| Background and Objective Asthma is a typical type 2 immune disease characterized by the development of high levels of Ig E and Th2 cells.Asthma mainly includes atopic asthma and non-atopic asthma.Atopic asthma is defined as Ig E-mediated degranulation of mast cells,while non-atopic asthma does not secret allergen specific Ig E,but more innate immune cells are activated,such as ILC2 s and EOS and IL-5 and IL-13 secreted by ILC2 s.Non-atopic asthma occurs mostly from January to April and is closely associated with seasonal air-borne allergens.Respiratory infections are the most common cause of death in asthma and are more common in non-atopic asthma.When the respiratory tract was stimulated by protease allergen,it caused the damage of airway epithelial barrier and the stress response of tissue damage,which further resulted in the release of cytokine IL-33,IL-25 and thymic stromal lymphopoietin(TSLP).When the allergen stimulated Rag2-/-mice lacking T and B cells,it was found that it could also induce the recruitment of EOS,secretion of mucus and airway hyperresponsiveness.This suggests that Th2 cells are not necessary during the acute phase of allergic inflammation.In addition,there was no eosinophilic inflammation and mucus production in lung tissues of mice with IL-33 deficiency during nasal injection of protease allergens,indicating the key role of IL-33 in allergen-induced asthma.In Rag2-/-Il2rg-/-mice lacking ILCs,it was further found that the level of EOS and the secretion of mucus decreased significantly,which demonstrated the key role of ILC2 s in the acute phase of allergic inflammation.Therefore,the IL-33-ILC2-IL-5/IL-13 axis is considered to be an important pathway in the early stage of asthma.At the beginning of the 21 st century,American scientist Tracey and others have discovered a new mechanism of inflammatory response regulation,that is,cholinergic anti-inflammatory pathway(CAP),mediates the vagus nerve,which distributes in every important organ of the whole body.Acetylcholine released from activated T cells inhibit the release of inflammatory factors,thus CAP plays an anti-inflammatory effect.Subsequently,the scientists found that α7 acetylcholine receptor(α7n ACh Rs)was expressed on macrophages and other immune cells,and α7n ACh Rs was demonstrated to be at the core of cholinergic anti-inflammatory pathway byα7n ACh Rs gene knockout mice.Recent experiments have confirmed that α7n ACh Rs is expressed not only on macrophages,T cells,B cells,but also on ILC2 s.More importantly,α7n ACh Rs expression on ILC2 s is significantly higher than that on other immune cells.Based on the core position of α7n ACh Rs in cholinergic anti-inflammatory pathway and the high expression of α7n ACh Rs on ILC2 s,we intend to use α7n ACh Rs agonists PNU-282987 to determine whether it can regulate allergic airway inflammation by acting on ILC2 s.α7n ACh Rs,as a therapeutic target of Parkinson’s disease,myasthenia gravis and neuropathic pain,has attracted more and more attention.The common agonists of α7n ACh R are acetylcholine and nicotine.However,they can also interact with other receptors while in combination with the other α7n ACh Rs,resulting in side effects such as headache,vomiting,and insomnia.Therefore,it can not be used in clinical treatment.Therefore,it is of great clinical significance to search for a new α7n ACh Rs highly selective agonist for the treatment of asthma.GTS-21,a spiral α7 n ACh Rs agonist developed by Astra Zeneca Company(Astra Zeneca),has a high affinity to α7n ACh Rs.Lauriane Galle-Treger et.al has shown that GTS-21 has significant inhibitory effect in ILC2s-mediated airway inflammation.Compared to GTS-21,PNU-282987 developed by Pfizer(Pfizer)have a higher affinity to α7nAChRs.The Ki value of PNU-282987 was 27 nmol / L,which had little effect on α1,β1,γδ and α3β4,and had no activation on monoamine,muscarinic acid,glutamate and GABA receptor except 5-HT3.In addition,PNU-282987 has been demonstrated to attenuate acute lung injury by altering macrophage proliferation in mice.Many animal experiments and clinical trials have shown that the application of α7n ACh R agonists in the treatment of inflammation has its unique advantages,which provides a new perspective for anti-inflammatory therapy.Therefore,we intend to use a powerful selective agonist PNU-282987 to develop new ideas and provide theoretical basis for the treatment of allergic airway inflammation mediated by ILC2 s and further verify whether PNU-282987 has stronger inhibitory effect on ILC2s-mediated airway inflammation than that of GTS-21.Materials and methods1.In vivo experiment: 6-8-week-old C57BL/6J female mice were randomly fall into PBS group,PNU-282987 group,GTS-21 group,IL-33 model group,IL-33+PNU-282987 intervention group and IL-33+ GTS-21 intervention group.The model of airway inflammation in mice was established by IL-33 nasal administration.The model was treated with PNU-282987/GTS-21.The infiltration of inflammatory cells in lung tissue was observed by hematoxylin eosin staining(HE),and the goblet cell proliferation and mucus secretion was observed by Periodic acid-Schiff staining(PAS).C57BL/6J female mice were randomly divided into PBS group,PNU-282987 control group,GTS-21 control group,Alternaria model group,Alternaria + PNU-282987 intervention group and Alternaria + GTS-21 intervention group.The infiltration of inflammatory cells in lung tissue was observed by HE staining.The proliferation of goblet cells and mucus secretions in airway epithelium were observed by PAS staining.Flow cytometry was performed to detect ILC2 s and EOS in lung tissue and BALF.IL-5 and IL-13 in BALF were detected by ELISA.The content of IL-5+ILC2 and IL-13+ILC2 in lung tissue of mice was analyzed by flow cytometry intracellular staining.Real-time quantitative PCR(q RT-PCR)was used to detect the expression of IL-5,IL-13,GATA3 and IL-33 m RNA.2.In vitro experiment: Selected female C57BL/6J mice for more than 6 months and the Lineage-lymphocytes in lung tissue were separated by density gradient centrifugation and magnetic beads separation,and then use flow cytometry to separate KLRG-1+Sca-1+cells.The ILC2 s isolated were divided into PBS group,PNU-282987 group,GTS-21 group,IL-33+ PNU-282987 group and IL-33+ GTS-21 group.After 72 h,the number of cells in different groups was detected.The expression of Ki67,GATA3,IKK-P and NF-κB p65 in ILC2 s cells was detected by flow cytometry.The levels of IL-5 and IL-13 in the supernatant were detected by ELISA,and the m RNA level of IL-5,IL-13,and GATA3 in ILC2 s cells was detected by q RT-PCR.Results1 Results in vivo1.1 PNU-282987 inhibited the infiltration of inflammatory cells around the bronchus and the secretion of mucus in the bronchial cavity of the lung tissue of asthma mice.Compared with PBS control group,a large number of inflammatory cells around the bronchioles,and a large number of goblet cells and mucus in the bronchial of IL-33/Alternaria model mice was detected.Compared with IL-33/Alternaria model group,inflammatory cell infiltration in lung tissue in IL-33/Alternaria+ PNU-282987 intervention group and IL-33/Alternaria+ GTS-21 intervention group was significantly reduced.The goblet cell hyperplasia and mucus secretion in the bronchial mucosa in IL-33/Alternaria+ PNU-282987 intervention group and IL-33/Alternaria+ GTS-21 intervention group are decreased significantly.1.2 PNU-282987 inhibited the recruitment of EOS and the proliferation of ILC2 s in BALF and lung tissue of asthma mice.There was no significant difference in the percentage and absolute values of ILC2 s and EOS in lung tissue and BALF between PNU-282987 group,GTS-21 group and PBS control group(p > 0.05).Compared with the PBS control group,the percentage and absolute values of ILC2 s and EOS of lung tissue and BALF in the Alternaria model mice were higher(p < 0.05).Compared with the Alternaria model group,the percentage and absolute value of ILC2 s and EOS of lung tissue and BALF in the Alternaria+ PNU-282987/GTS-21 intervention group were lower(p < 0.05).ompared with Alternaria+ GTS-21,the content of ILC2 s and EOS in the lung tissue and BALF of the Alternaria+ PNU-282987 intervention group were not significantly different(p > 0.05).1.3 PNU-282987 inhibited the functional activation of ILC2 s in lung tissue of asthma mice.There was no significant difference in the level of IL-5 and IL-13 in the supernatant of BALF between PNU-282987 and GTS-21 group and PBS control group(p> 0.05).Compared with the control group of PBS,the content of IL-5 and IL-13 in the supernatant of BALF in the Alternaria model group was higher(p < 0.05).Compared with Alternaria model group,the level of IL-5 and IL-13 in Alternaria+PNU-282987/GTS-21 intervention group was lower(p < 0 05).There was no significant difference in the protein level of IL-5 in BALF between the Alternaria+PNU-282987 group and the Alternaria+ GTS-21 group,but the protein level of IL-13 in the BALF in the Alternaria+ GTS-21 group was lower than that in the Alternaria+PNU-282987 group.1.4 PNU-282987 inhibited the expression of IL-5,IL-13,GATA3 and IL-33 gene in lung tissue of asthma mice.There was no significant difference in the relative expression of IL-5,IL-13,GATA3 and IL-33 m RNA between PNU-282987 group,GTS-21 group and PBS control group(p > 0.05).Compared with the PBS group,the relative expression of IL-5,IL-13,GATA3 and IL-33 m RNA in the Alternaria model group was increased(p< 0.05).Compared with Alternaria model group,the relative expression of IL-5,IL-13,GATA3 and IL-33 m RNA in Alternaria+ PNU-282987/GTS-21 intervention group was decreased(p < 0.05).1.5 PNU-282987 inhibited the contents of IL-5+ILC2s and IL-13+ILC2s in lung tissue of asthma mice.Compared with Alternaria model group,the percentage of IL-5+ ILC2 s and IL-13+ILC2s of lung tissue in Alternaria+PNU-282987/GTS-21 intervention group was significantly lower(p < 0.05).There was no significant difference in the number of IL-5+ILC2s and IL-13+ILC2s between Alternaria+PNU-282987 group and Alternaria+ GTS-21 group(p > 0.05).2 Results in vitro .1 PNU-282987 inhibited the proliferation of ILC2 s.After 72 hours of culture,the cells of each group were counted.The number of cells in IL-33 stimulated group was higher than that in PBS group,PNU-282987 group and GTS-21 group(p < 0.05).The number of cells in IL-33+PNU-282987 group and IL-33+ GTS-21 group was significantly lower than that in IL-33 group(p< 0 05).There was no significant difference in the number of ILC2 s between IL-33+PNU-282987 group and IL-33+ GTS-21 group(p > 0.05).2.2 PNU-282987 inhibited the expression of IL-5 and IL-13 in supernatant.The protein levels of IL-5 and IL-13 in supernatant of IL-33 group were significantly higher than those of PBS group,PNU-282987 group and GTS-21 group(p < 0.05).The protein levels of IL-5 and IL-13 in IL-33+PNU-282987/GTS-21 group were significantly decreased(p < 0.05),and there was no significant difference in the inhibition of IL-5 and IL-13 between PNU-282987 and GTS-21(p > 0.05).2.3 PNU-282987 suppressed the m RNA levels of IL-5 and IL-13.After 72 hours of culture,the m RNA levels of IL-5 and IL-13 in IL-33 group were significantly higher than those in PBS group,PNU-282987 group and GTS-21 group(p < 0.05).The level of m RNA in IL-5 and IL-13 in IL-33+ PNU-282987/GTS-21 group was significantly lower than that in IL-33 group(p < 0.05),and the inhibitory effect of PNU-282987 and GTS-21 on IL-5 and IL-13 was not significantly different(p > 0.05).2.4 PNU-282987 inhibited the expression of Ki67,GATA3,P-IKK and NF-κB p65.After 24 hours of culture,the expression levels of phosphorylated protein IKK-P and NF-κB p65 were detected by flow cytometry.Compared with PBS group,PNU-282987 group and GTS-21 group,IKK-P level and NF-κB p65 level were significantly increased in IL-33 group(p < 0.05).Compared with IL-33 group,the level of IKK-P and NF-κB p65 were significantly lower in IL-33+ PNU-282987 group and IL-33 +GTS-21 group(p < 0 05).But the inhibitory effect of PNU-282987 on IKK-P and NF-κB p65 was stronger than that of GTS-21(p < 0.05).After 72 hours of culture,the protein expression of transcription factor GATA3 and proliferating factor Ki67 were detected by intracellular staining.The expression of GATA3 in IL-33 group was significantly higher than that in PBS group,PNU-282987 roup and GTS-21 group(p< 0.05),and the expression of GATA3 in IL-33+PNU-282987 group and IL-33 +GTS-21 group was significantly decreased than that in IL-33 group(p < 0.05).The inhibitory effect of PNU-282987 and GTS-21 on GATA3 was not significantly different(p > 0.05).The percentage of Ki67+ILC2s in IL-33 group was higher than that in PBS,GTS-21 and PNU-282987 group(p < 0.05).The expression of Ki67 in IL-33+ PNU-282987 group and IL-33+ GTS-21 group was significantly lower than that in IL-33 group(p < 0.05),but the inhibitory effect of GTS-21 on Ki67 was more obvious(p < 0.05).Conclusion PNU-282987 attenuates the phosphorylation of key transcription factor GATA3 and inflammatory regulators IKK and NF-κB by binding to α7n ACh Rs on ILC2 s,thus inhibiting the functional effect of ILC2 s and ILC2s-mediated airway inflammation. |
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