| Objectives: Part 1.To investigate the mechanism of interleukin-27(IL-27)on airway inflammation,epithelial-mesenchymal transition(EMT),and Rho A/POCK signaling pathway by establishing asthmatic mice mouse model with intranasal administration of IL-27.To further explore the signal transduction mechanism of IL-27 in epithelial mesenchymal transformation of bronchial epithelial cells(16HBE).Part 2.To investigate the mechanism of artesunate on airway inflammation and epithelial-mesenchymal transformation in allergic asthma and the effect of different concentrations of artesunate on IL-27 expression levels in mouse BMDC in vitro,providing a new target and theoretical basis for artesunate in the prevention and treatment of asthma.Methods:1.Animal experiment: Female BALB/ C mice aged 6-8 weeks were selected to establish mouse asthma model by ovalbumin(OVA)method,and the mice were divided into Control group(Control),asthma model group(OVA),and IL-27 group(OVA +IL-27).airway responsiveness was measured by electronic mouse pulmonary function instrument within 24 hours after the last challenge(OVA or saline replacement solution).The required lung tissue,bronchoalveolar lavage fluid and spleen were collected after the mice were killed.The lung tissue was sectioned and stained separately with HE、PAS and Masson to assess the levels of airway inflammation,mucus secretion and collagen deposition.The levels of IL-4,IL-13,IFN-γ,IL-9,IL-17 A in bronchoalveolar lavage fluid were determined by enzyme-linked immunosorbent assay(ELISA).The ratio of Th17 and Th9 in spleen was measured by flow cytometry(FCM).The m RNA and protein expression levels of E-cadherin vimentin and α-SMA were detected by RT-PCR and Western blot respectively.Western blot was used to detect the expression level of Rho Aand ROCK1.Cell experiment:The EMT of bronchial epithelial cells(16HBE)induced by TGF-β1.The cells were divided into control group,IL-27 group,TGF-β1 group and IL-27+TGF-β1 group.The ability of cell migration was detected by transwell.The levels of E-Cadherin(molecular makers of EMT)、vimentin、α-SMA protein were determined by immunofluorescence.WB detected the protein levels of Rho A and ROCK1.2.Animal experiment: A mouse model of asthma was developed by OVA,the mice were randomized into negative control group(control),asthma model group(0VA),artesunate group(Art)and artesunate+anti-IL-27 group(Art+anti-IL-27).The airway responsiveness of mice in each group were detected within 24 hours after the final challenged by OVA solution or alternative saline solution.The levels of airway inflammation and mucus secretion of lung tissues in mice were analyzed by HE and PAS staining microscopically.The levels of IL-4,IL-13,IL-27,IL-9,IL-17 A in alveolar lavage fluid were determined by ELISA.The propotion of Th17 and Th9 in spleen was measured by FCM.RT-PCR and Western blot detectedthe m RNA and protein expression levels of E-cadherin vimentin and α-SMA respectively.Western blot analyzed the expression levels of Rho A and ROCK1 in lung tissues of each group.Cell experiment: Mouse BMDCwere induced and cultured in vitro.After one week of culture,artesunate and/or OVA with different concentration gradient were added,respectively.After 48 hours of intervention,the content of IL-27 in the supernatant of cell culture medium was determined by ELISA.Results:1.The mechanism of IL-27 in asthmatic airway inflammation and EMTAnimal experiment: Compared with the control group,the airway responsiveness of asthmatic mice was significantly altered,which showed increased airway resistance and decreased lung compliance(P < 0.01).Compared with asthma group,IL-27 can significantly reduce airway resistance and improve lung compliance(P<0.01).In pathological section staining,there were obvious inflammatory cell infiltration,mucus production and subepithelial collagen deposition in bronchial mucosa and airway of asthmatic mice.Compared with asthma group,IL-27 significantly reduced inflammatory cell infiltration,mucus production and subepithelial collagen deposition in asthmatic mice.There was a significant increase in IL-4,IL-13,IL-9 and IL-17 A in asthma group,a obvious decrease in IFN-γ(P<0.01).The intervention of IL-27 could cut down the levels of IL-4,IL-13,IL-9 and IL-17 A,and increase the level of IFN-γ(P<0.01).FCM showed that the propotionof Th17 and Th9 in the spleen from asthma group was significantly higher than control group(P<0.01).Compared with asthma group,the intervention of IL-27 could induce the propotion of Th17 and Th9 in the spleen(P<0.01).RT-PCR and WB showed the m RNA and protein expression of vimentin and α-SMA from asthmatic mice were increased,while the expression of E-Cadherin was decreased(P<0.01).The intervention of IL-27 could lower the levels of vimentin and α-SMA,increase the level of E-Cadherin(P<0.01).WB showed that the levels of Rho A and ROCK1(signaling pathway protein)from asthmatic mice were higher than control group(P<0.01),and they can be effectively reduced by the intervention of IL-27(P<0.05).Cell experiment:The migration rate of 16 HBE was obviously higher induced by TGF-β1(P<0.01),while the migration rate in IL-27 + TGF-β1 group significantly lower than that in TGF-β1 group.Immunofluorescence showed that compared with the control group,the expression levels of Vimentin and α-SMA protein in TGF-β1 group were significantly increased,while the expression of E-cadherin was significantly decreased(P<0.01).Vimentin and α-SMA were significantly decreased and E-cadherin expression was increased in IL-27+TGF-β1 group(P<0.01)WB analysis showed that the protein levels of Rho A and ROCK1 in TGF-β1group was significantly higher were obviously higher than these from control group(P<0.01).In IL-27+TGF-β1 group,the intervention of IL-27 can effectively reduce the protein expression of Rho A and ROCK.2.The mechanism of artesunate on airway inflammation and EMT by the pathway.Animal experiment: Compared with asthmatic mice,artesunate effectively reduced airway resistance and improved lung compliance(P<0.01);Airway resistance in artesunate+anti-IL-27 group was significantly higher than that in artesunate group,lower than that in asthma group,lung compliance was significantly lower than that in artesunate group,but higher than that in asthma group(P<0.05)The pathological changes of lung tissue showed the infiltration with inflammatory cells and mucus secretion were more obvious in asthma group,and artesunate could reduce the degree of inflammatory cells infiltration and mucus secretion effectively,while they were obviously higher in artesunate+anti-IL-27 group.ELISA assay showed that the expression of Th2 cytokines(IL-4、IL-13),IL-9IL-17 A and IL-27 in BALF of asthmatic mice induced by OVA was significantly increased compared with the control group(P<0.01);Compared with asthma group,the expression of IL-4,IL-13,IL-9,IL-17 A was significantly decreased and IL-27 was significantly increased in Art group(P<0.01);Compared with Art group,the expression of IL-4,IL-13,IL-9,IL-17 A in BALF of mice in Art+anti-IL-27 group was increased(P<0.01),Il-27 was below low limit of detection.FCM analysis showed that the proportion of Th17 and Th9 in spleen of asthmatic mice was significantly increased(P<0.01),The intervention of artesunate effectively reduced the proportion of Th17 and Th9 in the spleen(P<0.01),the proportion of Th17 and Th9 in artesunate +anti-IL-27 group was higher than that in artesunate group(P<0.01),but slightly lower than asthma group(P<0.05).PT-PCR and and Western blot showed that the m RNA and protein expression of Vimentin and α-SMA were significantly increased in asthmatic mice,while the expression of E-cadherin was significantly decreased(P<0.01),artesunate effectively decreased vimentin and α-SMA,and increased E-cadherin expression(P<0.01),compared with artesunate group,the expression of Vimentin and α-SMA was increased and the expression of E-cadherin was decreased in artesunate+anti-IL-27 group(P<0.01),artesunate can play the role of anti-asthmatic airway inflammation and EMT by interfering with the expression level of IL-27.Western blot showed that artesunate could significantly decrease the expression of Rho A and ROCK in lung tissues of mice(P<0.01).The expression levels of Rho A and ROCK proteinsin artesunate+anti-IL-27 group were significantly higher than those in artesunate group(P<0.05).Cell experiment: The level of IL-27 in BMDC culture medium was determined by ELISA.Compared with the control group,the level of IL-27 in OVA-induced cells was increased(P<0.01),which may be associated with promoting the maturationof BMDC,and artesunate combined with OVA significantly increased IL-27 expression in BMDC(P<0.01)in a dose-dependent manner.Conclusions:1.IL-27 can alleviate airway inflammation and airway hyperreactivity in asthmatic mice by downregulating the expression of IL-4,IL-13,IL-9,IL-17 A in BALF of asthmatic mice,upregulating the expression of IFN-γ,decreasing the proportion of Th9 and Th17 cells.2.In vivo and in vitro experiments have shown that IL-27 can inhibit airway epithelial-mesenchymal transformation and slow down the degree of airway remodeling by inactivating Rho A/ROCK signaling pathway,thus playing a therapeutic role in asthma3.Artesunate can alleviate airway inflammation,airway hyperreactivity and epithelial-mesenchymal transformation in asthmatic mice,by downregulating the expression of IL-4,IL-13,IL-9,IL-17 A in BALF of asthmatic mice,upregulating the expression of IL-27,decreasing the proportion of Th9 and Th17 cells,which provides a theoretical basis for artesunate in asthma treatment.4.After neutralizing IL-27 in airway,the effects of artesunate on airway inflammation and epithelial interstitial transformation were significantly reduced.In vitro studies reveal that artesunate can effectively improve the expression of IL-27 secreted by BMDC.Artesunate can exert its anti-asthma effect through IL-27 pathway,which provides a research basis for further effect target. |
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