| Objectives:Bronchial asthma (asthma for short) is a chronic airway inflammatory disease characterized with eosinophils infiltration [1]. The incidence rate of people with asthma has reached as high as 18% and there are about 200 million people with asthma worldwide [2], and the incidence rate of asthma in china has reached about 0.5-1.5% with 20 million people. It is now widely recognized that the allergens in normal environment can cause the inappropriate immune response in genetically susceptible people with asthma, which resulted in the occurrence and development of airway inflammation [3]. The role of house dust mites, endotoxins, viral infections and other factors in asthmatic airway inflammation has attracted more and more attention of scholars. The environments allergens can be combined with the main pattern recognition receptors of the body, and induce different patterns of gene expression through activating different intracellular signal transduction pathway. The release of various pro-inflammatory cytokines could partly induce dendritic cells (DC) activation in vivo and further activated the endogenous immune response and regulated T lymphocyte differentiation. Toll-like receptors (TLRs) are important transmembrane proteins. We have found that the Toll-like receptor 4 (TLR4) is the main pattern recognition receptors with house dust mite (HDM). HDM could activate TLR4/NF-κB signal transduction pathway and induced DC activation, which could induce T cell differentiation. Besides the interleukin (IL)-25, IL-33, thymic stromal lymphopoietin (TSLP), granulocyte-macrophage colony-stimulating factor release (GM-CSF) and other pro-inflammatory cytokines are promoted at the same time and prompted the differentiation of T lymphocytes to Th2 lymphocytes, which play an important role in asthmatic airway inflammation.Quercetin, the representative of flavonoids, is a common plant estrogen and widely presents in a variety of everyday fruits and vegetables such as onions, strawberries, apples, blueberries and so on. The average human daily intake of quercetin has been estimated to be approximately ～25 mg. At present, a large number of studies have shown that quercetin has a variety of biological anti-inflammatory activity in vivo such as anti-inflammatory, anti-oxidant, anti-tumor, anti-platelet aggregation, hypoglycemic, regulating blood lipids, dilation of blood vessels, etc. Quercetin has been considered one of the most promising diet drugs for the prevention and treatment of inflammation-related chronic diseases; however, the mechanism of anti-inflammatory activity of quercetin in reducing airway inflammation need to be further clarified. Therefore, our research focuses on the regulation of quercetin in asthmatic airway inflammation through TLR4/NF-κB-mediated signaling pathway.In this study, we investigated the effects of HDM-induced asthmatic airway inflammation in a mouse model of allergic asthma to define the molecular mechanism of TLR4/NF-κB signaling transduction pathway. The activation of TLR4/NF-κB signaling induced the expression of IL-25, IL-33 and other inflammatory factors and further activated DC as well as Th2 lymphocyte-associated factors expression, which thereby further clarify the important interaction of the innate immunity and acquired immune response in asthmatic inflammation. Meanwhile, quereetin was observed in mouse model of asthma. We also obtained the peripheral venous blood obtained from patients with exacerbating asthma and detected the changes in the expression levels of TLR4 and NF-KB(p65)mRNA as well as a variety of pro-inflammatory cytokines such as IL-6 and TNF-α. Therefore, this study further clarified the asthmatic inflammatory response mechanism mediated by TLR4/NF-κB signaling pathway and discussesed quercetin in regulating the T lymphocyte differentiation and inflammatory cytokines release. The quercetin may provide a new and effective way in the pathogenesis and treatment of bronchial asthma.Methods:Section one:30 BALB/c mice were randomly divided into control group A with PBS instead of HDM, asthma group B using HDM to sensitize and challenge and group C using quercetin and HDM. The histopathology change of pulmonary tissues was observed by light microscope. The total cell numbers were counted with a hemacytometer. Measuring the concentration of cytokineIL-25、IL-4, IL-5, IL-13, IFN-y and GM-CSF in supernatant of BALF by ELISA. The expression of TLR4 and NF-κB were analyzed by western blotting and quantitative real-time RT-PCR separately. The expression of CD80 and CD86 protein in DC were analyzed by Flow Cyto Meter.Section two:10ml of peripheral venous blood were obtained from 20 patients with exacerbating asthma, each to add EDTA 1.2ml for anticoagulation. PBMCs were isolated by density gradient centrifugation and incubated with phytohemagglutinin (PHA) in vitro for 24h in the absence (blank group) or presence (quercetin group) of quercetin. Supernatant and sediment were collected, respectively. Expression of IL-6 and TNF-a in supernatant were determined by ELISA. The expression of TLR4 and NF-κB (p65)mRNA in sediment were measured by RT-PCR.Results:1. The Mice Asthma ModelStudies in the HDM-induced model had demonstrated that a subset of overreaction as a major feature of asthma successful model. When compared with mice in saline group, the mice of HDM group showed dysphoria, more activity, upper limb raise, urinary and fecal incontinence and so on.2. Patho-manifestation by HE in lung tissuesWhen compared with mice in group A, the lung tissue in group B showed wall thickening, inflammatory cells infiltration, subepithelial flbrosis, mucous metaplasia, and myocyte hyperplasia and hypertrophy. However, we observed no obvious inflammation in group C.3. Analysis of airway responsivenessAirway responsiveness demonstrated that the expiratory resistance of HDM-induced mice was elevated, which indicates that the HDM-induced mice developed marked airway hyperresponsiveness.4. Immunohistochemical method was used to detect the expression intensity of TLR4 protein in the lung tissue.Compared with group A, the lung tissue of mice in group B showed significantly increased expression intensity of TLR4 protein (P<0.05). Compared with group B, the lung tissue of mice in group C showed significantly reduced expression of TLR4 protein (P<0.05).5. Detection of the concentration of IL-25、IL-4, IL-5, IL-13, IFN-y and GM-CSF in BALF with ELISA.The mice in group B developed eosinophilic inflammation with significant increased production IL-25、IL-4, IL-5, IL-13 and GM-CSF while decreased expression of IFN-y in BALF compared with group A (P<0.05). Compared with group B, we also detected decreased expression of cytokines IL-25> IL-4, IL-5, IL-13 and GM-CSF (P<0.05) while the IFN-y expression showed no significant differences (P>0.05) in BALF supernatants in group C. 6. Expression of TLR4mRNAThe lung tissue of mice in group B developed significantly increased expression of TLR4 mRNA (P=7.251×10-6) when compared with group A. Compared with group B, the lung tissue of mice in group C showed significantly reduced expression of TLR4mRNA(P=8.140×10-7).7. Expression of NF-κB (p65) mRNAWhen compared with group A, the lung tissue of mice in group B developed significantly increased expression of NF-κB (p65) mRNA (P=8.748×10-). While compared with group B, the lung tissue of mice in group C showed significantly reduced expression of NF-κB (p65) mRNA(P=2.194×10-6).8. Western-blotting method was used to detect the expression of TLR4 protein in the lung tissue.The lung tissue of mice in group Bshowed significantly increased expression intensity of TLR4 protein (P= 6.251×10-7) compared with group A. Compared with group B, the lung tissue of mice in group C showed significantly reduced expression of TLR4 protein (P= 4.947×10-7).9. The expression of NF-κB (p65) protein in the lung tissue was detected with Western-blotting.The lung tissue of mice in group B showed significantly increased expression intensity of NF-κB (p65) protein (P=8.013×10-7) compared with group A. Compared with group B, the lung tissue of mice in group C showed significantly reduced expression of NF-κB (p65) protein (P=3.846×10-7).10. Expression of CD80 protein in DCMice in group B showed the increased expression of CD80 protein (P=7.935×10-7) in DC compared with group A. When compared with group B, the significantly decreased expression of CD80 protein was detected in group C (P= 5.902×10-7).11. Expression of CD86 protein in DCMice in group B showed the increased expression of CD86 protein (P=2.964×10-6) in DC compared with group A. When compared with group B, the significantly decreased expression of CD86 protein was detected in group C (P= 7.531×10-8).12. Detection of IL-6 expression in supernatant of PBMCs with ELISAThe supernatant level of IL-6 in quercetin group was significantly reduced than that in blank group (P<0.05).13. Detection of TNF-a expression in supernatant of PBMCs with ELISAThe supernatant level of TNF-a in quercetin group was significantly reduced than that in blank group (P<0.05)14. Expresion of TLR4 mRNA in sediment was detected with RT-PCRThe expression of TLR4 mRNA in sediment of quercetin group was significantly decreased than that of blank group (P<0.05)15. Expresion of NF-κB (p65) mRNA in sediment was detected with RT-PCRThe expression of NF-κB (p65) in sediment of quercetin group was significantly decreased than that of blank group (P<0.05)Conclusions:1. The activation of TLR4/NF-κB signaling pathway plays a vital role in asthmatic inflammatory responses.2. DC, which is activated by TLR4 signaling pathway, is a key factor in the interaction of innat and acquired immunity in asthma pathology.3. Quercetin may reduce the asthmatic airway inflammation by down-regulating the expression of TLR4/NF-κB signaling pathway. |
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