The Preventive Effects Of Xiyanping Injection On Acute Lung Injury Induced By Lipopolysaccharide

Objective:Firstly,network pharmacology methods were used to preliminarily screen the targets and related pathways of Xiyangping injection for the prevention and treatment of acute lung injury(ALI).Then,a lipopolysaccharide(LPS)-induced acute lung injury model in mice was constructed to observe the preventive and therapeutic effects of Xiyanping injection on acute lung injury and preliminarily explore its mechanism of action by observating the effects of Xiyanping injection on pathomorphological changes of lung tissue,changes of inflammatory cytokines,oxidative stress indicators and expression of inflammatory signal pathway factors.Methods:Network pharmacology research methods:1.The targets of chemical active ingredients of Xiyanping injection were found and screened by the TCMSCP database analysis platform TCMSCP and the Bioinformatics Analysis Tool for Molecular mech ANism of Traditional Chinese Medicine(BATMAN-TCM)database,which were imported into the Unipot database for gene standardization processing.2.By searching for Gene Cards,human Mendelian genetic data OMIM,with "Acute Lung Injury" as the keyword,the targets related to the disease were obtained,all targets from two databases were integrated in the Excel,the duplicate genes were eliminated,and the target genes of acute lung injury were obtained by the Uniprot database.3.The screened drug ingredient targets and disease targets were input into the Venn diagram software for mutual mapping,and then a Venny diagram was made to obtain the intersection genes.Then,the "Drug-Active Ingredient-Target" network was constructed using Cytoscape3.7.2 software.4.The drug targets and disease targets were uploaded to the String protein-protein interaction database to construct a protein-protein interaction(PPI)network,with the species set as "Homo sapiens".Cytoscape3.7.2 software was used to construct the network diagram of Xiyangping injection and acute lung injury target proteins,and key targets for Xiyangping injection against acute lung injury were screened.5.GO enrichment analysis and KEGG pathway analysis were performed on key targets,and the results of GO and KEGG analysis were visualized after deleting non-pathway entries.6.The key target structures and drug ingredient structures were processed into "pdbqt" format files using Auto Dock Tools(v1.5.6),and the active pocket positions were defined separately.Then,molecular docking was performed using Auto Dock Vina 1.2.Finally,Py MOL 2.4.0 was used for the visualization.Experimental research methods:1.A single intratracheal instillation of LPS was used to induce an animal model of acute lung injury in mice.The mice were randomly divided into 6 groups: normal control group(NS),ALI model group(LPS),low(XYP-L),medium(XYP-M),and high(XYP-H)dosage groups of Xiyanping injection,and dexamethasone positive drug group(DXM).Xiyanping injection(75.83 mg/kg,37.92 mg/kg,18.96 mg/kg)and dexamethasone(3.03 mg/kg)were injected intraperitoneally once a day at the same time for 3 consecutive days before intratracheal instillation of LPS.LPS was instilled into the trachea to induce acute lung injury in mice 1hour after the last administration of the above drugs.The samples of lung tissue and BALF of mice were collected 24 hours after intratracheal instillation of LPS,and the average weight and lung coefficient of mice were calculated.2.The lung tissue sections were stained with HE,and the pathological injury of lung tissue in mice was observed under a light microscope,the inflammation degree of pathological tissue sections was scored to assess the degree of lung injury.3.BCA method was used to determine the content of total protein in BALF;Total cells in BALF were counted under microscope;ELISA(enzyme linked immunosorbant assay)was used to detect the contents of inflammatory cytokines TNF-α,IL-1β,and IL-8 in BALF.4.Malonaldehyde(MDA)and Superoxide Dismutase(SOD)kits were used to assess the content of MDA and the activity of SOD in lung tissue of mice.5.Western blot was used to detect the protein expression of inflammation signal pathways related factors including p38,JNK,Er K1/2,and NF-κB p65 in lung tissue of mice.Results:Network pharmacology research results:1.In the TCMSCP and BATMAN-TCM databases,four active ingredients2.related to andrographolide were found: 14-deoxy-11-oxo-andrographolide,14-deoxyandrographolide,14-deoxy-12-methoxyandrographolide,and3.Andrographolide-19-β-D-glucoside＿qt.They were imported into Swiss Target Prediction and Super Pred databases,and a total of 288 corresponding drug targets were obtained(excluding duplicates).Gene Cards and OMIM databases were used to search for gene targets of acute lung injury,and a total of 4960 disease targets were obtained after removing the duplicates.The analysis results of Wayne demonstrated that there were 192 key targets against acute lung injury with Xiyanping injection.4.According to the screening results of the STRING database,the core targets of Xipengling injection for anti-acute lung injury mainly include STAT3,EGFR,PIK3R1,MAPK1,PIK3 CA,NFKB1,ESR1,MAPK8,JAK2,FYN,etc.5.The results of GO and KEGG pathway enrichment analysis demonstrated that the anti-acute lung injury effect of Xiyanping injection may be related to signaling pathways such as MAPK,TNF-α,VEGF,c AMP,m TOR,AMPK,NOD,JAK-STAT,IL-17,and NF-κB.6.Molecular docking results demonstrated that the key targets MAPK1,IKBKB,MAPK8,NFKB1 had a strong binding affinity with the active ingredients 14-deoxy-12-methoxyandrographolide and 14-deoxyandrographolide.Experimental research results:1.The lung coefficients of ALI mice induced by LPS were significantly increased.Xiyanping injection effectively alleviated the edema of lung tissue in mice and significantly reduced the lung coefficients of ALI mice.2.Significant inflammatory reaction was found in the lungs of LPS-induced ALI mice,including a large number of inflammatory cells infiltration and the destruction of alveolar structure,and the pathological and histological inflammation scores were significantly increased.Xiyanping injection significantly improved the degree of lung injury,reduced the pathological and histological inflammation scores,and alleviated inflammatory reaction in the lungs of ALI mice.3.The total protein concentration,cell number and the contents of inflammatory cytokines TNF-α,IL-1β and IL-8 in BALF of ALI mice induced by LPS increased significantly.Xiyanping injection significantly reduced the total protein concentration and cell number in BALF of ALI mice,and decreased the levels of inflammatory cytokines TNF-α,IL-1β and IL-8.4.MDA content increased significantly in lung tissue of ALI mice induced by LPS,while endogenous antioxidant SOD activity decreased significantly.Xiyanping injection significantly lowered MDA levels while increased SOD activity in lung tissues of mice.5.LPS induced the activation of MAPK and NF-κB p65 signaling pathways in lung tissues of ALI mice,and significantly upregulated the expression of p38,JNK,Erk1/2 and NF-κB p65 proteins.Xiyanping injection significantly downregulated the expression of p38,JNK,Er K1/2 and NF-κB p65 proteins in lung tissues of ALI mice,and inhibited the activation of MAPK and NF-κ B signaling pathways.Conclusions:Xiyanping injection has a certain preventive effect on LPS-induced ALI in mice,and its mechanism may be to alleviate lung inflammation by inhibiting the activation of MAPK,NF-κB signaling pathway and releasing downstream inflammatory factors,and to inhibit oxidative stress in lung tissue caused by LPS through its antioxidant effect.This study can provide a reference for the ALI treatment with Xiyanping injection,and provide a new drug candidate for the prevention and treatment of acute lung injury.