The Effects Of SPHK1 In Asthmatic Airway Epithelium Barrier Injury And The Influence Of Gubenfangxiao Decoction

ObjectiveThis study is aimed to confirm the credibility of OVA-RSV co-stimulte asthmatic murine model.Observe the dynamic changes of pathology,inflammation cytokines release,airway barrier injury at different stages of asthma.Identification the components of Gubenfangxiao decoction(GBFXD)by HPLC-MS.Explore the mechanism of asthmatic airway barrier function damage at the cell leve,and explain the effects of GBFXD and ASIV on allivate airway inflammation and repaire airway barrier injury.MethodsIn vivo study,to investigate the dynamic changes of airway barrier injury in asthmatic mose and explore the mechanisms involved,mouse were stimulate by OVA and RSV,after adaptation stage,mice were divided into six groups:(1)control-1 group:mice were rearing under normal conditions,(2)persistent-model group:mice were rearing to establish chronic asthma model;(3)GBFXD-PM group:persistent asthmatic mice treated with GBFXD;(4)control-2:mice were rearing under normal conditions for remission asthma;(5)remittent-model:mice were rearing to establish remission asthma;(6)GBFXD-PM group:remittent asthmatic mice treated with GBFXD.At the 86th day,mice were sacrificed and the blood,bronchoalveolar lavage fluid(BALF),and lung tissue were collected.Pathological changes of lung tissue.ELISA kits were used to test the level of TNF-?,IL-4,IL-13 in mice lung and serum.real-time PCR and Western Blot were used for exam the genetic transcription and protein changes of Sphk1,S1P,E-cadherinand Claudin-18 in each groups mice lung tissue.Identification the alcohol soluble components of GBFXD by HPLC-MS.Analysis of the possible material of GBFXD for its anti-asthma functionThen,in vtrio study,to further verify whether TNF-? could activated Sphk1 to induce airway barrier injury,16HBE cells were treated with TNF-?,si-sphk1,and ASIV.After stimulated with the TNF-? for 12,24,48hours,the epithelium barrier function were examed by TER,and then cells were collected to exam Sphkl acticity,Sphk1,S1P,E-cadherin and Claudin-18 mRNA and protein expression.Immunofluroscence assay was performed to confirm the expressions and locations of Sphk1,E-cadherin,Claudin-16,Claudin-18,ZO-1.Finally,in order to find out the association between sphkl with airway barrier injury,and to explore the effects of ASIV on repaire airway barrier function damage,16HBE cells were intered with si-Sphk1 and ASIV.Changes in E-cadherin,Claudin-18 and EGFR/ERK pathway were observed by real-time PCR and Western Blot.ResultsIn an in vivo study,OVA-RSV induced long-term Inflammatory infiltration in asthmatic mice lung tissue,the inflammatory cytokine TNF-? keep in high level even in remittent-model group,the airway barrier damage was existed in every stage of asthma.GBFXD was shown to alleviate the pathology changes and airway barrier impairment.The alcohol soluble components of GBFXD contains:Calyconsin-7-O-beta-D-glucoside,Astragaloside ?,Isoliquiritigenin,Glycyrrhizic Acid and Liquiritin.In an in vitro study,the data indicated that TNF-? damage each kind of cell-cell junction proteins,and directly activate Sphk1 and ORMDL3 upregulation.Interfere Sphk1 or use ASIV downregulate ORMDL3expression,restraining TNF-? induced epithelium barrier damage and allivate EGFR/ERK pathway activation.Conclusions1 GBFXD can obviously alleviate the pathological changes and airway barrier injury of lung tissue of OVA-RAV treated asthma model mice.2 The alcohol soluble components of GBFXD are mainly from Astragalus membranaceus and liquorice.3 TNF-? directly induce Sphk1 and ORMDL3 expression.4 TNF-? lead to extensive and perisitent epithelium barrier injury.5 Sphk1 decrease ORMDL3 expression and involved the epithelium barrier damage6 Sphk1 involved in TNF-? induced airway barrier injury and ORMDL3 increase by by activate EGFR/ERK pathway.7 ASIV can deduce Sphk1and ORMDL3 expression,alivate EGFR/ERK phosphorylation,and repair TNF-? induced epithelium barrier damage.