| OBJECTIVE:1.To predict the active ingredients and potential mechanism of Jianpi Yiqi formula(JPYQF)in treating chronic atrophic gastritis(CAG)through network pharmacological methods.2.To explore the relationship between the pathogenesis of CAG and the proliferation and differentiationof gastric stem cells by observing the differences in-the expression of Wnt signaling initiators Wnt3A and Rspol,gastric stem cell markers Lgr5 and Sox2,cell proliferation markers Ki67 and PCNA,and cell differentiation marker Muc5AC in gastric tissues of patients with CAG and non-CAG(NCAG)people.3.To explored the new mechanism of CAG pathogenesis by establishing MNNG-induced CAG model in gastric organoid,and observing the effect of MNNG on the expression of gastric stem cell marker Lgr5,cell proliferation marker Ki67,cell differentiation marker E-Cadherin,Muc5AC,and Muc6 in CAG gastric organoid model.4.To explore the molecular mechanism of JPYQF on CAG,and provide a scientific basis for explaining the curative effect by observing the regulation of JPYQF on the expression of Wnt signaling initiators Wnt3A and Rspo1,gastric stem cell markers Lgr5 and Sox2,cell proliferation markers Ki67 and PCNA,and cell differentiation markers Muc5AC and Muc6 in CAG rats.METHODS:1.Screen the active ingredients of JPYQF using TCMSP database;Use PubChem,DisGeNET and GeneCards databases to screen the targets of active ingredient and CAG genes;Use the Venn diagrams tool to obtain the intersection targets of active ingredients and CAG genes;Use Cytoscape to construct H-C-T network to screen the main active components of JPYQF;Use STRING database and Cytoscape to conduct PPI analysis and PPI network construction to predict the core targets of JPYQF in treating CAG;Use the DAVID database for GO and KEGG enrichment analysis to obtain the signaling pathways related to the treatment of CAG with JPYQF.2.Collect gastric antrum tissue samples from patients with CAG and NCAG who underwent gastroscopy and pathological examination at the Gastrointestinal Endoscopy Center of Jiangsu Provincial Hospital of Integrated Traditional Chinese and Western Medicine from October 2021 to January 2022,30 cases in each;Use IHC method to detect and compare the protein expression of Wnt3A,Rspol,Lgr5,Sox2,Ki67,PCNA and Muc5 A in gastric tissue of two groups.3.Extract gastric stem cells in gastric antrum tissue from a 3-week-old SD rat to culture organoids;Observe morphological changes of gastric organoids with inverted microscope;Use HE staining,IHC and PCR to detect the expression of Lgr5,Ki67,E-Cadherin,Muc6,Muc5AC,Muc2,CDX2 in gastric organoids to confirm whether the culture was successful;Select the time points with better growth state of gastric organoids,and treat them with complete medium containing 10μmol/L,20 μmol/L,40 μmol/L,80 μmol/L MNNG,and observe the diameter and morphological changes of gastric organoids at 0,6,12,and 24 h;Select the MNNG concentration and time point with the most obvious morphological changes in gastric organoids,and use HE staining,IHC and PCR to detect and compare the morphological changes and expressions of Lgr5,Ki67,E-Cadherin,Muc5AC and Muc6 in normal growing and MMNG-treated gastric organoids.4.There were 70 healthy SD rats,10 were set as control group,and the remaining 60 were used to replicate the CAG rat model of spleen deficiency with MNNG+ranitidine+abnormal diet of hunger and satiety for a total of 24 weeks.Fifty rats with successful CAG modeling were randomly divided into model group(0.09%sodium chloride solution),positive drug group[(folic acid 2.7 mg/(kg·d)+teprenone 13.5 mg/(kg·d)],JPYQF low-dose group[13.1g/(kg·d)],middle-dose group[26.2g/(kg·d)],high-dose group[52.4g/(kg·d)],10 in each group.Each drug intervention group was given 1ml/100g by gavage,and the control group was given 1ml/100g 0.09%sodium chloride solution by gavage,for a total of 8 weeks.The general state and body weight of the rats were observed.HE staining was used to observe the pathological changes of gastric tissue.IHC,WB and RT-qPCR were used to determine the expression changes of Lgr5,Sox2,Wnt3A,Rspo1,Muc5AC,Muc6,Ki67 and PCNA in the gastric tissue of the rats in each group.RESULTS:1.Results of network pharmacology research:(1)191 active ingredients of JPYQF were obtained from TCMSP,literature and pharmacopoeia,and 173 ingredients had curative effects on CAG,and naringenin,quercetin and stigmasterol may be the key components of JPYQF in the treatment of CAG.(2)There were 167 effect targets of JPYQF in the treatment of CAG,and PPI analysis showed that VGEFA,AKT1 and IL6 may be the core targets of curative effect.(3)GO enrichment 442 BP items,104 MF items and 56 CC items,effector target products were mainly enriched in biological processes such as apoptosis,inflammatory response,protein phosphorylation,senescence,cell proliferation,and molecular functions were enriched in enzyme binding,protein binding,protein kinase activity,kinase activity,the effector target products are mainly active on the cell surface,cytosol,cell membrane and extracellular space.(4)KEGG enriched 122 signaling pathways,and the top 17 signaling pathways with the smallest P value included PI3K-Akt,TNF,HIF-1 and FoxO,etc.2.Results of clinical pathogenesis study:(1)Pathological characteristics:In the CAG group,22 cases with mild inflammation,accounting for 73.33%,7 cases with moderate inflammation,accounting for 23.33%,1 case with severe inflammation,accounting for 3.33%;In the non-CAG group,24 cases with mild inflammation,accounting for 80.00%,6 cases with moderate inflammation,accounting for 20.00%,0 cases with severe inflammation,accounting for 0.00%,there was no significant difference in the degree of inflammation between the two groups(P>0.05).In the CAG group,11 cases had active inflammation,accounting for 36.67%,and 19 cases were chronic,accounting for 63.33%;In the non-CAG group,8 cases had active inflammation,accounting for 26.67%,and 22 cases were chronic,accounting for 73.33%.There was no significant difference in activity(P>0.05).(2)Protein expression:Compared with the non-CAG group,the protein expressions of Wnt3A,Rspo1,Lgr5,Sox2,Ki67,PCNA and Muc5AC in the gastric tissue of the CAG group were significantly decreased(P<0.01).3.Results of molecular pathological mechanism study:(1)Establishment of gastric organoids:Rat gastric organoids are spherical structures composed of monolayer cells,expressing stem cell marker Lgr5,cell proliferation marker Ki67,epithelial cell marker E-Cadherin,and gastric mucosal pit cell marker Muc5AC,cervical mucus cell marker Muc6,but not intestinal epithelial cell markers Muc2 and CDX2.(2)Dynamic effects of different concentrations of MNNG on the diameter of gastric stem cells:at 0 and 12 hours after MNNG treatment,there was no significant difference in the diameter of gastric organoids between different concentrations(P>0.05);Compared with normal growth gastric organoids,the diameter decreased significantly at 40 μmol/L and 80 μmol/L concentrations,and the difference was statistically significant(P<0.05).The diameter of gastric organoids in normal growth gradually increased over time,and the diameter at 24 hours was significantly larger than that at 0 hours(P<0.01);treated with MNNG concentrations of 10 μmol/L and 20 μmol/L did not change significantly over time,and the difference in diameter at each time point was not statistically significant(P>0.05);the diameters of gastric organoids treated with MNNG concentration of 40μmol/L tended to decrease over time,but the difference was not statistically significant(P>0.05);the diameter of gastric organoids treated with MNNG concentration of 80 μmol/L decreased significantly over time,and compared with the 0th hour,the diameter at 24 hours was significantly reduced(P<0.05).(3)The effect of MNNG on the morphology of gastric organoids:Similar to the overall morphology of normal gastric organoids,gastric organoids treated with MNNG basically showed a balloon-like structure composed of single-layer cells,but the balloon wall was significantly thinner.(4)The effect of MNNG on the expression of gastric organoid cell markers:Compared with normal gastric organoids,the protein expression level of Ki67 in gastric organoids treated with MNNG was decreased(P<0.05),Lgr5 and E-Cadherin were significantly decreased(P<0.01),and the gene expression level of MucSAC was decreased(P<0.05),Muc6 decreased significantly(P<0.01).4.Results of animal experiment:(1)General situation:The body weight of rats in the model group was significantly lower than that in the blank group(P<0.01);the body weight of the rats in each intervention group were close to the blank group.(2)Macroscopic morphology of gastric mucosa:The gastric mucosa of the rats in the control group was light orange-red,shiny,and the mucosal folds were obvious,tight and regular;the gastric mucosa of the rats in the model group was dark red,the gastric antrum mucosa was gray,the gloss was poor,and the mucosal folds were significantly reduced or disappeared;the gastric mucosa in each intervention group was orange-red,shiny,with increased mucosal folds,and the arrangement tended to be regular.(3)Histopathological morphology:In the control group,the gastric mucosa epithelial structure was clear,the number and shape of glands were normal,the arrangement was compact and neat,and the infiltration of inflammatory cells was less;the gastric mucosal epithelium of rats in the model group was significantly thinner,the glands were significantly reduced,and the arrangement was loose and disordered,and goblet cells were seen,and there were many infiltration of inflammatory cells;the gastric mucosal epithelium of the rats in each intervention group was thickened,the glands increased,the shape tended to be normal,the arrangement tended to be neat,and the infiltration of inflammatory cells was reduced.(4)Expression levels of Wnt3A and Rspo1 proteins:Compared with the control group,the expression levels of Wnt3A and Rspo1 in the model group were decreased(P<0.05).Compared with the model group,the expression levels of Wnt3A and Rspol in the middle-and high-dose JPYQF group increased(P<0.05).(5)Expression levels of Lgr5 and Sox2 proteins:Compared with the control group,the expression levels of Lgr5 and Sox2 in the model group were significantly decreased(P<0.01).Compared with the model group,the expression levels of Lgr5 and Sox2 in the positive drug control group and high-dose JPYQF group were increased(P<0.05).(6)Expression levels of Ki67 and PCNA proteins:Compared with the control group,the expression level of PCNA in the model group was significantly decreased(P<0.01).Compared with the model group,the expression level of PCNA in each drug intervention group was significantly increased(P<0.01).There was no significant difference in the expression level of Ki67 among the groups(P>0.05).(7)Expression level of Muc5AC protein:Compared with the control group,the expression level of Muc5AC decreased in the model group(P<0.05).Compared with the model group,the expression level of Muc5AC in the high-dose JPYQF group increased(P<0.05).(8)Genes expression levels:Compared with the control group,the expression levels of Lgr5,Wnt3A,Rspol,Muc5AC and Muc6 genes in the model group all decreased,but the difference was not statistically significant(P>0.05).Compared with the model group,the expression levels of Lgr5,Wnt3A,Muc5AC and Muc6 genes in the high-dose JPYQF group were significantly increased(P<0.05).CONCLUSION:1.The network pharmacology study found that the curative effect of JPYQF on CAG is caused by various active ingredients such as naringenin,quercetin and stigmasterol in the formula,and through many signaling pathways such as PI3K-Akt,TNF,HIF-1,and acting on VGEFA,AKT1,IL6 and other effector targets,which provides a theoretical basis for exploring the mechanism of JPYQF in the treatment of CAG.2.Clinical and gastric organoid studies have found that the pathogenesis of CAG may be related to the inhibition of the proliferation and differentiation of gastric stem cells under the low activation or inhibition of Wnt signaling,and the inability to replenish glandular cells in time and repair the gastric mucosa.Its exploration of the new pathogenesis of CAG provides a new idea for the mechanism study of the biological effect of traditional Chinese medicine on CAG.3.The curative effect of JPYQF on CAG is related to regulating the abnormal expression of Wnt signaling pathway-related proteins.JPYQF can increase the expression of Wnt signaling initiators,activate Wnt signaling to a certain extent,promote the proliferation and differentiation of gastric stem cells,replenish glandular cells and repair mucosal damage.The research provides a certain basis for explaining the scientific connotation of the effect of JPYQF on CAG.4.The results of this study further explained the molecular mechanism of the therapeutic effect of JPYQF on CAG,and explored the research direction of the microscopic targets of Chinese medicine on CAG.The new molecular mechanism and its cutting-edge technology were introduced into the mechanism study of the therapeutic effect of traditional Chinese medicine,which provided a reference for the therapeutic effect of scientific,microscopic and empirical traditional Chinese medicine on CGA. |
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