Study On The Mechanism Of Weipiling In Regulating The PI3K/AKT/mTOR Signaling Pathway To Mediate Autophagy In Mice With Gastric Precancerous Lesions

ObjectiveIt is well known that the occurrence of gastric cancer is a slow and continuous multi-stage biological process starting from chronic superficial gastritis,atrophic gastritis,intestinal metaplasia,and finally to dysplasia and adenocarcinoma.Gastric precancerous lesions(GPL),which include incomplete intestinal metaplasia or dysplasia,are vital stages in the occurrence of gastric cancer.Autophagy is a process of cell catabolism which involves in the occurrence and development of tumors,but its role in the pathogenesis of GPL remains unknown.In order to clarify the effect of autophagy on GPL and the control mechanism of Weipiling,the GPL mouse models were constructed to observe the effect of regulating autophagy on GPL.Then,the effects of Weipiling on autophagy through the PI3K/AKT/mTOR signaling pathway on GPL mice were detected,which provided a basis for clinical application of this prescription.Methods1.Bioinformatics and network pharmacology research(1)Based on the GEO database under the NCBI platform,the GPL-related chip data was retrieved.The R language Limma package was used to analyze the differential genes of GPL.According to the differential genes related to autophagy,the expression of differential genes and the signaling pathways involved in the differential genes were obtained.(2)Network pharmacology was performed on 9 Chinese medicines contained in Weipiling to clarify the active ingredients and the targets involved in the treatment of GPL.After that,through the analysis of the protein interaction network of the drug target and the enrichment of GO and KEGG,the mechanisms involved in the treatment of GPL by Weipiling were analyzed,which provided a basis for subsequent experimental verification.2.Experimental research(1)Observe the autophagy activity of GPL mice and the effect of regulating autophagy on GPL miceGPL mouse models were replicated by combining the chemical modeling agent MNU with free drinking and hunger and dystrophy,and then 48 mice were randomly divided into blank control group,model group,autophagy inhibitor 3-MA group and autophagy activation RAPA group,after intervention for 10 weeks,HE staining was used to observe the pathological changes of gastric mucosa in each group of mice.ULK1,Atg7,Atg12,Atg16L1 mRNA expression were detected by PCR,and autophagy proteins LC3,p62,Beclin-1,ULK1,Atg13,Atg5,Atg12 were detected by Western blot.(2)The effects of Weipiling on autophagy of GPL mice and its effects on PI3K/AKT/mTOR Signaling pathwayThe GPL mouse model was replicated by the above method,and 72 mice were randomly divided into a blank control group,a model group,a low-dose Weipiling group,a high-dose Weipiling group,a Weipiling+3-MA group,and a RAPA group.The mice in each group were then intervened for 10 weeks.The gastric mucosal pathology of each group was observed by HE staining and AB-PAS staining.The expressions of Ki-67 and Caspase-3 were observed by immunohistochemistry.ALT,AST,creatinine,Urea nitrogen was used to observe the liver and kidney function of mice in each group,and the expressions of ULK1,Atg12,Atg16L1,PIK3CA,AKT,and mTOR were detected by PCR.Western blot was used to detect the expression of LC3,p62,Beclin-1,ULK1,Atg13,Atg5,Atg12 and upstream PI3K,AKT,p-AKT,mTOR,PTEN,p70S6k and 4E-BP1 proteins.Results1.Bioinformatics and network pharmacology research(1)By analyzing the gene chip data of 19 chronic gastritis cases and 39 GPL patients in GSE55696 data,9498 differential genes were obtained,and 77 genes involved in autophagy.Among the 77 autophagy-related differential genes,ULK1,ATG13,PIK3C3,ATG16L1,BAD,etc.are down-regulated genes,and BCL2,TNF,HIF1A,IL6,S100A8,etc.are up-regulated.These differential genes are mainly involved in 17 signal pathways including autophagy,PI3K/AKT,and apoptosis,etc.(2)113 active ingredients were obtained from TCMSP database and Chinese Academy of Sciences database.Through TCMSP database,SymMap database and SwissTargetPrediction database,107 targets were retrieved,of which 49 targets related to GPL.49 targets were involved in metabolic-related biological processes,and were related to 15 signal pathways including PI3K/AKT signaling pathway,TNF signaling pathway,and apoptosis,etc.2.Experimental results(1)Histopathological lesions of GPL mice and the effects of autophagic activationCompared with the blank group,the GPL model group had defect and disappearance of gastric mucosal barrier,disordered distribution of gastric mucosa epithelial glands,poorly defined glandular cavity,adjacent glands back to back,and lamina propria hemorrhage.Gastric mucosal epithelial cells have different morphology and size,and deep staining of the nucleus.The expression of LC3? and p62 in GPL model group were decreased significantly(P<0.05),the mRNA expression of ULK1,Atg7,Atg12,Atg16L1 were decreased significantly(P<0.05),and the protein expression of Beclin-1,ULK1,Atg13,Atg5,Atg12 were decreased significantly(P<0.05).In the 3-MA group,the structure of gastric mucosa glands was disordered,the shape and size of glandular ducts were irregular,the glands were crowded and dense,the cell morphology was different,the nucleus was deeply stained and enlarged.The expression of LC3II and p62 in GPL model group were decreased significantly(P<0.05),the mRNA expression of ULK1,Atg7,Atg12,Atg16L1 were decreased significantly(P<0.05),and the protein expression of Beclin-1,ULK1,Atg13,Atg5,Atg12 were decreased significantly(P<0.05).In the RAPA group,the glandular structure of the mice was intact,the boundaries were clear,and the lamina propria structure was well preserved.No changes in cell atypia and bleeding were observed.The expression of LC3II was increased and the expression of p62 was decreased in RAPA group significantly(P<0.05).At the same time,the expression of ULK1,Atg7,Atg12,Atg16L1 mRNA in RAPA group was increased significantly(P<0.05),Beclin-1,ULK1,Atg13,Atg5,Atg12 protein expression was increased significantly(P<0.05),suggesting that activated autophagy has a certain effect on the treatment of GPL.(2)Weipiling can activate GPL autophagy by inhibiting PI3K/AKT/mTOR signalingAfter 10 weeks of intervention,the results of HE staining showed that the gastric mucosa of the high-and low-dose Weipiling 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 Weipiling were mainly red stained and some blue stained.Immunohistochemical results showed that Ki-67 expression was reduced significantly(P<0.05)and Caspase-3 expression was increased significantly(P<0.05)in the low-dose and high-dose Weipiling group.After the treatment of Weipiling,the western blot showed that LC3?expression increased,p62 expression decreased significantly(P<0.05),and Beclin-1,ULK1,Atg13,Atg5,Atg12 protein expression increased significantly(P<0.05),and the PCR results showed that ULK1,Atg12,and Atg16L1 mRNA expression increased significantly(P<0.05).After intervention with Weipiling,the expressions of PIK3CA,AKT and mTOR mRNA were significantly reduced(P<0.05),and the expressions of PO3K,AKT,p-AKT,mTOR,p70S6k and 4E-BP1 proteins were significantly reduced(P<0.05),The expression of PTEN protein increased significantly(P<0.05).Conclusion1.GPL mice have autophagy inhibition.Activation of autophagy can improve pathological changes in GPL mice.2.Weipiling can activate autophagy by inhibiting the PI3K/AKT/mTOR signaling pathway and activating PTEN,thereby reversing the pathological changes of gastric mucosa in GPL mice and blocking the malignant progression of GPL.