| Part One Observation of synchronously 24-hour bile reflux and alkali reflux monitoring in primary pathological patients and the relationship with the clinical symptom Objective To explore the characteristic of primary duodeno-gastric reflux(DGR), and the relationship among clinical symptom, bile reflux and alkali reflux through synchronously monitoring 24-hour intro-gastric pH and bilirubin absorbance. Methods 1 Subjects: 43 patients in primary pathological DGR group, 25 males, 18 females, age 19-50 years old. 10 subjects in control group, 8 males, 2 females, age were 36-60 years old. All patients were randomly recruited from patients seek health consult in the Digestive Department of Fourth Hospital Attached to Hebei Medical University for upper gastro-duodenal symptoms. And all patients fell into corresponding registered standards. 2 Methods 2.1 Questionnaire filling: Each DGR patient filled a questionnaire recorded clinical symptom, habits of smoking, drinking, eating irritant food, medicine taken history and formerly medical history. According to the degree of six symptoms, upper-abdominal pain, bloating, belching, vomiting bitter water, anorexia, nausea, symptom scores were graded. The total symptom scores of each symptom was symptom integral. 2.2 24-hours intro-gastric pH and bile reflux synchronously monitoring: 24-hours intro-gastric pH and bile reflux were synchronously monitored in 43 DGR patients and 10 control subjects. Bilirubin absorbance≥0.25 time percentage, the number of bile refluxes ( the bile reflux time lasted less than 5 minites), the number of long bile refluxes ( the bile reflux time lasted more than 5 minites), the longest bile reflux time and the maximum, the mean and median of absorbance during total 24 hours, upright, supine and post-prandial phases were respectively calculated. The time percentage of pH≥4 in total, upright and supine phases were recorded. 2.3 Statistical analysis:The reflux parameters were expressed as mean±SD. T-test was used for comparison of data between groups. Every kind of symptom score was expressed as mean±SD. Spearman correlatin analysis was used for comparison of the total symoton integral and wvery kind of symptom score with tim percentage of bilirubin absorbance≥0.25 and time percentage of pH≥4 respectively. Results 1 The result of 24-hour intro-gastric bile reflux monitoring of DGR patients 1.1 The comparison of reflux parameters in total, upright and supine phases in DGR and healthy control groups: The mean 24 hours time percentage of intro-gastric bilirubin absorbance≥0.25, the number of bile refluxes , the number of long bile refluxes , the longest time of bile reflux, the maximum, mean and median of the bilirubin absorbance were respectively higher than those in control group(P<0.05). Compared with control group, the bilirubin absorbance≥0.25 time percentage is significantly higher in upright in DGR group while there were no significant difference in the number of bile refluxes, the number of long bile refluxes and the longest reflux time between the two groups. The time percentage of bilirubin absorbance≥0.25 and the longest reflux time in supine were higher than that in control group(P<0.05), while there were not significant difference in the number of bile refluxes and the number of long bile refluxes. And the time percentage of bilirubin absorbance≥0.25 of post-prandial in DGR group was higher than that in control group. 1.2 The comparison of reflux parameters in upright and supine phases in DGR group: Compared reflux parameters between upright and supine inDGR group, there were not significant difference in bilirubin absorbance ≥0.25 time percentage and the maximum, median and mean of bilirubin absorbance. And there were no significant difference in the number of bile refluxes and the number of long bile refluxes. But the longest bile reflux time in supine was significantly higher than that in upright position (P<0.05). 2 The result of 24-hour intro-gastric alkali reflux monitoring: The mean time percentage of intro-gastric pH≥4 was 22.97% in DGR group, which had no significant difference compared with control group. There were no significant difference in the time percentage of pH≥4 between the two groups during upright and supine phase ( P>0.05). 3 Comparison between symptoms and bile reflux in DGR patients: The scores of six kinds of symptoms were as follows: bloating 2.35±0.67, abdominal pain1.71±0.72, vomiting bitter water 2.17±0.71, nausea 2.00±0.65, belching1.38±0.50, anorexia 1.78±0.80. Among them, the degree of bloating, vomiting bitter water, nausea were comparatively severe. The analysis results showed that bloating, vomiting bitter water, nausea had positive correlate to the bilirubin absorbance≥0.25 time percentage. And nausea had positive correlate to bilirubin absorbance≥0.25 time percentage in upright. Bloating and vomiting bitter water had positive correlate to bilirubin absorbance≥0.25 time percentage in supine. The total symptom score had positive correlation to the bilirubin absorbance≥0.25 time percentage of 24 hour, upright and supine. The total symptom integral and the six kinds of score had no correlate to the number of bile refluxes, the number of long bile reflux respectively in different phases. And belching was correlated to the longest bile reflux time in total and supine. Anorexia was correlated to the longest bile reflux time in upright. 4 Comparison between symptoms and alkali reflux in DGR patients: Each symptom score had no correlate to the time percentage of pH≥4 respectively in total, upright, and supine phase(P>0.05). The total symptom integral had no correlation to time percentage of pH≥4 respectively in total, upright, and supine phase(P>0.05).Conclusion 1 Primary pathological DGR occured on the basis of physiological DGR with increased reflux frequency, prolonged reflux time and increased reflux amounts. The degree of bile reflux between in upright and supine position is primarily the same. Long time bile reflux often appears in supine position. 2 When primary pathological DGR occurs, bloating, vomiting bitter water, nausea are related closely to the degree of bile reflux. So these symptoms can become the clinical clue of pathological DGR. The clinical symptoms of primary Pathological DGR are mainly caused by bile reflux. 3 The symptoms of DGR primarily are caused by bile reflux and the clinical has less relationship with alkali reflux. Bile reflux is the main cause of clinical symptoms. Part Two The relationship among gastric mucosa lesion, H.pylori infection, bile reflux and alkali reflux in primary pathological duodenogastric reflux patients Objective To explore the relationship among the gastric mucosa lesion caused by primary DGR, the H.pylori infection, the bile reflux and the alkali reflux, through synchronously monitoring 24-hour intro-gastric pH and bilirubin absorbance. Methods 1 Subjects: The same as the frist part above. 2 Methods 2.1 H.pylori infection determination and mucosa biopsied under gastroscope: Each DGR patient underwent 14C-urase expiration test and gastric mucosa biopsy under gastroscope. Five gastric tissue biopsied specimens were taken, two from antrum, two from gastric body and one from gastric anguli and underwent Giensa dyeing and HE dyeing respectively. H.pylori infection was assessed and the degree of chronic inflammation, activity, atrophy and intestinal metaplasia of gastric mucosa were respectively observed according to the new Sydney system criteria. The lesions of gastricmucosa were classified into four degrees, ie, normal, mild, moderate and severe. And each degree corresponded to 0, 1, 2, 3 scores. The total scores of gastric mucosa lesions degree were tissue pathological integral. 2.2 24-hours intro-gastric pH and bile reflux synchronously monitoring: 24-hours intro-gastric pH and bile relux were synchronously monitored in 43 DGR patients and 10 control subjects. The same as Part One described above. 2.3 Statistical analysis: The reflux parameters were expressed as mean±SD. T-test and rank test were used for comparison between groups. Spearman correlation analysis was used for comparison of H.pylori infectin, time percentage of pH≥4 and time percentage of bilirubin absorbance ≥0.25 with the pathological score of different part of gastric mucosa respectively. Result 1 Gastric mucosa lesion in different part of DGR patients: Among 43 patients, there were little nertraphil infiltration on the basis of chronic inflammation. Chronic inflammation and atrophy were the main pathological change. There was no obviously active gastritis. Antrum chronic inflmmation occurred in 34 patients. Among them, 13(30.23%) were mild, 16(37.21%) were moderate, and 5(11.63%)were severe. Gastric anguli area chronic inflmmation occurred in 35 patients. Among them, 17(39.53%) were mild, 13(30.23%) were moderate, and 5(11.63%)were severe. Gastric body chronic inflmmation occurred in 34 patients. Among them, 21(48.83%) were mild, 9(20.93%) were moderate, and 4(9.3%)were severe. Antrum atrophy occurred in 18 patients. Among them, 14(32.55%) were mild, 3(6.975%) were moderate. Gastric anguli area atrophy occurred in 17 patients. Among them, 14(32.55%) were mild, 3(6.97%) were moderate. Gastric body atrophy occurred in 6 patients. Among them, 5(11.62%) were mild, 1(2.3%) were moderate. 2(4.65%) mild intestinal mataplasia occurred in antrum. 2 Gastric mucosa lesion in primary DGR patients and the results of intro-gastric bilirubin monitoring: Pathological scores of gastric antrum and gastric anguli were positively correlated to the bilirubin absorbance≥0.25 time percentag. Pathological scores of gastric body was not correlated to thebilirubin absorbance≥0.25 time percentage, see figure 2,3. The comparison of 24-hour bile reflux parameters among DGR patients with mild, moderate and severe chronic inflammation in antrum, see table 2. Tested by Wilcoxn rank test, the bilirubin absorbance≥0.25 time percentage of three groups of patients had significant difference(P<0.05). With the increased of bilirubin absorbance≥0.25 time percentage, the degree of chronic inflammation in antrum was heavy. Other bile reflux parameters had no significant difference(P>0.05). The bilirubin absorbance≥0.25 time percentage in patients with gastric atrophy and intestinal metaplasia were significantly higher than that in patients with gastric mucosa chronic inflammation(P<0.05). 3 The correlation of H.pylori infection and the gastric mucosa lesion in DGR patients: Among 43 patients, the H.pylori infection rate in antrum, gastric body and gastric aguli area were 18.6%, 18.6%and 23.2%respectively. Mild chronic inflammation in antrum mucosa was observed in 6 patients, moderate in 2 patients, and severe in 0 patients. Gastric anguli area mild chronic inflammation occurred in 3 patients, moderate in 5 patients, and severe in 0 patients. Gastric body mucosa mild chronic inflammation occurred in 3 patients, moderate in 6 patients, and severe in1 patients. Among patients with negative H.pylori infection, 7 patients had mild, 14 had moderate and 5had severe chronic inflammation in gastric antrum14 patients had mild, 8 had moderate and5 had severe chronic inflammation in gastric anguli; 18 patients had mild, 4 had moderate and 5 had severe chronic inflammation in gastric body. There was significant difference of mild, moderate, severe chronic inflammation in antrum between H.pylori positive patients and negtive patients. The degree of chronic inflammation in antrum in H.pylori pasitive patients is milder than that in H.pylori negative patients. There was no difference of chronic inflammation in gastric body and anguli area between H.pylori positive and negative patients. There were no atrophy and intestinal metaplasia occurred the patients with positive H.pylori infection. Atrophy and intestinal metaplasia only observed in patients with negative H.pylori infection.4 The correlation of H.pylori infection and bile reflux monitoring: Gastric antrum H.pylori infection was negatively correlated to the time percentage of bilirubin absorbance≥0.25. Gastric anguli and gastric body H.pylori infection were not correlated to the time percentage of bilirubin absorbance≥0.25. Time percentage of bilirubin absorbance≥0.25 in DGR patients with positive H.pylori infection is higher than that with negative H.pylori infection. Other bile reflux parameters had no statistical difference (P>0.05). 5 The relationship among alkali reflux, gastric mucosa lesion and H.pylori infection: The time percectage of pH≥4 was not correlated to H.pylori infection and pathological scores respectively in different part of gastric mucosa. There was no significant difference of time percentage of pH≥4 between patients with mild chronic inflammation and severe chronic inflammation in antrum. There was no significant difference of time percentage of pH≥4 between patients with chronic inflammation and atrophy in gastric mucosa. Conclusion 1 Primary DGR contributes to chronic inflammation, gastric antrum atrophy, intestinal metaplasia of gastric mocosa. Antrum lesion is heavier than gastric body mucosa. 2 Of primary DGR reflux components, bile may be the main cause to the lesion of gastric mucosa. And alkali reflux components mainly include pancreatic juice may play less important role in gastric mucosa lesion. 3 H.pylori infection is not the main pathogenesis to lesion of gastric antrum in primary DGR. And the lesion in gastric body mucosa may has the relationship to H.pylori infection. 4 Bile may inhibit H.pylori to locate in gastric antrum; When primary pathological DGR happens, the environment for H.pylori location is changed which weaken H.pylori infection.Part Three The effect of Qingreliqi Granules on bile caused gastric mucosa lesion of rat model Objective To establish the rat model of gastric mucosa lesion caused by bile orally poured into the stomach and observe the therapy effect of Qingreliqi Granules on experimental rat gastric mucosa lesion caused by bile. Methods 1 Subjects: 50 clean level male SD rats, body weight 180~220g, were provided by the Hebei medical university experimental animal center. (eligibility number: DK0041-0018) 2 Methods 2.1 Bile collection: Other 30 clean level male SD rats, body weight >250g, were provided by the Hebei medical university experimental animal center. (eligibility number: DK0041-0018) Weighted after 12hr's fast (permitted drinking water). After aneasthesia, the common hepatic duct was separated, and a plastic duct was inserted into the common hepatic duct to collect bile. Closed the plastic duct, and fixed it to the back of the rat. And the plastic duct was opened periodically. 2.2 Medicine preparation: Qingreliqi Granules was made by the medicine preparation department of the Fourth Hospital, Hebei medical university. And cisapride was made by Xi'an Yangsen medicine limited corporation. 2.3 The establishment of gastric mucosa lesion rat model caused by bile: 40 rats were randomly chosen orally poured with bile, three times per day, 0.4ml/100g per time, and continuously poured for two weeks to establish the rat gastric mucosa lesion model caused by bile. 2.4 Experiment groups determination: 10 were randomly chosen as normal control group. 40 bile caused experimental gastric mucosa lesion model rats were randomly divided into 4 groups. There were 10 rats in each group. 3 groups were orally poured with Qingreliqi Granules equivalent dosage, large dose and cisapride equivalent dosage respectively. The rats in other one group were as pathological model group, which orally poured with 0.9% saline. The rats were orally poured one time daily and continuouslypoured for two weeks. After two weeks all rats were killed. The gastric tissue specimens were taken and processed for later assessed of the degree of gastric mucosa lesion with light-microscope and electric-microscope. The degree of inflammation cell infiltration was devided into 4 grades: 0: normal. â… : The inflammation cell infiltrated the superficial mucosa. â…¡: The inflammation cell infiltrated the two-thirds of the mucosa. â…¢: The inflammation cell infiltrated the whole layer of the mucosa or the muscle. 2.5 Statistical analysis: Raddit analysis was used for data comparison among groups. Results 1 Pathological change under light-microscope: Pathological change in control group showed that submucosa was bloodshot and edema, hyperplasia in gastric pit and infiltration of inflammatory cell. In Qingreliqi Granules group, gastric mucosa epithelia array in order; glands were the same in size and in shape; no remarkable necrosis appeared; a little infiltration of inflammatory cell occurred in mucosa layer; and close to the normal mucosa. 2 Pathological change under electric-microscope: The results under electric-microscope showed that gastric mucosa cell tight conjugation was lesiond; mitochondrion swelled; rough reticulum expended; microvillus was shorten or disappeared. In Qingreliqi Granules group, gastric mucosa epithelia microvillus array densely in order; organelle abounded; mitochondrion and rough reticulum were normal; secrete Granules did not decrease; cell tight conjugationwas normal. The number of chronic inflammation cell in the â…¢degree was significenantly decreased in Qingreliqi Decotion compared with the saline group. Conclusion Qingreliqi Granules can improve microstructure lesion of gastric mucosa caused by bile and protect gastric mucosa barrier. Part Four The effect of Qingre Liqi Decotion on gastric motility and gastrointestinal hormone in patients with functional dyspepsia Objective To study clinical effect of Qingre Liqi Decotion on gastricmotility and gastrointestinal hormone level in patients with functional dyspepsia (FD). Methods 1 Subjects: 30 patients in FD group, 11 males, 19 females, mean age was 42 ±9.5 years old. 18 healthy volunteers in control group, mean age was 39 ±8.9 years old. All patients were randomly recruited from patients seeking health consult in the Digestive Department of Fourth Hospital, Hebei Medical University for upper gastro-duodenal symptoms. And all patients fell into corresponding registered standards. 2 Methods 2.1 Clinical symptoms integral of FD: Targeted 7 kinds of symptoms of gastric disorders: epigastralgia or discomfort, satiety, post-prandial abdominal distension, belching, anorexia, nausea, vomiting. According to different symptom degree, no symptoms (does not have symptom), slight (patient may realize symptom existence after reminding), moderate (patient realizes symptoms existence, but daily life has not been affected), severe (patient have symptoms, also daily life comes under influence), recorded 0, 1, 2, 3scores respectively. Each kind of symptom scores was added together for integral symptom scores. 2.2 Administration methods for FD patients: After performed gastric empty examination and the gastrointestinal hormone testing, FD group patients took Qingre Liqi Decotion 250ml , twice a day for continually 5 days, the patients re-undertook gastric empty examination and the gastrointestinal hormone testing. During the therapy, no other pro-motility medicines were taken. 2.3 Liquid phase of half gastric empty time examination: The instrumentation of WDL-2000 gastric motility examination diagnosis meter was made by Weige science and technology limited company Jiangxi province. After the electrode laying correct position, the patient takes half supine (45。). After the baseline impedance was stable for 2 min, started to take liquid test meal (5% glucose 500ml, 35 ~ 40 ℃), after then continuously records theepigastrium impedance for 20min. Calculated the half of gastric empty time and the antrum motility frequency based on the instrument analysis. 2.4 The determination of plasma MTL,CCK,VIP and serum GAS level: The plasma MTL,CCK,VIP and serum GAS level was investigated before and after therapy respectively in FD patients. Join 0.3mol/L EDTA 60μL and anti-peptide enzyme 60μL in advance into the test tube to prevent clotting. On the gastric empty examination date, phlebotomized venous blood 4ml in the morning after a night fasting. Set the blood into the preventive clotting tube and set it immediately to the ice and water mixture. Centrifuged at 2500r/min for 20min under 4。C. Took the blood plasma and preserved it at 70℃for later measurement. The method of radio-immunal test performed according to the operation explanation. 2.5 Observing the safety of the medicine: Recorded the side effects of the medicine during therapy. 2.6 Staisical analysis: Data were expressed as mean±SD. T-test was used for data comparison between groups. Results 1 The improvement of clinical symptom score of FD patients before and after therapy: Qingreliqi Decotion could improve the symptom of FD patients. The symptom score was 9.16±3.66 before therapy, and decreased to 6.48±3.23 after therapy(P<0.05). 2 The change of gastric empty time of FD patients: Compared to control group, liquid gastric half-empty time in FD patients was higher(,17.03±2.76)min vs(12.8±3.57)min,P<0.05. After treatment liquid gastric half-emptying of FD group significantly decreased(P<0.01). 3 The change of plasma MTL level: Compared with control group, plasma MTL level was significantly lower(250.2±43.21) pg/ml vs(301.1±48.78) pg/ml,(P<0.01). And plasma MTL level significantly increased after therapy(P<0.05). 4 The change of serum GAS level: Compared with control group, serum GAS level was(44.80±6.34)pg/ml vs( 22.16±3.56)pg/ml,(P<0.01).
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