Study On The Immune Regulation Mechanism Of Jia-Wei-Yu-Ping-Feng-San On ILC2-Mediate Airway Inflammation In Allergic Asthma

Objective:Asthma is a common chronic airway inflammation disease with a high incidence and an increasing trend.The introduction of GINA step-up therapy has improved asthma control,but achieving overall control of asthma remains a huge challenge.Jia-Wei-Yu-Ping-Feng-San(JWYPFS)is an effective Chinese medicine compound for clinical treatment of asthma,which is optimized by the combination of the classic prescription"Yu-Ping-Feng-San"of traditional Chinese medicine"invigorating qi and consolidation of superficies"and the empirical prescription"Bu-Shen-Yi-Qi-Fang"of famous Chinese medicine professor Shao Changrong.However,its mechanism of action is still unclear.Type 2 innate lymphoid cells(ILC2)is a new type of lymphocyte discovered in the past decade.Its important contribution in mediating airway inflammation in allergic asthma has attracted more and more attention from researchers at home and abroad.After the body is exposed to allergens,epithelial cells will release a large number of"alarm elements"such as IL-33,IL-25,TSLP,etc.These cytokines directly cause the activation of ILC2 cells,and then secrete IL-5,IL-13 and other type 2inflammatory factors.Finally,it leads to typical asthmatic pathophysiological changes such as eosinophils(EOS)infiltration in the airway,goblet cell proliferation and mucus secretion.Based on this,this study proposed that the mechanism of the effective treatment of asthma by JWYPFS may be closely related to the regulation of ILC2activation,and planned to achieve the following research objectives through a series of in vivo and in vitro experiments:1.To clarify the mechanism of JWYPFS in regulating ILC2-mediated airway inflammation of allergic asthma,and to provide more traditional Chinese medicine(TCM)treatment methods and pathological mechanism theoretical support for the treatment of allergic asthma;2.To explore the role of relevant signaling pathways in the activation of ILC2 and the possible intervention targets of JWYPFS,deepen the study on the pathogenesis of ILC2in asthma,and enrich the traditional Chinese medicine theory of JWYPFS in the treatment of asthma.3.To analyzed the composition of JWYPFS and provide the basis for further elucidating the possible pharmacodynamic substance basis of JWYPFS in the treatment of asthma.Methods:1.In vivo experiment:C57BL/6 mice were taken as the research object,and the allergic asthma mouse model was established by the method of OVA sensitization and stimulation(atomization combined with nasal drops).Mice were divided into 6 groups:Control group,OVA asthma model group,OVA+JWYPFS low-dose group,OVA+JWYPFS medium-dose group,OVA+JWYPFS high-dose group,OVA+Dexamethasone(DEX)group,all treatment groups were given different doses of JWYPFS or positive control drug DEX at the same time of OVA stimulation to intervene.The effect of JWYPFS on airway hyperresponsiveness was evaluated by respiratory function test.HE and PAS staining were used to observe the effects of JWYPFS on lung tissue inflammation and mucus secretion.The inflammatory cell count was used to classify and analyze the white blood cells in the alveolar lavage fluid,and the effects of JWYPFS on white blood cells,EOS and lymphocytes(LYM)were evaluated.The levels of OVA specific Ig E,Ig G1 and other immunoglobulin and the expression of type 2 inflammatory factors such as IL-5 and IL-13 were detected by ELISA kit.ILC2,IL-5~+ILC2,IL-13~+ILC2 in lung tissues of mice were detected by flow cytometry to observe the effect of JWYPFS on ILC2.PCR was used to detect the expression levels of ILC2 transcription factors IRF4,GATA3 and IL-33 receptor ST2m RNA in the lung tissues of mice and the intervention effect of JWYPFS.2.Asthma mouse model with highly activated ILC2 was established by OVA induction combined with nasal stimulation of recombinant mouse IL-33 protein,and a high dose of JWYPFS was given for intervention.HE staining was used to evaluate the infiltration of inflammatory cells in the lung tissues of mice.The single cell suspension was extracted from the lung tissues of mice,and the ILC2 cells were sorted by flow cytometry.Then,the double-ended sequencing mode of Illumina Hiseq sequencing platform was applied to carry out high-throughput sequencing of ILC2 cells in the blank control group,model group and JWYPFS high-dose treatment group.In vitro experiment:ILC2 in lung tissue of mice were sorted by flow cytometry for in vitro culture,and the recombinant mouse IL-33 protein was given to stimulate ILC2.After different periods of IL-33 stimulation,the peak time of ILC2 expression of IL-5 was explored.After the peak expression time was determined,the drug-containing serum of JWYPFS was added for intervention.ELISA was used to detect the levels of type 2inflammatory factors such as IL-5 and IL-13 in the supernant of cells,and PCR was used to detect the m RNA expression levels of ILC2 transcription factors IRF4 and GATA3 and IL-33 receptor ST2.3.The chemical constituents and quality control of JWYPFS were analyzed by UPLC-Q-TOF-MS,combined with the data of natural product high resolution mass spectrometry database and related literatures.Results:1.Compared with the control group,mice in the OVA-induced asthma model group showed significantly increased airway hyperresponsiveness,severe inflammatory cell infiltration in lung tissue,airway stenosis,goblet cell proliferation,mucus secretion,significantly increased EOS in alveolar lavage fluid,and increased serum OVA-specific Ig E and Ig G1.Type 2 inflammatory cytokines,such as IL-5 and 13,were upregulated in lung tissue,indicating that the OVA induced allergic asthma mouse model was successfully established,and the airway inflammation was dominated by type 2inflammatory response.2.When acetylmecholine concentration was 25mg/m L atomized,compared with model group,airway resistance of mice treated with different doses of JWYPFS decreased,and the increase of airway resistance in the JWYPFS high-dose group was the most significant,and its effect was close to that of positive control drug DEX.3.After treated with JWYPFS or DEX,lung tissue inflammation and airway mucus secretion were reduced in all mice.With the increase of the dosage of JWYPFS,inflammatory cell infiltration in lung tissue and airway mucus secretion were gradually improved,and the improvement of inflammation in the JWYPFS high-dose group was weaker than that in the DEX group.Compared with the model group,the pathological inflammation score and PAS score of HE decreased after treatment with different doses of JWYPFS.4.After the intervention of JWYPFS,the total number of white blood cells,EOS,Lym,neutrophils and macrophages in the alveolar lavage fluid of mice decreased compared with the model group,and the effect was most obvious in the JWYPFS high-dose group.5.Compared with the model group,the serum Ig E and Ig G1 of mice in the medium-high dose treatment group and the DEX treatment group were significantly decreased,and the Ig E and Ig G1 levels in the high-dose treatment group were close to those in the Dex group;The Ig E and Ig G1 levels of mice in the low-dose treatment group were slightly decreased,but there was no significant statistical difference compared with the model group.6.Compared with mice in the asthma model group,IL-5 level in lung tissue of mice in the medium-high dose group and the DEX group was decreased,and IL-5 content in the low-dose group was slightly down-regulated,but the difference was not statistically significant.IL-13 levels were significantly reduced in all treated mice.7.The levels of ILC2,IL-5~+ILC2 and IL-13~+ILC2 in lung tissues of model group were significantly higher than those in blank group,while oral DEX and medium and high doses of JWYPFS significantly inhibited the upregulation of ILC2,IL-5~+ILC2 and IL-13~+ILC2.After treatment with JWYPFS,the number of ILC2,IL-5~+ILC2,IL-13~+ILC2decreased,and its effect was more obvious with the increase of administration dose.8.The m RNA levels of ST2,IRF4 and GATA3 in OVA asthma model group were significantly higher than those in control group.After treatment with different doses of JWYPFS and Dex,the m RNA levels of ST2,IRF4 and GATA3 were decreased,and the JWYPFS high dose had the best treatment effect.9.In the asthmatic mouse model with highly activated ILC2 stimulated by OVA combined with IL-33,JWYPFS can inhibit the infiltration of inflammatory cells in lung tissue,the proportion and quantity of ILC2 in asthmatic mice.The MAPK signaling pathway may be involved in the activation of ILC2 during the detection of ILC2m RNA-seq in mouse lung tissue.After PCR verification,the expression of MAPK-related genes My D88 and MAPK10 were significantly different between the mice treated with JWYPFS and the model group.10.In the in vitro experiment,IL-5,IL-13,ST2,IRF4 and GATA3 levels were significantly increased after IL-33 stimulation(cell model group).When 10%and 20%of the drug-containing serum of JWYPFS were given to interfere with activated ILC2,compared with the group of cell model and blank serum,the drug-containing serum of JWYPFS could reduce the expressions of IL-5,IL-13,ST2,IRF4 and GATA3,and the effect was enhanced with the increase of the drug-containing serum volume of JWYPFS.11.UPLC-Q-TOF-MS analysis and preliminary identification of 102 kinds of chemical components in JWYPFS,including protocatechuic acid,neochlorogenic acid,neoaconitine,etc.After comparing with the standard substance,the 21 chemical components in JWYPFS were confirmed and identified:psoralenoside,isopsoralenoside,prim-O-glucosylcimifugin,orientin,liquiritin,liquiritin apioside,vitexin,ellagicacid,4‘-O-β-glucopyranosyl-5-O-methylvisamminol,benzoylmesaconine,benzoylhypaconine,liquiritigenin,calycosin,hypaconitine,astragaloside IV,glycyrrhizic acid,astragaloside II,schisandrol A,schisandrol B,tigloylgomisin H and atractylenolide I.Conclusion:1.JWYPFS can reduce airway inflammation in OVA induced allergic asthma mice.2.JWYPFS can regulate the activation of ILC2 in allergic asthma by down-regulating ILC2 transcription factors IRF4 and GATA3,inhibiting the activation of MAPK signaling pathway in ILC2 cells.3.102 kinds of chemical components were identified by UPLC-Q-TOF-MS,among which 21 kinds of monomers,such as psoralinin,were confirmed by comparison with standard substances,providing a basis for clarifying the basic research on the pharmacological substances of JWYPFS.