Study On The Mechanism Of Fusu Mixture In The Treatment Of Extrapulmonary Acute Respiratory Distress Syndrome Based On Transcriptome Analysis

Purpose:Based on Transcriptome technology,explore the changes of Transcriptome level in peripheral blood of patients with pulmonary exogenous acute respiratory distress syndrome treated with Resuscitation Mixture.Based on these changes,analyze the possible mechanism of action with Shengxin language,then,Lipopolysaccharide(LPS)induced inflammation of human lung microvascular endothelial cells(HPMEC)as a model,and the serum containing Resuscitation Mixture as an intervention measure,to verify its mechanism of action,and provide a basis for the subsequent clinical application of Resuscitation Mixture.Methods:Screening patients with ARDSexp based on diagnostic,inclusion,and exclusion criteria,they were randomly divided into a control group and a Fusu mixture group.The control group was treated with conventional Western medicine,the Fusu mixture group was treated with Fusu mixture on the basis of conventional Western medicine treatment,the healthy subjects were in the blank group,peripheral blood samples were collected from three groups of subjects,use the common transcriptome sequencing method to determine the m RNA in the whole blood cells,and obtain the original data.Use HISAT2 software to compare the original data with the data in the Illumina Nova Seq sequencing platform,and obtain the effective data.Use HISAT2 software to obtain the counts and FPKM values of m RNA.Then use DESeq2 and Edge R software for differential gene analysis.and P <0.05 and |log2FC|≥0 were used as the thresholds to evaluate m RNA expression differences was statistically significant.Finally,based on DAVID,functional annotation of differential genes was performed on the three levels of GO,and the pathway enrichment of differential genes was performed on KEGG.Using HPMEC inflammatory injury as a model and resuscitation mixture containing serum as an intervention measure,the signaling pathway of its action was verified,the specific methods are as follows:(1)To explore the LPS concentration in HPMEC inflammatory models:Culturing and identifying human pulmonary microvascular endothelial cells in vitro,RT-PCR was used to determine the content of IL-1β、TNF-α in the HPMEC inflammatory model treated with different concentrations of LPS,Determine the LPS concentration required for HPMEC injury models.(2)To explore the mechanism of Fusu mixture in treating HPMEC inflammatory model:(1)Preparation of medicated serum;(2)HPMEC was randomly divided into 5 groups: blank control group,LPS group,low dose Fusu mixture group(LPS+2% medicated serum),medium dose Fusu mixture group(LPS+5% medicated serum),and high dose Fusu mixture group(LPS+8%medicated serum).Except for the control group,the other groups were incubated with50μg/ml LPS in conventional culture medium for 24 hours,and then given corresponding drug-containing serum for 24 hours in groups.The content of IL-1β、TNF-α in each group was detected by ELISA.The transcription level of IL-1β、TNF-αm RNA in each group was detected using RT-PCR.Results:(1)Reversed m RNA expression by Fusu mixture: Compared with the blank group,447 of the 2189 up-regulated m RNA in the control group were restored to down-regulation in the Fusu mixture treatment group.In the control group there are2330 m RNA was downregulated which have 307 m RNA was restored to upregulation in the Fusu mixture treatment group.A total of 754 m RNA were reversely expressed by the Fusu mixture.GO function enrichment analysis: From the enriched TOP20 entries,it can be seen that BP is mainly enriched in the positive regulation of inflammatory response,innate immune response,and phagocytosis;MF is mainly concentrated in protein binding,protease binding,and cadherin binding;CC is mainly localized in extracellular bodies,special granular cavities,and extracellular regions.KEGG pathway enrichment analysis: From the TOP30 enrichment pathway histogram,it can be seen that in lysosomes,apoptosis,leukocyte migration across endothelium,Nod-like receptor signaling pathway,MAPK signaling pathway,NF-κ The B signal pathway is significantly enriched.From the bubbles Chart of the TOP30 enrichment pathway,it can be seen that it is mainly enriched in lysosomes,Nod-like receptor signaling pathways,leukocyte migration across endothelium,MAPK signaling pathways,and NF-κ B signal path,etc.(2)LPS concentration is 1μ g/ml、5μ g/ml、20μg/ml,IL-1β、TNF-α m RNA in sample transcription level did not increase significantly;When LPS is 50μg/ml IL-1β 、 TNF-α m RNA at sample,The transcription level of was significantly increased(P <0.001).The HPMEC injury model concentration induced by LPS is 50μg/ml。Compared with LPS group,the serum containing Fusu mixture in each group down-regulated IL-1 β、TNF-α The level of m RNA expression and the reduction of its content play a protective role in the inflammatory injury of HPMEC,and the 8% Fusu mixture containing serum is the most significant(P <0.01).Compared with LPS group,after treatment with 8% Fusu mixture medicated serum,The transcription level of NF-κB(P =0.0111)、IκBα(P=0.0271)m RNA returned to down-regulation.Conclusion:(1)The mechanism of Fusu mixture in treating pulmonary exogenous ARDS may be related to Nod-like receptor signaling pathway,MAPK signaling pathway,NF-κ B is associated with inflammatory signaling pathways.(2)Fusu mixture down-regulates IL-1β、 TNF-α MRNA transcriptional expression reduces The content of inflammatory factors of IL-1β、TNF-α plays a protective role on human microvascular endothelial cells;Fusu mixture may protect human microvascular endothelial cells from inflammation by inhibiting NF-κB signal path.