Document Research And The Effects And Mechanism Of Houpo Mahuang Decoction In The Environmental Pollutant Aggravated Asthma

ObjectiveHoupo Mahuang Decoction,from the "Essentials from the golden cabinet",is one of the classical formulas.The present study aimed to comb and analyze the key information including efficacy,indications,the origin,processing methods,and dosages of drugs in this prescription,to provide reference for clinical application and the development of the formula preparation.Environmental pollutants are the common inducing and triggering factors of asthma.Thus,in this study,Sulfur Dioxide derivatives(SDD)were used as the trigger and aggravating factor to explore the aggravating effects and mechanisms of SDD on asthma through in vivo and in vitro experiments.Combined with metabolomics research methods,the therapeutic effects and mechanisms of Houpo Mahuang Decoction and decomposed recipes of the prescription on SDD aggravated OVA-induced asthma were explored in vivo and vitro.Methods1.The relevant records and discussions of Houpo Mahuang Decoction in past dynasties were collected and sorted out for combing and analyzing the origin,efficacy,indications and decoctions.The records and discussions on the dosages of herbs were also collected,in combination with the discussions of the ancient medical physicians with the dosage and proportion of medicines in the Zhang Zhongjing’s formula of similar functions and efficacy,the herbs in the prescription were examined with the use of scales measuring vessels and the comparing objects.2.The model of OVA-induced asthma aggravated by SDD was created by airway dripping with SDD.The number of eosinophils in peripheral blood,total white blood cell(WBC),and percentage of various cells in bronchoalveolar lavage fluid(B ALF)of animals were counted and compared at the experiments treating with different experimental periods and SDD doses.The differentiation of Th2 and Th17 in splenic lymphocytes,airway hyperresponsiveness(AHR)and pathological injury of lung tissue were detected by ELISA kit.The levels of relevant verification factors in BALF of rats in each group were detected by WB and qPCR,and the expressions of TRPV1,tight junction protein(TJs),including ZO-1,Occludin in lung tissue of rats were detected to investigate the aggravation effects and mechanisms of SDD on OVA-induced asthma.3.Wild Type(WT)mice and TRPV1 knockout(TRPV1-/-)C57BL/J6 mice were then intervened by SDD and OVA to explore the effect of TRPV1 in the aggravating procedure of asthma.The differences in inflammatory cells,AHR and pathological injury of lung tissues were compared,and the changes of TRPV1 and TJs expressions in lung tissues were also compared between the WT and TRPV1-/-mice.To further analyze the influence of TRPV1 on TJs expression in this type of asthma,human bronchial epithelial cells(16HBE)were treated with SDD and IL-4 alone or in combination to detect changes in intracellular Ca2+concentration and expression changes of TRPV1 and TJs,and TRPV1 receptor antagonist AMG was used to intervene SDD+IL-4 damaged cells,then the expression and morphological changes of TJs in 16HBE cells were observed.4.Houpu Mahuang Decoction was used for drug intervention in rats with OVA induced asthma aggravated by SDD.The effects of Houpu Mahuang Decoction intervention on inflammatory cells,AHR and pathological injury of lung tissues in rats were counted and observed,the expression of TRPV1 and TJs in lung tissue were also detect for the investigating of effects and mechanisms of the prescription.SDD and IL-4 were then used to intervene 16HBE cells in vitro,and the effects of Houpu Mahuang Decoction on cell survival,changes of Ca2+ concentration were measured,and effects on TRPV1 and TJs proteins were also detected to investigate the protective effects and mechanisms of Houpu Mahuang Decoction on TJs of 16HBE cell.5.Serum metabolic changes of rats were detected by metabonomics method to analyze the metabolic characteristics of this type of asthma and the metabonomics changes of houpu Ephehuang decoction in the treatment of this type of asthma.Results:1.Houpu Mahuang Decoction has the effect of promoting lung and eliminating phlegm,reducing qi and relieving asthma.The treatment of Houpu Mahuang Decoction is cough,throat adverse such as water chicken sound,pulse floating caused by cold drink stop,with heat-obstruct in lung tissues.There are 9 herbs in the prescription:dried raw products of dried bark,root bark and branch bark of Magnolia officinalis Rehd.et Wils.;dried raw grass stems of Ephedra sinica Stapf;Gypsum Fibrosum;Processed seeds of Prunus armeniaca L.var.ansu Maxim.;dried raw tubers of Pinellia ternata(Thunb.)Breit;rhizome dry raw of Zingiber officinale Rosc.;dried raw root of Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag.;dried raw seed of Triticum aestivum L.;crushed dried raw fiuit of Schisandra chinensis(Turcz.)Baill.In the prescription,the drugs measured by weighing instrument were Magnolia officinalis Rehd.et Wils.,Ephedra sinica Stapf,Zingiber officinale Rose.,Asarum heterotropoides Fr.Schmidt varmandshuricum(Maxim.)Kitag.In the prescription,Pinellia ternata(Thunb.)Breit,Prunus armeniaca L.var.ansu Maxim.,Schisandra chinensis(Turcz.)Baill.,Triticum aestivum L.were measured by measuring apparatus.Combined with the literature reports in past dynasties and the weighing apparatus recording the herbs,the dosages of the medicine were measured.The dosage ratio of drugs in the formula is Magnolia oficinalis Rehd.et Wils.:Ephedra sinica Stapf,:gypsum:Pinellia ternata(Thunb.)Breit.:Zingiber officinale Rosc.:Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag:Schisandra chinensis(Turcz.)Baill.:Triticum aestivum L.(5:4:3:8:3:2:2:2:2:10).The dosage of Magnolia officinalis Rehd.et Wils.was about 15-18 g,and other drugs could be converted according to this.The decocting method was:boiling the Triticum aestivum L.with 2400 mL of water,and add the other drugs after removing the dregs.Decocting the soup to 600 mL,and then take the pharmaceuticals in 3 times,200 mL each time.2.Exposure to environmental pollutant SDD could aggravate asthma of OVA-induced rats,increase the numbers of eosinophils in peripheral blood(P<0.05,P<0.01),and total number of WBC(P<0.05,P<0.01).The percentages of eosinophils and neutrophils,and the levels of IL-4 and IL-13 in BALF were also increased(P<0.05,P<0.01).Increased the levels of IL-4 and IL-13 in BALF(P<0.05,P<0.01),aggravated airway hyperresponsiveness and lung tissue pathological injury,and increased the differentiation of spleen lymphocytes Th2 and Th17 cells(P<0.05).It increased the expression of TRPV1 gene and protein,and decreased the expression of ZO-1,Occludin and Claudin-3 gene and protein.Compared with WT mice,after TRPV1 knockout,the number of eosinophils in peripheral blood decreased(P<0.01),while the total number of WBC,the percentage of eosinophils and neutrophils in BALF decreased(P<0.01).Airway reactivity was decreased,pathological injury of lung tissue was alleviated,and TJs expression was reversed(P<0.05).3.In vitro experiment,results showed that SDD exposure could enhance the activation and increment of TRPV1 induced by IL-4,induce the concentration of intracellular Ca2+(P<0.05,P<0.01),and decrease the expressions of ZO-1,Occludin,Claudin-3(P<0.05),the continuity of TJs was broken.TRPV1 receptor antagonists increased the expression of ZO-1,Occludin and Claudin-3 proteins(P<0.05,P<0.01).4.Houpu Mahuang Decoction and its decoctions could reduce eosinophil in peripheral blood(P<0.01),WBC in BALF(P<0.05,P<0.01),and the percentages of eosinophil and neutrophil in BALF(P<0.05,P<0.01).It alleviated airway reactivity and pathological injury of lung tissue in rats,reduced IL-4 and IL-13 levels in BALF(P<0.01),reduced the expression of TRPV1 genes and proteins(P<0.05,P<0.01),and reduced the gene and protein expressions of ZO-1,Occludin and Claudin-3(P<0.05,P<0.01).In vitro experiment,results showed that Houpu Mahuang Decoction could reverse the increase of TRPV1 expression caused by SDD combined with IL-4,reduce the concentration of intracellular Ca2+(P<0.05,P<0.01),and reverse the expression of TJs(P<0.05,P<0.01).The whole part is better than the demolition groups.The level of Chai-1 inflammatory cells decreased compared with Chai-2.Compared with Chai-1,Chai-2 decreased airway reactivity and the expression of Occludin,Occludin,claudin-3 gene and protein were increased.Chai-3 was superior to Chai-4 in reducing airway reactivity and in regulating ZO-1 and Claudin-3 protein expressions.5.The results of serum metabolic analysis of rats showed that Houpo Mahuang Decoction was involved in the metabolic processes of amino acids,glycerol phospholipids,fatty acids,organic heterocyclic compounds,alkaloids,organic acids,sugars,aliphaloids,bile acids,phytohormones and sphingolipids in asthmatic rats.For example,arginine,glycine,arachidonic acid metabolism,histidine,dihydrosphingol,sphingol,vitamin K,pantothenic acid,etc.,are related to airway inflammation,airway hyperreactivity,airway remodeled,etc.Houpu Mahuang Decoction participates in multiple metabolic pathways related to TJs and Ca2+.Conclusion:1.Houpu Mahuang Decoction has the effect of promoting lung and eliminating phlegm,reducing qi and relieving asthma.The adaptation symptoms of Houpu Mahuang Decoction are cough,throat adverse such as water chicken sound,pulse floating.The prescription consists of nine herbs.Its weighing apparatus and measuring apparatus are converted by referring to ancient and modern weights and measures.The dosage ratio of drugs in the formula is Magnolia officinalis Rehd.et Wils.:Ephedra sinica Stapf,:gypsum:Pinellia ternata(Thunb.)Breit.:Zingiber officinale Rosc.:Asarum heterotropoides Fr.Schmidt var.mandshuricum(Maxim.)Kitag:Schisandra chinensis(Turcz.)Baill.:Triticum aestivum L.(5:4:3:8:3:2:2:2:2:10).The dosage of Magnolia officinalis Rehd.et Wils.was about 15-18 g,and other drugs could be converted according to this.The decocting method follows zhongjing’s original record:boil the Triticum aestivum L.with 2400 mL of water,remove the dregs,add the other drugs,decoct to 600 mL,and take the pharmaceuticals in 3 times,200 mL each time.2.Environmental pollutant SDD can aggravate OVA-induced asthma,and its effect is more serious when its concentration increases.And long-term exposure,even at low doses,can make asthma worse.After SDD exposure,the expression of ZO-1,Occludin and Claudin-3 decreased,the expression of TRPV1 increased,and the intracellular Ca2+concentration increased.TRPV1 receptor antagonists can reverse the expression of TJs protein,so the mechanism of SDD aggravating OVA-induced asthma and TJs destruction may be related to the regulation of TRPV1/TJs protein expression.3.Houpu Mahuang Decoction can improve the inflammatory response,airway hyperresponsiveness and lung tissue pathological damage caused by environmental pollutant SDD in OVA-induced asthmatic rats,reduce the expression of TRPV1 and regulate the expression of ZO-1,Occludin and Claudin-3.At the same time,the survival rate of 16HBE cells was increased,the intracellular Ca2+ concentration was decreased,and the expression of TJs protein was reversed.The efficacy of the whole prescription was better than that of the prescription removal group,and each prescription removal group played synergistic effect in inhibiting inflammation,protecting lung function,and callback of TRPV1 and TJs.4.Houpu Mahuang Decoction can participate in the metabolic processes of multiple metabolites,and the metabolic pathways involved in sphinolipid signaling pathway metabolism,arachidonic acid metabolism,histidine-metabolism and other metabolic pathways are related to intracellular Ca2+concentration,TRP channel and TJs.