To Observe The Efficacy Of TGF-β1Combine With Anti-CCL20Antibody In Asthma Mice And Illustrate The Mechanisms

PARTⅠInhibition of CC chemokine ligand20monoclonal antibody onairway inflammation and airway hyperresponsiveness ofasthmatic miceOBJECTIVE: To observe expression of chemokine ligand20(CCL20)in lung tissue of asthmatic mice and the effect of anti-chemokine ligand20monoclonal antibody （MCA-CCL20） on inflammation of lung tissue andairway hyperresponsiveness (AHR) in BALB/c mice.METHODS: A total of32BALB/c mice at the age of6~8weeks weredivided into four groups: asthma group, normal control group, CCL20monoclonal antibody treatment group and therapeutic control group. Micein asthma group were sensitized and challenged with ovalbumin (OVA) toestablish asthma model, while mice in normal control group weresensitized and challenged with PBS. The sensitive and challenge methodsof MCA-CCL20treatment group and therapeutic control group were the same as asthma group, but since the first time challenge, mice of the former2groups respectively received an intraperitoneal injection of MCA-CCL20or PBS before an hour of atomization inhalation for5d.Within24hoursafter the final OVA challenge, the airway hyperresponsiveness(AHR) wasmeasured by Spirometer, the degree of inflammation in lung tissue wasevaluated by HE, the expressions of CCL20mRNA in lung homogenateand protein in lung tissue section were detected by real-time PCR andimmunochemistry.RESULTS: Compared with the normal control group, the score ofinflammation in lung tissue, AHR, the expression of CCL20m RNA in lungtissue homogenate and Mean Density for CCL20of asthmatic model groupwere significantly higher. In CCL20antibody group, pulmonary cellinfiltration were alleviated when compared with the asthmatic model group（p＜0.05）. Lung function Penh values were significant lower in CCL20antibody group than asthmatic model group（p＜0.05）. The Mean Densityfor CCL20expression were significant lower in CCL20antibody groupthan model group（p＜0.05）,but had no effect on the mRNA expression.CONCLUSION: MCA-CCL20can attenuate OVA-induced miceasthma inflammation and airway hyperresponsiveness, its inhibition may beinvolved anti-CCL20monoclonal antibody can partially block CCL20activity. PARTⅡTo observe the efficacy of TGF-β1combine with anti-CCL20antibody in asthma mice and illustrate the mechanismsOBJECTIVE: To evaluate the combined effects of TGF-β1andanti-CCL20antibody (MCA-CCL20) on airway inflammation and AHR ofBALB/c mice, and preliminary reveal the possible mechanisms.METHODS: A total of40BALB/c mice at the age of42~56dayswere divided into asthma group, control group, MCA-CCL20group,TGF-β1group and MCA-CCL20+TGF-β1joint group. Mice in asthmagroup were sensitized and challenged with ovalbumin (OVA), while incontrol group were sensitized and challenged with PBS. The sensitive andchallenge methods of MCA-CCL20group, TGF-β1group andMCA-CCL20+TGF-β1group were the same as asthma group, but sincethe first time challenge, mice of the MCA-CCL20group received anintraperitoneal injection of MCA-CCL20before an hour of atomizationinhalation for5d; TGF-β1group received an intratracheal instillation ofTGF-β1prior to an hour of the first atomization inhalation; mice of jointgroup with combined intervention of TGF-β1and MCA-CCL20. Within24hours after the final OVA challenge, the AHR was measured, the numberof total cells and differential leukocyte count in was tested, inflammation oflung tissue was evaluated by HE, expressions of CCL20, Smad6andTGF-β1mRNA were detected by real-time PCR, protein of CCL20and Smad6in lung tissue were detected by immunochemistry and theconcentration of TGF-β1, TNF-α, IL-1β were measured by ELISA.RESULTS: MCA-CCL20enhanced the expression of CCL20in lungtissue sections, reduced the level of IL-1β and TNF-α of BALF (p <0.01);Expression of Smad6in lung tissue section was markedly enhanced and theexpression of Smad6mRNA was significantly increased in lunghomogenates after TGF-β1intervention. All changes above can be seenafter TGF-β1+MCA-CCL20intervention, but the level of TNF-αsignificantly decreased in BALF. The number of total cells and theproportion of eosinophils, lymphocytes in BALF, the inflammation of lungtissue and the AHR were significantly decreased in TGF-β1group as wellas MCA-CCL20group than in asthma group. Compare with aloneintervention, TGF-β1combine with MCA-CCL20can greatly improveairway inflammation and AHR in mice.CONCLUSION: TGF-β1and MCA-CCL20joint intervention canup-regulate the expression of Smad6, reduce IL-1β and TNF-α levels,neutralize or reduce the release of CCL20, and can significantly alleviateinflammation in airway and reduce AHR, improve lung function, providean trial evidence for biological agents to treat asthma.