| Objective:To study the efficacy,best compatibility,compatibility effects and mechanism of action of the combination of Huisheng Powder（HSP）in the treatment of gastric ulcer（GU）.Methods:1.Study on the efficacy and composition of HSP in treating GU:Prepare the decoction of HSP,the essential oil of the tangerine peel and patchouli,which are composed of HSP,and give rats the water decoction and main components of HSP.Essential oil and its different proportions of the composition（1:2,2:1,1:1）were administered by gavage.After 7 days of administration,the acute GU model was established by water-immersion and restraint stress（WIRS）.The protective effect of HSP on GU was observed with naked eyes,and the pH value of gastric acid was measured with precision test paper;HE stain staining was used to observe the effect of HSP on gastric mucosal tissue morphology;Serum GAS,PGC,PGE2,and 5-HT were detected by ELISA.2.Study on the mechanism of the combination of HSP against GU:After obtaining the best ratio of the components of HSP for the treatment of GU,use this ratio to set different high,medium and low dose groups（600 mg/Kg,300 mg/Kg,150mg/Kg）for administration After administration for 7 days,a rat model of acute GU was established by WIRS,the effects of the composition of HSP on the morphology of gastric mucosa were observed,and the pH value of gastric juice was measured with precision test paper;Serum GAS,PGC CCK,PGE2,Motilin,c AMP and c GMP were detected by ELISA;Gstric epidermal growth factor and trefoil factor 2 protein expression were detected by immunohistochemical;Western blot detects gastric tissue JNK,p-JNK,p53,Bax,Caspase-3 protein expression;transmission electron microscopy to observe the effect of the composition of HSP on the ultrastructure of rat gastric chief cells and parietal cells.3.Study on the regulation of gastric acid secretion in rats by the combination of HSP:Following the best ratio and dosage of the HSP obtained in the experiment to treat GU,gavage for 7 days,then give histamine injection to induce gastric acid secretion in rats,collect the gastric acid by ligating the pylorus,observe the effective group of HSP.Analyze the influence of gastric acid secretion gastric juice volume and pH value in rats,and use the kit to determine the H+-K+-ATPase activity of gastric tissue.4.Study on the mechanism of protecting gastric mucosal epithelial cells GES-1injury with HSP components:In vitro culture of human gastric mucosal epithelial cells GES-1,using ethanol-induced cell damage model,MTT was applied to detect the effects of the components and compatibility of HSP on cell viability and morphology;kits were applied to determine the ROS,GSH,SOD content or activity;Flow cytometry was used to detect cell apoptosis rate;Western blot was used to detect cell JNK,p-JNK,c-Jun,p53,Bax,Bcl-2,Caspase-3,Caspase-8 protein expression;RT-q PCR detection of cells JNK,c-Jun,p53,Bax,Bcl-2,Caspase-3,Caspase-8m RNA expression.Results:1.Effects of HSP and its components on rats with acute gastric ulcer The rat model of acute gastric ulcer was successfully prepared by WIRS.The rats in the model group showed obvious bleeding,erosion and ulcer of gastric mucosa.Compared with the blank group,the gastric mucosal pathological damage score of the rats in the model group was significantly increased,the serum GAS and PGC contents were significantly increased,and the PGE2 content was significantly decreased（P<0.01）.Compared with the model group,the gastric mucosal pathological damage scores of the rats in the 2:1 group and the 1:1 group were decreased（P<0.05）,and the pathological gastric mucosal pathological damage scores in the 1:2 group were significantly decreased（P<0.05）.Compared with the model group,the serum GAS content of the rats in each treatment group was significantly decreased（P<0.01）,and the 1:2 group could also reduce the serum PGC content and increase the PGE2content（P<0.05）;Compared with the water decoction group,the effect of the 1:2group on the GAS,PGC and PGE2 of the rats was more significant（P<0.05 or 0.01）.2.The mechanism of the HSP composition treatment on acute gastric ulcer in rats Compared with the blank group,the pH value of gastric juice in the model group was significantly decreased,and the gastric mucosal pathological injury score was significantly increased（P<0.01）,serum GAS,PGC,SP,CCK content and c AMP/c GMP value increased（P<0.05 or 0.01）,PGE2 content was significantly decreased（P<0.01）,and gastric tissue EGF and TFF2 protein expressions were significantly decreased（P<0.01）,the relative expressions of p-JNK,p53,Bax and Caspase-3 proteins in gastric tissue increased(（P<0.05 or 0.01）.Compared with the model group,the high-dose group could significantly increase the pH value of gastric juice（P<0.01）,reduce the serum GAS and SP contents（P<0.05）,and significantly reduce the serum PGC,CCK contents and c AMP/c GMP value（P<0.01）,significantly increased the expression of EGF and TFF2 protein in gastric tissue（P<0.01）,and inhibited the relative expression of p-JNK protein in gastric tissue（P<0.05）.The dose group could significantly increase the pH value of gastric juice（P<0.01）,reduce the gastric mucosal pathological damage score（P<0.05）,reduce the serum GAS,PGC,SP content（P<0.05）,significantly reduce the serum CCK content and c AMP/c GMP value（P<0.01）,significantly increased the expression of EGF and TFF2 protein in gastric tissue（P<0.01）,inhibited the relative expression of p-JNK protein in gastric tissue（P<0.05）and the relative expression of p53,Bax and Caspase-3 proteins（P<0.01）;compared with the model group,the low-dose group could reduce the serum GAS and PGC contents（P<0.05）,and significantly reduce the serum CCK contents and c AMP/c GMP values（P<0.01）,increased the expression of EGF,TFF2protein in gastric tissue（P<0.05）,inhibited the relative expression of p53 protein in gastric tissue（P<0.05）and the relative expression of p-JNK,Bax and Caspase-3protein（P<0.01）;The middle-dose group（300mg/kg）had the best effect,and the electron microscope test showed that this dose could significantly correct the endoplasmic reticulum expansion of gastric chief cells in rats with acute gastric ulcer,and reduce mitochondrial swelling,membrane loss and cristae fusion and other injuries.Thereby inhibiting the activity of endocrine tubules of gastric parietal cells and reducing gastric acid secretion.3.The effect of HSP composition on gastric acid secretion in rats After subcutaneous injection of histamine in the model group,the gastric juice secretion was significantly increased（P<0.01）,and the gastric juice pH value was significantly decreased（P<0.01）;compared with the model group,the 1:2 compatibility of HSP can significantly reduce gastric fluid secretion（P<0.01）and increase gastric juice pH（P<0.05）,and can significantly inhibit gastric tissue H+-K+-ATPase activity（P<0.05）;and 1:2 compatibility group was significantly better than the single component group in increasing the pH value of gastric juice and inhibiting the activity of H+-K+-ATPase in rats（P<0.01）.4.The results of the protective mechanism of HSP composition on gastric epithelial cell injury The combination of HSP composition can inhibit the GES-1 cell damage induced by ethanol at 25μg/m L;compared with the blank group,the adherent cells in the model group decreased,ROS signal expression increased,GSH content significantly decreased,and cell apoptosis rate increased significantly（P<0.01）.The relative expressions of p-JNK,c-Jun,p53,Caspase-3,and Caspase-8 proteins enhanced,Bcl-2 protein expression decreased,JNK,Bax,Caspase-3,Caspase-8m RNA relative expression increased,Bcl-2 m RNA relative expression decreased（P<0.05 or 0.01）.Compared with the model group,the levels of ROS in GES-1 cells decreased,while the contents of GSH and MOD increased significantly（P<0.01）in the GES-1 cells treated with HSP（P<0.01）.It can reduce the apoptosis rate of GES-1cells induced by ethanol（P<0.05）,reduce the relative expressions of p-JNK,p53,Caspase-3 and Caspase-8 proteins,increase the relative expression of Bcl-2 proteins,and can inhibit JNK,The relative expression of Bax,Caspase-3 and Caspase-8 m RNA significantly increased the relative expression of Bcl-2 m RNA（P<0.05 or 0.01）.Conclusion:1.HSP has a therapeutic effect on acute gastric ulcer,and essential oil is its main medicinal substance.This group has better therapeutic effect on gastric ulcer than water decoction,and the therapeutic effect after compatibility is better than that of single use,when the patchouli essential oil and dried tangerine peel essential oil are compatible with 1:2 at the dosage of 300mg/kg,it exerts the best therapeutic effect on acute gastric ulcer model rats.2.The mechanism of the combination of HSP in the treatment of GU may be related to inhibiting gastric acid secretion,repairing gastric mucosal structure and resisting gastric mucosal cell apoptosis. |
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