| Objective:The traditional Chinese medicine theory was applied to explain the prescription mechanism of the classical Uygur medicine Zukamu granules,and modern scientific and technological methods were used to explore the immunomodulatory effect and mechanism of Zukamu granules.Methods:(1)Study on the prescription mechanism of Zukamu granules:by consulting Uygur medicine books such as the "Chinese Materia Medica-Uyghur Medicine Volume","The Pharmacopoeia of the People's Republic of China"(2020 edition),CNKI,Wanfang and other major databases,the records of Zukamu granules and their constituent drugs were reviewed to sort out the basic sources,drug parts,properties and efficacy of Zukamu granules.And draw lessons from traditional Chinese medicine "seven harmonious relations" and "monarch,minister,assistant and envoy" theory to analyze the compatibility of Zukamu granules.(2)Prediction of immunomodulatory targets and pathways of Zukamu granules:?Based on TCMSP database and the literatures published in open source databases of major journals,the active components and action targets of 10 drugs of Zukamu granules were searched,and the data set of "active components-potential action targets" of Zukamu granules was constructed.?PPI network and module analysis were constructed by using STRING 11.0 database and Cytoscape 3.7.3 software.GO functional enrichment analysis,signal pathway analysis and disease prediction were carried out on the key module targets with the help of DAVID v6.8 database,in order to screen and predict the potential targets and mechanisms of Zukamu granules immunomodulatory effect.The potential targets and pathways of immunoregulation of Zukamu granules were predicted by network.(3)Study on immunomodulatory activity and target verification of Zukamu granules:the immunomodulatory effect of Zukamu granules in different stages of lung injury was studied based on the pathological model of acute lung injury induced by lipopolysaccharide in mice and chronic lung fibrosis induced by bleomycin in rats,and the regulation of Thl/Th2 balance as the breakthrough point.? The effects of Zukamu granules on lung pathology and myeloperoxidase in mice with acute lung injury were observed,the characteristic cytokines of Thl and Th2 in tissue and serum were determined,and the ratio was calculated to analyze the immunomodulatory effect of Zukamu granules in the early stage of acute lung injury.?The effects of Zukamu granule on the general condition,pathological changes of lung tissue and expression of HYP in rats with chronic idiopathic pulmonary fibrosis were observed,and the levels of serum Thl,Th2 characteristic cytokines,related immunoglobulin and the expression of GATA3 and STAT6 were measured to explore immunomodulatory effect of Zukamu granules in the late stage of pulmonary fibrosis.Results:(1)Study on the prescription mechanism of Zukamu granules:?Through the carding of ancient books and modern research literature,Zukamu granules had certain characteristics of Uighur medicine.it was mainly reflected in the fact that the prescription contained two kinds of Uygur medicinal herbs,including Cordiae Dichotomae Fructus and Nymphaea flos,as well as the use of Rhei Radix et Rhizoma and Chamomilae Flos were different from those of traditional Chinese medicine.?Both Uygur medicine and traditional Chinese medicine reckoned that Zukamu granules was cold and non-toxic,had the effects of clearing heat,relieving cough,tonifying lung and stomach,and can treat wind-heat cold.? For wind-heat cold,it should be treated mainly by dispelling wind and relieving the exterior,supplemented by clearing heat and detoxification,relieving cough and asthma,so Mentha Haplocalycis Herba and Nymphaea flos in the prescription were used as monarch medicine,mainly dispelling wind and heat;Papaveris Pericarpium,Cordiae Dichotomae Fructus and Matricaria chamomile were used to relieve cough and asthma as ministerial medicine,Rhei Radix et Rhizoma and Althaea rosea(L.)Cav.were used to enhance the effect of clearing away heat and purging fire,Jujubae Fructus and Kaempferiae Rhizoma were used to protect the spleen and stomach together.Four medicines were assistant medicine;Glycyrrhizae Radix et Rhizoma was used to coordinate the property of all medicines as conductant medicine.(2)Prediction of immunomodulatory mechanism of Zukamu granules:? Combined with database screening and literature resource supplement,a total of 174 active components of Zukamu granules were obtained,and the corresponding potential targets were 249.?The PPI network consisted of 207 proteins,and 4 key protein modules were obtained.There were 12 pathways related to immune regulation in the key protein modules.The top three pathways in the number of enriched genes were Toll-like receptor signal pathway.NOD-like receptor signal pathway and T cell receptor signal pathway.30 diseases were predicted according to Zukamu granules key proteins,of which 5 were respiratory diseases.Based on the previous study of Zukamu granules on TLR4/MyD88/NF-?B signal pathway,starting with the effect of T cell receptor signal pathway-regulating T cell differentiation,the potential target on T cell receptor signal pathway was selected as the evaluation index of biological effect of Zukamu granules in regulating Th1 and Th2 immunity.(3)Study on immunomodulatory activity and target verification of Zukamu granules:?The pathological results of HE staining in mice with acute lung injury showed that the degree of edema and hyperemia of lung tissue and the number of inflammatory cell infiltration in Zukamu group were less than those in the model group,and the lung injury score and MPO level were significantly lower than those in the model group(P<0.01),indicating that Zukamu granules can reduce the degree of acute lung injury induced by lipopolysaccharide.As for the characteristic cytokines of Thl and Th2,Zukamu granules could significantly inhibit the secretion of IFN-y and the expression of IL-4 in lung tissue and serum compared with the model group.There was significant difference in the ratio of IFN-y/IL-4 in serum.?The experiment of rats with pulmonary fibrosis showed that on the 28th day,the respiratory rate in the Zukamu group was significant lower than that in the model group(P<0.01);in terms of HYP content,it was significantly lower than that in the model group on the 14th and 28th day,and the results of HE staining of pathological sections of lung tissue also showed that Zukamu granules could delay the progress of pulmonary fibrosis to some extent.In terms of Thl and Th2 cytokines,compared with the model group,Zukamu granules could significantly increase the secretion of serum IFN-y,inhibit the expression of IL-4 and IL-13,and increase the ratio of IFN-y/IL-4 on the 14th day.On the 28th day,compared with the model group,Zukamu granules could promote the secretion of IFN-y,significantly inhibit the expression of IL-4 and IL-13,and significantly increase the ratio of IFN-y/IL-4.In terms of immunoglobulin,compared with the model group,the contents of IgG1 and IgE in Zukamu group decreased significantly on the 14th day(P<0.01),the content of IgA also decreased(P<0.05).On the 28th day,the contents of IgA and IgE in Zukamu group were significantly lower than those in model group(P<0.01),and the content of IgG1 in Zukamu group was lower than that in model group(P<0.05).The results of correlation analysis showed that IgA,IgE and IgG1 were positively correlated with IL-4 and IL-12(P<0.01),but negatively correlated with IFN-y and IL-13(P<0.01).In terms of the expression of pathway protein,Zukamu granules could significantly inhibit the expression of GATA3 and p-STAT6 in lung tissue on the 28th day(P<0.01).Conclusion:Zukamu granules could regulate lung injury,inhibit Th1 cytokine secretion in acute lung injury,inhibit Th2 cytokine secretion,GATA3 expression and STAT6 phosphorylation,and up-regulate Th1 cytokine level in the fibrotic stage of lung injury.Combined with theoretical research and network prediction,Zukamu granules for immune regulation was mainly carried out by restoring the dynamic balance of Th1/Th2 cytokines. |
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