Clinical And Experimental Study On Treatment Of Slow Transport Type Constipation Based On "Chronic Blood Stasis And Blood Stasis Syndrome"

Purpose:There are many slow transmit constipation( STC) patients who are blood stasis type in clinic. Based on traditional Chinese medicine dialectical, tutor professor Yu Yongduo puts forward “long illness blood stasis, blood stasis toxin impairing collaterals”pathogenesis hypothesis and put the rule of law “removing toxin and activating collaterals”into use to form Huayu Tongbian decoction to use in clinic.To observe clinical effect of Huayu Tongbian decoction to treat slow transmit constipation; To discuss the effects of Huayu Tongbian decoction on slow transmit constipation rates colon in interstitial cells of cajal( ICC); To discuss the effects of Huayu Tongbian decoction on slow transmit constipation rates colon in Substance P( SP) and vasoactive intestinal polypeptide( VIP);To discuss the effects of Huayu Tongbian decoction on slow transmit constipation rates colon in aquaporin-1( AQP 1) and aquaporin-3( AQP 3). From the clinical research and experiental research on two parts, to discuss Huayu Tongbian decoction hypothesis from a variety of mechanisms jointly for the treatment of blood stasis type slow transmit constipation.Material and method:Paper One :Clinical Research on Slow Transmit Constipation Using Huayu Tongbian Decoction63 slow transmit constipation cases which are from The third affiliated hospital of liaoning university of Chinese medicine( Anorectal Hospital in Liaoning Province)are randomly divided by random number table method into treatment group which are 32 cases and control group which are 31 cases.The treatment group uses Hua yu Tong bian decoction and control group Ma ren Run chang Wan for 14 days. To observe before and after treatment in patients with defecation frequency, feces traits, the use of laxative, abdominal distension and integral time of defecation.To record treatment group and control group before and after treatment the main symptom integral. To calculate the integral. To observe clinical effect of treatment group and control group.Paper Two:Experimental Research on Slow Transmit Constipation Using Huayu TongbianDecoction Part One: The Preparation of Slow Transit Constipation Model in RatsAccording to random number table and body mass,72 rats were randomly divided into 6groups, which were 5 model groups of 60 and 1 blank control group of 12. Model groups were given rhubarb powder, which initial measurement of 3 mg /( 10 g · d). For drug suspension liquid, diluted with distilled water to fill the stomach once a day. First round :model groups were given rhubarb powder daily dose in the increase on the basis of 3mg /( 10 g · d) until more than 50 % rats were in atherosclerosis and maintain the dose until80% of rat atherosclerosis disappears. Second round :based on the first round,model groups were given rhubarb powder daily dose in the increase on the basis of 3 mg /( 10 g · d) until more than 50 % rats were in atherosclerosis and maintain the dose until 80% of rat atherosclerosis disappears. Third round :based on the sceond round,model groups were given rhubarb powder daily dose in the increase on the basis of 3 mg /( 10 g · d) until more than50% rats were in atherosclerosis and maintain the dose until 80% of rat atherosclerosis disappears.Completing the whole experiment conducted three rounds of circulation to induce experimental slow transmit constipation model.To observe model group model preparation time and rhubarb powder dosage of each round; To observe model group and blank control group rats body mass, dung texture, hair and activity.To observe rats first black stool time; To observe 24 h stool weight of two groups.Part Two: Effects of Huayu Tongbian Decoction on Slow Transmit Constipation Rates Colon in Interstitial Cells of Cajal60 model rats were randomly divided into model control group, Hua yu Tong bian decoction group(low, medium, high dose)and western medicine control group, each group of 12 rates were given by gavage according to the animal and human body per kilogram of body weight dose conversion coefficients table to calculate rats dose.1 times a day, for 14 day.Rates of high dose group were given 96 g / kg/ d( content of crude drugs), midium dose group 48 g / kg/ d and low dose group 24 g / kg/ d. Rats Gastric volume was 0.1 ml / 10 g.The western medicine control group rates were given mosapride by gavage 2.5 mg / kg/ d, 1 timesa day, for 14 day.Model control group and blank control group will not be given any drug,distilled water on the same volume, 1 times a day, for 14 day.15 days all rats were not given food but water for 24 h and given by gavage 5 % carbon+ 10 % gum arabic of 1 ml / 100 g.After 30 mins, rates were put to death for the colon specimens. Colon specimens is divided into two parts: measurement of quick rinse after use0.9% sodium chloride solution, placed in the specimen volume of more than 10 times of 4%paraformaldehyde fixed; The rest put in – 80 ℃ refrigerator, for measurement.Measuring carbon propulsion rate: at the end of the measurement of pylorus to carbon( carbon forward distance) at the end of the front end at the end of the distance and the pylorus to the end of the rectum bowel loops( the total length of the intestinal length).Carbon propulsion rate = at the end of the forward distance at the end of the total length( cm)/ intestinal X 100 %( cm).To test C-kit expression in rat colon using immunofluorescence method.Part Three: Effects of Huayu Tongbian Decoction on Slow Transmit Constipation Mice Colon in Substance P and Vasoactive Intestinal60 model rats were randomly divided into model control group, Hua yu Tong bian decoction group(low, medium, high dose)and western medicine control group, each group of12 rates were given by gavage according to the animal and human body per kilogram of body weight dose conversion coefficients table to calculate rats dose.1 times a day, for 14 day.Rates of high dose group were given 96 g / kg/ d( content of crude drugs), midium dose group 48 g / kg/ d and low dose group 24 g / kg/ d. Rats Gastric volume was 0.1 ml / 10 g.The western medicine control group rates were given mosapride by gavage 2.5 mg / kg/ d, 1 times a day, for 14 day.Model control group and blank control group will not be given any drug,distilled water on the same volume, 1 times a day, for 14 day.15 days all rats were not given food but water for 24 h and given by gavage 5% carbon +10 % gum Arabic of 1 ml / 100 g.After 30 mins, rates were put to death for the colon specimens. Colon specimens is divided into two parts: measurement of quick rinse after use0.9% sodium chloride solution, placed in the specimen volume of more than 10 times of 4%paraformaldehyde fixed; The rest put in- 80 ℃ refrigerator, for measurement.To test substance P and vasoactive intestinal poly peptide mRNA expression in rat colonusing RT- PCR method.Part Four: Effects of Huayu Tongbian Decoction on Slow Transmit Constipation Mice Colon in Aquaporin-1 and Aquaporin-360 model rats were randomly divided into model control group, Hua yu Tong bian decoction group(low, medium, high dose)and western medicine control group, each group of12 rates were given by gavage according to the animal and human body per kilogram of body weight dose conversion coefficients table to calculate rats dose.1 times a day, for 14 day.Rates of high dose group were given 96 g / kg/ d(content of crude drugs), midium dose group48 g / kg/ d and low dose group 24 g / kg/ d. Rats Gastric volume was 0.1 ml / 10 g.The western medicine control group rates were given mosapride by gavage 2.5 mg / kg/ d, 1 times a day, for 14 day.Model control group and blank control group will not be given any drug,distilled water on the same volume, 1 times a day, for 14 day.15 days all rats were not given food but water for 24 h and given by gavage 5% carbon +10 % gum Arabic of 1 ml / 100 g.After 30 mins, rates were put to death for the colon specimens. Colon specimens is divided into two parts: measurement of quick rinse after use0.9% sodium chloride solution, placed in the specimen volume of more than 10 times of 4 %paraformaldehyde fixed; The rest put in- 80 ℃ refrigerator, for measurement.To test aquapori n-1 and aquapori n-3 protein expression in rat colon using Western blot method.Results:1. Clinical Research on Slow Transmit Constipation Using Huayu Tongbian DecoctionThere was no statistically significant difference by statistical tests for two groups in terms of gender, age and illness course(P > 0.05), the condition of equilibrium between groups.The self-total integral of the treatment group and control group before and after treatment are statistically significant(P < 0.01).The total integral of two groups after treatment is statistically significant(P < 0.01).The total integral reduce amplitude of treatment group is larger than the control group.The treatment are effective in both groups.The treatment group total effective rate is 78.1 % and the control group is 71.0 %. Clinical curative effect of two groups is statistically difference(P < 0.05). The treatment groupcurative effect is better than the control group.2. The preparation of Slow Transit Constipation Model in RatsThe first round of the cycle, a total of 24 days and rhubarb dose escalation to 21 mg/(10g, d); The second round of cycle for a total of 31 days and rhubarb dose escalation to 37 mg/(10 g, d); The third round of a total of 32 days and rhubarb dose escalation to 57 mg/(10 g,d). The whole experiment for three rounds of circulation, a total of 87 days.Compared with blank control group, body mass of model group rates is less( P < 0.05), stool crack、shorten,a gray hair, dark and inactive. First black row of model group rats was prolonged( P < 0.05);24h stool weight of model group rats feces is light( P < 0.05).3. Effects of Huayu Tongbian Decoction on Slow Transmit Constipation Mice colon in Interstitial Cells of CajalCompared with blank control group, model control group C- kit(Cajal interstitial cells)expression decreased significantly( P < 0.01), the model control group C- kit number decreased significantly. Each dosage group compared with model control group, the C- kit different degree increased( P < 0.01), in which the Hua yu Tong bian decoction high dose group and western medicine control group increased degree is higher, can explain the drug therapy group were different degree C- kit number, Hua yu Tong bian decoction high dose group and western medicine control group has an obvious effect to raise C- kit number.Compared with the western medicine control group, Hua yu Tong bian decoction high dose group C- kit expression has no difference( P > 0.05), Hua yu Tong bian decoction low dose group, middle dose group C- kit expression has difference( P < 0.05), Hua yu Tong bian decoction high dose group and western medicine control group have the same impact on the C- kit number. Hua yu Tong bian decoction low and middle dose group C- kit number is not obvious.4. Effects of Huayu Tongbian Decoction on Slow Transmit Constipation Mice Colon in Substance P and Vasoactive Intestinal Polypeptide4.1 Effects of Huayu Tongbian Decoction on Substance P ExpressionCompared with blank control group, model control group substance P purpose gene and the internal integral optical density ratio increased significantly(P < 0.01), the model control group substance P content increased significantly. Compared with model control group, thetreatment group substance P integral optical density ratio varying degrees lower(P < 0.01), in which Hua yu Tong bian decoction high dose group and western medicine control group a large integral ratio decline. Various treatment group drugs can reduce the content of substance P for different level. Hua yu Tong bian decoction high dose group and western medicine control group has an obvious effect to reduce content of substance P. Compared with the western medicine control group, Hua yu Tong bian decoction substance P integral optical density ratio, high and medium dose group no difference(P > 0.05), low dose group blood purge soup substance P integral optical density ratio difference(P < 0.05). Indicates Hua yu Tong bian decoction high dose group and western medicine control group to reduce content of substance P no difference, low dose group of lower blood purge soup substance P content is not obvious.4.2 Effects of Huayu Tongbian Decoction on Vasoactive Intestinal Poly peptide ExpressionCompared with blank control group, the vasoactive intestinal poly peptide model control purpose gene and the internal integral optical density ratio significantly decreased(P < 0.01),the model control group vasoactive intestinal poly peptide content decreased significantly.Compared with model control group, Hua yu Tong bian decoction medium、high dose group and western medicine control group vasoactive intestinal poly peptide integral optical density ratio of different level increased(P < 0.01), in which Hua yu Tong bian decoction high dose group and western medicine control group integral ratio higher degree. The drug therapy group(except the low dose group) all vasoactive intestinal polypeptide content to varying degrees, Hua yu Tong bian decoction high dose group and western medicine control group has an obvious effect of vasoactive intestinal poly peptide content. Compared with model control group, low dose group blood purge soup P = 0.051 > 0.05 and is different from the early stage of the consult literature material.It may be related to no significant effects on vasoactive intestinal poly peptide express of low doses of Hua yu Tong bian decoction on slow transmit constipation rats. Compared with the western medicine control group, Hua yu Tong bian decoction high dose group of VIP integral optical density ratio no difference(P >0.05), low and medium dose Hua yu Tong bian decoction group of VIP integral optical density ratio difference(P < 0.05). It indicates that effect of Hua yu Tong bian decoction highdose group is the same with the western medicine control group to reduce the vasoactive intestinal poly peptide content.Low and medium Hua yu Tong bian decoction dose group which decreased vasoactive intestinal poly peptide content is not obvious.5. Effects of Huayu Tongbian Decoction on Slow Transmit Constipation Mice Colon in Aquaporin-1 and Aquaporin-3Compared with blank control group, model control group significantly increased relative grey value expression of aquaporin-1 and aquaporin-3(P < 0.01), the model control group aquaporin-1 and aquaporin-3 content increased significantly. Each dosage group compared with model control group, aquaporin-1 and aquaporin-3 relative degree of grey value different reduced(P < 0.01), in which the blood purge decoction high dose group and western medicine control group relative extent of the grey value is big, can explain the drug therapy group were different degree reduce aquaporin-1 and aquaporin-3 content, blood purge decoction high dose group and western medicine control group has an obvious effect to reduce aquaporin-1 and aquaporin-3 content. Compared with the western medicine control group, high dose group of aquaporin-1 and aquaporin-3 blood purge soup relative grey value no difference(P > 0.05), low blood purge soup, middle dose group of aquaporin-1 and aquaporin-3 relative grey value have difference(P < 0.05). Indicates blood purge decoction high dose group and western medicine control group to reduce content of substance P no difference, low blood purge soup, middle dose group reduce aquaporin-1 and aquaporin-3content is not obvious.Conclusion:1.Clinical effect of Huayu Tongbian decoction is remarkable.There are many slow transmit constipation patients who are blood stasis type in clinic.Clinical effect of Hua yu Tong bian decoction which is “ removing toxin and activating collaterals ” is remarkable.2. Slow transmit constipation model in rats can be successfully made by Rhubarb powder.Rhubarb powder preparation of slow transmit constipation model in rats is in line with the patients disease formation process.It can be copied and used for animal experiment.3. The occurrence of slow transmit constipation rats may be related to the change ofinterstitial cells of cajal、substance P、vasoactive intestinal poly peptide 、aquaporin-1 and aquaporin-3 expression.The occurrence of slow transmit constipation rats may be related to the decreases of interstitial cells of cajal and vasoactive intestinal poly peptide expression and the increases of substance P、aquapori n-1 and aquapori n-3 expression in the colon tissue.4.Huayu Tongbian decoction impact rates colon interstitial cells of cajal、substance P、vasoactive intestinal poly peptide、aquapori n-1 and aquapori n-3 expression.Pathogenesis of curing slow transmit constipation may be related to the increase of C-kit、vasoactive intestinal poly peptide expression and lower substance P、aquapori n-1 and aquapori n-3 expression.5. Huayu Tongbian decoction acts on slow transmit constipation rats colon in interstitial cells of cajal, substance P, vasoactive intestinal and aquaporins.The relevance of the mutual influence and specific mechanism deserves further research.From the clinical research and experimental research of two parts, respectively for Hua yu Tong bian decoction treating blood stasis type slow transmit constipation were studied.Clinical research part pays attention to the clinical curative effect of observation,a variety of mechanisms to explore experimental section of Hua yu Tong bian decoction for the treatment of blood stasis type slow transmit constipation. Hua yu Tong bian decoction acts on slow transmit constipation rats colon in interstitial cells of cajal, substance P, vasoactive intestinal and aquaporins.The relevance of the mutual influence and specific mechanism deserves further research.