Study On The Effects And Mechanisms Of Shuang-Huang-Lian On Experimental Allergic Asthma

Bronchial asthma(referred to as "asthma") is a widespread and reversible airway stenosis disease that occurs in airway hyperresponsiveness condition caused by allergens or other factors.The latest research data published on Lancet about "Chinese Adult Lung Health Study" indicated that the overall prevalence of asthma among Chinese adults is 4.2%.Allergic asthma is the most common asthma phenotype,accounting for as much as 50%of adult asthmatic patients.Allergic asthma is characterized by eosinophilic inflammation caused by type 2 T helper(Th2)immune responses and IgE-mediated degranulation of mast cells.Shuang-Huang-Lian(SHL),an herbal formula from Chinese Pharmacopoeia,consists of Lonicerae Japonicae Flos,Scutellariae Radix and Forsythiae Fructus.As a commercialized preparation,SHL has different formulations,including oral solutions,injections,granules and nebulizers.Although SHL has been reported to be effective in the treatment of bronchial asthma,its underlying mechanism remains to be elucidated.Thus,in this study,we systematically investigated the effects of SHL(powder)injection on allergic asthma and revealed the related mechanism.We used homemade shrimp protein(SP) with strong sensitization as the allergen and established a murine allergic asthma model by intraperitoneal injection for 4 weeks plus aerosol challenge for 1 week.Based on this model,SHL powder injection(150 mg/kg,300 mg/kg,600 mg/kg)was orally administered daily throughout the course to investigate its effect on allergic asthma.Our studies showed that SHL significantly inhibited SP-induced airway inspiratory and expiratory resistance,reduced thickness of airway smooth muscle and infiltration of inflammation cells.Besides,SHL could also alleviate eosinophilic airway inflammation,including reducing the number of eosinophils and decreasing eotaxin and eosinophil peroxidase levels in the bronchoalveolar lavage fluid(BALF).Further studies indicated that SHL suppressed SP-elevated total IgE and SP-specific IgE in serum and total IgE in BALF,and also lowered Th2 cytokine(e.g.,interleukine(IL)-4,IL-5,and IL-13)production in both BALF and the supernatant of cultured mediastinal lymph nodes.These results highly suggested that SHL has the effect on suppressing Th2 immunity.Considering the key role of early IL-4 secreted by basophils in the initiation phase of Th2 immunity,we isolated basophil-rich splenocytes using magnetic activated cell sorting.Our results showed that SP robustly stimulated the release of early IL-4 from basophils,while SHL could significantly reduce IL-4 secretion,thus inhibiting Th2 differentiation and Th2 immune response.Mouse mast cell protease-1(mMCP-1) is one of the biomarkers of the degranulation of mast cells.SHL significantly suppressed SP-elevated the mMCP-1 level in BALF,suggesting that it may exert a direct effect on mast cell stabilization.Our data showed that either in in vitro models(Compound 48/80 or IgE-FcεRI activated mast cells)or in vivo models(Compound 48/80-induced anaphylactic shock mice,IgE-FcεRI-mediated active and passive systemic allergic mice),SHL could significantly prevent mast cell degranulation.Mechanism study showed that SHL could dampen the level of cytosolic calcium in mast cells in a quick and reversible way.This effect was achieved by directly enhancing mitochondrial calcium uptake and it could be blocked by ruthenium red,a specific inhibitor of mitochondrial calcium uniporter(MCU).Accordingly,in MCU-deficient mast cells and mice,both the effects of SHL on stabilizing mast cells and reducing cytosolic calcium disappeared.These results indicated that by directly activating MCU,SHL increased the calcium uptake ability of mitochondrial,thereby decreasing cytosolic calcium level and exerting a remarkable effect on stabilizing mast cells.In conclusion,this study systematically reveals the anti-allergic effect of SHL for the first time.Long-term administration of SHL can reduce early IL-4 secretion from basophils,prevent Th0 cells from differentiating into Th2 type,reduce the release of Th2 cytokines,and inhibit IgE production.Meanwhile,single dose administration of SHL can enhance the mitochondrial calcium uptake by direct activation of MCU,and thus reduce the cytosolic calcium level to prevent mast cells from degranulation.Therefore,SHL can distinctly suppress both the early phase(Th2 differentiation and IgE production)and the late phase(mast cell degranulation)of allergic reactions,showing a comprehensive effect on type I hypersensitivity.In particular,its prominent inhibition effect on the degranulation of mast cells suggests that SHL may also have considerable application value in mast cell activation-associated diseases.