| Background: "Aging" as the most important risk factor and pathogenesis of many chronic diseases,seriously threatens the life and health of the elderly.As an important organ for human food intake,digestion,and absorption,the stomach is a key source of energy for the body.In traditional Chinese medicine,the stomach is the place where postnatal water and grain essence is generated,and it is also the key to continuously nourishing the congenital essence and the essence of the five organs and six viscera in the kidney.Its weakness is an important cause of human aging.The speed of stomach aging directly determines the accumulation of essence,qi,blood,and kidney essence in the body.Therefore,if people want to achieve the wish of "healthy and long life" and delay the aging of the body,they must first take care of their stomach to achieve their "long-term gastrointestinal health".It can be seen that the resistance of stomach aging by middle-aged and elderly people is a fundamental measure to delay aging and increase longevity.The gastric mucosa is an important site of stomach aging,and the gastric mucosal epithelial cells are the main cells of the gastric mucosa,and the cell aging mediated by them is the basis of stomach aging.Therefore,studying the drug intervention and mechanism of gastric mucosal epithelial cell aging is of important theoretical and practical significance for combating stomach aging.Previous studies have found that Chaenomeles speciosa(Sweet)Nakai triterpenes(CST)have a good protective effect on the gastric and intestinal mucosal injury in mice/rats,and can effectively inhibit the apoptosis of human gastric mucosal RGM-1 and GES-1 cells stimulated by indomethacin and lipopolysaccharide,showing good anti-gastric mucosal epithelial cell damage and aging activity.Clinical trials have also confirmed its therapeutic effect on gastric and intestinal mucosal injury.However,its mechanism of anti-stomach aging is not clear,which to some extent limits its clinical application and further development.Objective: To study the molecular mechanism of CST delaying stomach aging from the cellular and animal levels,and provide a solution to break through the bottleneck of anti-stomach aging drug development,and provide a new path for the development of anti-stomach aging drugs.The research will lay a solid theoretical foundation for the future clinical application of triterpenes from Chaenomeles speciosa.Methods:1.In vitro experiments:(1)The effect of CST on the viability of D-galactose(D-gal)-induced aging GES-1 cells was detected by MTT assay;(2)Scratch and Transwell assays were used to detect the effect of CST on the migration of aging GES-1 cells;(3)SA-β-gal staining was performed to evaluate the effect of CST on cellular senescence of GES-1 cells;(4)Flow cytometry was used to detect the effect of CST on reactive oxygen species(ROS),mitochondrial membrane potential(MMP),cell cycle,and apoptosis of GES-1 cells;(5)Western blot was used to detect the expression of PI3K/AKT/GSK3β/β-catenin pathway-related proteins and Bcl-2/Bax proteins in GES-1 and GSK3β-overexpressing GES-1cells treated with CST.2.In vivo experiments:C57/BL6 J mice were used to establish a D-gal and high-fat diet(HFD)-induced aging model.CST(50 and 100 mg/kg)was administered by gavage once a day for 8 weeks.Blood samples were collected from the eye and gastric tissues were harvested 6 hours after the last administration under pentobarbital anesthesia.(1)HE staining was used to examine the morphological changes of gastric tissues in mice;(2)Transmission electron microscopy was used to observe the changes in gastric cell and organelle structures in mice;(3)Western blot was used to detect the expression of PI3K/AKT/GSK3β/β-catenin signaling pathway-related proteins and Bcl-2/Bax proteins in the gastric tissues of aging mice.Results:1.In vitro experiments:(1)MTT results showed that CST significantly increased the viability of aging GES-1cells in a dose-dependent manner within the concentration range of 12.5-100 μg/m L(P<0.01 compared to the D-gal group);(2)CST significantly promoted the migration of aging GES-1 cells(P<0.05 or P<0.01 compared to the D-gal group);(3)CST significantly reduced the proportion of SA-β-gal-positive cells(P<0.05 or P<0.01 compared to the D-gal group);(4)CST significantly reduced ROS and the proportion of cell cycle arrest in aging cells(P<0.05 or P<0.01 compared to the D-gal group).CST had no effect on apoptosis or MMP in normal GES-1 cells but promoted apoptosis and MMP depolarization in aging GES-1 cells;(5)Western blot results showed that compared to the control group,the expression of p-PI3K/PI3 K,p-AKT/AKT,p-GSK3β/GSK3β,β-catenin,Cyclin D1,CDK4,and p-Rb/Rb proteins were significantly decreased in the D-gal group,and the expression of P16 protein was significantly increased(P<0.05 or P<0.01).Treatment with CST(25,50,and 100 μg/m L)significantly increased the expression of p-PI3K/PI3 K,p-AKT/AKT,p-GSK3β/GSK3β,β-catenin,Cyclin D1,CDK4,and p-Rb/Rb proteins,and reduced the expression of P16protein(P<0.05 or P<0.01 compared to the D-gal group);2.In vivo study:(1)The HE staining results showed that,in the Control group,the gastric mucosal epithelial layer boundary in mice was clear and intact,with a single layer of columnar epithelial cells and intrinsic gland cells orderly arranged,small and tightly connected.In the D-gal group,the gastric epithelial layer boundary was blurred,with single-layer columnar epithelial cells degenerating and necrotizing,shedding,chief cells and parietal cells enlarging,and disordered loose arrangement,with a small amount of inflammatory infiltration.Compared with the D-gal group,the mucosal epithelial layer morphology in CST-treated group(50 and 100 mg/kg)was clearer and more intact,and the chief and parietal cell morphology tended to be normal.(2)Ultrastructure detection of gastric tissue showed that,in the D-gal group,the cell membrane of gastric wall cells in mice was damaged,cytoplasmic dissolution occurred,mitochondria deformed,and intracellular tubules disordered.chief cells manifested significant endoplasmic reticulum expansion,which improved in the treatment group.In the D-gal group,the gastric columnar epithelial cells showed cytoplasmic dissolution and deepened nuclear staining,while the CST-treated group(50 and 100 mg/kg)had intact columnar epithelial morphology,orderly cell arrangement,and abundant mucus granules.In the D-gal group,the neck mucous cell volume increased,and the cell membrane was damaged,without a complete shape,while the CST-treated group(50 and 100 mg/kg)had intact neck mucous cell morphology,orderly arrangement,and a clear migration trend.(3)Western blot results showed that,compared with the Control group,the expression of p-PI3 K,p-AKT,p-GSK3β,β-catenin,cyclin D1,CDK4,and p-Rb proteins in the D-gal group was significantly reduced(P<0.01).Compared with the D-gal group,CST-treated group(50and 100 mg/kg)increased the expression of p-PI3 K,p-AKT,p-GSK3β,β-catenin,cyclin D1,CDK4,and p-Rb proteins(P<0.05 or P<0.01).In addition,CST-treated group(50 and 100mg/kg)inhibited the expression of pro-apoptotic protein Bax in mouse gastric tissue while also inhibiting the expression of anti-apoptotic protein bcl-2.Conclusion:CST can delay D-gal-induced aging of GES-1 cells and mouse stomach,and its mechanism of action is confirmed to be through the activation of the PI3K/AKT/GSK3β/β-catenin pathway,promoting the migration of aging GES-1 cells,delaying cell cycle arrest,and improving the gastric tissue,cell morphology,and subcellular structure of aging mice.These results demonstrate the good prospects of CST in delaying gastric aging. |
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