| ObjectiveChronic atrophic gastritis(CAG)is a common and difficult disease of the digestive system and a major precancerous disease of gastric cancer.Traditional Chinese medicine has manifested its superiority to CAG and Jianpi-Qingre-Huoxue Formula(JQHF)is an effective clinical prescription for CAG.The objective of this study was to elucidate the pharmacodynamic material basis of JQHF and further reveal its mechanism in the treatment of CAG.Methods1.JQHF decoction,drug-containing serum of JQHF,and normal serum of rats were analyzed and identified by ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry(UHPLC-QTOF-MS).The components in plasma of JQHF were screened by comparison.2.Based on network pharmacology and bioinformatics method,the target of the components in plasma of JQHF was retrieved through the public database,and the disease target of CAG was retrieved through GEO chip data set,team sequencing data,and public database,so as to obtain the target of the components in plasma of JQHF in the treatment of CAG.Based on the effect target,protein-protein interaction network was constructed,core proteins were screened out,GO function and KEGG pathway enrichment analysis were conducted.The core target was docked with the main components in plasma of JQHF.3.The rat model of CAG was established by free drinking of N-Methyl-N’-nitro-N-nitrosoguanidine(MNNG)solution+irregular eating+0.05%ranitidine hydrochloride diet+10%sodium chloride solution intragastric administration.The rats were divided into blank group,model group,Chinese medicine group,and folic acid group.HE staining and AB-PAS staining were used to detect the gastric mucosa pathology.The expression of serum pepsinogen,serum gastrointestinal hormone,and serum inflammatory factors was measured by enzyme-linked immunosorbent assay.The expression of the markers of intestinal metaplasia(IM)in gastric mucosa was detected by RT-qPCR and immunohistochemistry.The expression of key targets in PTEN/PI3K/AKT pathway was determined by RT-qPCR,immunohistochemistry,and WB.4.IM cell model was induced by MNNG,and IM cell was intervened by the JQHF drug-containing serum.Cell proliferation rate was detected by CCK-8 and the expression of the IM markers was detected by RT-qPCR and immunofluorescence,which determined the optimal concentration of MNNG in model construction and the optimal concentration of drug-containing serum in cell intervention.PI3K/AKT pathway agonist(740 Y-P)and inhibitor(LY294002)were used to intervene in cells,and JQHF drug-containing serum was also used to intervene in cells in the meantime.Then,the expression of the IM markers of cells was detected by RT-qPCR and immunofluorescence.The cells were divided into blank group(GES-1 cell+normal serum medium),model group(IM cell+normal serum medium),and Chinese medicine group(IM cell+drug-containing serum medium).The expression of key targets in PTEN/PI3K/AKT pathway was determined by RT-qPCR and WB.Results1.By UHPLC-QTOF-MS analysis,a total of 931 compounds were identified in JQHF decoction,1230 compounds were identified in JQHF drug-containing serum and normal serum of rats.By comparing the compounds of JQHF decoction and removing the compounds of normal serum of rats,75 components in plasma of JQHF,including hyperoside,isoliquiritin,scutellarin,kaempferol,acacetin,naringin,and so on,were screened.2.Using network pharmacology and bioinformatics,1426 pharmacodynamic targets of the components in plasma of JQHF and 3410 disease targets of CAG were obtained,and 549 effect targets were obtained after the intersection.Through the construction of protein-protein interaction network and topological analysis,the core proteins were selected as MAPK3,MAPK1,STAT3,JUN,SRC,and so on.By GO enrichment analysis,3512 biological processes,119 cell compositions and 300 molecular functions were obtained,which were closely related to the response to oxidative stress,membrane raft,and protein serine/threonine kinase activity.KEGG pathway enrichment analysis showed that 193 pathways were closely related to JQHF pharmacodynamic effect,which included PI3K/AKT signaling pathway,FoxO signaling pathway,apoptosis,and HIF-1 signaling pathway.The main components in plasma of JQHF,such as hyperoside,isoliquiritin,scutellarin,kaempferol,and acacetin,had a strong binding affinity with PIK3CA and AKT1.3.After 8 weeks of intervention,the weight gain in Chinese medicine group was higher than that in model group(P>0.05).HE staining and AB-PAS staining showed moderate to severe atrophy and IM in the model group,and mild atrophy and IM in the Chinese medicine group.For serum pepsinogen,the PGⅠ,PGⅡ and PGR levels in Chinese medicine group was higher than that in model group(P>0.05).For serum gastrointestinal hormone,gastrin level in Chinese medicine group was significantly lower than that in model group(P<0.05).Motilin and prostaglandin E2 levels in Chinese medicine group was higher than that in model group(P>0.05).For serum inflammatory factors,the level of TNF-α,IL-6 and IL-1β in Chinese medicine group was lower than that in model group(P>0.05).For the IM markers,the mRNA expression levels of CDX2,KLF4,MUC2 and VILLIN in Chinese medicine group were lower than those in model group(P>0.05).The protein expression levels of MUC2 and VILLIN in Chinese medicine group were lower than those in model group(P<0.001).For the key target of PTEN/PI3K/AKT pathway,the mRNA expression levels of PTEN(P>0.05),PI3K(P<0.01)and AKT(P>0.05)in Chinese medicine group were lower than those in model group.The results of immunohistochemistry showed that the positive expression of PTEN,p-PI3K and p-Akt mainly concentrated in IM region,and the protein expression levels of PTEN(P>0.05),p-PI3K(P>0.05)and p-Akt(P<0.05)in Chinese medicine group were lower than those in model group.The results of WB showed that the protein expression levels of PTEN(P>0.05),PI3K(P>0.05),p-PI3K(P<0.05),AKT(P>0.05),p-Akt(P>0.05),the ratio of p-PI3K/PI3K(P<0.05)and p-Akt/AKT(P>0.05)in Chinese medicine group were lower than those in mode group.4.MNNG at the concentration of 10μM had little cytotoxicity on GES-1 cells(P>0.05),and could significantly promote the mRNA expression of IM markers CDX2(P<0.05),KLF4(P<0.001),MUC2(P<0.001)and VILLIN(P<0.001),and the protein expression of MUC2(P<0.01)and VILLIN(P<0.05).JQHF drug-containing serum medium at the concentration of 10%could significantly inhibit the proliferation of IM cells(P<0.05)and decreased the mRNA expression of IM markers CDX2(P>0.05),KLF4(P<0.01),MUC2(P<0.05)and VILLIN(P<0.001),and the protein expression of MUC2(P<0.05)and VILLIN(P>0.05).After intervention with 740 Y-P,the mRNA expression of IM markers CDX2(P<0.05),KLF4(P<0.05),MUC2(P<0.01)and VILLIN(P>0.05)in GES-1 cells increased.After intervention with LY294002,the mRNA expression of IM markers CDX2(P<0.01),KLF4(P<0.05),MUC2(P>0.05)and VILLIN(P>0.05)in IM cells decreased.JQHF drug-containing serum could reduce the mRNA expression of CDX2(P<0.001),KLF4(P<0.05),MUC2(P>0.05)and VILLIN(P>0.05)and the protein expression of MUC2(P<0.01)and VILLIN(P>0.05)in GES-1 cells treated with 740 Y-P.In the blank group,the model group and the Chinese medicine group,the mRNA expression levels of PTEN(P>0.05),PBK(P>0.05)and AKT(P<0.01)in the Chinese medicine group were lower than those in the model group.The protein expression levels of PTEN,PI3K,p-PI3K and AKT,the ratio of p-PI3K/PI3K and p-Akt/AKT in the Chinese medicine group were lower than those in the model group,with no statistical difference(P>0.05),and the protein expression level of p-Akt in the Chinese medicine group was significantly lower than that in the model group(P<0.05).Conclusion1.The main components in plasma of JQHF included hyperoside,isoliquiritin,scutellarin,kaempferol,acacetin,naringin,and so on.2.The components-in plasma of JQHF could treat CAG through multiple targets,approaches,and pathways,which were closely related to PI3K/AKT signaling pathway.3.JQHF was effective in improving body weight,gastric mucosa pathology,and gastric digestive function in CAG rats,and its therapeutic effect was related to the inhibition of PI3K/AKT signaling pathway.4.The JQHF drug-containing serum could inhibit the proliferation of IM cells and the expression of the IM markers,and its intervention effect was related to the inhibition of PI3K/AKT signaling pathway. |
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