| ObjectiveGastric cancer ranks third in the number of cancer incidence and deaths in China,and it has become a difficulty in diagnosis and treatment due to lacking of specific treatment methods.Gastric Precancerous Lesions(GPL),as a key stage with a higher risk of cancer in the process of gastric "inflammatory-cancer" transition,is a key node in the study of gastric cancer prevention and treatment.The current clinical GPL is mostly symptomatic and supportive treatment without specific diagnosis and treatment methods.Studies have confirmed that traditional Chinese medicine can significantly improve the symptoms of GPL patients.Studies have shown that the imbalance of GPL microenvironment can show marked abnormalities in hypoxia markers such as HIF-1 α,VEGF,and P53,and activation of AEG.Hypoxic microenvironment can also induce or alter oxidative stress,hypoxia-inducible factors(HIFs)and other pathways to regulate the energy metabolism and malignancy of tumor cells.Therefore,in this study,GPL rat and mouse models were constructed,taking the"hypoxic microenvironment" as the breAKThrough point,and based on the regulation of AKT/FOXO3a signaling pathway,to observe the effect of Jianpi Huayu Jiedu recipe on oxidative stress,glycolysis-The role of oxidative phosphorylation,thereby revealing the mechanism of action of Jianpi Huayu Jiedu Fang in the treatment of GPL,and providing a basis for clinical medication.(1)Intervention effect of Jianpi Huayu Jiedu Fang on GPLThe GPL rat model was replicated by the method of "MNNG(200μg/mL)+abnormal hunger and satiety",and the experiment was divided into control group,model group.Jianpi Huayu Jiedu Fang low-dose group,Jianpi Huayu Jiedu Fang high-dose group and maintenance group.Model and administration group used MNNG to drink freely+fasting every other day for 28 weeks,and drug intervention started from 13 weeks for 16 consecutive weeks.The ATP4a-/-GPL mouse model was replicated by gene knockout technology,and the experiment was divided into blank group(wild-type mice),model group,low-dose Jianpi Huayu Jiedu Fang group,Jianpi Huayu Jiedu Fang high-dose group,Verbinin group,model and administration group were given free drinking water+fasting every other day for 20 weeks,and drug intervention lasted for 10 weeks from the 11th week.HE staining,AB-PAS staining and IHC were used to observe the pathological changes of gastric mucosa and the expression of MUC2 in the two models.Tunel staining was used to observe the gastric mucosal apoptosis.Western blot was used to detect the expressions of apoptosis-related proteins BCL-2 and BAX.(2)Effects of Jianpi Huayu Jiedu Fang on GPL oxidative stressThe expression of HIF-1α in gastric mucosa tissue of GPL rats and mice was detected by IHC and PCR,the expression of oxidative stress-related enzymes/proteins and/or intermediate products SOD,GSH and MDA were detected by the kit,and the oxidative stress signaling pathway was detected by Western blot.The expression of KEAP1-NRF2/HO-1 related proteins,and the expression of HO-1 and NRF2 was detected by immunohistochemistry.(3)Effects of Jianpi Huayu Jiedu Fang on GPL aerobic glycolysis and oxidative phosphorylationWestern blot was used to detect the expressions of glycolysis-related proteins GLUT1,PFKFB3 and HK2;PCR was used to detect the mRNA expressions of glycolysis-related genes PFKFB3,PKM2 and PDK1;Expression of IDH3β,UQCRC2 protein and mRNA.(4)Effects of Jianpi Huayu Jiedu Fang on AKT/FOXO3a signaling pathway in GPLThe expressions of AKT,P-AKT,FOXO3a and p-FOXO3a proteins were detected by Western blot,and the activation and nuclear entry of FOXO3 a were detected by immunofluorescence.Results(1)Jianpi Huayu Jiedu Fang improves GPL gastric mucosal lesions and promotes apoptosisHE staining,AB-PAS staining and immunohistochemical staining(IHC)in two GPL model mice showed that MNNG and ATP4a knockout could induce gastric mucosal atrophy,intestinal metaplasia,dysplastic lesions and The high expression of MUC2 with Ki-67 indicated that the model was successfully replicated.The results of Tunel and WB showed that apoptosis was inhibited in the GPL model.Jianpi Huayu Jiedu Fang can effectively improve the histopathological changes of GPL gastric mucosa,reduce the expression of MUC2,at the same time promote the expression of the pro-apoptotic protein BAX,inhibit the expression of the anti-apoptotic protein BCL-2,promote the apoptosis of GPL cells,and play the role of gastric mucosa.Protective effects.(2)Jianpi Huayu Jiedu Fang activates GPL to resist oxidative stressThe results of IHC and PCR showed that Jianpi Huayu Jiedu Decoction effectively reduced the high expression of HIF-1α induced by MNNG and ATP4a knockout,and the kit test showed that Jianpi Huayu Jiedu Decoction promoted antioxidant stress related enzymes SOD and GSH in GPL.The results of Western blot and IHC showed that Jianpi Huayu Jiedu Recipe activated GPL oxidative stress signaling pathway KEAP1-NRF2/HO-1,promoted the increase of NRF2 into the nucleus,and activated its downstream antioxidant stress-related proteins.Express.(3)Jianpi Huayu Jiedu Fang inhibits GPL glycolysis and activates oxidative phosphorylationWestern blot results showed that Jianpi Huayu Jiedu Fang inhibited the expression of glycolysis-related proteins GLUT1,PFKFB3 and HK2 in GPL,and PCR results showed that Jianpi Huayu Jiedu Fang inhibited the expression of GPL glycolysis-related genes PFKFB3,PKM2,PDK1 mRNA,the kit results showed that Jianpi Huayu Jiedu Fang significantly reduced the expression of serum lactic acid increased in GPL,Western blot and PCR results showed that Jianpi Huayu Jiedu Fang significantly activated oxidative phosphorylation in GPL,up-regulated its related enzymes IDH3β,UQCRC2 protein and mRNA expression.(4)Study on the mechanism of Jianpi Huayu Jiedu Fang inhibiting AKT/FOXO3a signaling pathway in GPLWestern blot results showed that Jianpi Huayu Jiedu Fang inhibited the activity of AKT/FOXO3a signaling pathway in GPL,inhibited the phosphorylation of AKT,alleviated its inhibitory effect on FOXO3a,decreased the expression of p-FOXO3a protein,and upregulated the expression of FOXO3a.Further verification by immunofluorescence showed that Jianpi Huayu Jiedu Fang promoted the increased nuclear transcription and expression of FOXO3a in GPL,thereby initiating its regulation of downstream oxidative stress and glucose metabolism balance.Conclusion1.The improvement of GPL by Jianpi Huayu Jiedu Prescription may be to block its malignant progression by promoting the apoptosis of GPL cells.2.Jianpi Huayu Jiedu Prescription activates the KEAP1-NRF2/HO-1 signaling pathway in GPL to resist oxidative stress,regulate the redox balance of the microenvironment,correct the hypoxic microenvironment of GPL,and exert an antioxidant effect.3.Jianpi Huayu Jiedu Prescription inhibits the aerobic glycolytic activity of GPL,promotes the selective oxidative phosphorylation of GPL cells,and regulates the imbalance of glucose metabolism induced by GPL hypoxic microenvironment.4.Jianpi Huayu Jiedu Prescription regulates the balance of aerobic glycolysis-oxidative phosphorylation in the downstream GPL by inhibiting the activation of AKT/FOXO3a signaling pathway in GPL,and activates the antioxidant response of the downstream KEAP1NRF2/HO-1 signaling pathway.It stimulates and corrects the hypoxic microenvironment of GPL,thereby exerting its therapeutic effect on GPL. |
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