Study on mechanism of Miao medicine Aletris spicata regulating NF-κB/JAK pathway to improve acute lung injury in rats
Objective To explore the mechanism of the preventive and therapeutic effects of Aletris spicata on acute lung injury(ALI)induced by lipopolysaccharide(LPS)in rats through network pharmacology and experimental verification.Methods Review the relevant literature on A.spicata and use the SwissADME database to screen the active components of A.spicata.Predict the potential targets of the active components of A.spicata using the Swiss Target Prediction platform.Use the GeneCards database,DrugBank database,and OMIM database to search for genes related to ALI,compare them with the targets of the active compounds,and draw a Venn diagram to obtain the intersection targets.Input the intersection target information into the String database,select targets with a comprehensive score of 0.9 or above,and use Cytoscape 3.9.0 software to obtain a protein-protein interaction(PPI)network.Use the Metascape database to perform Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis on key targets,and select pathways related to ALI.Draw a"key component-target-pathway"network diagram using Cytoscape 3.9.0 software.Randomly divide SD rats into a control group,a model group,a dexamethasone(positive drug,5 mg·kg-1)group,and A.spicata water extract(ASWE)high,medium,and low dose(9.00,4.50,2.25 g·kg-1)groups.Continuously administer the rats with the drugs by ig for 7 days,ip LPS(5 mg·kg-1)to induce the ALI model 7 days later,and observe and analyze the pathological changes in the lung tissue.Detect the expression levels of inflammatory factors in the serum and oxidative stress indicators in the lung tissue using kit assays,and detect the mRNA levels of β-activated protein kinase 2(TAK2),Janus kinase 2(JAK2),nuclear factor kappa B(NF-κB),Nras,signal transducer and activator of transcription(STAT),and Ensa in the lung tissue using real-time quantitative PCR(qRT-PCR),and detect the expression levels of NF-κB and JAK2 proteins in the lung tissue using Western blotting experiments.Results Ten main active compounds of A.spicata were screened out,including methyl salicylate,coumarin,ursolic acid,etc.A total of 189 key targets were identified for A.spicata acting on ALI,with MAPK1,MAPK8,STAT3,AKT1,PIK3R1,TP53,ESR1,and RELA as core targets.The KEGG enrichment analysis showed that A.spicata may exert its therapeutic effects through MAPK,NF-κB,and JAK-STAT signaling pathways.Compared with the model group,the pulmonary tissue pathological manifestations of inflammatory cell infiltration in the ASWE treatment groups were significantly improved.The TNF-α level was significantly lower in the middle and high-dose groups(P＜0.05),and the IL-1β level was significantly lower in the middle-dose group(P＜0.05).The IL-6 level showed a downward trend in all dosage groups;the SOD and GSH-Px levels were significantly higher in the high and middle-dose groups(P＜0.05);The Nras,STAT3,MAPK1,JAK2,Ensa,and NF-κB mRNA levels were significantly lower in the high and middle-dose groups(P＜0.05,0.01).The expression levels of NF-κB and JAK2 proteins were significantly lower in the middle-dose group(P＜0.05,0.01).Conclusions A.spicata can inhibit the excessive secretion of inflammatory mediators,regulate the balance of oxidative stress and have a preventive and therapeutic effecton ALI.The mechanism may be related to inhibiting the expression of MAPK1,NF-κB,STAT3 and other genes,thereby inhibiting the expression of NF-κB/JAK and other inflammatory signaling pathways.
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