| Gastroesophageal reflux disease(GERD)is a kind of symptoms caused by the reflux of gastric juice and/or duodenal juice into esophagus,which can cause reflux esophagitis,as well as tissue damage outside the esophagus such as throat and trachea.The prevalence of GERD in China is increasing year by year,and it has become the most common chronic disease of digestive system.The pathogenesis of GERD has not been fully elucidated.With the popularization and application of high-resolution esophageal manometry,in-depth study of GERD from the perspective of esophageal dynamics,the reduction of contraction integral at the gastroesophageal junction,transient relaxation of the lower esophageal sphincter,low pressure of the lower esophageal sphincter and ineffective esophageal movement are considered as the main esophageal motility disorders in GERD.GERD can be classified into "choking diaphragm","chest pain","vomiting acid","noisy","epigastralgia" and other categories in traditional Chinese medicine.On March 4,1997,the national standard "terminology of clinical diagnosis and treatment of traditional Chinese medicine·disease department" issued by the State Bureau of technical supervision named the disease "esophageal obstruction".According to traditional Chinese medicine,this disease is mainly caused by deficiency of spleen and stomach elements,and the attack of internal and external evils,so that "Qingqi" does not rise and "turbid Qi" does not fall.According to the consensus opinion on TCM diagnosis and treatment of gastroesophageal reflux disease(2017),liujunhe Xuanfu Daizhe Decoction can mainly treat the syndrome of deficiency Qi,which can improve the symptoms such as acid regurgitation,panting,salivation clearing,belching and stomachache dull pain caused by deficiency Qi.Interstitial cells of Cajal(ICC)are the intermediate media between gastrointestinal nerve and smooth muscle,which play an important role in signal transmission from nerve to smooth muscle.Gastrointestinal nerve,ICC and smooth muscle cells form a complex control system together.ICC is widely distributed in the gastrointestinal tract in the form of network.The decrease of ICC number and structural abnormality are related to GERD and other gastrointestinal motility disorders.Groneber et al.Proved that ICC differentiation is an important cause of GERD.The level of Ca2+and calcium oscillations in ICC are the premise of the generation and conduction of slow waves in ICC.Some scholars have proved that inositol triphosphate receptor(IP3Rs)can cause the release of Ca2+from endoplasmic reticulum calcium pool and the calcium oscillations(regular intracellular Ca2+concentration changes)caused by mitochondrial Ca2+reuptake are the basis of ICC pacing function.Therefore,the Ca2+concentration in ICC plays an irreplaceable role in maintaining ICC pacing function and gastrointestinal motility function.Cajal stromal cells are pacemaker cells of gastrointestinal tract,which are closely related to esophageal motility disorder of GERD.There is no report about the effect of spleen invigorating and Qi regulating herbs on ICC activity and Ca2+concentration at home and abroad.The advantages of traditional Chinese medicine intervention in GERD are good curative effect and few side effects,which can reduce the recurrence risk of GERD to a large extent.However,the correlation between the curative effect of GERD intervention and esophageal motility and the specific cellular mechanism have not been reported.Through this study,we preliminarily revealed the correlation between the changes of GERD related objective indicators and the changes of curative effect after the intervention of traditional Chinese medicine,so as to lay a foundation for the research of GERD's prevention and treatment effect and mechanism of traditional Chinese medicine.Part ? isolation,culture and identification of interstitial cells of Cajal in rat stomachObjective:to isolate,culture and identify interstitial cells of Cajal in rat stomach.Methods:three Wistar rats were selected from 10-15 days of birth.After 18 hours of fasting,the gastric antrum tissues were taken out aseptically,and smooth muscle strips were isolated.ICC was extracted by type ? collagenase enzymolysis,and then inoculated on the six pore plate containing M199 medium nutrient solution.After 7-8 days of stable culture,it was identified by c-kit immunofluorescence method.Results:under inverted microscope,ICC was triangular,fusiform or polygonal,with large nucleus.The process of cell body was connected with the cell body and process of surrounding cells.Immunofluorescence staining showed that c-kit was positive on ICC surface.Conclusion:the culture and identification of ICC were successful,which laid a foundation for further study on the relationship between ICC and gastroesophageal motility disorders.Part 2 Preparation the Serum Containing of Liujunzi Decoction and Xuanfudaizhe Decoction and Study on Its Active IngredientsObjective:to prepare the serum containing Liujunzi Decoction and Xuanfu Daizhe Decoction,and to explore the content of atractylode ?,quercetin and poria acid in compound preparation and serum containing rats.Methods:Wistar rats were divided into low,medium and high dose groups and blank groups.The concentrations were 0.846,1.728 and 3.456 g/ml of Liujunzi Decoction Combined with Xuanfu Daizhe Decoction and normal saline for 5 days after gavage.Based on liquid chromatography-mass spectrometry(LC-MS),the active components of compound preparations and serum containing drugs were determined.Results:the rat serum containing the compound was prepared,and the average concentration of each active component in the test solution of the compound reagent was determined:atractylode? 4109.12mg;quercetin 50.70mg;Poria acid 0.666mg;the average concentration of atractylode ? in the serum containing the compound was 55.75mg.Conclusion:baizhuolide ? is one of the material bases of Liujunzi Decoction.Part 3 The Effect of Liujunzi Decoction Combined with Xuanfu Daizhe Decoction Containing Serum on the Activity and Calcium Concentration of Caj al interstitial Cells in Rat StomachObjective:To explore the possible mechanism of Liujunzi Decoction Combined with Xuanfu Daizhe Decoction in the treatment of gastroesophageal reflux disease.Methods:after 7-8 days of ICC stable culture,5 × 104/ml of ICC was inoculated into 96 well plates,which were divided into low,medium and high dose groups of traditional Chinese medicine,mosapride group and blank group.After 12 hours,the corresponding serum containing medicine was added,200 UL/well.The activity of ICC cells was detected by MTT method after 24,48 and 72 hours of ICC culture respectively.The change of Ca2+ concentration in cells was detected by fluorescence of fluo-3-am under confocal laser microscope.Results:compared with the blank group and the low-dose group,the activity of ICC and the fluorescence intensity of Ca2+ in the middle,high-dose group and Mosapride group increased significantly(P<0.05);at 48h and 72h,the activity of ICC in mosapride group was significantly higher than those in the middle and high-dose groups,and the fluorescence intensity of Ca2+ was also higher than those in the middle and high-dose groups(P<0.05).Conclusion:Liujunzi Decoction Combined with Xuanfu Daizhe Decoction can enhance the concentration of Ca2+ in ICC,thus enhancing the activity of ICC,which may be one of the possible mechanisms of its treatment of gastroesophageal reflux disease.Part 4 Effects of Liujunzi Decoction and Xuanfu Daizhe Decoction on Esophageal Motility in Patients with Deficiency SyndromeObjective:To observe the effect of Liujunzi Decoction and Xuanfu Daizhe Decoction on esophageal motility in patients with gastroesophageal reflux disease.Methods:from March 2017 to March 2019,33 cases of GERD were randomly divided into the traditional Chinese medicine group and the control group,17 cases in the traditional Chinese medicine group and 16 cases in the control group.The traditional Chinese medicine group was treated with Liujunzi Decoction Combined with Xuanfu Daizhe Decoction,and the control group was treated with Mosapride tablet.Esophageal manometry was performed before and 14 days after the treatment.Results:After treatment,the esophageal sphincter resting pressure and the esophageal sphincter residual pressure increased significantly after treatment in the two groups,and the difference was statistically significant(P<0.05).After treatment,the esophageal sphincter residual pressure in the Chinese medicine group was lower than the control group,and the difference was statistically significant(P<0.05).The distal esophageal systolic integral and distal systolic delay time were significantly increased after treatment in the two groups,and the differences were statistically significant(P<0.05);the contraction frontal speed decreased after treatment in both groups,and the difference was statistically significant(P<0.05);The score of distal esophageal contraction in the Chinese medicine group was lower than that in-the control group after treatment,and the difference was statistically significant(P<0.05).Conclusion:Liujunzi Decoction and Xuanfu Daizhe Decoction can significantly improve the resting sphincter pressure and residual pressure of patients with insufficiency of Qi deficiency in GERD,and enhance the peristaltic function of the esophagus. |
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