Study On Mechanism Of MaXingShiGan Decoction(MXSGD)in Intervention Of Asthma Airway Inflammation And Its Compatibility

Objective:Ma Xing Shi Gan Decoction(MXSGD),which originates from Zhang Zhongjing’s Shanghan Lun,is a classic prescription for the treatment of lung diseases.By replicating the ovalbumin(OVA)induced asthma airway inflammation and compound respiratory syncytial virus(RSV)aggravated asthma model,the effect of relieving asthma by MXSGD and the underlying mechanism was explored.Besides,its compatibility characteristics were analyzed.Combined with TMT quantitative proteomics technology,systematic analysis was made on the characteristics of MXSGD and its disassembled prescriptions in intervening RSV aggravated asthma.The above-mentioned study would not only further explain the scientific connotation of MXSGD for relieving asthma and its compatibility,but also provide a scientific basis for the theory of classical prescriptions and clinical treatment of asthma.Methods:1.The related literature on asthma and MXSGD was collected and sort out,and the knowledge of Chinese medicine on asthma and MXSGD was reviewed.2.The OVA-induced asthma model and the OVA plus RSV aggravated asthma model were replicated.The former model was intervened with MXSGD,San Ao Decoction(SAD),Ma Huang Decoction(MHD),and the latter one was intervened by the whole MXSGD and its decomposed recipes.The Penh variation after Ach stimulation,the level of inflammatory cells in EOS and BALF,as well as the lung pathological damage were observed after the intervention of MXSGD.The levels of IL-4,IL-13,SP,and PGE2in BALF were detected by ELISA.The TRPV1,TRPA1 mRNA,and protein expressions in lung tissue and CD4+T cells were examined via q-PCR and Western Blot method.The results were also validated and compared through the experiments in TRPV1-knocked out and-inhibited mice.3.The 16HBE cell inflammatory injury model stimulated by IL-4 combined with RSV was replicated in vitro.Through the comparison and verification with normal,shRNA-targeted silencing TRPV1 cells,and TRPV1 inhibitor intervention cells,the TRPV1 expression and changes in intracellular Ca2+levels after the stimulation of TRPV1 agonist was detected.4.Proteomic studies:The lung tissue samples from mice in groups of control,model,and MXSGD were collected,and detected by the TMT technology.Proteomics analysis in terms of significantly different targets,biological processes,main enrichment pathways,protein interactions,et al.was conducted.The pathogenesis mechanism of RSV aggravated asthma,as well as the intervention mechanism of MXSGD,were comprehensively investigated.Results:1.Doctors in different dynasties acknowledged the complexed pathogenesis of asthma.Its pathogenic factor was mainly related to phlegm,and were triggered by external sensations,internal injuries from diet and emotional disorders.Asthma syndrome types included cold,hot,cold-packed-heat,salt,alcohol,et al.Based on the characteristics of the disease,several treatment methods,such as promoting cold in the lung,clearing phlegm in the lung,dispersing cold and clearing heat,inducing and directing phlegm were proposed.In the past dynasties,the doctors’understanding of the functional characteristics of MXGSD can be used to treat typhoid fever,febrile disease,febrile malaria,asthma,cough,et al.MXSGD was also a common prescription for modern clinical treatment of asthma.Modern pharmacological studies showed that MXGSD had the effects of anti-inflammatory and immunomodulatory.Ephedrine,pseudoephedrine,,et al.are the main efficacy substance composition in the prescription.Bioinformatics studies also confirmed the multi-component,multi-target and multi-pathway intervention mechanism of MXSGD.2.MXSGD,MHD and SAD all significantly reduced the number of peripheral blood EOS(P<0.05)and WBC in BALF(P<0.05),decreased airway hyperresponsiveness(P<0.05,P<0.01)and relieved lung histopathological damage in OVA-induced asthmatic mice.MXSGD and SAD reduced TRPV1 and TRPA1 protein expression in model mice(P<0.05,P<0.01).The regulation effect of MXSGD on TRPV1 was better than that of MHD(P<0.01).The high dose of MXSGD also showed a better regulation effect than that of low dose(P<0.01).MXSGD also significantly downregulated TRPV1 expression in splenic CD4+T cells of asthmatic mice(P<0.01)and decreased IL-4,IL-13,NGF,PGD2 levels in BALF of model mice(P<0.05,P<0.01).3.Compared with OVA or RSV intervention alone,the mice in the OVA+RSV group had significantly higher numbers of EOS in peripheral blood,WBCs and various types of inflammatory cells in BALF(P<0.01).Besides,significantly enhanced airway hyperresponsiveness(P<0.05,P<0.01),severe lung histopathological damage,significantly increased levels of IL-4,IL-13,PGE2 and SP in BALF(P<0.01),and higher expression of TRPV1 protein and mRNA in lung tissue(P<0.05,P<0.01)were observed in the OVA plus RSV aggravated asthma mice.With the intervention of inhibitor Capsazepine,the number of EOS in peripheral blood(P<0.01),the number of WBC and various types of inflammatory cells in BALF(P<0.01)in asthmatic mice were reduced.Besides,the decreased airway hyperresponsiveness(P<0.05,P<0.01),improved lung injury,and reduced levels of IL-4,IL-13,PGE2,and SP in BALF(P<0.05,P<0.01)were detected.In TRPV1 knockout mice,the number of WBC and various types of inflammatory cells in the peripheral blood EOS and BALF did not increase significantly,no obvious airway hyperresponsiveness and lung tissue pathological damage,and no increased levels of various inflammatory mediators in BALF were observed.Intervention with high(13.2 g/kg),medium(6.6 g/kg)and low(3.3 g/kg)doses of MXSGD significantly reduced the number of EOS in peripheral blood(P<0.01),the number of WBC and various types of inflammatory cells in BALF(P<0.05,P<0.01)and reduced airway hyperresponsiveness(P<0.05,P<0.01)in model mice.Improved lung histopathological damage,reduced IL-4,IL-13,PGE2,and SP levels in BALF(P<0.05,P<0.01),and down-regulated TRPV1 protein and mRNA expression in lung tissue(P<0.05,P<0.01)can also obtained with these interventions.MXSGD-containing serum increased the survival rate of IL-4 combined with RSV-stimulated 16HBE cells in vitro,down-regulated TRPV1 protein and mRNA expression(P<0.05,P<0.01),and decreased intracellular Ca2+fluorescence intensity after stimulation by TRPV1 agonist(P<0.01).4.The whole prescription of MXSGD,the disassembled recipes of Ma Xing Cao(MXC)and Gypsum can all reduce the number of EOS in peripheral blood and the number of EOS and neutrophil in BALF in RSV aggravated asthma mice(P<0.01).The intervention of MXC and Gypsum can also reduce the number of WBCs in BALF(P<0.01),and the intervention effect of the whole prescription group is better than that of the disassembled recipes group(P<0.01).The three administration groups reduced airway hyperresponsiveness to varying degrees and improved lung injury.MXSGD can significantly reduce the levels of IL-4,IL-13,PGE2,and SP in BLAF(P<0.05,P<0.01),while MXC decreased the levels of IL-13 and PGE2 in BALF(P<0.05,P<0.01),and gypsum reduced the levels of IL-13,PGE2,and SP(P<0.05,P<0.01).In addition,MXSGD down-regulated the expression of TRPV1 protein and mRNA in lung tissues(P<0.05,P<0.01),and MXC down-regulated the expression of TRPV1 protein(P<0.01).The medicated serum of MXSGD,MXC,and Gypsum all improved the survival rate of 16HBE cells stimulated by IL-4 combined with RS V.After the intervention of MXSGD medicated serum,the expression of TRPV1 protein and mRNA in 16HBE cells showed a lower level that that intervened by MXC.Lower level of fluorescence intensity of intracellular Ca2+after stimulation by TRPV1 agonist was also observed in MXSGD intervened group than that intervened with MXC and gypsum(P<0.05).5.The results analyzed by the proteomics indicates that:(1)59 differential proteins was obtained in the lung tissues in RSV aggravated asthma model,including 16 down-regulated proteins and 43 up-regulated proteins.These proteins were mainly involved in biological processes including biological regulation,regulation of immune system processes,response to stimuli such as exogenous,oxide,cytokines,antigen processing and presentation,and participated in catalysis,antioxidant,transcriptional regulation,molecular sensing and other functions.The KEGG pathway was mainly enriched in antigen processing and presentation,Thl and Th2 cell differentiation,and arachidonic acid metabolism.These were mainly involved in infectious diseases,asthma and other immune-related diseases,and related to immune response,inflammatory response and other pathways.(2)There were 6 differential proteins in the intervention of MXSGD in RSV aggravated asthma,including 2 up-regulated proteins and 4 down-regulated proteins.The differential targets of MXSGD in intervening RSV aggravated asthma were enriched in the biological processes of cell process,response to stimulation,regulation of membrane potential,membrane permeability and functions of contributing to adrenocorticotropic hormone releasing hormone receptor binding,neuropeptide receptor binding.These targets also acted on AMPK signaling pathway,relaxing signaling pathway,estrogen signaling pathway,oxytocin signaling pathway,Rapl signaling pathway,et al.,and played a role in resisting viruses,regulating immune response,regulating transmembrane signals,regulating calcium ion transport and so on.(3)There were 1 differential protein between MXSGD and MXC,5 differential proteins specific to MXSGD and 1 differential protein specific to MXC.MXC can play a role in defense against viruses,anti-inflammation,induction of apoptosis,and regulation of autophagy,while MXSGD showed effects in defense against viruses,regulation of immune response,regulation of cross-modal signaling,and regulation of calcium ion transport.Conclusion:1.In this study,we systematically reviewed the understanding of the name,etiology.classification and treatment of asthma in ancient and modern literature.MXGSD is mainly used to treat the syndrome of wind-cold,febrile disease,asthma and cough.The four herbs in the prescription showed the synergistic effect of clearing the lungs and relieving asthma.In modern literature,it was found that MXGSD is a common prescription for clinical treatment of asthma.It is suggested that MXSGD can play a multi-pathway and multi-target role in anti-inflammatory,and immune regulation.2.MHD,SAD and MXSGD had different regulatory effects on TRPs,in which MXSGD regulated TRPV1 more obviously.MXSGD can regulate the expression of TRPV1 in CD4+T cells,as well as the level of Th2 cytokines and neurogenic inflammatory mediators in BALF,suggesting that its anti-asthmatic mechanism may be related to the regulation of the neuro-immune interaction involving TRPV1.3.The results of in vivo and in vitro experiments verified the protective effect of MXSGD on RSV aggravated airway inflammation in asthma.The underlying mechanism was related to the regulation of TRPV1 channels,the levels of Th2 cytokines,and neurogenic inflammatory mediators.MXC can significantly reduce the airway hyperresponsiveness,and the plaster exert anti-inflammatory effects.The intervention effect of the whole prescription is better than the disassembled recipes,and showed the synergistic effects.4.The analysis results of quantitative proteomics indicated that the mechanism of RSV aggravating asthma may be related to antigen presentation and processing,immune response,inflammatory response,signal transduction and so on.MXC played a role in immune regulation,regulating cell apoptosis and cycle.The whole prescription of MXSGD may take effect on defending against viruses,regulating immune response,regulating transmembrane signals,and regulating calcium ion transport.