The Effect Of Herbs Tonifying Lung And Kidney On Steroid-Resistant Asthma And Its Action Of Mechanism

ObjectiveGlucocorticoid is an effective anti-inflammatory drug and the major drug for the treatment of asthma.However,some patients are not clinically controlled even after getting high dose of glucocorticoid,suggesting that these asthmatics may be resistant to the glucocorticoids.This type of asthma is called steroid-resistant asthma.Steroid-resistant asthma could be attributed to“stubborn asthma”or“deficient gasp”in Traditional Chinese Medicine.The pathologic feature of“stubborn asthma”or“deficient gasp”bases on lung deficiency and kidney deficiency,accompanied by wind,cold,heat,wet,phlegm,blood stasis.Basing on the pathologic feature of steroid-resistant asthma,we put forward the hypothesis that herbs tonifying lung and kidney could be used to treat this disease.There are two herb formulas has been proved to effectively treat asthma in clinic.One is Chuankezhi(CKZ)which has the tonifying kidney effect.Another is ASHMI which has the tonifying lung effect.CKZ is a national class II new drug consisting of yinyanghuo and bajitian.It has the effect of tonifying kidney yang and relieving cough and asthma.This research will explore the effect of CKZ on steroid-resistant cell model,and study the material basic and mechanism of its anti-inflammatory effect.ASHMI is a Traditional Chinese Medicine formula approved by the US FDA for clinical trials for the treatment of asthma.It is consisted of lingzhi,gancao and kushen.The major herb lingzhi has the effect of tonifying lung qi and relieving cough and asthma.It could be used to treat deficient cough and asthma.In further studies,we identified Ganoderic Acid Cl(GAC1)as a key active compound in lingzhi to exert anti-inflammatory effect in vitro.Present study will assess the effect of GAC1 on mice steroid-resistant asthma model to develop a natural product for this type of disease,and.to study its underlying mechanism.Methods1.Construct a cell model of steroid resistance in vitro.RAW 264.7 cells were stimulated with LPS/IFN y and treated by dexamethasone.ELISA was used to detect TNF-α content and western blot glucocorticoid receptor nuclear transfer.2.Construct a steroid-resistant mouse model in vivo.A mouse model of asthma was established by intraperitoneal injection of ragweed and intranasal challenge of Balb/c mice.Mice were given oral acute dexamethasone treatment at 24 and 48 hours prior to challenge.After 48 hours of the last ragweed challenge,methacholine was used to induce airway hyperresponsiveness.The airway hyperresponsiveness of the mice was detected.The cytokines TNF-α,IL-4,IL-5 and IFN-y in bronchoalveolar lavage fluid were measured by ELISA.HE staining of cells in bronchoalveolar lavage fluid were used to observe the classification of inflammatory cells.3.Observe the effect of CKZ and its component on the steroid-resistant cell model in vitro:LPS was used to induce the inflammatory response in macrophage cell line Raw 264.7.ELISA was used to detect the inhibition effect of CKZ and its composition of Epimedium and Morinda officinalis on inflammatory factor TNF-α.Western blot was used to investigate the effect of Epimedium on the protein expression of p-IkBa,IkBa,p-JNK,JNK,p-p38,p38,p-ERK,ERK proteins in NF-κB and MAPK signaling pathways.LPS/IFN-γ was used to induce cell model with steroid resistance in Raw 264.7 cells.The effect of epimedium combined with dexamethasone on TNF-α was observed by ELISA,and the effect of epimedium combined with dexamethasone on glucocorticoid receptor nuclear translocation was detected by western blot.The liquid chromatography method was used to identify several compounds with the highest content in Epimedium.The active compounds were screened by detecting the inhibition effect on production of TNF-α of Raw 264.7 induced by LPS.The effects of this monomer on LPS-induced IkB-α phosphorylation and NF-κB p65 nuclear translocation were detected by Western blot and immunofluorescence assay.The molecular docking method was used to predict the binding of compound to the protein on Toll-like receptor/NF-κB signaling pathway.CETSA technology was used to verify the binding of Icarrin and TAB2 protein.The effect of Icarrin on TAK1,TAB1,TAB2 mRNA expression was detected by Real-time PCR.4.Observe the effect of GAC1 on the ragweed-induced asthma mouse model in vivo:a mouse model of asthma was established by intraperitoneal injection of ragweed and intranasal challenge of Balb/c mice.After sensitization,mice were given oral GAC1 treatment for 4 weeks or acute dexamethasone treatment at 24 and 48 hours prior to challenge.After 48 hours of the last ragweed challenge,methacholine was used to induce airway hyperresponsiveness.The airway hyperresponsiveness of the mice was detected.The cytokines TNF-α,IL-4,IL-5 and IFN-y in bronchoalveolar lavage fluid were measured by ELISA.HE staining of cells in bronchoalveolar lavage fluid were used to observe the classification of inflammatory cells.The HE staining of lung sections was used to observe the infiltration of trachea and vascular inflammation.Phosphorylated-p65(p-p65),p-PI3K,p-Akt,p-STAT6 和 HDAC2 protein expression in the lung tissue were determined by western blot.Results1.The ragweed sensitized mouse asthma model was successfully established.The active compound GAC1 isolated from ASHMI could reduce airway and vascular inflammatory cell infiltration in asthmatic mice,reduce airway hyperresponsiveness,and reduce IL-4,IL-5 and TNF-α and increase IFN-γcytokines in bronchoalveolar lavage fluid.inhibited phosphorylation of NFκB,PI3K and Akt in lung,and restore HDAC2.2.CKZ and its component Epimedium could inhibit LPS induced-TNF-α and inhibit the phosphorylation of IκB-α and JNK.LPS/IFN-γ induced dexamethasone-resistant TNF-α in Raw 264.7 cells,while Icarrin combined with dexamethasone inhibited TNF-α production.Combination of Epimedium and dexamethasone promotes nuclear translocation of glucocorticoid receptor,whereas Epimedium alone did not promote nuclear translocation of glucocorticoid receptor.3.The highest content of 8 compounds in Epimedium was identified by liquid chromatography.Icarrin had the strongest inhibitory effect on TNF-α.Icarrin dose-dependently inhibited mRNA and protein levels of TNF-α and IκB-αphosphorylation and NF κB p65 protein nuclear translocation.Molecular docking revealed that Icarrin has 16 binding sites for the TAB2 protein on the Toll-like receptor/NF-κB signaling pathway.CETSA result showed that Icarrin binded to TAB2 without affecting the expression of the TAB2 gene.GonclusionGAC1 may be a promising therapeutic agent for asthma,which can be used to treat neutrophil-dominant and steroid-resistant asthma.Epimedium could reverse the hormone resistance caused by LPS/IFN-γ,and provide a basis for its clinical treatment with dexamethasone in the treatment of hormone-resistant diseases.Icarrin is an active compound in Epimedium that have anti-inflammatory effect.Its anti-inflammatory mechanism may be through binding TAB2 protein,affecting the formation of TAK1-TAB1-TAB2 complex,inhibiting the activation of NF-κB pathway,thereby inhibiting production of inflammation factors.1.A cell model of hormone resistance was successfully construct.Dexamethasone could not inhibit the TNF-α induced by LPS/IFN-γ in RAW 264.7 cells,and could not significantly promote the glucocorticoid receptor nuclear translocation.2.A ragweed-sensitized asthma mice model was successfully constructed.The airway responsiveness of mice increased.White blood cells,neutrophils and eosinophils increased,and IL-4,IL-5,IFN-y and TNF-α cytokines increased in BALF.Dexamethasone did not reduce airway hyperresponsiveness or neutrophil inflammation and TNF-α cytokines in BALF.3.Epimedium increases hormone sensitivity.CKZ and its constituent Epimedium can inhibit LPS-induced TN F-α and inhibit the phosphorylation of IκB-α and JNK.Combination of Epimedium and Dexamethasone inhibited TNF-α which is resistant in LPS/IFN-γ-stimulated Raw 264.7 cells.Combination of Epimedium and Dexamethasone promotes nuclear translocation of glucocorticoid receptor,whereas Epimedium alone did not promote nuclear translocation of the glucocorticoid receptor.4.Anti-inflammatory substance basis and mechanism of action of Epimedium.Eight kinds of compounds with the highest content in Epimedium were identified by liquid chromatography,and Icarrin had the strongest inhibitory effect on LPS-induced TNF-α.Icarrin dose-dependently inhibits mRNA and protein levels of TNF-α and inhibits IκB-α phosphorylation and NF κB p65 protein nuclear transfer.Molecular docking revealed that Icarrin has 16 binding sites for the TAB2 protein on the Toll-like receptor/NF-κB signaling pathway.CETSA results showed that Icarrin binds to TAB2 without affecting the expression of the TAB2 gene.5.The therapeutic effect of GAC1 on a hormone-resistant asthma model.The active compound GAC1 isolated from ASHMI reduced airway hyperresponsiveness and airway and vascular inflammatory cell infiltration,and IL-4,IL-5 and TNF-α cytokines in airway alveolar perfusate.It inhibited phosphorylation of NF κB,PI3K and Akt proteins in the lung and restored HDAC2 protein expression.ConclusionConstruction of a cell model and mouse model of steroid resistance facilitates the development of drugs for the treatment of steroid-resistant diseases.Epimedium can reverse the steroid resistance caused by LPS/IFN-γ,and provide a basis for its clinical treatment with dexamethasone in the treatment of sterlid-resistant diseases.Icarrin is an active compound that plays an anti-inflammatory role in Epimedium.Its anti-inflammatory mechanism may be through the binding of TAB2 protein,affecting the formation of TAK1-TAB1-TAB2 complex,affecting the activation of NF-κB pathway,thereby inhibiting the generation inflammation of factors.GAC1 may be a promising therapeutic agent for asthma,which can be used to treat neutrophil-dominant and steroid-resistant asthma.