| Objective:(1)To explore the mechanism of Jianpi Qingre Huoxue Prescription in treating chronic atrophic gastritis(C AG)by network pharmacology;(2)To investigate the phenotypic characteristics of MDSCs,S100A8 and S100A9 in patients with CAG.(3)To investigate the intervention effect of Jianpi Qingre Huoxue Prescription on CAG patients.Methods:(1)The TCMSP database was used to screen the active components of JianpiQingre Huoxue Prescription.The CAG-related disease genes were searched in Gene Cards,OMIM and Pharm Gkb databases,and the TCM-disease-target regulatory network andprotein-protein interaction network were constructed.GO analysis and KEGG analysis were performed.(2)According to the results of pathology,48 subjects were divided into chronic non-atrophic gastritis group(CNAG)(control group)and C AG group(experimental group).The blood and gastric mucosa tissues of the two groups were collected.Thephenotypic characteristics of MDSCs,S100A8 and S100A9 in the blood and the recruitment of MDSCs in the gastric mucosa were detected.(3)The pathological slices preserved in the 2015 project“Multicenter,randomized,controlled clinical study of Weiweiqing Granule in the treatment of chronic atrophicgastritis”(the approval number of the Ethics Committee is“ZYYECK【2015】024-ND-01”)were selected.The pathological slices of gastric mucosa of CAG patients before and aftertaking Jianpi Q ingre Huoxue Prescription for 24 weeks were detected byimmunofluorescence staining to explore the intervention effect of Jianpi Q ingre Huoxue Prescription on the recruitment of MDSCs in gastric mucosa of CAG patients.Results:(1)Network pharmacology researchA total of 64 effective active ingredients and 245 drug targets were screened out by Jianpi Q ingre Huoxue Prescription.A total of 759 disease-related genes were screened out by CAG.93 genes were obtained by combining drug targets and disease genes,that is,Jianpi Q ingre Huoxue Prescription may play a therapeutic role by intervening in the expression of 93 genes in CAG.The Jianpi Q ingre Huoxue Prescription-CAG-target regulatory network was constructed.The core target genes were PTGS2/COX2,PTGS1/COX1,NOS2/INOS,AR and PGR,respectively.The active components of the core drugs were quercetin,luteolin,kaempferol,baicalein and baicalin.By constructing a protein interaction network,12 core proteins including JUN,TP53,and MAPK3 are finally screened out and constructed as a core protein network.GO enrichment analysis showed that the biological process of Jianpi Q ingre Huoxue Prescription interfering with the potential gene targets of C AG is mainly enriched in processes such as the response to oxidative stress and regulation of apoptotic signaling pathway.And it also closely related to the body’s immune function.KEGG analysis showed that PI3K-Akt signaling pathway,IL-17 signaling pathway,and TNF signaling pathway are the key signaling pathways for Jianpi Qingre Huoxue Prescription to treat CAG.The IL-17 signaling pathway and the TNF signaling pathway are important pathways in the immune mechanism.The latest research shows that the PI3K-Akt signaling pathway is closely related to MDSCs.Therefore,we will validate the intervention effect of Jianpi Q ingre Huoxue Prescription on MDSCs through clinical specimens.(2)Clinical trial researchA total of 48 subjects were collected in this study,which were divided into CNAG group(control group)and C AG group(experimental group)according to pathological results,with 24 cases in each group.According to the chi-square test,there was no statistically significant difference in the baseline levels of age and gender between the two groups,and the two groups were comparable.The content of MDSCs in the blood of the two groups was tested by Wilcoxon rank sum test,and the content of MDSCs in the experimental group was significantly higher than that in the control group(P=0.000).According to the results of the patient’s Hp infection,the control group and the experimental group were divided into Hp+and Hp-groups respectively.According to the Kruskal-Wallis H test,the Hp+and Hp-groups of the experimental group were higher than those of the control group Hp+and Hp-groups(P<0.05),and the median MDSCs content of the Hp+group in the experimental group was higher than that in the Hp-group,but the difference was not statistically significant.The gastric mucosal pathological score was combined with the MDSCs in the blood of CAG patients for correlation analysis.Spearman rank correlation analysis showed that the expression of MDSCs in the blood of patients increased gradually with the pathological score of CAG patients(r_s=0.554,P=0.005),showing a significant moderate correlation.Immunofluorescence staining of the pathological sections of the gastric mucosa showed that a large number of MDSCs were recruited in the gastric mucosa of the experimental group.The t-test of two independent samples showed that the average expression of MDSCs in the gastric mucosa of the experimental group was significantly higher than that in the control group(P=0.006).The two groups of plasma were subjected to the Elisa test.The Wilcoxon rank sum test found that the levels of S100A8 and S100A9 in the experimental group were significantly higher than those in the control group(P=0.000,P=0.000).Spearman rank correlation analysis showed that as the expression of MDSCs in blood gradually increased,S100A8 and S100A9 in blood also increased(r_s=0.426,P=0.003;r_s=0.458,P=0.001).After taking Jianpi Q ingre Huoxue Prescription for 24 weeks,the pathological sections of CAG patients were stained by immunofluorescence.The results showed that the recruitment of MDSCs in gastric mucosa after treatment was significantly less than that before treatment(P=0.037),and the pathological score was significantly decreased(P=0.008),indicating that Jianpi Q ingre Huoxue Prescription can inhibit the recruitment of MDSCs in gastric mucosa and improve gastric mucosal lesions.Conclusion:(1)Jianpi Q ingre Huoxue Prescription exerts the therapeutic effect of CAG through multiple targets and multiple pathways.Especially in terms of immune mechanism,it has obvious therapeutic effect.(2)The blood levels of MDSCs,S100A8 and S100A9 in CAG patients were significantly higher than those in CNAG patients,indicating that CAG patients are in a weakened state of immunity,while a large number of MDSCs are recruited in the gastric mucosa of CAG patients to form an immunosuppressive microenvironment to promote inflammation.(3)Jianpi Q ingre Huoxue Prescription can effectively reduce MDSCs recruitment in gastric mucosa to resist immunosuppression and improve the pathological state of gastric mucosa. |
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