0.05). Compared with asthma model group, there was less eosinophils infiltration around bronchi and pulmonary vessels in drug groups (n=6,P<0.05) .2. The expression of IL-10 mRNA and VCAM-1 mRNA in the lung by RT-PCRThe expression of VCAM-1 mRNA and IL-10 mRNA in the lung were measured by reverse trancription-polymerase chain reaction ( RT-PCR). Compared with the control group, there was statistical significance of IL-10 mRNA in asthma model group, BCG10⁵/OVA group and BCG10^5-3/OVA group (n=6,P<0.05 ) . However, there was no statistical significance of VCAM-1 mRNA (p>0.05) among all groups.3. The expression of IL-10 in BALF by ELISAIL-10 was measured by ELISA. Compared with asthma model group and BCG10^5-3/OVA group, there were obviously heightened in BCG10⁵/OVA group (n=6,p<0.05) ; Compared with control group, there were heightened in asthma model group and BCG10^5-3/OVA group, though there was no statistical significance (p>0.05) .4.The expression of VCAM-1 in the lung by immunohistochemistryThe expression of VCAM-1 was observed by immunohistochemistry. VCAM-1 was in cytoplasm of many cells, such as airway epithelial cells, bronchial smooth muscle cells, blood vessel endothelium cells and lymphocytes around bronchi and pulmonary vessels. There were different degree from dark brown to fawm and from strong to weak masculine expression of VCAM-1 in airway epithelial cell in asthma model group, BCG10⁵/OVA group and BCG10^5-3/OVA group; There were fawn and weak masculine expression of VCAM-1 in airway epithelial cell in the control group. With rank sum test, there were statistical significance of all sample as a whole (n=6,p<0.05) . Compared with two different samples one after another, there were statistical significance between BCG10⁵/OVA group and the other 3 groups (n=6,p<0.05) .Conclusion1. BCG vaccination of mice at forepart life has long-term effect on the prevention of asthma. The effect is related to different BCG dosage.(25 weeks old mice)2. IL-10 has a protective and mediating effect on asthma, which might be related to the immune mediation of APC, the activation of Treg, the inhibition of Th2 cells, etc.3. VCAM-1 may have complicated effect on the occurrence of asthma in that it takes part in the pathological course of EOS infiltration into mice lung' tissue on one hand and it activates CD4⁺T cell and plays an immune and supervising role as a stimulating signal on the other.