| Background:Asthma is a chronic lung disease characterized by variable airflow limitation.In addition to bronchoconstriction and airway inflammation,airway remodeling is also a key mechanism involved in disease development.Airway remodeling,which caused by repeated inflammatory injury stimulation and abnormal repair,is closely related to the severity of the disease and pulmonary dysfunction.It is the pathological basis of refractory asthma.Airway remodeling refers to the progressive recombination of various airway components,including airway wall thickening,lumen narrowing,epithelial cell damage and detachment,goblet cell proliferation,fibroblast accumulation,and smooth muscle hyperplasia and hypertrophy.Epithelial cells are the key point in the initial response to asthma.With the stimulation of various cytokines,epithelial cells lose cell polarity,cross the basement membrane and migrate to the subepithelial region,and become myofibroblasts.This process is called epithelialmesenchymal transition（EMT）.The occurrence of EMT is not only one of the main factors of epithelial barrier injury,but also an important source of airway submucosal myofibroblasts.EMT is not only one of the main causes of epithelial barrier dysfunction in asthma,but also an important source of myofibroblasts around the airway.The effect of bronchial epithelial cells in asthmatic airway remodeling through EMT has been demonstrated in vitro,in animal models,and in human histological specimens.Transforming growth factor β1（TGF-β1）,as a classic cytokine that induces EMT,has been proved to be able to induce EMT in bronchial epithelial cells and promote airway fibrosis remodeling in asthma.Interleukin-27（IL-27）is a member of the IL-12 family.Consisting of two subunits,EBI3 and P28,IL-27 is a heterodimeric cytokine and its receptor IL-27R consists of gp130 and IL-27Rα（WSX-1）.After activated,the antigen presenting cells produce IL-27,which binds to IL-27R on the target cell membrane.Then they activate specific cellular signaling pathways,such as Janus kinase（JAK）/signal transduction and activator of transcription（STAT）signaling pathways to exert biological effects.Previous studies have showed that IL-27 could attenuate airway inflammation and improves airway hyperresponsiveness in an OVA-induced mouse model.OVAchallenged IL-27Rα-/-mice showed significant enhancement of airway inflammation.However,previous studies were limited to the effect of IL-27 on airway inflammation in asthma,and whether IL-27 has an effect on airway remodeling in asthma is still unclear.Other studies have shown that IL-27 can inhibit EMT process in different cells,such as alveolar epithelial cells,non-small cell lung cancer cells and trophoblast cells.However,its effect on EMT of human bronchial epithelial cells involved in asthmatic airway remodeling is unclear.As a downstream pathway of IL-27 signaling,STAT3 pathway also plays an important regulatory role in EMT.Provirus Integration Site for Moloney Murine Leukemia Virus 1（PIM1）is a proto-oncogene.Its encoding protein PIM1 kinase is an effector gene product of STAT3 pathway.Stimulation of GP130 cytokine receptor can activate STAT3 and up-regulate PIM1 expression.Recent studies have shown that PIM1 protein not only has anti apoptotic function,but also promotes EMT.Therefore,we speculate that IL-27 may regulate EMT through STAT3/PIM1 pathway,and then participate in the regulation of airway remodeling.Objective:To study the effect of IL-27 on EMT of bronchial epithelial cells and explore the specific signaling mechanism,to find to find an effective target for asthma airway remodeling therapy.Methods:1.Establish a mouse model of asthma,detect the expression of IL-27 and analyze the correlation between IL-27 and EMT markers in lung tissue of asthmatic mice.1.1 Group the following twenty mice according different treatment:control group（10 mice）and OVA group（10 mice）.1.2 The airway responsiveness of mice was measured by mouse lung function instrument 24 h after the last challenge.1.3 Bronchoalveolar lavage fluid（BALF）was gathered.The statistics of total cells was made using a hemocytometer.The statistics of eosinophils was obtained after cell smear stained with Wright-Giemsa.1.4 The airway inflammation was observed and scored by Hematein＆Eosin（H＆E）.Perimeter of basement membrane（Pbm）,the total epithelial length,epithelial detachment length（ED）,airway wall aera（Wat）and smooth muscle area（Wam）were observed by Image Pro Plus 6.0 software.The ratio of ED to the total epithelial length（ED%）,airway wall thickness（Wat/Pbm）and smooth muscle thickness（Wam/Pbm）were calculated.1.5.Periodic acid-Schiff（PAS）staining was used to examined the goblet cell hyperplasia in airway mucosa.Image Pro Plus 6.0 software was used to detect the Pbm and PAS staining areas（APAS+）,and the PAS staining area/Pbm（APAS+/Pbm,μm2/μm）was calculated.1.6 Masson staining was performed to examined collagen fiber production around the airway.Image Pro Plus 6.0 software was used to measure Pbm and Masson staining area（AMasson+）,and the Masson staining area/Pbm（AMasson+/Pbm,μm2/μm）was calculated.1.7 ELISA was used to test the levels of IL-27 in BALF supernatant.1.8 The distribution of E-cadherin and α-SMA in the lung tissues was revealed through immunohistochemical staining.The intergrated option density（IOD）of α-SMA and E-cadherin protein in the lung tissues was detected with Image Pro Plus 6.0 software.The correlation between the level of IL-27 in BALF and IOD of Ecadherin and α-SMA in lung tissue was analyzed.2 Detect the effect of IL-27 on airway inflammation,airway hyperresponsiveness,airway remodeling and EMT in asthmatic mice2.1 Group the following thirty mice according different treatment:control group（10 mice）,OVA group（10 mice）and OVA+IL-27 group（10 mice）.2.2 The airway responsiveness of mice was measured by mouse lung function instrument 24 h after the last challenge.2.3 After collecting the BALF,we detected the concentrations of IL-4,IL-5 and IL-13 in BALF supernatant using ELISA kit.We counted the total number of cells using a hemocytometer and the number of eosinophils after Wright-Giemsa staining.2.4 The airway inflammation was observed and scored by Hematein＆Eosin（H＆E）.Pbm,the total epithelial length,ED,Wat and Wam were measured by Image Pro Plus 6.0 software.ED%,Wat/Pbm and Wam/Pbm were calculated.2.5 PAS staining was used to examined the goblet cell hyperplasia in airway mucosa.in airway mucosa.We chose Image Pro Plus 6.0 software to obtain Pbm and PAS staining areas（APAS+）,and the PAS staining area/Pbm（APAS+/Pbm,μm2/μm）was calculated.2.6 Masson staining was selected to text collagen fiber deposition surrounding the airway.We chose Image Pro Plus 6.0 software to obtain Pbm and Masson staining area（AMasson+）,and the Masson staining area/Pbm（AMasson+/Pbm,μm2/μm）was calculated.2.7 Immunohistochemical staining was used to evaluate the expression of EMT markers E-cadherin and α-SMA in the lung tissues.The Intergrated option density（IOD）of α-SMA and E-cadherin protein was measured by Image Pro Plus 6.0 software.2.8 The mRNA and protein expression levels of EMT markers E-cadherin and α-SMA in the lung tissues were performed through qPCR and Western blot.2.9 The protein expression levels of phosphorylated STAT3（p-STAT3）,STAT3 and PIM1 in lung tissues were performed through Western blot.3.The impact and mechanism of IL-27 on TGF-β1-induced EMT in BEAS-2B cells3.1 Detect the impact of IL-27 at different concentrations on TGF-β1-induced EMT in human bronchial epithelial（BEAS-2B）cells.（1）Cells were grouped as follow:control group;TGF-β1 group;TGF-β1+IL27（10 ng/ml）group;TGF-β1+IL-27（20 ng/ml）group;TGF-β1+IL-27（40 ng/ml）group.（2）The mRNA and protein expression of E-cadherin and α-SMA in BEAS2B cells were tested by Quantitative real-time PCR（qPCR）and Western blot.3.2 Detect the effects of IL-27 at different concentrations on TGF-β1-induced STAT3/PIM1 pathway in BEAS-2B cells.（1）Cells were grouped as follow:control group;TGF-β1 group;TGF-β1+IL27（10 ng/ml）group;TGF-β1+IL-27（20 ng/ml）group;TGF-β1+IL-27（40 ng/ml）group.（2）The protein expression of p-STAT3,STAT3 and PIM1 in BEAS-2B cells were tested by Western blot.3.3 Detect the role of STAT3/PIM1 pathway in the regulation of IL-27 on TGF-β1induced EMT in BEAS-2B cells.（1）Cells 1 were grouped as follow:control group;TGF-β1 group;TGF-β1+IL-27 group;TGF-β1+IL27+WP1066（STAT3 inhibitor）group;TGF-β1+WP1066 group.Cells 2 were grouped as follow:control group;TGF-β1 group;TGF-β1+IL-27 group;TGF-β1+IL27+SMI-4a（PIM1 inhibitor）group;TGF-β1+SMI-4a group.（2）The mRNA and protein expression of E-cadherin and α-SMA in BEAS2B cells were tested by qPCR and Western blot.（3）The protein expression of p-STAT3,STAT3 and PIM1 in BEAS-2B cells was tested by Western blot.Results:1.Mice model of asthma was successfully established.The expression of IL-27 in BALF of OVA-induced mice was decreased and related to the expression of EMT markers.1.1 Compared with the control group,the airway resistance of OVA-induced mice was increased.1.2 The number of total white cells and eosinophils in BALF of OVA-induced mice were increased obviously than those in the control.1.3 H＆E staining showed that regular bronchial lumen is regular in the control group.Lung tissue of OVA-induced mice showed narrowing of lumen,exfoliation of epithelial cells,thickening of airway wall and airway smooth muscle layer.Compared with the control group,inflammation score,ED%,Wat/Pbm and Wam/Pbm of OVA-induce mice were increased significantly.1.4 PAS staining showed that APAS+/Pbm in lung tissues of OVA-induced mice was increased apparently than that in the control group.1.5 Masson staining showed that AMasson+/Pbm in lung tissues of OVA-induced mice was increased distinctly that in the control group.1.6 The level of IL-27 in BALF of OVA-induced mice was significantly decreased that in the control group.1.7 Sensitization and challenge with OVA decreased the IOD of E-cadherin in lung tissue,while increased the IOD of α-SMA.The level of IL-27 in BALF was positively correlated with the IOD of E-cadherin and negatively correlated with the IOD of α-SMA in lung tissue of asthmatic mice.2.The effect of IL-27 on airway hyperresponsiveness,airway inflammation,airway remodeling and EMT changes in OVA-induced mice.2.1 The effect of IL-27 on airway hyperresponsiveness in OVA-induced mice.The airway resistance of OVA-induced mice increased obviously than that in the control group.IL-27 administration reduced airway resistance of OVA-induced mice.2.2 The effect of IL-27 on Th2 cytokines levels in BALF of OVA-induced mice.The levels of IL-4,IL-5 and IL-13 in BALF were rising clearly after OVA stimulation compared to non-treated mice.After IL-27 administration,the levels of those cytokines began to descend than those in OVA group.2.3 The effect of IL-27 on the number of inflammatory cells in BALF of OVA-induced mice.The number of total cells and eosinophils in BALF of OVA-induced mice were obviously increased than that in the control group.IL-27 administration reduced the number of total cells and eosinophils respectively in BALF of OVA-induced mice.2.4 The effect of IL-27 on the histopathology in lung tissues of OVA-induced mice.H＆E staining showed lung tissue of asthmatic mice exhibited luminal stenosis,airway wall thickening,disordering and rupturing,trachea epithelial cell shedding,airway smooth muscle thickening and inflammatory infiltration in the peribronchial and perivascular areas.These changes were significantly alleviated after the intervention of IL-27.The inflammation score,ED%,Wat/Pbm and Wam/Pbm were obviously increased than that in the control group.IL-27 administration reduced the inflammation score,ED%,Wat/Pbm and Wam/Pbm of OVA-induced mice.2.5 The impact of IL-27 on goblet cell hyperplasia in airway epithelium of OVAinduced mice.We used PAS staining to examined the goblet cell hyperplasia.APAS+/Pbm of OVAinduced mice was increased than that in the control group.IL-27 administration reduced APAS+/Pbm of OVA-induced mice.2.6 The effect of IL-27 on collagen deposition in lung tissues of OVA-induced mice.We used Masson staining to observe the subepithelial collagen deposition.AMasson+/Pbm of OVA-induced mice was increased than that in the control group.IL-27 administration reduced AMasson+/Pbm of OVA-induced mice.2.7 The effect of IL-27 on the expression of EMT molecular markers in lung tissues of OVA-induced mice.（1）Immunohistochemical staining showed that compared with the control group,OVA stimulation decreased the IOD of E-cadherin in lung tissue,while increased the IOD of α-SMA.Compared with the OVA group,IL-27 administration increased the IOD of E-cadherin in the lung tissues and decreased the IOD of α-SMA.（2）PCR and Western blot results showed that compared with the control group,OVA stimulation made the expression of E-cadherin on a downward trend,while made the levels of α-SMA on an upward trend in lung tissue.Compared with the OVA group,IL-27 administration boosted the levels of E-cadherin in the lung tissues,while downgraded the levels of α-SMA.2.8 The effect of IL-27 on the protein level of p-STAT3,STAT3 and PIM1 in lung tissues of OVA-induced mice.OVA promoted the protein level of p-STAT3 and PIM1 in lung tissues compared to control.IL-27 administration reduced the change of p-STAT3 and PIM1 levels induced by OVA.Meanwhile,the expression of total STAT3 in the lung tissue of mice in each group was not changed.3.The effect and mechanism of IL-27 on TGF-β1-induced EMT process in BEAS-2B cells.3.1 The effect of IL-27 on TGF-β1-induced EMT in BEAS-2B cells.PCR and Western blot results showed that compared with the control group,TGFβ1 stimulation gave rise to E-cadherin mRNA and protein downregulation,while led to the upregulation of α-SMA;IL-27 revealed E-cadherin upregulation and α-SMA repression compared to TGF-β1 stimulation.3.2 The effect of IL-27 on the expression of p-STAT3,STAT3 and PIM1 induced by TGF-β1 in BEAS-2B cells.Western blot showed that TGF-β1 stimulation gave rise to p-STAT3 and PIM1 expression upregulation compared with non-treated group.IL-27 downregulated the level of p-STAT3 and PIM1 protein expression level compared with TGF-β1 group,while the expression of total STAT3 in each group was not changed.3.3 The role of STAT3/PIM1 pathway in the regulation of IL-27 on TGF-β1-induced EMT in BEAS-2B cells.Compared to the absence of WP1066,the presence of WP1066 abrogated the surge of E-cadherin protein expression and the decline of α-SMA,p-STAT3,PIM1 protein expression induced by IL-27.Compared to the absence of SMI-4a,the presence of SMI-4a abrogated the rise of E-cadherin protein expression and the decline of α-SMA and PIM1 protein expression induced by IL-27,but had no effect on the expression of p-STAT3 induced by IL-27.Conclusion:IL-27 inhibits TGF-β1-induced EMT in human bronchial epithelial cells through STAT3/PIM1 pathway,and then inhibits airway remodeling in asthma. |
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