| Objective: In this study,the main active components and protein targets in XCD were sorted out by network database Integrative Pharmacology-based Research Platform of TCM(TCMIP)and Traditional Chinese Medicine Systems Pharmacology Database(TCMSP),and the key target genes of XCD in treatment of ALI were screened by RNA sequencing.Further the key core target genes and related pathways were verified by animal experiments,the mechanisms of XCD in the treatment of ALI were explored.Methods:(1)The main blood active components and action targets of XCD were screened by TCMIP and TCMSP platform,and human related genes were screened by Uni Prot,and the genes related to ALI in human disease gene database were intersected to obtain the potential targets of XCD in the treatment of ALI,and to construct the key potential gene target network of the main active components of XCD.The potential mechanism of XCD decoction in the treatment of ALI was analyzed by protein-protein interaction(PPI)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)and Gene Ontology(GO)enrichment analysis.(2)The expression of mRNAs in normal group,model group and XCD group were detected by RNA-seq,and the obtained mRNAs were compared by pairwise to screen the differentially expressed genes(DEGs).The human related target genes in the DEGs of mRNA,XCD group and model group were screened by Uni Prot,and the related target genes were analyzed by PPI analysis,KEGG and GO enrichment analysis,to analyze the pathogenesis of ALI induced by LPS and the mechanism of XCD in the treatment of ALI.(3)The potential target genes of XCD in the treatment of ALI were intersected with the DEGs obtained by RNA-seq between XCD and the model group,and the key target genes of XCD in the treatment of ALI were obtained.PPI analysis,GO and KEGG enrichment analysis were carried out,the core key targets and key signal pathways were screened.(4)The quality control of XCD was carried out by high performance liquid chromatography(HPLC),and its main active components were analyzed.(5)The LPS-induced ALI model was established.TNF,IL-1 β and IL-6 in bronchoalveolar lavage fluid of rats in each group were determined by ELISA.Comparing with the dry-wet weight ratio of lung tissue to detect the degree of lung tissue oedema.hematoxylin-eosin(HE)staining was used to detect the morphological changes of lung tissue,and immunohistochemistry,Western blot(WB)and Polymerase Chain Reaction(PCR)were used to detect the key target genes and its related proteins.To investigate the effects of XCD on protein and gene expression of core key targets and related signal pathways in rat mouse with LPS-induced ALI.Results:(1)According to the TCMSP and TCMIP network database,the main blood components and target genes of XCD decoction were predicted and screened by network pharmacology,and a total of 206 active components were obtained,including87 in R.officinale Baill,63 in Semen Armeniacae Amarum,56 in T.kirilowii Maxim and5222 target genes.Oral bioavailability(OB)≥ 30% and drug similarity(DL)≥ 0.18 were regarded as selection criteria.A total of 46 active components and 281 potential target genes were obtained.Through PPI,KEGG and GO analysis of 281 potential target genes,the key targets of XCD in the treatment of ALI were TP53,JUN,AKT1,PIK3 CA,MAPK1,HSP 90,PTGS,etc.,which are closely related to HIF-1 signal pathway,TNF signal pathway and PI3K/AKT signal pathway.(2)The lung tissues of rats in normal group,model group and XCD group were sequenced with high throughput.Compared with the normal group,there were 1085up-regulated and 1768 down-regulated DEGs,in the model group,and there were 361up-regulated and 392 down-regulated DEGs in XCD group.Through PPI analysis,KEGG and GO enrichment analysis of DEGs between normal group and model group,model group and XCD group,86 related signal pathways and 356 related GO terms were found,which were related to the pathogenesis of ALI induced by LPS.And its mechanisms were closely related to cellular immunity,cellular inflammation and apoptosis.It was mainly realized by regulating tumor necrosis factor(TNF)signaling pathway,NF-κB signaling pathway and cancer signaling pathway.There were 55 related pathways and 192 related GO terms were related to XCD in the treatment of LPS-induced ALI,and its mechanism was mainly related to the regulation of cellular immune function,slowing down of apoptosis and inhibition of cellular inflammation.The main related pathways include PI3K(Phosphatidylinositide 3-kinases)/AKT(protein kinase B)signaling pathway,HIF-1(hypoxia inducible factor)signaling pathway,TNF signaling pathway,PPAR(peroxisome proliferators-activated receptor)signaling pathway and so on.(3)The potential target genes of XCD for the treatment of ALI as described by the database screen were intersected with the DEGs between the XCD group and the model group obtained by RNA-seq,and a total of 57 key target genes were obtained,and KEGG and GO analyses were performed on these genes to obtain six key core targets including PI3 k,AKT,mTOR,PTEN,VEGF and HIF-1a,and four key signaling pathways including PI3K/AKT,mTOR,VEGF and HIF-1.(4)There were total l137 chemical components were identified in XCD by HPLC analysis,mainly including emodin,quercetin,cepharanthine and amygdalin.(5)As for the experimental in vivo study,compared with the normal group,the dry/wet weight ratio of lung tissue in the model group was significantly up-regulated,and the expression of inflammatory factors such as TNF-ɑ,IL(Interleukin)-1β and IL-6 in the alveolar lavage fluid was significantly increased(P<0.05),and the lung tissue appeared significant pathological damage;compared with the model group,the dry to wet weight ratio of lung tissue in the DEX(dexamethasone)group and the XCD high and low dose groups was significantly decreased.The levels of TNF-ɑ,IL-1β and IL-6 in the BALF(bronchoalveolar lavage fluid)were significantly reduced(P<0.05),and the lung tissue damage was also improved significantly.Immunohistochemistry results showed that PTEN and mTOR protein expression were significantly lowered and PI3 K,AKT,HIF-1a and VEGF protein expression were significantly lifted in the model group compared with the normal group(P < 0.05).PTEN and mTOR expression had no significant change in the DEX group compared with the model group(P>0.05),and XCD could significantly increase PTEN and mTOR protein expression(P<0.01),and significantly decreased PI3 K,AKT,HIF-1a and VEGF protein expression(P<0.05);The PCR results showed that compared with the normal group,PTEN mRNA and mTOR mRNA expression in lung tissue in the model group were significantly decreased(P < 0.05).The expression of PI3 K mRNA and AKT mRNA were significantly increased(P<0.05),while the expression of PTEN mRNA and mTOR mRNA were significantly increased(P <0.05)and the expression of PI3 K and AKT mRNA were significantly decreased(P<0.01)in the lung tissues of the treated rats compared with the model group;WB results showed that the expression of p-PI3 K,p-mTOR,HIF-1a and VEGF in the model group were significantly increased compared with the normal group(P<0.05),and the expression of p-PI3 K,p-mTOR,HIF-1a and VEGF in lung tissues of rats in each treatment group were significantly decreased compared with the model group(P<0.05).Conclusion:(1)XCD could effectively relieve the clinical symptoms of rats with ALI induced by LPS,decrease the expression of inflammatory cytokines such as TNF-a,IL-6 and IL-1 β,alleviate pulmonary edema and mitigate the pathological injury of lung tissue.(2)Through the analysis of network pharmacology and RNA sequencing,the mechanism of XCD in the treatment of ALI was closely related to the regulation of cellular immunity,cellular inflammation and apoptosis,and the key targets of XCD in the treatment of ALI might be AKT1,VEGFA,PTEN,MTOR,HIF1 A and PIK3 CA.(3)The mechanism of XCD on treating ALI might involve with inhibiting PI3K/AKT signal pathway and reducing the expression of downstream mTOR,thus reducing the stimulation of HIF-1a/VEGF signal pathway and reducing inflammatory reaction and edema in ALI rats. |
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