| ObjectiveTo investigate the mechanism of blocking the "inflammation-cancer" transformation of the stomach in the treatment of chronic atrophic gastritis with the Yi Qi Hua Yu Detoxification Formula,and to provide a theoretical basis for the clinical treatment of Chinese medicine.Methods1.Rats with chronic atrophic gastritis were modelled by 20 mmol/L deoxycholic acid and 0.1%ammonia ad libitum with hunger and satiety disorders for 16 weeks,and the groups were set up as control group,model group,high-dose Yi Qi Hua Yu Detoxification Formula group,regular-dose Yi Qi Hua Yu Detoxification Formula group,AG490 group,AG490+Yi Qi Hua Yu Detoxification Formula group,Colivelin group,Colivelin+Yi Qi Hua Yu Detoxification Formula group,Colivelin+Yi Qi Hua Yu Detoxification Formula group,and the interventions corresponding to 4 weeks were carried out for each group.The general condition of the rats was evaluated by the modelling results of spleen deficiency,and the pathological changes of gastric mucosa were assessed by HE staining,and the genes related to JAK2/STAT3 signalling pathway and Epithelial-Mesenchymal Transition(EMT)were detected by protein immunoblotting,immunohistochemistry and immunofluorescence.expression changes.2.GES-1 was stimulated with Chenodeoxycholic acid to construct a model of gastric precancerous disease,and the effect of the model was evaluated by the expression of CDX2,KLF4,Villin and other genes related to gastric precancer.After successful modelling,the groups were set up as control group,model group,high-dose Yi Qi Hua Yu Detoxification Formula with drug serum group,regular-dose Yi Qi Hua Yu Detoxification Formula with drug serum group,AG490 group,AG490+Yi Qi Hua Yu Detoxification Formula with drug serum group,and the STAT3 overexpression cell line was constructed.3.To screen the effective blood components of Yi Qi Hua Yu Detoxification Formula by serum mass spectrometry,and to predict their possible targets by network pharmacology analysis,and to screen the differentially expressed genes in the cells of the gastric precancer model by transcriptome sequencing,and to compare the differentially expressed genes in gastric cancer tissues.Results1.In the control group,the epithelial cells in the gastric mucosa were neatly arranged in a columnar shape with uniform size and shape,and the gastric mucosal glands were of moderate density,uniform in size and shape,neatly arranged,without obvious heterogeneous glands,and without inflammatory cell infiltration in the mucosa.After 16 weeks of modelling intervention,the rats in the model group had different sizes of glands in the lamina propria of the gastric mucosa,and the number of glands was significantly reduced.The nuclei of the epithelial cells in the gastric mucosa became smaller,the cytoplasm became larger,the epithelial cells were highly reduced,and the mucosal interstitium was infiltrated by inflammatory cells;the rats were significantly thinner than the control group,with reduced activity and rotten stools.After the intervention of the formula,the number of gastric mucosal glands increased,the height of epithelial cells increased,and the inflammatory cell infiltration was relieved;the rats were obese,more active and responsive than the control group,and their stools were better formed.2.The expression levels of p-STAT3/STAT3 and p-JAK2/JAK2 in the SD rat model group were significantly higher compared to normal(P<0.05);the expression levels of pSTAT3/STAT3 and p-JAK2/JAK2 were significantly inhibited after intervention with high dose of Yi Qi Hua Yu Detoxification Formula(P<0.05);the regular-dose of Yi Qi Hua Yu Detoxification Formula could inhibit the expression of p-STAT3/STAT3 after intervention(P<0.05)but the inhibition of p-JAK2/JAK2 expression was not statistically significant(P>0.05);AG490 group could inhibit the expression of p-STAT3/STAT3 and p-JAK2/JAK2(P<0.05);the AG490+Yi Qi Hua Yu Detoxification Formula group was not statistically different from the AG490 group(P>0.05);In the Colivelin group,the expression levels of pSTAT3/STAT3 and p-JAK2/JAK2 were higher than those in the control group(P<0.05),while the expression levels of p-STAT3/STAT3 and p-JAK2/JAK2 were reduced after the intervention with Yi Qi Hua Yu Detoxification Formula(P<0.05).3.The protein expression of EMT-related genes Vimentin,Snail and β-Catenin was significantly elevated in the SD rat model group compared to the control group(P<0.05);the expression of Vimentin,Snail and β-Catenin was significantly suppressed in the highdose Yi Qi Hua Yu Detoxification formula compared with the model group(P<0.05).The expression of Vimentin and β-Catenin could be inhibited by the regular-dose of Yi Qi Hua Yu Detoxification Formula compared with the model group(P<0.05),but the expression of Snail was not significantly inhibited(P>0.05);The expression of β-catenin,Vimentin,and Snail was reduced in the AG490 group compared with the model group(P<0.05),and the expression of β-catenin,Vimentin,and Snail was significantly higher in the Colivelin group compared with the control group(P<0.05),while the Colivelin+Yi Qi Hua Yu Detoxification Formula group showed that the expression of EMT-related proteins βcatenin,Vimentin,and Snail expression decreased compared with the agonist group(P<0.05).The expression level of E-cadherin was not significantly different in expression between the groups(P>0.05).4.In the gastric precancerous disease cell model,the protein results showed that the expression of CDX2,VILLIN and KLF4 was most significant at a concentration of 400μmol/mL of CDCA compared to the control group(P<0.05),and the cellular immunofluorescence results showed that the fluorescence expression of KLF4 and MUC2 was strongest at 400μmol/mL of CDCA,and the differences were statistically significant compared to the control group(P<0.05).5.In in vitro experiments,the expression of p-JAK2/JAK2 and p-STAT3/STAT3 was significantly higher in the gastric precancer model group compared with the control group(P<0.05);the expression of p-JAK2/JAK2 and p-STAT3/STAT3 was reduced after the intervention with high dose of Yi Qi Hua Yu Detoxification Formula-containing serum compared with the model group(P<0.05);the expression of p-STAT3/STAT3 could also be reduced by using the regular-dose of Yi Qi Hua Yu Detoxification Formula-containing serum(P<0.05),but the inhibition effect on p-JAK2/JAK2 was not stable(P>0.05);The AG490 group could inhibit the expression of p-JAK2/JAK2 and p-STAT3/STAT3(P<0.05);there was no statistical difference in the expression of p-JAK2/JAK2 and p-STAT3/STAT3 after the intervention with Yi Qi Hua Yu Detoxification Formula on top of AG490 compared with AG490(P>0.05);In the STAT3 overexpression group,p-STAT3/STAT3 expression was significantly higher compared to the control group(P<0.05),while pSTAT3/STAT3 expression decreased in the STAT3 overexpression+Yi Qi Hua Yu Detoxification Formula containing serum group relative to the STAT3 overexpression group(P<0.05).The p-STAT3/STAT3 expression in the null group was not significantly elevated and the difference was not statistically significant compared with the control group(P>0.05).6.In in vitro experiments,the expression of β-catenin,Vimentin and Snail were significantly higher in the gastric precancerous disease cell model group compared with the control group(P<0.05);high-dose of Yi Qi Hua Yu Detoxification Formula intervention reduced the expression of β-catenin,Vimentin and Snail in all groups compared with the model group(P<0.05);regular-doses reduced the expression of Vimentin and Snail(P<0.05),but the inhibition of β-catenin expression was not statistically significant compared with the model group(P>0.05);the expression of β-catenin,Vimentin and Snail in the overexpression group was significantly higher compared with the control group(P<0.05);while after the intervention of high-dose Yi Qi Hua Yu Detoxification Formula containing serum,the expression of β-catenin,Vimentin and Snail compared with the STAT3 overexpression group showed a decreasing trend(P<0.05);the differences in the expression of EMT-related genes in the null group compared with the control group were not statistically significant(P>0.05).There was no statistically significant difference in the expression of E-cadherin in each group(P<0.05).7.In this study,765 annotated metabolites were screened,among which 68 bloodentering compounds of Yi Qi Hua Yu Detoxification Formula were screened.The results of the network pharmacological analysis showed that the targets of the blood-entering compounds of Yi Qi Hua Yu Detoxification Formula were mainly concentrated in the pathways of chemo-carcinogenesis-receptor activation,gastric cancer,MAPK signaling pathway and JAK-STAT signaling pathway;transcriptomic sequencing of cell models of pre-cancerous gastric disease identified 4861 differentially expressed genes.The transcriptomic sequencing of the pre-cancerous gastric disease cell model identified 4861 differentially expressed genes,and compared the differentially expressed genes with the effective compounds of Yi Qi Hua Yu Detoxification Formula,and screened out three core targets of the blood-incorporated compounds of Yi Qi Hua Yu Detoxification Formula,namely BCHE,SNCA and PRNP,which may interfere with the transformation process of gastric "inflammatory cancer".Conclusions1.In the in vivo experiment,a rat model of chronic atrophic gastritis was successfully constructed;by detecting the protein expression of JAK2/STAT3 pathway and EMT-related genes,it was found that the JAK2/STAT3 pathway was abnormally activated in the chronic atrophic gastritis model,and the expression of EMT-related proteins was positively correlated with the activation of JAK2/STAT3 pathway.However,the intervention of Yi Qi Hua Yu Detoxification Formula could inhibit the activation of JAK2/STAT3 and suppress the expression of EMT-related genes,thus suggesting that Yi Qi Hua Yu Detoxification Formula could inhibit the progression of EMT by regulating the JAK2/STAT3 pathway,thus achieving the treatment of chronic atrophic gastritis and blocking the process of gastric"inflammation and cancer" transformation.2.In an in vitro experiment,a gastric pre-cancer cell model was successfully constructed using CDCA;the protein expression of JAK2/STAT3 pathway and EMT-related genes was detected,suggesting that the JAK2/STAT3 pathway was activated in the gastric pre-cancer cell model,while the drug-containing serum of Yi Qi Hua Yu Detoxification Formula could inhibit the abnormal activation of JAK2/STAT3.At the same time,the STAT3 overexpression cell line was constructed,and it was found that Yi Qi Hua Yu Detoxification Formula could inhibit the expression of EMT-related genes in the overexpression cell line with the inhibition of p-STAT3/STAT3 expression,thus suggesting the important mediating role of JAK2/STAT3 pathway in the regulation of EMT by Yi Qi Hua Yu Detoxification Formula.It was further demonstrated that Yi Qi Hua Yu Detoxification Formula could interfere with the process of gastric "inflammatory cancer"transformation by inhibiting the JAK2/STAT3 pathway.3.Through the analysis of the material basis and bioinformatics of the effect of Yi Qi Hua Yu Detoxification Formula,this study clarified the effective blood-entering components of this formula,and also revealed the potential mechanism of its therapeutic effect by combining and analyzing the targets of the effective components of Yi Qi Hua Yu Detoxification Formula and the genes with the same trend of differential expression in the cell model of gastric pre-cancer and gastric cancer,which provides a basis for further research on the mechanism of blocking gastric "inflammation-cancer" transformation by Yi Qi Hua Yu Detoxification Formula in the treatment of chronic atrophic gastritis. |
PDF Full Text Request