Role Of Liujunzi Combined With Xuanfu Daizhe Decoction On ICC And SCF/c-kit Signal Pathway In RE Rats’ Esophagus

Gastroesophageal reflux diseases(GERD)was a common digestive system disease.According to the difference in endoscopy,it could be divided into three types:reflux esophagitis(RE),non erosive gastroesophageal reflux disease and Barrett’s esophagus.The morbidity of GERD was increasing gradually around the global.Antiacid treatment based on Proton pump inhibitors(PPI)remained the main program.However,about 40%to 60%of the patients with GERD were ineffective when given PPI treatment or Relapsed after drug withdrawal.What’s worse,the recurrence rate of RE was as high as 90%,so how to solve the high recurrence problem of RE was the important tasks of current research.It was believed that the occurrence of this disease was mainly related to the drop of basal lower esophageal sphincter pressure,the transient relax of lower esophageal sphincter and the disorder of esophageal motility,so the anti-reflux treatment which was based on the functional restoration of the lower esophageal sphincter had been attended widely and attempted actively.A batch of new treatment modalities such as transoral fundoplication,magnetic sphincter augmentation,Radiofrequency ablation and LES electrical stimulation had been founded,however their feasibility,safety and long-term efficacy evaluation had not yet reached a consensus.In traditional Chinese medicine(TCM),RE could be classified as "chest pain","spit acid" and "epigastric pain",Among which,most of the patients blonged to the deficiency in origin and the excess in superficiality.Our previous study found that the spleen deficiency syndrome in patients with RE relapse accounted for 37.6%.The treatment we advocated to choose the Liujunzidecoction,Zuojin pill,Xuanfu daizhedecoction and so on.Although TCM had an outstanding anti-reflux effect,with less side effects,the mechanism research on this subject was still relative weak around the world.Interstitial cell of Cajal(ICC)distributed in the gastrointestinal tract widely,having an indispensable role in the mechanism of gastrointestinal dynamic adjustment,and the normal cellular phenotype and function of them depend on the normal expression of SCF/c-kit signal pathway.Previous studies found that ICC also existed in the lower esophagus,which may become a potential target for GERD treatment.Our experiment aimed to study the role of Liujunzi combined with xuanfu daizhe decoction on the ICC and SCF/c-kit signal pathway of the esophagus in RE rats with spleen deficiency syndrome to reveal the new target of TCM and to open up new ways to solve the problem of high recurrence rate in RE.Part one Explore the method of establishing RE rat model with TCM syndromeAims:Used the method of continuous perfusion with different concentrations of acid pepsin combining with rhubarb gavage and the mothod of pylorus semi-ligation combined with anterior gastric ligation by different way of pylorus ligation combineing with rhubarb gavage to eastablish the RE rat model with spleen deficiency syndrome respectively,then compared them to each other to screen a perfect method to creat the RE rat model with TCM syndrome to provide theoretical basis for the later research.Methods:Wistar rats were randomly divided into six groups.First,we established the RE model:Rats in Group A were given with 0.25mol/L HCl + 0.5mg/ml pepsin suspension(the rate was 75μl/min for 30 min for a total of 14 days)by gastrogavage continuously.The same,Group B were continuously perfused with 0.5mol/L HCl +0.5mg/ml pepsin suspension.Group C were set as blank control group one using the same equivalent physiological saline perfused to the stomach.The D Group were used the mothod of 1/4 pyloric suture combined with Anterior gastric ligation.The Group E were used the mothod of traditional pylorus half of the ligation combined with anterior gastric ligation to create RE rats models.The F Group were the contrast group two,which only performed laparotomy,and closed the abdominal cavity again after fifteen minutes.After two weeks,the RE rats models were finished.At the number of the 15th day,we started to establish the spleen deficiency syndrome model.The A、B、D、E four groups were reference as model groups,who were used the percent of 100 Rhubarb decoction to perfuse the stomach by 2ml/count,2 times/day for continuous 7 days.The C,F were the contrast groups,using the same equivalence normal saline to perfuse the stomach.Then compared the general state,the death rate,the content of the blood serum IL-6、IL-1β and IFN-y of the rats,as well as the pathwayological feature of the lower esophageal tissue to evaluate the rats model from safety,efficacy and so on.Result:1)Rats in the Model groups were less active,less dynamic,lazy,flocking together,and arching the back;the spirit was indifference and easily frightened;the response to external stimuli was weakened and the coat was brown and unkempt;the diet was gradually reduced with Loose stool or dry knot.2)The content of blood serum IL-6、IL-1β、IFN-γ in the model group rats were obviously increased.3)The display of the pathwayology:the model group rats could be obvioused the different degree papillary hyperplasia,hyperplasia of squamous epithelium,inflammatory cell infiltration and mucosal ulceration.4)The rate of effective and death:the effective rate of Group A was 42.8%,and the death rate was 30%;the Group B was 60%and 50%respective;the Group C was blank control group,with a 10%death rate.The effective rate of Group D was 75%,and the death rate was 20%;the Group E was 100%and 50%;the Group F rat was set as contrast,whose death rate was 14.3%.Part two Role of Liujunzi combined with xuanfu daizhe decoction on ICC and SCF/ckit signal pathway in esophagus of RE rats with spleen-deficiency syndrome Aims:Studied the effect of SCF/c-kit signal pathway on the number,the morphological structure and the function of ICC to explore the potential target of Liujunzi combined with xuanfu daizhe decoction in the treatment of RE spleen deficiency syndrome.Method:Used the method of pylorus half of the ligation combined with Anterior gastric ligation andrhubarb gavage to eastablish the model of RE splenasthenic syndrome rat model.Then the experimental animals were randomly divided into six groups:the negative contrast group,the positive contrast group,the western medicine group,the traditional Chinese medicine group with high,middle and low dosage respectively,among which the western medicine group was given rabeprazole and moxapride suspension by intragastrically;the three groups of Chinese medicine were given Liujunzi combined with xuanfu daizhe decoction with high,middle and low doses respectively.All the animals were administered for 14 days,1 ml each one per day.The negative and positive contrast groups were given the same amount of normal saline.At the end of the experiment,the lower segment of the rat esophagus was taken.The ultrastructure of ICC in lower esophageal tissue was observed by transmission electron microscope.The content of c-kit and SCF protein in the lower esophageal tissue were detected by Western blot.The relative expressions of c-kit and SCF mRNA in the lower esophageal tissue of rats were detected by RT-PCR.Results:1)ICC existed in the rat’s esophagus tissue.In the negative control group,ICC were characterized by complete and clear structure,spindle shape,large nucleosome,less cytoplasm,large mitochondria and abundant endoplasmic reticulum,and having more synapses of the cell body,forming close gap junctions with the surrounding cells.In the positive control group,the number of ICC was obviously reduced,the morphology and structure of the cells was vague,the endoplasmic reticulum was dilated obviously,the vacuolization of mitochondria was serious,the synaptic structure of the cells was less and the space between the surrounding cells was larger with a loose connection.In the low dose group of Liujunzi combined with xuanfu daizhe decoction,the number of ICC was less,the structure was not clear,the cytoplasmic mitochondria crista was dissolved in the cytoplasm,the endoplasmic reticulum was expanded,the synapse of the cell body was less and the connection was not close to the surrounding cells.The number of ICC in the high dose and middle dose groups was less than that of the negative control group with basically complete cell structure.It could be seen the endoplasmic reticulum and mitochondria damaged in the cytoplasm.The gap between the surrounding cells was closely connected.The performance of the western medicine group was basically the same as that of the negative control group.2)WB:Compared with the negative contrast group,the content of c-kit and SCF protein was decreased significantly in the positive control group(P<0.01).The expression level of c-kit and SCF protein in western medicine group was significant higher than that in positive contrast group(P<0.01),and the expression level of c-kit and SCF protein in TCM groups was higher than that in positive contrast group(P<0.05).The expression level of c-kit and SCF protein in the high dose group was slightly higher than that in the middle dose group(P<0.05).3)Rt-PCR:Compared with the negative contrast group,the relative expression level of c-kit and SCF mRNA was decreased significantly in the positive control group(P<0.01).The relative expression level of c-kit and SCF mRNA in western medicine group was significant higher than that in positive contrast group(P<0.01)and in the negative control group(P<0.05).The relative expression level of c-kit and SCF mRNA in TCM groups was higher than that in positive contrast group(P<0.01).There was no difference in the expression of c-kit and SCF mRNA between middle dose group with high dose group(P>0.05).Conclusion:1)The RE rats model with spleen deficiency syndrome could be successfully established by acidified pepsin continuous perfusion and gastric ligation before pyloric half ligation in combination with rhubarb gavage,and the effect of latter was better than the former.2)The mortality in rats was related to the concentration of hydrochloric acid and the mode of pyloric ligation.3)Although the 1/4 pyloric suture combined with Anterior gastric ligation was less severe than traditional pylorus half ligation combined with Anterior gastric ligation,the method was simple,Moreover,the injury was small and the animals were easy to survive after operation.If the time of modeling was extended properly,the severity of RE was serious.Therefore,it was more suitable for eastablishing rats model massively and for studying intervention treatment of drugs after operation long-term.4)It could successfully establish a rat model of spleen deficiency syndrome via rhubarb gavage.5)ICC was existed in rat’s esophageal tissue,and the occurrence of spleen deficiency syndrome of RE was related to the structure、the number and the function of ICC.6)The decreasing of c-kit、SCF protein and its gene expression which induced the down-regulation of SCF/c-kit signal pathway was the most direct cause of the number change and structural abnormality of ICC.7)Traditional Chinese medicine "Liujunzi combined with xuanfu daizhe decoction "could maintain the normal function of the SCF/c-kit pathway by promoting the expression of c-kit,SCF mRNA and its protein to make the damaged ICC restore to normal,and further regulates the LES function via the ENS-ICC-SMC signal network system to play an anti reflux role and reduce the reflux recurrence.