Xiaojianzhong decoction prevents gastric precancerous lesions in rats by inhibiting autophagy and glycolysis in gastric mucosal cells

BACKGROUND Gastric precancerous lesions(GPL)precede the development of gastric cancer(GC).They are characterized by gastric mucosal intestinal metaplasia and dysplasia caused by various factors such as inflammation,bacterial infection,and injury.Abnormalities in autophagy and glycolysis affect GPL progression,and their effective regulation can aid in GPL treatment and GC prevention.Xiaojianzhong decoction(XJZ)is a classic compound for the treatment of digestive system diseases in ancient China which can inhibit the progression of GPL.However,its specific mechanism of action is still unclear.AIM To investigate the therapeutic effects of XJZ decoction on a rat GPL model and the mechanisms underlying its effects on autophagy and glycolysis regulation in GPLs.METHODS Wistar rats were randomly divided into six groups of five rats each and all groups except the control group were subjected to GPL model construction for 18 wk.The rats’body weight was monitored every 2 wk starting from the beginning of modeling.Gastric histopathology was examined using hematoxylin-eosin staining and Alcian blue-periodic acid-Schiff staining.Autophagy was observed using transmission electron microscopy.The expressions of autophagy,hypoxia,and glycolysis related proteins in gastric mucosa were detected using immunohistochemistry and immunofluorescence.The expressions of the following proteins in gastric tissues:B cell lymphoma/Leukemia-2 and adenovirus E1B19000 interacting protein 3(Bnip-3),microtubule associated protein 1 light chain 3(LC-3),moesin-like BCL2-interacting protein 1(Beclin-1),phosphatidylinositol 3-kimase(PI3K),protein kinase B(AKT),mammalian target of rapamycin(mTOR),p53,AMP-activated protein kinase(AMPK),and Unc-51 like kinase 1(ULK1)were detected using western blot.The relative expressions of autophagy,hypoxia,and glycolysis related mRNA in gastric tissues was detected using reverse transcription-polymerase chain reaction.RESULTS Treatment with XJZ increased the rats’body weight and improved GPL-related histopathological manifestations.It also decreased autophagosome and autolysosome formation in gastric tissues and reduced Bnip-3,Beclin-1,and LC-3II expressions,resulting in inhibition of autophagy.Moreover,XJZ down-regulated glycolysis-related monocarboxylate transporter(MCT1),MCT4,and CD147 expressions.XJZ prevented the increase of autophagy level by decreasing gastric mucosal hypoxia,activating the PI3K/AKT/mTOR pathway,inhibiting the p53/AMPK pathway activation and ULK1 Ser-317 and Ser-555 phosphorylation.In addition,XJZ improved abnormal gastric mucosal glucose metabolism by ameliorating gastric mucosal hypoxia and inhibiting ULK1 expression.CONCLUSION This study demonstrates that XJZ may inhibit autophagy and glycolysis in GPL gastric mucosal cells by improving gastric mucosal hypoxia and regulating PI3K/AKT/mTOR and p53/AMPK/ULK1 signaling pathways,providing a feasible strategy for the GPL treatment.

Effect of bile salts and bile acids on human gastric mucosal epithelial cells

Objective：To explore the effect of bile salt and bile acid on cultured eternalized human gastric mucosa epithelium GES-1 cells. Methods：Cultured eternalized human gastric mucosa epithelium GES-1 cells were treated with media containing 6 different kinds of bile salts and 3 different kinds of bile acids and their mixture with different concentrations： GCDC（glycochenodeoxychoμte）, GDC （glycodeoxychoμte）, GC（glycochoμte）, TCDC（taurochenodeoxychoμte）, TDC（taurodeoxychoμte）, TC （taurochoμte）, LCA （lithocholicacid）, CA（cholic acid）, DCA（deoxycholic acid）（50 μ mol/L,250 μ mol/L,500 μ mol/L,1000 μ mol/L）, DY（mixture of bile salts） and DS（mixture of bile acids）（250 μ mol/L,500 μ mol/L,1000 μ mol/L,1500 μ mol/L, 2000 μ mol/L）, in comparison with the control group（in normal media without bile salts and bile acids）. Cell proliferation was assessed by MTT（3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide） assay for 72 hours with different concentrations and the apoptotic cells were assayed by flow cytometry （FCM） with Annex V-FITC conjugated with propidium iodide（PI） staining for 24 hours with different concentrations（1500,2000 μt mol/L）. Results：There was no significant difference in morphology and cell proliferation in GC group after 24-72 h. Low concentration（50 μ mol/L） of GCDC, GDC, TCDC, TDC and TC accelerated gastric epithelial cell growth in a dosage-time dependent manner. At middle concentration （250-500 μ mol/L）, it showed positive effect after 24-48 h, while negative effect after 72 h. At high concentration（1000 μ mol/L）, it accelerated gastric epithelial cell growth after 24h and show consistent inhibition even leading to necrosis after 48-72 h. LCA and CA showed a positive effect on the concentration of 50 μ mol/L after 24-72 h, while 250-1000 μ mol/L showed a trend towards apoptosis after 24-72 h. At 50-500 μmol/L, DCA showed proliferation after 24 h and apoptosis after 48-72 h, but showed necrosis after 24-72 h at 1000 μmol/L. DY and DS could facilitate normal gastric mucosa epithelial cell growth at low concentration （250-500 μ mol/L）, however at 1000-2000 μ mol/L the trend shifted from apoptosis to necrosis. FCM with Annexin-V conjugated with PI staining revealed that GCDC, GDC, GC, TCDC, TDC, TC, LCA, CA, DCA, DY and DS induced apoptosis of human gastric mucosal epithelial cells. They were all significantly higher than that of the control（P 〈 0.05）, but there was no significant difference in GC group （P 〉 0.05）. The bile salts induced apoptosis in a time-dose-dependent manner. Conclusion：Our results suggested that bile acid and bile salt is the trigger of injury in human gastric mucosal epithelial cells.