Predicting The Of Jie Geng Tang Q-marker And Exploring The Improvement Mechanism Of Acute Lung Injury

JGT was first recorded in the Shang-Han-Lun.It is efficacious in improving lung diseases.However,the quality markers(Q-markers)and pharmacological mechanism of JGT are not clear.This has seriously hindered the scientific and accurate application of JGT.This study aimed to investigate the Q-markers of JGT through network pharmacology and experimental validation.The details are as follows:1.Prediction of potential Q-markers of JGT1.1 Scientometric-based analysis of the literature with JGT and acute lung injury(ALI)This study was based on the core ensemble database of Web of Science to collect and organize the previously published literature related to JGT and ALI.The keyword enrichment co-occurrence network analysis was performed using Citespace on the 381 retrieved documents,and the co-occurrence network results showed that: 1.Glycyrrhetinic acid,18β-glycyrrhetinic acid,Licochalcone A,glycyrrhizin,and Platycodin D,which are characteristic components of licorice and Platycodon grandiflorus,have been paid more attention by scholars and maybe the more critical active components in JGT.2.Lipopolysaccharide(LPS),Klebsiella,Staphylococcus aureus,etc.are commonly used as modeling agents in ALI models.When the above substances are exposed to the organism or cells,they often induce pro-inflammatory factor release,ROS overproduction,and apoptosis.3.Targets such as NF-κB,TNF-α,COX-2,NLRP3,IL-1β,IL-17,ASC,TLR4,IL-18,and Bcl2 are widely enriched in the co-occurrence network,while NF-κB signaling pathway,inflammation,oxidative stress,apoptosis,focal death,autophagy,cell cycle block,neutrophil recruitment,ROS production,and other keywords were enriched co-present.The improvement of ALI by JGT may be closely related to these enriched targets,signaling pathways,and biological processes.1.2 Network pharmacological analysis of JGT and potential Q-markersBy compiling disease-related databases,human gene database(Gene Cards),online Mendelian genetic database for humans(OMIM),pharmacogenetics and pharmacogenomics database(Pharm Gkb),therapeutic target database(TTD),and drug bioinformatics common database(Drug Bank),Chinese medicine related databases,Chinese medicine systemic pharmacology(TCMSP)and the Bioinformatics Analysis Tool for Molecular Mechanisms in Chinese Medicine(BANTMAN-TCM)databases were collated to obtain a total of 1386 disease targets and 1170 component targets,of which 94 targets were the intersection of the two.The 94 intersecting targets were analyzed for KEGG and GO enrichment.There were 3673 biological process entries,356 molecular function entries,238 cellular component entries,and 227 signaling pathways.The biological processes involved were mainly related to lipopolysaccharide response,oxidative stress response,regulation of apoptotic signaling pathways,and regulation of NF-κB transcription factor activity.This may be related to the molecular functions involved,such as cytokine activity,signaling receptor activator activity,transcriptional core factor binding,and antioxidant activity.In addition,the 94 targets mentioned above were significantly enriched in the TNF signaling pathway,apoptosis,NF-κB signaling pathway,etc.1.3 Screening analysis of the active ingredients of JGTA combination of scientometric and TCM-related databases(TCMSP and BANTMAN-TCM)was used to collect the active ingredients of JGT.The results showed that a total of 34 active ingredients in JGT were obtained from the scientometric analysis;191ingredients were obtained from the TCMSP database.Of these 191 components,165 were derived from licorice and 26 from Platycodon grandiflorus.Comparative analysis of the active ingredients derived from the two fractions yielded 13 intersecting components.Through further screening,five active ingredients were found to meet the requirements of quality markers(specificity,traceability,and association with TCM theory).These five active ingredients were liquiritin,glycyrrhizic acid,platycodin D,glycyrrhetinic acid,and Licochalcone A.They may be potential Q-markers for JGT.1.4 Comparison of GO and KEGG enrichment analysis of potential Q-marker targets with the active ingredient targets of JGTThe results of GO and KEGG enrichment analysis of Q-markers of JGT with disease intersection targets and the results of GO and KEGG enrichment analysis of intersection targets of active ingredient targets of JGT with disease targets were compared.The results of biological processes showed that there were 2818 intersection entries between the two,accounting for70.41% of all biological process results.The results of the molecular function fraction showed238 intersecting entries for both,accounting for 53.85% of all molecular function results.The results of the cellular fraction section showed that both had 160 entries,accounting for 59.47%of all cellular fraction results.the results of the KEGG section showed that both had 206 intersecting signaling pathways,accounting for 86.55% of all KEGG results.Combined with the analysis of both GO and KEGG enrichment results,the potential Q-marker targets overlap well with the targets of JGT components.This indicates that the potential Q-markers obtained from the screening of JGT may be highly similar to the process of JGT in ameliorating acute lung injury,tentatively indicating the accuracy of the potential Q-markers predicted by the analysis.1.5 Preparation of JGT and determination of its potential Q-markers contentThe decoction was made up of 1 part of Platycodon grandiflorus and 2 parts of licorice,with the material-liquid ratio of 1:10,and decocted twice in boiling water.the fingerprint results of 10 batches of samples showed that the relative standard deviations of peak retention time and peak area were less than 0.56% and 15.91%,respectively.The correlation coefficients between the chromatograms of different batches and the chromatograms of selected samples ranged from0.894 to 0.992,and the peaks of herbs from different batches had high similarity with small differences in the contents of samples from different batches.Next,we clarified the optimal detection wavelengths for the potential quality markers.The results showed that the optimal detection wavelengths were 201,270,250,250 and 375 nm for platycodin D,liquiritin,glycyrrhizic acid,glycyrrhetinic acid,and licochalcone A.The contents of the potential Q-markers in the lyophilized powder of JGT were 4.2 mg/g,7.36 mg/g,12.1 mg/g,76.7 ug/g and 4.14 ug/g,respectively.The results indicate that the potential Q-markers satisfy the measurability and traceability of the Q-markers.1.6 Molecular Docking and Molecular Dynamics Simulation of Potential Q-markers and Core TargetsCombined network pharmacology and scientometric results identified NF-κB and Caspase3 as possible key targets of JGT for ameliorating acute lung injury.Molecular docking and molecular dynamics simulations of potential mass markers with NF-κB and Caspase 3,respectively,were performed.The results showed that the potential Q-markers could stably bind to NF-κB and Caspase 3.The molecular docking results showed that the receptor protein binds to the small molecule ligands as long as they are bound by van der Waals forces and hydrogen bonds.Combining the results of RMSD,RMSF,Rg,SASA,and the number of hydrogen bonds involved in the binding process,it was found that liquiritin and platycodin D bound more stably to Caspase 3,and licochalcone A bound more stably to NF-κB.It is hypothesized that liquiritin and platycodin D may be the key active ingredients in JGT to inhibit the onset of apoptosis during the improvement of ALI,and licochalcone A may be the key active ingredient in JGT to inhibit the further increase of inflammation during the improvement of ALI.2.Experimental validation of potential Q-markers in vivoIntratracheal drops of LPS were first used to establish an in vivo ALI mouse model.Histopathological examination revealed edema,congestion,alveolar collapse,and inflammatory cell infiltration observed in the model mice,and these pathological changes were significantly improved in the treatment groups with Kratom soup(250 or 500 mg/kg)and potential mass markers(5 and 10 mg/kg).In addition,treatment with JGT and potential Q-markers significantly reversed the LPS-induced lung wet weight to dry weight ratio and the expression of inflammatory factors and proteins(i NOS,COX2,TNF-α,and IL-1β)in alveolar lavage fluid and serum.In addition,JGT and potential Q-marker treatment groups improved LPS-induced apoptosis(Cleaved-Caspase 3,Bax and Bcl2),inflammation(TLR4,p-IKKβ,p-IKKα,and p-IκBα),scorching(p-NF-κB,NLRP3,ASC,Cleaved-Caspase 1 and Cleaved-GSDMD)-related protein expression.The results of in vivo experiments showed that both JGT and potential Q-markers could ameliorate LPS-induced acute lung injury through the modulation of apoptosis,focal death,and inflammation.3.Experimental validation of potential Q-markers in vitroThe results of in vitro experiments showed no significant toxic and value-added effects of tangerine broth(5 and 10 ug/ml)and potential mass marker(0.5 and 1 ug/ml)administration groups on RAW264.7 cells.In addition,the administration groups inhibited LPS-induced ROS production,mitochondrial membrane potential disturbance,LDH,NO,cytokine secretion and inflammatory factor proteins(i NOS,COX2,TNF-α,and IL-1β),apoptosis(Cleaved-Caspase 3,Bax and Bcl2),inflammation(TLR4,p-IKKβ,p-IKKα,and p-I κBα),and scorch death(p-NF-κB,NLRP3,ASC,Cleaved-Caspase 1,and Cleaved-GSDMD)-related protein expression levels.The results of in vitro experiments showed no significant toxic and value-added effects of tangerine broth(5 and 10 ug/ml)and potential mass marker(0.5 and 1 ug/ml)administration groups on RAW264.7 cells.In addition,the administration groups inhibited LPS-induced ROS production,mitochondrial membrane potential disturbance,LDH,NO,cytokine secretion and inflammatory factor proteins(i NOS,COX2,TNF-α,and IL-1β),apoptosis(Cleaved-Caspase 3,Bax and Bcl2),inflammation(TLR4,p-IKKβ,p-IKKα,and p-I κBα),and scorch death(p-NF-κB,NLRP3,ASC,Cleaved-Caspase 1,and Cleaved-GSDMD)-related protein expression levels.A combination of in vivo and in vitro experiments further validated the Q-markers.In conclusion,we clarified the Q-markers of JGT by network pharmacology,scientometrics,HPLC analysis,in vivo experimental validation,and in vitro experimental validation strategies.The results clearly showed that platycodin D,liquiritin,glycyrrhizic acid,glycyrrhetinic acid,and licochalcone A met the requirements as Q-markers of JGT.Meanwhile,this study is expected to provide new ideas and methods for quality control of Chinese medicine and provide strong support for clinical application and promotion of Chinese medicine.