Study On The Protective Effect Of Volatile Oil From Three Chinese Medicines Of Alpinia On GES-1 Cell Injury And Its Mechanism Of Anti Gastric Ulcer Cold Syndrome

Objective:1.Objective to investigate the protective effect and mechanism of volatile oil from Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd on gastric epithelial cell（GES-1）injury induced by ethanol,and to evaluate the biological activity of volatile oil from three Chinese medicines of Alpinia.2.Objective to establish the rat model of gastric ulcer cold syndrome,and to explore the pharmacodynamic effect and mechanism of volatile oil from Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd on rats with gastric ulcer cold syndrome.3.By discussing the effect of the volatile oil of three Chinese medicines of Alpinia on gastric mucosal epithelial cells in vitro and the in vivo efficacy and mechanism of anti-cold syndrome of gastric ulcer in rats,the correlation between the volatile oil and the material basis of the efficacy was analyzed.Methods:1.Protection and mechanism of GES-1 cell injury.（1）Model establishment:GES-1 cells were treated with 0-9%ethanol for2,3,4,5 and 6 hours,respectively.（2）Viability test:the normal GES-1 cells were treated with different concentrations of volatile oil solution,the cell survival rate was determined,and the effect of volatile oil on the activity of normal GES-1 cells was observed.（3）Protective effect:the cell survival rate of GES-1 cells treated with volatile oil of three Chinese medicines of Alpinia was determined by ethanol induced cell injury model.（4）Mechanism:the levels of IL-8,TNF-αand VEGF secreted in the cell supernatant were measured by ELISA method;the expression of b FGF protein was measured by Western-Blot method.2.Pharmacodynamic study on cold syndrome of gastric ulcer.（1）Grouping and administration:90 SD rats were randomly selected as blank control group,and the remaining rats were divided into model group,positive drug gingerol group,high-dose and low-dose groups of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil,with 10 rats in each group.The blank control group and model group were given 0.5%Tween 80 aqueous solution,the positive group was given gingerol solution,and the other test groups were given high and low dose volatile oil solution of three Chinese medicines of Alpinia.（2）Efficacy index:The overall state,food intake,water intake,body weight and Anal temperature of rats were observed and recorded.The morphological and pathological changes of gastric tissue were observed by naked eye and he staining.Gastric juice volume,gastric acid p H and pepsin activity were measured.3.Study on pharmacodynamic mechanism of cold syndrome of gastric ulcer.The levels of SOD,MDA,GAS,SS,6-keto-pgf1αin gastric tissue and IL-6,IL-10,ET,NO,COR,MTL and TXB₂in serum were detected by ELISA.Results:1.Protection and mechanism of GES-1 cell injury.（1）GES-1 cell injury model:the results showed that 6%ethanol solution for 3 h was the best condition to induce GES-1 cell injury.（2）Cell viability test:when the concentration of each volatile oil reached320μg/ml,there was no obvious cytotoxicity on GES-1 cells.（3）Protective effect on cell injury:the survival rate of GES-1 cells was significantly increased in low,medium and high dose groups of volatile oil of three Chinese medicines of Alpinia（P<0.01）.（4）Protection mechanism:After the volatile oil is administered,the levels of IL-8 and TNF-αin the cell supernatant were significantly reduced（P<0.01or P<0.05）,and the level of VEGF was significantly increased（P<0.01 or P<0.05）;b FGF protein expression in cells increased（P<0.01 or P<0.05）.2.Effects of volatile oil from three Chinese medicines of Alpinia on the overall state,eating,drinking,body weight,Anal temperature,gastric juice,gastric acid,pepsin,morphology and pathology of rats with gastric ulcer cold syndrome.（1）Compared with the model group,the rats in the three Chinese medicines of Alpinia volatile oil administration group were in good mental state,their hair condition recovered,their activity level increased,and their stools were dry and formed.（2）Compared with the model group,rats in the high-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil and the low-dose red cardamom volatile oil group increased their food intake（P<0.01）.The water consumption of rats in the high-dose group of Alpinia officinarum Hance volatile oil and the high and low-dose groups of seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil increased significantly（P<0.01 or P<0.05）.The body weight of rats in the high-dose group of the volatile oil of three Chinese medicines of Alpinia increased significantly（P<0.05）.The rectal temperature of rats in the high and low dose groups of the volatile oil of three Chinese medicines of Alpinia increased significantly（P<0.01 or P<0.05）.（3）Compared with the model group,rats in the high-dose Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil groups had a significant increase in gastric acid p H（P<0.01）,and a significant decrease in gastric juice volume and pepsin activity（P<0.01 or P<0.05）.The p H value of gastric acid in rats in the low-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil increased significantly（P<0.01 or P<0.05）.The amount of gastric juice and pepsin activity in the low-dose Alpinia officinarum Hance volatile oil group were significantly reduced（P<0.01 or P<0.05）.（4）The mucosal injury of rats in the high-dose group of the volatile oil of three Chinese medicines of Alpinia was significantly improved,with slight bleeding,and the ulcer surface was not obvious;pathological section observation showed that the mucosa was repaired well,the structure was clear,the cell shedding was slight,hemorrhage,edema and inflammatory infiltration The situation is significantly reduced.The gastric mucosa of rats in the low-dose group of volatile oil of three Chinese medicines of Alpinia has a small amount of bleeding points,and the ulcer surface is reduced;pathological section observation shows that the gastric mucosa is in a repaired state,and the structure is clearer than the model group,Edema and inflammatory infiltration.3.Study on the pharmacodynamic mechanism of volatile oil from three Chinese medicines of Alpinia in rats with gastric ulcer cold syndrome.（1）Effect on oxidative stress:Compared with the model group,the MDA content of the rats in the high-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil significantly decreased（P<0.01 or P<0.05）.The SOD activity of rats in the high and low dose groups of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil increased significantly（P<0.01 or P<0.05）.（2）Effect on inflammation:Compared with the model group,the IL-6content of rats in the high-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil significantly decreased（P<0.01）.The levels of IL-10 in the high-dose and low-dose groups of Alpinia officinarum Hance volatile oil and the high-dose seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil groups were significantly increased（P<0.01 or P<0.05）.（3）Effects on Hemorheology:Compared with the model group,the ET content of rats in the high-dose Alpinia officinarum Hance volatile oil group and the high and low-dose groups of seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil significantly decreased（P<0.01 or P<0.05）.The NO content of rats in the high-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil increased significantly（P<0.05）.The TXB₂content of rats in the high-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil was significantly reduced（P<0.01）.The levels of 6-keto-PGF1αin the high and low dose groups of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil increased significantly（P<0.01 or P<0.05）.（4）Effects on endocrine:Compared with the model group,the GAS content of rats in the high-dose group of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil significantly decreased（P<0.05）.The SS content of rats in the high and low dose groups of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil increased significantly（P<0.01 or P<0.05）.The COR content of rats in the high-dose group of Alpinia officinarum Hance and Alpinia galanga Willd volatile oil and the high and low-dose seeds of Alpinia galanga Willd volatile oil groups were significantly reduced（P<0.01 or P<0.05）.The MTL levels of rats in the high and low dose groups of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd volatile oil were significantly reduced（P<0.01）.Conclusion:1.The volatile oil of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd can protect GES-1 cells damaged by ethanol,reduce the degree of cell damage,and improve cell survival.Its mechanism of action may be related to inhibiting the release of inflammatory factors,promoting the secretion of growth factors,and up-regulating the expression of b FGF protein in cells.2.The volatile oil of Alpinia officinarum Hance,seeds of Alpinia galanga Willd and Alpinia galanga Willd can improve the overall state,behavioral signs,and secretion of gastric digestive juice in rats with gastric ulcer cold syndrome,reduce gastric mucosal damage,and accelerate gastric ulcer heal.Its pharmacodynamic mechanism may be to reduce the levels of MDA,IL-6,ET,TXB₂,GAS,MTL,COR,increase the activity of SOD,and increase the levels of IL-10,NO,SS,and 6-keto-PGF1α.It is speculated that it may play the anti-cold syndrome effect of gastric ulcer by reducing the erosion of gastric digestive juice,anti-oxidation,reducing inflammation,improving gastric mucosal microcirculation,and regulating the balance of gastrointestinal and neuroendocrine system.3.In vitro cell experiments show that the volatile oil of three Chinese medicines of Alpinia has certain biological activity and can protect the damaged gastric mucosal epithelial cells.The study of the efficacy and mechanism of action in vivo found that the volatile oil of three Chinese medicines of Alpinia can treat the cold syndrome of gastric ulcer in rats and repair the damage of gastric mucosa.It indicates that the volatile oil may be the effective material basis of the three-flavored mountain ginger in the anti-gastric ulcer cold syndrome.