| Objective: The effects of IL-37 on the airway hyperresponsiveness,the inflammatory pathology of bronchial-lung tissue and inflammatory cytokines in the bronchoalveolar lavage fluid of mice induced by PM2.5 exposure that were observed by constructing IL-37 transgenic mice and using recombinant human IL-37 protein pre-treatment,and the molecular mechanisms in the above processes were explored.Methods:1.Experimental animal grouping and experimental conditions: Thirty two healthy wild-type C57BL/6 female mice were randomly categorized into four groups,respectively clean air(FA)group,PM2.5 group,PBS+PM2.5 group and IL-37+PM2.5 group;16 transgenic mice using a CMV promoter to express human IL-37b(h IL-37tg-heterozygote)were randomly divided into clean air(IL-37tg-FA)group and PM2.5(IL-37tg-PM2.5)group(8 in each group).Except for the clean air filtered by the filter membrane in the FA group and IL-37-tg-FA group,the other groups all used the PM2.5 online enrichment oral and nose exposure system.In addition,30 minutes before the daily PM2.5 exposure,wide-type mice in the IL-37+PM2.5 group and PBS+PM2.5 group will be separately given 200 ng recombinant human IL-37 protein and 20 μl comfort agent(phosphate buffer saline,PBS)intra-nasally.2.Observation aspects and methods: Twenty four hours after PM2.5exposure,we adopted the small animal pulmonary function instrument to measure the respiratory mechanics and airway compliance of all experimental groups;we employ the enzyme-linked immunoassay to compare the level of inflammatory factors(interleukin-1β,IL-1β;interleukin-6,IL-6;tumor necrosis factor-α,TNF-α)in bronchoalveolar lavage fluid(BALF);we use hematoxylin-eosin staining(HE)and light microscope to detect the pathological form of the bronchi-pulmonary tissues of all experimental groups;Western Blotting(WB)was to explore the expression of protein of proliferation related PCNA,migration associated MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of bronchial-pulmonary tissues in each group of mice3.Statistical analysis: Data were expressed as Mean ± Standard Error of Mean(SEM)and analyzed using SPSS version 21.0 software(SPSS Inc.,Chicago,IL,USA).One-way analysis of variance(One-Way ANOVA)was adopted to detect whether there are significance.P<0.05 was implied that the difference of different groups was statistically significant.Results:1.Airway reactivity measurements in each experimental group of mice:Compared to FA group(>6.25 mg/ml),the acetylcholine concentration required to increase the initial airway resistance by 1-fold in the PM2.5exposure group was 1.56-3.125 mg/ml,and its airway reactivity was significantly increased(p <0.05);Compared to IL-37tg-FA group,the acetylcholine concentration required to increase the initial airway resistance by 1-fold in the IL-37-tg-PM2.5 group was 3.125-6.25 mg/ml and its airway reactivity was also significantly increased(p <0.05);Compared to PBS+PM2.5 group(1.56-3.125 mg/ml),given 200 ng recombinant human IL-37 protein intra-nasally 30 min before PM2.5 exposure could alleviate airway hyperresponsiveness induced by PM2.5;Compared to PM2.5 group,IL-37tg-PM2.5 group reduced airway resistance by PM2.5 exposure;Compared to IL-37tg-PM2.5 group(3.125-6.25 mg/ml),the effects of pre-treatment of recombinant human IL-37 protein on airway reactivity by PM2.5 induction was more obvious(p <0.05);2.Bronchial-pulmonary histopathology: FA group showed that the terminal bronchioles and alveolar epithelium were basically intact,with almost no inflammatory response;PM2.5 group had thickened tracheal wall,small bronchial smooth muscle hyperplasia,mild loss of tracheal epithelial cells,infiltration of inflammatory cells filled in the bronchi and perivascular area,normal alveolar structure damaged,fused,enlarged alveolar septum;Compared to PM2.5 group,IL-37tg-PM2.5 group and IL-37+PM2.5 group reduced the inflammatory cell infiltration in the bronchial and perivascular regions caused by PM2.5exposure and significantly improved the alveolar structure,manifested by narrowing of the alveolar septum,reduced small bronchial smooth muscle hyperplasia,reduced tracheal wall thickening and reduced loss of tracheal epithelial cells;3.Determination of inflammatory factors in BALF: Compared to FA group,the levels of IL-1β,TNF-α and IL-6 were significantly increased after PM2.5 exposure;Compared to IL-37tg-FA group,the levels of IL-1β,TNF-α and IL-6 were also significantly increased in the IL-37-tg-PM2.5 group;Compared to PBS+PM2.5 group,given 200 ng recombinant human IL-37 protein intra-nasally 30 min before PM2.5 exposure could reduce the levels of IL-1β,TNF-α and IL-6 induced by PM2.5;Compared to PM2.5 group,IL-37tg-PM2.5 group also reduced the levels of IL-1β,TNF-α and IL-6 by PM2.5 induction;Compared to IL-37tg-PM2.5 group,the effects of pre-treatment of recombinant human IL-37 protein on the reduction of levels of IL-1β,TNF-α and IL-6 by PM2.5 induction was more obvious;4.Detection of the expression levels of protein of proliferation related PCNA,migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8: Compared to FA group,the expression levels of proliferation related PCNA,migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 were significantly increased after PM2.5 exposure;Compared to IL-37tg-FA group,the expression levels of proliferation related PCNA,migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 were also significantly increased in the IL-37-tg-PM2.5 group;Compared to PBS+PM2.5 group,given 200 ng recombinant human IL-37 protein intra-nasally 30 min before PM2.5 exposure could reduce the expression levels of proliferation related PCNA,migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8;Compared to PM2.5 group,IL-37tg-PM2.5 group also reduced the expression levels of proliferation related PCNA,migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 by PM2.5 induction;Compared to IL-37tg-PM2.5 group,the effects of pre-treatment of recombinant human IL-37 protein on the reduction of expression levels of proliferation related PCNA,migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 by PM2.5 induction was more obvious.Conclusions: PM2.5 exposure caused airway hyperresponsiveness of mice of different experimental groups,exacerbated bronchial-lung inflammatory pathology changes,increased levels of inflammatory factor release in BALF,and increased proliferation-related protein PCNA,migration-related proteins MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 protein expression levels of bronchial-lung tissues in mice,but IL-37 transgenic and pre-treatment of recombinant human IL-37 protein intra-nasally decreased above processes.This research will clarify a novel druggle target for the therapy of PM2.5 with airway hyperresponsiveness or even asthma.Objective: The aim of this study is to observe the effect of IL-37 on contraction,proliferation and migration function of h ASMCs after PM2.5 incubation collected by the enrichment system by pre-treatment with recombinant human IL-37 protein and silencing IL-37 receptor IL-1R8 and detect the role of the IL-37 receptor IL-1R8 in above processes.Methods: 1.Experimental cell grouping and experimental conditions: control group: cells were cultured with complete medium containing 10% FBS;PM2.5 group: cells were incubated with PM2.5 suspension(12.5 μg/ml)for 24 h;IL-37+PM2.5 group: cells were pre-treated with recombinant human IL-37 protein(200 ng/ml)30 minutes before PM2.5 incubation(12.5 μg/ml);PBS+PM2.5 group: cells were pre-treated with PBS 30 minutes before PM2.5 incubation(12.5 μg/ml);si+IL-37+PM2.5 group: after IL-37 receptor IL-1R8 of h ASM cells were silenced,cells were pre-treated with recombinant human IL-37 protein(200 ng/ml)30 minutes before PM2.5 incubation(12.5 μg/ml);scr+IL-37+PM2.5 group: after IL-37 receptor IL-1R8 of cells were not silenced,cells were pre-treated with recombinant human IL-37 protein(200 ng/ml)30 minutes before PM2.5 incubation(12.5 μg/ml).2.Cell Counting Kit-8 kit detects the effects of IL-37 on proliferative function of h ASMCs after incubation with PM2.5(12.5 μg/ml);Cyto Select TM 48-Well Cell Contraction Assay Kit explores the role of IL-37 on contractile function of h ASMCs after incubation with PM2.5(12.5 μg/ml);Cell migration assay detects the effects of IL-37 on migratory function of h ASMCs after incubation with PM2.5(12.5 μg/ml);Western Blotting is used to detect the effects of IL-37 on expression of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of h ASMCs after incubation with PM2.5(12.5 μg/ml);Using probe Flu-4 fluorescent staining to explore the role of of IL-37 on the total calcium level after incubation with PM2.5(12.5 μg/ml)by flow cytometry.3.Using Lipo Rimax and three kinds of small interfering RNAs(si RNAs)and scramble si RNA to transfect h ASM cells to silence the IL-37 receptor IL-1R8 gene,pre-incubation with the recombinant human IL-37 protein(200 ng/ml)for 30 minutes,incubation with PM2.5(12.5 μg/ml)for another 24 h to detect the changes of cell contraction,proliferation,migration function,the expression of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 and the level of total calcium released of h ASMCs by flow cytometry.4.Statistical analysis: Data were expressed as Mean±Standard Error Mean(M±SEM)and analyzed using SPSS version 21.0 software(SPSS Inc.,Chicago,IL,USA).One-way analysis of variance(One-Way ANOVA)was adopted to detect whether there are significance.P<0.05 implies that the difference between the different groups was statistically significant.Results: 1.Changes of contractile function of h ASMCs: Compared with control group,incubation with PM2.5(12.5 μg/ml)promoted further contraction of h ASMCs,consistence with our previous results;Compared with PBS+PM2.5 group,pre-incubation of the recombinant human IL-37 protein(200 ng/ml)for 30 min reduced the contraction amplitude of h ASMCs after incubation with PM2.5;Compared with scr+IL-37+PM2.5 group,silencing the IL-37 receptor IL-1R8 on the membrane of h ASMCs,pre-incubation with recombinant human IL-37 protein(200 ng/ml)for 30 minutes and then incubation with PM2.5(12.5 μg/ml)for another 24 h,the protective effect of IL-37 on reducing the amplitude of contraction caused by PM2.5 was not anymore;There was no statistical difference between PBS+PM2.5 group and PM2.5 group;There was no statistical difference between IL-37+PM2.5 group and scr+IL-37+PM2.5 group;2.The changes of proliferative function of h ASMCs: Compared with control group,incubation with PM2.5(12.5 μg/ml)promoted proliferation of h ASMCs,consistence with our previous results;Compared with PBS+PM2.5 group,pre-incubation of the recombinant human IL-37 protein(200 ng/ml)for 30 min reduced the proliferation of h ASMCs after incubation with PM2.5;Compared with scr+IL-37+PM2.5 group,silencing the IL-37 receptor IL-1R8 on the membrane of h ASMCs,pre-incubation with recombinant human IL-37 protein(200 ng/ml)for 30 minutes and then incubation with PM2.5(12.5 μg/ml)for another 24 h,the protective effect of IL-37 on reducing the proliferation caused by PM2.5 was not anymore;There was no statistical difference between PBS+PM2.5 group and PM2.5 group;There was no statistical difference between IL-37+PM2.5 group and scr+IL-37+PM2.5 group;3.The changes of migratory function of h ASMCs: Compared with control group,incubation with PM2.5(12.5 μg/ml)promoted migration of h ASMCs;Compared with PBS+PM2.5 group,pre-incubation of the recombinant human IL-37 protein(200 ng/ml)for 30 min reduced the migration of h ASMCs after incubation with PM2.5;Compared with scr+IL-37+PM2.5 group,silencing the IL-37 receptor IL-1R8 on the membrane of h ASMCs,pre-incubation with recombinant human IL-37 protein(200 ng/ml)for 30 minutes and then incubation with PM2.5(12.5 μg/ml)for another 24 h,the protective effect of IL-37 on reducing the migration caused by PM2.5 was not anymore;There was no statistical difference between PBS+PM2.5 group and PM2.5 group;There was no statistical difference between IL-37+PM2.5 group and scr+IL-37+PM2.5 group;4.The changes of the level of total calcium of h ASMCs: Compared with control group,incubation with PM2.5(12.5 μg/ml)increased total calcium level of h ASMCs,consistence with our previous results;Compared with PBS+PM2.5 group,pre-incubation of the recombinant human IL-37 protein(200 ng/ml)for 30 min reduced the total calcium level of h ASMCs after incubation with PM2.5;Compared with scr+IL-37+PM2.5 group,silencing the IL-37 receptor IL-1R8 on the membrane of h ASMCs,pre-incubation with recombinant human IL-37 protein(200 ng/ml)for 30 minutes and then incubation with PM2.5(12.5 μg/ml)for another 24 h,the protective effect of IL-37 on reducing the total calcium level caused by PM2.5 was not anymore;There was no statistical difference between PBS+PM2.5 group and PM2.5 group;There was no statistical difference between IL-37+PM2.5 group and scr+IL-37+PM2.5 group;5.The changes of the expression level of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of h ASMCs: Compared with control group,incubation with PM2.5(12.5 μg/ml)increased the expression level of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of h ASMCs;Compared with PBS+PM2.5 group,pre-incubation of the recombinant human IL-37 protein(200 ng/ml)for 30 min reduced the the expression level of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of h ASMCs after incubation with PM2.5;Compared with scr+IL-37+PM2.5 group,silencing the IL-37 receptor IL-1R8 on the membrane of h ASMCs,pre-incubation with recombinant human IL-37 protein(200 ng/ml)for 30 minutes and then incubation with PM2.5(12.5 μg/ml)for another 24 h,the protective effect of IL-37 on reducing the expression level of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of h ASMCs caused by PM2.5 was not anymore;There was no statistical difference between PBS+PM2.5 group and PM2.5 group;There was no statistical difference between IL-37+PM2.5 group and scr+IL-37+PM2.5 group.Conclusions: In cellular experiments,pre-treatment with IL-37 reduced the abnormal contraction,proliferation and migration,cytoplasmic calcium levels,and the expression level of cell proliferation related PCNA,cell migration related MMP-2,MMP-9,Vimentin and IL-37 receptor IL-1R8 of h ASM cells induced by PM2.5 incubation.Above processes was correlation with the receptor of IL-37,IL-1R8. |
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