| The research backgroundGastric cancer is characterized by high morbidity,high metastasis rate and high mortality rate,and its early diagnosis rate is low,which seriously affects human life and health.The occurrence and development of gastric cancer is a complex biological process.According to the classical Correa hypothesis,it develops along the direction of "normal gastric mucosa chronic non-atrophic gastritis→chronic atrophic gastritis→intestinal metaplasia→dysplasia→gastric adenocarcinoma".Wherein the incomplete intestinal metaplasia,dysplasia due to the higher risk of cancer,it is called precancerous lesions(GPL).In view of the fact that there is no specific therapeutic drug for gastric cancer,it is an effective measure to prevent and delay the progression of GPL to gastric cancer.The target drugs of modern medicine for GPL are still blank.Traditional Chinese medicine(TCM)can effectively improve the clinical symptoms of GPL patients,block and improve the malignant progress of GPL pathology.In addition,the characteristics of multi-target and safe use of traditional Chinese medicine make the treatment of GPL receive more and more attention.Previous studies found that although the pathology of GPL model rats induced by nitrosoguanidine(MNNG)was similar to that of clinic,the individual differences of animals were large and the replication cycle of the model was long.The GPL model of spleen deficiency syndrome with small individual differences and short cycle could be replicated by gene knockout combined with fasting every other day.Phase studies have also confirmed that TCM could inhibit the proliferation,apoptosis and autophagy of gastric mucosal epithelial cells and AEG by regulating PI3K/AKT/mTOR pathway,but the exact mechanism is still unclear.Therefore,this research aims to find new molecular targets for the effective treatment and regulation of GPL,and to provide new ideas for the development of preventive and therapeutic drugs for GPL by exploring the regulating effect of Weipiling(WPL)on mTOR/HIF-1a/SIRT6 signaling pathway mediated aerobic glycolysis and regulating the local microenvironment of gastric mucosa.Objective1.Atp4a-/-mice were constructed by knocking out H+-K+-ATPase a in gastric parietal cells of C57BL/6J mice by genetic engineering.By observing the pathological changes of gastric mucosa,the biological behavior of gastric mucosal epithelial cells(proliferation,apoptosis,differentiation,etc.)and the expression of GPL molecular markers(MUC2,Ki-67 etc.),a GPL mouse model was established and screened.2.On the basis of previous studies on the metabolism of aerobic glycolysis(AEG)in gastric mucosal cells of GPL rats,the pathogenesis of GPL was studied by observing the expression of key rate-limiting enzymes and transporters in the glycolysis of Atp4a-/-mice.3.Based on the study of the regulation mechanism of PI3K/AKT/mTOR signaling pathway in AEG metabolism of GPL rats in the early stage,the regulation mechanism of AEG signaling pathway in GPL was explored from the downstream of glycolysis mTOR signaling pathway by observing the activity of Atp4a-/-mice mTOR/HIF-1a/SIRT6.4.On the basis of down-regulation of PI3K/AKT/mTOR by traditional Chinese medicine of invigorating spleen,removing blood stasis and detoxifying,which inhibits the proliferation,apoptosis imbalance and autophagy inhibition of GECs induced by AEG in GPL rats,the pharmacodynamics and molecular mechanism of Weipiling’ s intervention on glycolysis and reversal of atrophy and Intestinalization of gastric mucosa in Atp4a-/-mice downstream of mTOR signaling pathway were discussed.Methods1.Reproduction of GPL animal model Atp4a-/-mice:① Construction of Atp4a-/-mice:By searching the coding gene structure of H+-K+-ATPase a in gastric parietal cells of C57BL/6J mice,the transcripts,translation start-stop sites and coding proteins were determined.Atp4a gene was edited by CRISPR/Cas9 technology,sg RNA and Cas9 RNA were prepared,and sg RNA and Cas9 RNA were injected into fertilization of C57BL/6J mice.In the ovum,the injected fertilized eggs were transplanted into the fallopian tubes of pseudopregnant female mice to obtain and screen positive FO mice,and the F1 hybrid mice were obtained by mating with wild type mice.The homozygous mice of F3 generation were obtained and their genotypes were identified by PCR.② Reproduction and identification of animal models of GPL mice:Thirty F3 and subsequent Atp4a-/-mice of the same generation were selected,and 20 WT mice were taken as blank control.Ten Atp4a-/-mice and five WT mice were randomly selected to observe the pathological changes of gastric mucosa at 10,12,14 and 16 weeks of age:atrophy,intestinal metaplasia and dysplasia(degree,range and grade),and gastric mucosa was determined simultaneously.Epithelial cell proliferation,apoptotic molecular markers(MUC2,Ki-67,p53),AEG metabolism(LDHA),mTOR/HIF-la signaling pathway(PI3K,AKT,mTOR,HIF-la).2.Effects of WPL on GPL animal model Atp4a-/-mice:Ten WT mice were taken as blank group and 50 Atp4a-/-mice were randomly divided into four groups:model group,vitamin group(VIT,0.2g.kg-1),WPL high dose group(WPL-H,15g.kg-1),low dose group(WPL-L,7.5g.kg-1).The mice were given orally for 10 weeks and 1.5-2 hours after the last administration.The gastric mucosa of dead mice was taken from the antrum of stomach and used to determine the signal pathways of mTOR/HIF-la/SIRT6(PI3K,p-AKT,mTOR,HIF-1a,SIRT6,AMPK,TSC1,TSC2,Rheb,NF-kappa B),AEG metabolic pathways(HK-II,PKM2,EN01,MPC1 and LDHA),intestinal metaplasts(CDX2,MUC2),proliferation(Ki-67),apoptosis(p53)and metastasis(Lgr5+).Results(I)Pathological changes and phenotypic characteristics of gastric mucosa in Atp4a-/-mice modelCompared with WT mice,10-week-old Atp4a-/-mice had defect and disappearance of gastric mucosal barrier,disordered gland arrangement,enlarged lacunae between glands,and increased intestinal metaplasia goblet cells.Histopathic intestinal metaplasia of large intestine type.The basement membrane was thicker and uneven in thickness.Inflammatory cells infiltrated the submucosa and formed anterior lymph node corpuscles.With the increase of age,intestinal metaplasia became more obvious,gland atrophy became more serious,basement membrane became thicker and cell atypia became more obvious.AB-PAS staining showed that the gastric mucosal epithelial cells secreted acid glycoprotein and proteoglycan mucus 10 weeks later.There were obvious blue staining foci.The gastric mucosal intestinal metaplasia foci in Atp4a-/-mice were wider and deeper stained,mainly small intestinal metaplasia.The extent and degree of intestinal metaplasia increased with the age of mice.Immunohistochemical results showed that MUC2,Ki-67 and p53 were positive in gastric mucosa of Atp4a-/-mice at 10 weeks.Western blot results showed that LDHA expression of key AEG enzymes(PI3K,p-AKT,mTOR,HIF-la)in mTOR/HIF-1a signaling pathway was significantly higher than that in WT model group(P<0.05).(Ⅱ)Effect of WPL on Pathological Histomorphology of Gastric Mucosa in Atp4a-/-Mice Model of GPLBased on the above results,The 10-week-old Atp4a-/-mice were used as animal models of GPL.Different doses of WPL acted on Atp4a-/-mice.The results of H&E and AB-PAS staining showed that the gastric mucosal tissues in each group were more orderly than those in the model group,with regular gland morphology and rare "back-to-back" or stratification changes.Goblet cells were seen in the gastric mucosal epithelium,with deep staining,enlargement,overlap,polarity weakening,prominent nucleoli,obvious nuclear division,interstitial edema and congestion on the basal side.A small amount of neutrophils and eosinophils infiltrated,but significantly less than the model group.AB-PAS staining showed that metaplasia area of intestinal epithelium of mice in each dose group of WPL decreased and the degree of metaplasia decreased.(Ⅲ)Biological effects of WPL on gastric mucosal cells of GPLImmunohistochemical results showed that compared with the model group,the expression levels of CDX2,MUC2,Ki-67,p53 and Lgr5+ in gastric mucosa of Atp4a-/-mice in high and low dose groups were significantly lower(P<0.05).(IV)Regulation of WPL on AEG Key Molecules Mediated by mTOR/HIF-1a/SIRT6 Signaling Pathway in Gastric Mucosa of GPLWestern Blot results showed that the expression of transporters such as HK-II,PKM2,ENO1,MPC1 and LDHA decreased significantly(P<0.05).And PI3K,p-AKT,mTOR,HIF-la,SIRT6,MAPK,Rheb,NF-kappa B protein,SIRT6,TSC1 and TSC2 protein in gastric mucosa of Atp4a-/-mice in high and low dose groups were significantly lower than those in model group(P<0.05),while the expression of SIRT6,TSC1 and TSC2 protein was significantly higher(P<0.05).Conclusion1.Atp4a-/-mice of 10 weeks old could simulate the model of precancerous lesions of gastric cancer;2.GPL model Atp4a-/-mice have abnormal AEG metabolism and are regulated by mTOR/HIF-1 a/SIRT6 signaling pathway;3.WPL may prevent gastric carcinogenesis by improving histological lesions,atrophy,intestinal metaplasia,dysplasia,inhibiting hyperproliferation of dedifferentiated cells,inhibition of apoptosis,super survival and other malignant biological behaviors in mice with precancerous lesions.4.WPL could inhibit the abnormality of AEG metabolism induced by activation of mTOR/HIF-1α/SIRT6 signaling pathway in gastric mucosa,and initiate the gastric mucosal protective cascade of GPL animal model Atp4a-/-mice,anti-GPL and prevent gastric cancer.In conclusion,this study successfully replicated a 10-week-old spontaneous GPL model of Atp4a-/-mice.WPL can inhibit the activity of AEG by regulating the mTOR/HIF-1α/SIRT6 signaling pathway,and exert anti-GPL effect to prevent gastric cancer. |
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