| Background:Zukamu granules(ZKMG)is a classical compound Uygur medicine commonly used in clinical practice,which is included in the "National Model Drug Catalogue for Direct Network Procurement of Emergency(Rescue)Drugs".Clinical studies have shown the efficacy of Zukamu granules in acute upper respiratory tract infections.Acute lung injury(ALI)is an acute and persistent respiratory emergency with high morbidity and mortality,which seriously threatens human health.This research paper,supported by the National Natural Science Foundation of China(81860803),further explores the pulmonary protective mechanism of Zukamu granules at the molecular level,which is important for the clinical promotion and application of Zukamu granules as well as the search for new therapeutic strategies for ALI.Objective:Bioinformatics was used to predict the targets and potential pathways of Zukamu granules in the treatment of ALI.Pharmacodynamic experiments were conducted to explore the effects of Zukamu granules on NLRP3 inflammatory vesicle activation and its mediated cell scorching in ALI mice,with a view to validating the targets and pathways predicted by the network.Methods:(1)Prediction of key targets and signaling pathways of Zukamu granules for the treatment of ALI:①The chemical composition and targets of the 10 flavors of Zukamu granules were collected through TCMSP database and literature search,and the active ingredients of Zukamu granules were screened with OB%≥30%and OL≥0.18.Target replenishmet and standardization through Swiss Target Prediction database and UniPort database.②GeneCards,OMIM database were used to obtain the disease targets of ALI,GEO database to obtain the differentially expressed genes of ALI,and jvenn online tool was used to obtain the common targets of Zukamu granules and ALI.③Construction of PPI networks of commons targets and module analysis by STRING database and Cytoscape 3.7.2 software,and KEGG and GO enrichment analysis of key targets and key target protein modules by DAVID database.To build "drug-active ingredient-critical target-disease" and "critical target-pathway" networks.(2)The effect of Zukamu granules on the pathology of ALI mice:The protective effect of Zukamu granules on ALI mice was verified by observing the histopathological changes of lung,MPO activity and macrophage F4/80 protein expression in each group of mice.(3)Effect of Zukamu granules on the balance of pro-inflammatory and anti-inflammatory factors in ALI mice:The effect of Zukamu granules on immune and inflammatory homeostasis in ALI mice was analyzed by measuring the levels of Th17 characteristic cytokine IL-17A and Treg characteristic cytokine IL-10 in lung tissue and serum to validate the IL-17 signaling pathway and Th17 cell differentiation predicted by the network.(4)Effects of Zukamu granules on activation of NLRP3 inflammasome and pyroptosis in ALI mice:①Based on the network-predicted NOD-like receptor signaling pathway,the NLRP3 inflammatory vesicle activation mechanism and its mediated classical cell scorching pathway in ALI were used as entry points in conjunction with literature studies.The mRNA and protein expression levels of NLRP3 and the protein expression levels of Caspase-1,Cleaved-caspase-1 p20,ASC and GSDMD were measured in the lung tissue of each group of mice.②The expression levels of IL-1β and IL-18 in the lung tissues and serum were measured to investigate the effect of Zukamu granules on the maturation of IL-1 family cytokines mediated by NLRP3 inflammatory vesicle activation in ALI.Results:(1)Prediction of the key targets and signaling pathways of Zukamu granules in the treatment of ALI:①The drug components and targets were collected through various databases and literatures,and a total of 174 active ingredients and 825 targets of Zukamu granules were obtained.②1253 targets of ALI were screened from the disease database.Three datasets(GSE10474,GSE3037 and GSE2322)were downloaded from the GEO database,and a total of 1084 significantly differentially expressed genes were obtained after analysis and screening.After intersection of drug targets,disease targets and differential genes,46 common targets were obtained.③The PPI network contained 46 nodes and 487 edges,and MCODE analysis yielded 2 key protein modules.KEGG and GO enrichment analysis of the 46 key targets and 2 protein modules respectively show that the predicted pathways are enriched in various aspects such as inflammatory response,immune regulation,anti-infection,anti-cancer,anti-apoptosis and oxidative stress.The number of signaling pathways related to immune and inflammation was the largest.Immune-related pathways were mainly enriched in IL-17 signaling pathway,NOD-like receptor signaling pathway,etc.GO analysis suggested that biological processes such as inflammatory response,immune response,apoptosis,and cellular response to reactive oxygen species played key roles.In the "drug-active ingredient-key target-disease" network,the top 7 active ingredients with degree value included quercetin,rutin,kaempferol,luteolin,and naringenin,apigenin,and sennoside,the above ingredients were derived from the 9 herbs in the prescription.The "key target-pathway"network suggested that Zukamu granules may act on key targets such as RELA,TNF,MAPK1,MAPK3,IL-6,IL-1β through IL-17 signaling pathway and NOD-like receptor signaling pathway to play a protective effect on ALI lung.(2)Effect of Zukamu granules on lung histopathology in ALI mice:①The lung tissue of the model mice showed obvious inflammatory cell infiltration,alveolar structural disorder and interstitial thickening,indicating successful ALI modeling.The number of inflammatory cell infiltration and alveolar structural changes in the lung tissues of the Zukamu group were remarkably decreased compared with the model group.②The lung tissue scores and MPO levels were significantly lower than the model group(P<0.01).③Immunohistochemical results of F4/80 in lung tissue shows that the positive expression of F4/80 in lung tissue of the model group was significantly higher than that of the sham-operated group,and the expression level of F4/80 in the Zukamu group was significantly lower than that of the model groups(P<0.01).(3)Effect of Zukamu granules on the balance of pro-inflammatory and anti-inflammatory factors in ALI mice:Zukamu granules could significantly reduce the expression of IL-17A in lung tissues and serum of ALI mice(P<0.01)and promote the expression of IL-10 in lung tissues(P<0.05),but had no significant effect on the expression of IL-10 in serum(P>0.05).Zukamu granules reduced IL-17A/IL-10 ratio in lung tissue(P<0.01)and IL-17A/IL-10 ratio in serum of ALI mice(P<0.05),and restored the balance of inflammatory and anti-inflammatory cytokines in ALI mice.(4)Effects of Zukamu granules on activation of NLRP3 inflammasome and pyroptosis in ALI mice:①In terms of NLRP3 inflammatory vesicle activation and mediated pyroptosis pathway,Zukamu granules significantly inhibited the expression levels of NLRP3 mRNA in lung tissues of ALI mice(P<0.01)and significantly inhibited the expression of NLRP3,ASC,Caspase-1 and GSDMD protein(P<0.01).The protein expression of Cleaved-caspase-1 p20 was down-regulated,but the difference was not statistically significant(P>0.05).②Zukamu granules reduced the expression levels of IL-1β and IL-18 in lung tissue and serum of ALI mice(P<0.01).Conclusions:Zukamu granules can reduce macrophage and neutrophil infiltration in the lung tissue of ALI mice and restore the balance of inflammatory and anti-inflammatory factors in the ALI mice.and its mechanism of action is related to the inhibition of excessive activation of NLRP3 inflammatory and pyroptosis,it will provide a new therapeutic strategy for clinical treatment of ALI. |
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