Study On The Mechanism Of Shenling Baizhu San In The Treatment Of COPD

Objective: To investigate the material basis and possible mechanism of Shenling Baizhu San in the treatment of chronic obstructive pulmonary disease(COPD).Methods: Ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-Q-TOF-MS/MS)technology was used to qualitatively analyze the chemical compositions of Shenling Baizhu San medicated serum and network pharmacology was applied to predict the possible mechanism of Shenling Baizhu San in the COPD by network pharmacology.Pub Chem and Swiss Target Prediction database were adapted to predict the targets of chemical components in Shenling Baizhu San medicated serum.Gene Cards and TTD database were used to predict COPD disease targets.Venny2.1 online mapping tool was applied to obtain the potential targets of Shenling Baizhu San in treating COPD.STRING database and Cytoscape 3.7.2 software were used to construct PPI protein interaction network.Metascape database was used to perform GO and KEGG analysis of potential targets.MOE software was used to perform molecular docking between potential targets(ALB,AKT1,TNF,IL-6,VEGFA)and compounds(Quercetin,Protopanaxatriol,Apigenin).Based on the predicted targets and signaling pathway of network pharmacology,human bronchial epithelial cells(16HBE)were induced by cigarette smoke extract(CSE)solution to establish a COPD cell model in vitro,and the cells were intervented with Shenling Baizhu San medicated serum.CCK-8 and ELISA were used to screen the optimal concentration and time of CSE solution and Shenling Baizhu San medicated serum for the intervention of cells.High connotation cell imaging system was applied to detect cell apoptosis.The levels of TNF-α,IL-6 and IL-8 secreted by 16 HBE cells were detected by ELISA,while the nuclear translocation of NF-κB p65 protein was detected by immunofluorescence.q RT-PCR and Western Blot technology were used to detect the m RNA and protein expression of TLR9/NF-κB signaling pathway related molecules.Results:1.A total of 108 chemical components(30 prototype components and 78 metabolites)in the Shenling Baizhu San medicated serum were identified and analyzed,such as ginsenoside Rb1,ginsenoside Rg1,pachymic acid,ysin Ⅰ,bamboo ginsenoside V methyl ester,glycyrrhizic acid,platycodin A,etc.2.A total of 292 potential targets of Shenling Baizhu San in the treatment of COPD were predicted and obtained.GO enrichment results showed that biological processes mainly included inflammatory response and response to oxygen level,etc.Cell composition mainly included membrane rafts,perinuclear regions of cytoplasm,etc.The molecular functions were primarily involved in protein kinase binding and nuclear receptor activity and so on.KEGG pathway included regulation inflammatory mediator in TRP channel,necroptosis,NF-κB signaling pathway,etc.The results of molecular docking showed that 5 targets had good binding with 3 molecular compounds.3.The optimal concentration of CSE was 6%,and the action time was 24 hours.The optimal concentration and time of Shenling Baizhu San medicated serum on 16 HBE cells were 10% and 24 h,respectively.Compared with the CSE group,the apoptosis rate of Shenling Baizhu San medicated serum group was significantly decreased(P<0.05),and the levels of TNF-α,IL-6 and IL-8 in the culture supernatant declined observably(P<0.01).The expression of NF-κB p65 protein was significantly increased markedly in the cytoplasm while decreased in the nucleus.The expressions of TLR9,My D88,TRAF6 and RELA m RNA in 16 HBE cells were significantly decreased(P<0.05)and the NFκBIαexpression was significantly increased(P<0.01).The relative expressions of TLR9,My D88 and TRAF6 protein were significantly decreased(P<0.01).The ratios of p-NF-κB p65/ NF-κB p65 and p-IκBα/ IκBα were decreased significantly(P<0.01).Conclusion:1.The UPLC-Q-TOF-MS/MS method could be commendably used for the identification of the chemical components of Shenling Baizhu San medicated serum.2.Network pharmacology studies suggest that Shenling Baizhu San may act on TNF,IL-6,TLR9 and other targets,and play an anti-inflammatory and anti-apoptosis effect through NF-κB signaling pathway,necroptosis and other pathways,which may be the mechanism of action of this recipe in the treatment of COPD.3.The Shenling Baizhu San medicated serum can reduce the levels of inflammatory factors and cell apoptosis by regulating TLR9/NF-κB pathway,which may be one of the mechanisms of Shenling Baizhu San in the treatment of COPD.