Study On The Mechanism Of Qing-Jin-Hua-Tan Decoction Inhibiting The Formation Of NETs And Alleviating Acute Lung Injury

BackgroundIn recent years,the global spread of COVID-19 has aroused people’s attention to lung diseases.The pathogenesis of acute lung injury(ALI)is very complex,with high morbidity and mortality.The classic famous prescription Qing-Jin-Hua-Tan-Decoction(QJHTD)has the effect of clearing heat and nourishing the lungs,and it is mainly used for lung diseases caused by phlegm-heat stagnation in the lungs.The clinical curative effect of QJHTD is clear,but the mechanism of QJHTD in the treatment of ALI is still unclear.Therefore,modern pharmacological research methods are urgently needed to evaluate the protective effect of QJHTD on ALI,reveal its mechanism of action,and provide a theoretical basis for the clinical application of QJHTD in ALI.Research contentChapter 1 Experimental results of the protective effect of QJHTD on LPS-induced ALI in mice132 healthy male C57BL/6J mice were randomly divided into the following 6 groups:Control group,ALI group,QJHTD low,medium and high dose groups and dexamethasone(DEX)group.The drug was administered once a day,and PBS(Control group)or LPS(other groups)was instilled into the trachea 1 hour after the administration on the 7th day,and the samples were collected 1 hour after the administration on the 8th day.HE staining was used to observe the pathological changes of the lung tissue of the mice in each group and score the degree of lung injury.The infiltration of neutrophils and platelets in the lung tissue was observed by immunohistochemistry,and the number of white blood cells and platelets in the blood was measured by a blood cell counter.Dry weight ratio and MPO activity were measured,Western blot was used to detect the expression of CD41 in lung tissue,BCA method was used to measure the total protein concentration of BALF,ELISA was used to measure TNF-α,IL-1β,IL-6 and TAT complexes content.By evaluating these indicators,the protective effect of QJHTD on LPS-induced ALI in mice was explored.Chapter 2 Exploring the Mechanism of QJHTD in the Treatment of ALI Based on Network Pharmacology and TranscriptomicsIn the network pharmacology experiment,22 prototype components of QJHTD that entered the blood were taken as the research objects,and the chemical component targets and ALI disease targets were queried through the database,and the functional enrichment analysis was performed on the targets of QJHTD for ALI.To explore its possible mechanism for the treatment of ALI,build a "component-target-pathway" network,use molecular docking technology to carry out molecular docking of QJHTD blood components and core targets,and further verify the molecular docking results through SPR technology.Using the transcriptomics method to screen out the differential genes in the lung tissue of ALI mice treated with QJHTD,and through the enrichment analysis of GO and KEGG signaling pathways,analyze which biological processes,molecular functions,cellular components and signaling pathways QJHTD mainly affects to prevent and treat ALI.Finally,combined with the prediction results of network pharmacology,the possible pathways of QJHTD were selected for verification.Chapter 3 Verification of the Mechanism of QJHTD in Treating ALIFirst,PMA,the most commonly used inducer of neutrophil extracellular traps(NETs)in vitro experiments,was used to induce neutrophils to produce NETs.The effects of QJHTD on the release of NETs induced by PMA were studied by means of fluorescence microplate reader,immunofluorescence staining and flow cytometry.Secondly,in vivo experiments were conducted to study the ability of QJHTD to inhibit the release of NETs in ALI mice induced by LPS by detecting the specific marker citrullinated histone(CitH3)of lung tissue NETs formation(CitH3)and the content of free DNA in BALF,and used Spearman Correlation analysis method The correlation analysis was carried out between NETs and inflammatory cytokines TNF-α,IL-6,IL-1β and coagulation marker TAT Complexes.Finally,LPS-stimulated platelets were used as inducers to induce NETs formation.The effects of QJHTD on the formation of platelet-induced NETs and whether QJHTD inhibits the formation of platelet TLR4-reduced NETs were investigated under flow and static conditions.ResultsChapter 1 Experimental results of the protective effect of QJHTD on LPS-induced ALI m miceThrough experiments,it was found that QJHTD can improve the pathological changes of lung tissue in ALI mice,inhibit the infiltration of neutrophils and platelets in lung tissue,increase the number of white blood cells and platelets in peripheral blood,and reduce the wet-dry weight ratio and MPO activity of lung tissue.The total protein concentration,TNFa,IL-1β,IL-6 and TAT complexes in BALF of ALI mice were reduced.These evaluation indexes indicate that QJHTD has a protective effect on LPS-induced ALI in mice.Chapter 2 Exploring the Mechanism of QJHTD in the Treatment of ALI Based on Network Pharmacology and TranscriptomicsThe results of network pharmacology showed that baicalin,oroxylin A-7-glucuronide,hispidulin-7-O-β-D-glucuronide,wogorin,baicalein,wogonin and mangiferin might be the main active components of QJHTD in the treatment of ALI,CDK2,EGFR,AKT1,F2,SRC and MAPK14 are considered as the main targets.At the same time,SPR experiment also confirmed that baicalein,wogonin and baicalin had strong affinity with F2.The results of KEGG enrichment analysis suggested that the treatment of ALI by QJHTD was closely related to pathways such as lipid and atherosclerosis,HIF-1 signaling pathway,reninangiotensin system and NETs formation.The results of transcriptomic showed that GO enrichment biological process analysis,it was found that the differential genes of QJHTD treatment of ALI were mainly enriched in the regulation of cell activation,regulation of defense response,neutrophil migration and regulation of neutrophil degranulation,and regulation of platelet aggregation,leukocyte cellcell adhesion and inflammatory response,etc.KEGG pathway enrichment results related to osteoclast differentiation,TNF signaling pathway,NETs formation,JAK-STAT signaling pathway,IL-17 signaling pathway,etc.KEGG pathway analysis of network pharmacology and transcriptomics indicated that NETs formation pathway was significantly enriched,suggesting that it may be the main regulatory pathway for QJHTD treatment of ALI.Chapter 3 Verification of the Mechanism of QJHTD in Treating ALIIn vitro experiments showed that QJHTD could inhibit the release of NETs induced by PMA,and in vivo experiments showed that QJHTD could reduce the content of Citrullinated Histone H3(CitH3)in lung tissue and cell free DNA(cf-DNA)in BALF,indicating that QJHTD could inhibit the release of NETs induced by LPS in ALI mice.Correlation analysis results showed that the content of NETs in the process of ALI was closely related to TNF-α,IL-6,IL-1β and TAT Complexes,indicating that QJHTD may reduce the inflammation and coagulation of ALI by inhibiting the formation of NETs.In order to reflect the whole process of NETs formation in vivo,LPS-stimulated platelets were used as inducer to induce NETs formation.The results showed that QJHTD reduced the combination of LPS and platelets,inhibited LPS-stimulated platelet NETs formation and decreased the supernatant vWF level during NETs formation.Conclusion1.This study found that the administration of QJHTD has a significant therapeutic effect on LPS-induced ALI mice,can improve the pathological changes of lung tissue caused by LPS,reduce inflammatory cell infiltration,reduce pulmonary edema and lung tissue damage,reduce the content of inflammatory factors and TAT complexes.2.Comprehensive analysis showed that the enrichment of KEGG pathways in network pharmacology and transcriptomics indicated that the NETs formation pathway was significantly enriched,suggesting that the NETs formation pathway played an important role in the treatment of ALI by QJHTD.3.In vitro and in vivo experiments have shown that QJHTD can inhibit the formation of NETs.The correlation analysis of in vivo experiments shows that the content of NETs in BALF is positively correlated with TNF-α,IL-6,IL-1β and TAT Complexes,indicating that QJHTD may inhibit the formation of NETs to alleviate the inflammation and coagulation of ALI.Studies have found that QJHTD can reduce the combination of LPS and platelets,speculating that QJHTD can inhibit the combination of LPS and platelets and reduce the generation of NETs.