| BackgroundThe transition from gastric precancerous lesions(GPL)to gastric cancer is a gradual process,with a certain reversal possibility,especially when its pathology is in low-grade intraepithelial neoplasia.Therefore,blocking or delaying the progression of GPL into gastric cancer can be an effective measure to prevent gastric cancer.Although GPL takes"spleen deficiency,poison,and stasis" as its pathogenesis characteristics,it takes deficiency as its root,while "toxin" and " stasis" are secondary.The deficiency of the spleen is the basis of the disease and the key to causing "precancerous disease→precancerous lesion→gastric cancer".Moreover,spleen-qi deficiency,as one of the three common subtypes of spleen deficiency,is considered to be the key to the prolonged and difficult recovery of GPL.With the development of systems biology,the research of spleen-qi deficiency syndrome has gradually transitioned to different biological aspects such as metabolomics,proteomics,genomics,and transcriptomics.microRNA has received more attention in the study of TCM syndromes.The introduction of microRNA is beneficial to the standardization and objectification of TCM syndrome diagnosis,and provides a certain basis for the diagnosis and treatment of TCM clinical diseases.ObjectiveThe specific microRNAs of spleen-qi deficiency syndrome in GPL can be mined through high-throughput sequencing and bioinformatics.The purpose is to provide new ideas and methods for standardization and objectification of spleen-qi deficiency syndrome.The mechanism of Astragaloside Ⅳ(ASIV)in the treatment of GPL were analyzed through network pharmacology and molecular docking technology.The GPL mice with spleen-qi deficiency syndrome were treated by ASIV,and the expression changes of differential microRNAs in GPL spleen-qi deficiency syndrome were observed.The study intended to clarify that ASIV may target PTEN by regulating mmu-miR-215-3p,thereby regulating the PI3K/Akt signaling pathway to intervene in the development of the disease course in GPL mice with spleen-qi deficiency.Methods① High-throughput sequencing:SPF male C57BL/6 mice were selected in the study,and they were divided into blank control group,SQD(spleen-qi deficiency)group,and GPL-SQD group.In the course of the study,SQD mouse models were replicated by combining hunger with swimming fatigue.On the basis of SQD mouse models,GPL-SQD mouse models were replicated by combining the chemical modeling agent MNU(120μg/mL)with free drinking.The high-throughput sequencing platform(Illumina)was used to detect microRNA expression profiles of each group and screen microRNAs with significant differences in expression.GO enrichment and KEGG pathway analysis were used to predict the target genes of microRNA and the pathways involved.The RT-qPCR method was used to test the expression changes of the specific micorRNAs.② Network pharmacology and molecular docking:GPL disease targets were collected through the Genecards database and GenCLiP3 database,and the "Score≥5" screening condition was set to obtain the GPL target set.PharmMapper,TCMIP,Swiss Target Prediction,STITCH 5.0 and Binding Database were used to predict ASIV candidate targets.Then ASIV candidate targets were imported into the UniProt database for target standardization.The intersection of the GPL targets and the ASIV candidate targets were imported into the CTD database for GO and KEGG analysis.The CB-Dock website was used for molecular docking between ASIV and key target proteins.③ ASIV intervention mechanisms:The TargetScan database was used to connect the key target genes of the PI3K/Akt signaling pathway with microRNAs of potential GPL spleen-qi deficiency syndromes.SPF male C57BL/6 mice were selected.The method of obtaining GPL-SQD mice was as described above.SPF mice have been divided into blank control group,model group,ASIV-L group,ASIV-H group,Sijunzi decoction Group and vitacoenzyme group.Mice in each group were treated with ASIV gavage for 9 weeks,and then the mice were sacrificed to obtain experimental samples.HE staining and AB-PAS staining were used to observe the pathological changes of gastric mucosa in each group of mice.Serum D-xylose and serum salivary amylase activity were used to observe the improvement of spleen-qi deficiency syndrome.Real-time fluorescent quantitative PCR was used to detect the mRNA expression changes of differential microRNA,PTEN,PI3K,and Akt.Immunohistochemistry was used to detect the protein expression of PI3KResults①The microRNA expression profiles of the gastric mucosa from each group were constructed by high-throughput sequencing,and the test samples were expanded after screening.After quantitative real-time PCR detection,it was found that mmu-miR-1 94-1-3p,mmu-miR-215-3p,mmu-miR-669c-5p,mmu-miR-5114 and mmu-miR-8019 were differences in expression levels in the gastric mucosa of SQD group and GPL-SQD group(P<0.05),which was basically consistent with the sequencing results.②According to network pharmacology analysis,ASIV involves 141 targets.After mapping with 613 disease targets of GPL,36 cross-targets were obtained as potential targets.The PPI network suggested that 34 targets participate in the protein interaction network,involving 1 62 edges.The key sub-networks were constructed by MCC topology in cytoHubba,and key targets were obtained including PIK3CA,PIK3R1,VEGFA,STAT3,HSP90AA1,MTOR,IL2,AKT1,EGFR and PTPN11.The key targets were entered into the Kaplan-Meier website to analyze the relationship between key targets and the survival of gastric cancer patients,and to explore the impact of key proteins on the "inflammation-cancer" transformation process.The results showed that with the exception of MTOR(P>0.05),the overexpression of the other 9 key genes was related to a significant reduction in the overall survival time of GC patients(P<0.05).Molecular docking technology can dock ASIV with 10 key targets,with a view to exploratory research on the interaction mechanism between ASIV and targets at the microscopic level.The results show that ASIV has the lowest binding energy with PIK3CA,PIK3R1,AKT and MTOR,which means better affinity with a good binding score.These four proteins are the main genes of the PI3K/Akt signaling pathway.③ASIV intervention mechanism:Targetscan database analysis showed that mmu-miR-215-3p can bind to the 3’UTR of PTEN,PIK3CA,AKT1 and mTOR.Animal experiment was designed for ASIV intervention in GPL mice with spleen-qi deficiency syndrome,and it was found that the D-xylose and serum salivary amylase activities in the ASIV-L combined ASIV-H group were improved(P<0.05).The results of HE staining showed that the gastric mucosa of the high-and low-dose ASIV groups of mice was arranged more neatly than the model group.The nuclei and the nuclear-to-cytoplasm ratio were increased.AB-PAS staining showed that the low-and high-dose groups of ASIV were mainly red stained and some blue stained.Quantitative Real-time PCR showed that ASIV could down-regulate the mRNA expression of mmu-miR-215-3p in mice with GPL spleen-qi deficiency syndrome(P<0.05).In addition,it was observed that ASIV could down-regulate the mRNA levels of mmu-miR-194-3p and mmu-miR-8019(P<0.05),but has no intervention effect on mmu-miR-669-3p and mmu-miR-5114(P>0.05).Furthermore,ASIV could up-regulate PTEN mRNA expression(P<0.05)while inhibit mRNA expression of PI3K and AKT(P<0.05)in gastric mucosa of mice with GPL spleen-qi deficiency syndrome.Immunohistochemistry indicated that ASIV could up-regulate the protein expression of PTEN in the gastric mucosa of mice while inhibit protein expression of PI3K and AKT(P<0.05).Conclusions① mmu-miR-194-1-3p,mmu-miR-215-3p,mmu-miR-669c-5p,mmu-miR-5114 and mmu-miR-8019 were found as the specific microRNAs in GPL with spleen-qi deficiency syndrome through high-throughput sequencing and bioinformatics.② The results of network pharmacology and molecular docking suggested that ASIV may interfere with the development of GPL by regulating the PI3K-Akt signaling pathway.③ ASIV can relieve the intestinal metaplasia and dysplasia of gastric mucosa in GPL mice with spleen-qi deficiency syndrome to a certain extent,and improve the related indexes of spleen-qi deficiency syndrome.④ ASIV may regulate with mmu-miR-215-3p,thereby regulating PTEN to participate in the PI3K/Akt signaling pathway and intervene in the development of the disease process in mice with GPL spleen-qi deficiency syndrome. |
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