| Objective:Bronchial asthma(Asthma for short)is a heterogeneous disease characterized by chronic airway inflammation and airway hyperresponsiveness.More than 300 million people around the world suffer from asthma,which has become a global health problem.Inhaled corticosteroids(inhaledcorticosteroids,ICS)combined with long-acting beta agonists(longactingβ-agonists,LABA)can relieve asthma symptoms and reduce the frequency of wheezing attacks.But for those asthma patients who are not controlled by ICS and LABA,treatment options are limited.The increased dose of ICS usually does not bring additional benefits,and the risk of side effects increases.Therefore,it is necessary to seek new drugs for treatment and intervention in order to overcome the disease.Micheliolide(MCL)is a guaiac sesquiterpene lactone compound that can be obtained semi-synthetically from parthenolide(PTL).In recent years,many studies at home and abroad have shown that MCL has potential anti-inflammatory biological activity and can be used to treat various diseases such as inflammatory bowel disease and rheumatoid arthritis.It also has good antioxidant and immunomodulatory effects in the treatment of breast cancer and colon cancer.However,there is no report on the intervention of MCL on inflammation and oxidative stress in asthma models.Therefore,through the establishment of asthma animal and airway epithelial cell model intervened by MCL,we tried to clarify the ameliorative effect of MCL on asthma inflammation and oxidative stress,and further explore the possible mechanism of MCL in asthma,in order to provide experimental reference for the future drug treatment of asthma.Methods:1.Effects of MCL on Th1/Th2 imbalance,oxidative stress and inflammatory body activation in asthmatic mice(1)Preparation and grouping of asthmatic mouse model.Thirty BALB/C mice were divided into 5 groups with 6 mice in each group.They were:normal control group(Control group),asthma group(OVA group,50μL DMSO intraperitoneally injected with OVA);asthma+MCL12.5mg/kg group(OVA+MCL12.5 group,mice were sensitized with OVA,intraperitoneal injection of MCL,MCL dissolved in 50μL DMSO was 12.5mg/kg per mouse);asthma+MCL25 mg/kg group(OVA+MCL25 group,mice were sensitized with OVA,intraperitoneal injection of MCL,MCL dissolved in50μl DMSO was 25mg/kg per mouse);asthma+MCL50 mg/kg group(OVA+MCL50group,mice were sensitized with OVA,intraperitoneal injection of MCL,MCL dissolved in 50μl DMSO was 50mg/kg per mouse).(2)Effect of MCL on airway hyperresponse in asthmatic mice.24 hours after the last OVA or saline challenge,the airway hyperresponsiveness of mice in each group was indirectly evaluated by a non-invasive single-compartment whole body plethysmography system.(3)Effect of MCL on airway inflammation in asthmatic mice.The inflammatory cells in the bronchoalveolar lavage fluid(BALF)of mice in each group were counted by Wright Giemsa staining and the percentage of eosinophils in the total inflammatory cells was calculated.The levels of serum OVA-s Ig E and cytokines IL-4,IL-5,IL-13and IFN-γwere detected by ELISA method,and the pathological changes of airway were observed by HE and PAS staining.The m RNA levels of cytokine IL-4,IL-5,IL-13,IFN-γand chemokine CCL11,CCL24 in lung tissue of mice in each group were detected by q RT-PCR,and the proportion of T lymphocytes in spleen of mice in each group was detected by flow cytometry.(4)Effect of MCL on airway oxidative stress in asthmatic mice.In this study,we used a kit to detect the effect of MCL on the levels of oxidative stress indexes MDA,SOD and GSH in lung tissue of asthmatic mice.(5)Effects of MCL on NLRP3 inflammasome activation in lung tissue of asthmatic mice.The levels of IL-1βand IL-18 in mice were detected by ELISA,and the expression of NLRP3 inflammasome activation-related protein in mouse lung tissue was detected by Western Blot.2.Study on the regulatory effect of MCL on inflammation,oxidative stress and activation of NLRP3 inflammatory bodies in human bronchial epithelial Beas-2B cells.(1)Effects of MCL on IL-4+TNF-αco-induced inflammation in human bronchial epithelial Beas-2B cells.Firstly,CCK8 kit was used to detect the viability levels of Beas-2B cells treated with different concentrations of MCL.Then the levels of cytokines IL-6,IL-8,CCL11 and CCL24 in Beas-2B cells co-stimulated by MCL and IL-4+TNF-αwere detected by ELISA method.(2)Effects of MCL on IL-4+TNF-αco-induced oxidative stress in human bronchial epithelial Beas-2B cells.In this study,DCFH-DA probe was used to detect the effect of MCL on reactive oxygen species(ROS)in Beas-2B cells co-stimulated by IL-4+TNF-α,and the content and activity of oxidative stress indexes MDA,GSH and SOD in Beas-2B cells were detected by kit.(3)Effect of MCL on the activation of NLRP3 inflammatory bodies in Beas-2B cells co-stimulated by IL-4+TNF-α.ELISA method was used to detect the effect of MCL on the levels of IL-1βand IL-18 in the supernatant of Beas-2B cells co-stimulated by IL-4+TNF-α.The expression of NLRP3 inflammatory body activation related proteins ASC,caspase-1,cleaved-caspase-1,IL-1βand NLRP3 in human bronchial epithelial Beas-2B cells co-stimulated by IL-4+TNF-αwere detected by Western Blot method.3.Mechanism of MCL on the activation of NLRP3 bodies in the asthmatic airway and the protective effect of oxidative stress.(1)Bioinformatics data analysis was used to screen genes closely related to the role of MCL in asthma.(2)Western Blot was used to detect the expression levels of MAPK/NF-κB signaling pathway-related proteins p-ERK,ERK,p-JNK,JNK,p-P38,P38,p-IκBα,IκBα,p-P65 and P65 in lung tissue of mice in each group.(3)Western Blot method was used to detect the expression of MAPK/NF-κB signal pathway related proteins p-ERK,ERK,p-JNK,JNK,p-P38,P38,p-IκBα,IκBα,p-P65 and P65 in human bronchial epithelial Beas-2B cells induced by IL-4+TNF-α.To determine whether MCL might affect the activation of MAPK/NF-κB signal pathway in asthma model in vitro.(4)The NF-κB activator betulinic acid(BA)was added to human bronchial epithelial Beas-2B cells co-stimulated with IL-4+TNF-αunder the action of MCL,and the changes of reactive oxygen species levels in the cells were detected by DCFH-DA probe.The expression of NLRP3 inflammasome activation-related protein ASC,caspase-1,cleaved-caspase-1,IL-1βand NLRP3 in Beas-2B cells was detected by Western Blot.Results:1.MCL may modulate airway hyperresponsiveness,inflammation,oxidative stress,and NLRP3 activation-related markers in an OVA-induced allergic asthma model.(1)The non-invasive single-chamber whole-body plethysmography system detected the airway hyperresponsiveness of the mice in each group.The results showed that the Penh value of asthmatic mice after administration of MCL was significantly lower than that of OVA group.(2)The results of Wright-Giemsa staining showed that the number of leukocytes,eosinophils,neutrophils and the percentage of eosinophils in the total inflammatory cells in the BALF of asthmatic mice treated with MCL were significantly lower than those in the OVA group;HE and PAS The staining results showed that the infiltration of para-airway inflammatory cells and the secretion of airway mucus in the lung tissue of asthmatic mice after MCL were significantly reduced compared with the OVA group.The results of q RT-PCR showed that the m RNA levels of cytokines IL-4,IL-5,IL-13and chemokine CCL11,CCL24 in lung tissue of asthmatic mice treated with MCL were significantly lower than those of OVA group,while the m RNA level of IFN-γwas significantly higher than that of OVA group.The results of ELISA detection showed that the levels of serum OVA-s Ig E and cytokines IL-4,IL-5 and IL-13 in BALF of asthmatic mice treated with MCL were significantly lower than those of OVA group,while the level of IFN-γwas significantly higher than that of OVA group.The results of flow cytometry showed that the proportion of CD4~+IL-4~+Th2 cells in the spleen of asthmatic mice treated with MCL was significantly lower than that of OVA group,and the proportion of CD4~+IFN-γ~+Th1 cells was significantly higher than that of OVA group.(3)The test results of the kit showed that the level of oxidative stress index MDA in the lung tissue of asthmatic mice was significantly decreased after MCL administration compared with the OVA group;the activity of SOD and the level of GSH were significantly increased compared with the OVA group.(4)The results of ELISA showed that the levels of IL-1βand IL-18 in the serum of asthmatic mice treated with MCL were significantly lower than those of the OVA group;The results of Western Blot showed that the protein expressions of NLRP3inflammasome activation-related proteins ASC,caspase-1,cleaved-caspase-1,IL-1βand NLRP3 in the lung tissue of asthma mice after administration of MCL were significantly decreased compared with those in the OVA group.2.MCL may regulate inflammation,oxidative stress and NLRP3 inflammasome activation in human bronchial epithelial BEAS-2B cells co-interfered with IL-4+TNF-αin vitro asthmatic model.(1)The results of ELISA detection showed that the levels of cytokines IL-6,IL-8and chemokine CCL11 and CCL24 in the supernatant of Beas-2B cells treated with MCL were significantly lower than those without MCL treatment.(2)The results of DCFH-DA probe detection showed that the intracellular ROS of Beas-2B cells co-induced by IL-4+TNF-αafter MCL was significantly lower than that of the untreated group,and the MDA level of oxidative stress index of Beas-2B cells co-induced by IL-4+TNF-αafter MCL was significantly lower than that of the untreated group,while the SOD activity and GSH level were significantly increased compared with the non-MCL treated group.(3)The levels of IL-1βand IL-18 in the supernatant of Beas-2B cells treated with IL-4+TNF-αafter MCL treatment were significantly lower than those in the untreated group.The results of Western Blot detection showed that the expression of NLRP3inflammatory body signal activation related proteins ASC,caspase-1,cleaved-caspase-1,IL-1βand NLRP3 in human bronchial epithelial Beas-2B cells treated with MCL were significantly lower than those in the untreated group.3.MCL may regulate MAPK/NF-κB signal related proteins in asthma models in vivo and in vitro.(1)Western Blot detection results showed that the protein expression levels of MAPK/NF-κB signaling pathway-related proteins p-ERK,p-JNK,p-P38,p-IκBα,and p-P65 in the lung tissue of asthma mice after MCL treatment were significantly lower than those in the OVA group.The protein expression of IκBαwas significantly higher than that in the OVA group.(2)The results of Western Blot detection showed that the protein expression of MAPK/NF-κB signal pathway related proteins p-ERK,p-JNK,p-P38 and p-P65 in human bronchial epithelial Beas-2B cells treated with IL-4+TNF-αafter MCL treatment was significantly lower than that in non-MCL group,while the protein expression of IκBαwas significantly higher than that in non-MCL group.(3)The results of DCFH-DA probe detection showed that the ROS level of MCL10μM+BA group treated with NF-κB agonist betulinic acid was significantly higher than that of MCL 10μM group,and Western blot detection showed that the protein expressions of ASC,caspase-1,cleaved-caspase-1,IL-1βand NLRP3 in MCL 10μM+BA group were significantly higher than those in MCL 10μM group.Conclusions:1.MCL may reduce airway hyperresponsiveness,regulate Th1/Th2 imbalance,improve oxidative stress and inhibit the activation of NLRP3 in OVA-induced asthma mouse model.2.MCL may inhibit inflammation,oxidative stress and activation of NLRP3inflammatory bodies in human bronchial epithelial Beas-2B cells co-induced by IL-4+TNF-αin vitro.3.The protective effect of MCL on asthma airway may achieved by inhibiting MAPK/NF-κB signal pathway. |
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