Experimental Study On The Effect Of Maslinic Acid On Airway Inflammation In Asthma Model Mice

Objective:Asthma is a heterogeneous disease caused by a combination of inflammatory cells and airway epithelial cells and their secreted cytokines,and its pathological features are mainly chronic airway inflammation.It has been found that long-term exposure of asthmatic patients to PM_2.5 suspended in the air can lead to increased release of inflammatory mediators,thereby exacerbating asthma airway inflammation.Therefore,suppressing airway inflammation in asthma may be an important measure to alleviate the disease.Maslinic acid is a natural plant-derived pentacyclic triterpene,mainly found in plants such as olive and hawthorn,with various biological activities,such as anti-apoptotic,antioxidant and anti-inflammatory.Recent studies have found that maslinic acid has anti-inflammatory effect in respiratory diseases,and its mechanism is related to the regulation of inflammatory cytokines and NF-κB related protein expression.However,it is not clear whether maslinic acid can prevent airway inflammation in asthma.Therefore,the aim of this study was to observe the effect of maslinic acid on the airway inflammatory response induced by the combination of ovalbumin and PM_2.5 in asthmatic mice,and to further investigate the effect of maslinic acid on Toll Like Receptor-4/Nuclear factor kappa-B signaling pathway-related proteins.Methods:Seventy 5-week-old male Balb/c mice were randomly divided into five groups（14 mice per group）according to body weight:Control;OVA+PM_2.5(4.0μg OVA/mouse,0.05 mg PM_2.5/mouse);OVA+PM_2.5+MA₁(4.0μg OVA/mouse,0.05 mg PM_2.5/mouse,1 mg MA/kg);OVA+PM_2.5+MA₅(4.0μg OVA/mouse,0.05 mg PM_2.5/mouse,5 mg MA/kg);OVA+PM_2.5+MA₂₅(4.0μg OVA/mouse,0.05 mg PM_2.5/mouse,25 mg MA/kg).The OVA+PM_2.5 and maslinic acid intervention groups were poisoned with OVA and PM_2.5 via intratracheal drip,and the Control group was given saline drip for 4 interventions every 2 weeks;the Control and OVA+PM_2.5 groups were given 0.5%sodium carboxymethylcellulose via gavage 30 min before the tracheal drip,and each maslinic acid group was given different doses of MA.After 24h of the last drip in each group of mice,heart blood was collected and serum was separated for the detection of OVA-specific immunoglobulin;bronchoalveolar lavage fluid was collected for inflammatory cell counting.Mouse lung tissues were embedded and stained with Hematoxylin-eosin staining and Periodic Acid-Schiff staining to assess the pathological changes in mouse lungs.Enzyme linked immunosorbent assay was used to detect interleukin（IL）-4,IL-5,IL-6,IL-13,IL-1β,Eotaxin,TNF-α,and INF-γin BALF.Western blot assay was used to detect the expression levels of TLR-4,NF-κB p65,IκB-αand P-IκB-αproteins.Results:1.Inflammatory cell counts in BALF:Compared with the Control group,the total number of leukocytes and the numbers of eosinophils,neutrophils,lymphocytes and macrophages in the OVA+PM_2.5 group increased significantly;the counts of each inflammatory cell in the BALF of mice in the maslinic acid intervention group decreased significantly,and the differences were all statistically significant.2.Lung histopathological changes:（1）HE staining:No inflammatory cell infiltration under the airway mucosa in Control group mice;In OVA+PM_2.5 group,inflammatory cell infiltration around the fine bronchus and under the airway mucosa in the lung tissue;these pathological changes were significantly reduced after the administration of maslinic acid pretreatment.（2）PAS staining:Compared with the Control group,the secretion of goblet cells and mucus increased significantly in the OVA+PM_2.5 group;maslinic acid pretreatment could significantly inhibit the above pathological changes.3.Cytokine levels in BALF:Compared with the Control group,the OVA+PM_2.5 group resulted in significantly higher levels of IL-4,IL-5,IL-6,IL-13,Eotaxin,TNF-αand IL-1βcytokines in BALF,and maslinic acid pretreatment significantly decreased the levels of these cytokines,and the differences were statistically significant;meanwhile,the OVA+PM_2.5 group resulted in lower levels of the Th1-type cytokine IFN-γ,while maslinic acid pretreatment elevated the levels of INF-γ.4.OVA specific antibodies in serum:Compared with the Control group,the serum concentrations of OVA-Ig E and OVA-Ig G1 in the OVA+PM_2.5group were significantly increased,and the difference was statistically significant.Compared with the OVA+PM_2.5 group,maslinic acids significantly reduced their serum levels.5.Western blot detection results:Compared with the Control group,the protein expression levels of TLR-4,NF-κB p65 and P-IκB-αin the OVA+PM_2.5 group were significantly increased,and the difference was statistically significant.Maslinic acid pretreatment significantly decreased the expression levels of TLR-4、NF-κB p65 and P-IκB-α,the differences were statistically significant.Conclusion:Maslinic acid prevents airway inflammation in asthmatic mice induced by the combination of OVA and PM_2.5,which may be mediated by down-regulation of TLR-4,NF-κB p65 and P-IκB-αprotein expression levels and reduction in the number of inflammatory cells and inflammatory cytokine levels in the lung.