| Purpose：Constipation is one of the most common complications of hemorrhoidsafter operation. Patients will suffer severe negative consequences if it happens.The purpose of this study is to observe the therapeutic efficacy and side effectof Acesodyne and Lubricating Gut Condensed Decoction (ALGCD) in treatment ofconstipation after operation of mixed hemorrhoids through clinical research,to test the possible toxicity of ALGCD through animal experimentation, toinvestigate the method of setting up rat model which is consistent with theclinical characters of constipation after operation of mixed hemorrhoids, tofind out the possible mechanism of ALGCD in prevention and treatment ofconstipation after operation of mixed hemorrhoids through experimental researchat the level of gene and protein expression, and to provide theoretical basisfor its wide application to clinical therapy.Material and method：The first part: Clinical research on ALGCD in prevention and treatment ofconstipation after operation of mixed hemorrhoidsPatients with hemorrhoids received operation in hospital were divided intotwo groups randomly, all of the patients had related medical examination, andthen the cases who did not meet the standards were excluded. The patients ofthe two groups all received open haemorrhoidectomy of internal ligation andexternal excision (Milligan-Morgan) under the caudal anesthesia, and injectionof Xiaozhiling was added if needed. Besides the routine management afteroperation, treatment group (n=41) took ALGCD orally, control group (n=37) tookMaren Soft Gelatin Capsule. The time of first defecation post operation wasobserved. The stool form scale and pain scale of defecation at first time,3-4thday,6-7th day and9-10th day post operation were also observed。The second part: Experimental research on ALGCD in prevention and treatment of constipation after operation of mixed hemorrhoidsExperiment1Experimental study on acute toxicity of ALGCD by intragastricadministration30Kunming mice of SPF grade were divided into three groups randomly, groupA, B and C,10mice in each group, half female and half male for preliminaryexperiment. All animals were fasted except drinking for16h before drugadministered. Group A administered drug once daily, group B twice daily at aninterval6h, group C3times daily at an interval4h.The mice were orallyadministered concentrate liquid (3.5g crude drug/ml)of ALGCD40ml/kg every time.After that all mice were raised conventionally. The mice’s appearance, behavior,respiration, secretion, defecation and death were observed for7days. Therewere no dead mouse was found as result. And the lethal median dose (LD50) ofALGCD orally administered to mice can’t be found. So we do the experimentalresearch of mice intragastrically administered ALGCD with maximum amount.40Kunming mice of SPF grade were divided into two groups randomly, the blankcontrol group and ALGCD administration group,20mice in each group, half femaleand half male. The mice were orally administered maximum concentrated liquid(3.5g crude drug/ml) of ALGCD40ml/kg, the maximum volume of mouse intragastricadministered one time,3times daily at an interval4h, the rats of blank controlgroup were administered purified water with the same method of ALGCDadministration group. The mice of two groups were given normal diet after thethird times of intragastric administration, raised for14d. The Mice’sappearance, behavior, respiration, spirit, secretion, defecation, diet andtoxic symptom were observed, the body weight was weighted before gave medicineand7,14d after gave medicine, the survival animals were anatomized at the endof observation period, and recorded relative information, if there were abnormalorgan histopathological examination would be done.Experiment2Setting up rat model of constipation after operation of hemorrhoids24SD rats were divided into three groups randomly: normal group, model groupand model recovery group,8rats in each group, half male and half female. The method of making model accorded to the references and preliminary experiment.The1st-3rdday the rats of model group and model recovery group were gaven nowater but enough food and loperamide suspension3mg/kg, through intragastricadministration, twice a day at an interval6hours. The rats of normal groupwere gaven enough water and food, and purified water with same volume, by thesame method of model group. At the4thday, the rats of model group and modelrecovery group received operation on anuses, and normal control group receivedsham operation. Model group was gaven loperamide as before,3mg/kg, throughintragastric administration, twice a day at an interval6hours, gaven drinkwater1/6volume of normal needed (8ml) and enough food. Model recovery groupand normal control group were no limited of food and drink. At the5thand laterdays, the rats of every group took drug, water and food same with the4thday.The procedure of anal operation: After anaesthesia with aether, rat wasfixed on the dissecting table; perianal skin was disinfected with iodophor. Theright rear and left of anal canal each was made a fusiformis cut, deep to thesubcutaneous tissue,0.6cm length,0.2cm width, inner to the juncture of rectumand anal canal, outer to the anal edge. Sham operation method is the same asabove exclude making cut.Observe weight of food intake, volume of drinking, weight of defecation,the difficulty of defecating and the quality of fresh feces, and calculate therate of water content in fresh feces at the day before making model and everyday since begin making model.Experiment3: experimental research on ALGCD in prevention and treatment ofconstipation after operation of mixed hemorrhoids48SD rats were randomized into6groups: group A (normal control group),group B (model group), group C (Maren Soft Gelatin Capsule group), group D (lowdose therapy group), group E (middle dose therapy group), group F (high dosetherapy group), each group8rats, half male and half female.Models of constipation after operation of hemorrhoids were made accordingto the method of experiment2, and then therapy medicine was given after2h loperamide intragastric administration. Saline2ml were administered to groupA and B, twice a day, Maren Soft Gelatin Capsule suspension（0.0075g/ml）2mlwas given to group C, twice a day, equal effective dosage of human, ALGCD（0.6g crude drug/ml）2ml was given to group D,1/2equal effective dosage ofhuman, ALGCD（1.2g crude drug/ml）2ml was given to group D, equal effective dosageof human, ALGCD（2.4g crude drug/ml）2ml was given to group D, double equaleffective dosage of human. The weight of defecation and the rate of water contentin fresh feces were measuredAt48h after operation the rats of all groups were given the5ththerapymedicine, and then after forbidden drinking and eating for4h all rats were orallyadministered activated carbon gel,4ml/rat.Then the rats were anaesthetized withaether after30min of intragastric administration, abdomen was opened througha inverted “T” cut, then drew blood from abdominal aorta until the rat died.Placed the blood tube at room temperature for20min, centrifuged,3000rpm,20min,drew0.5ml serum into EP tube, and then was stored in ultra low temperaturefreezer. Took out the whole small intestine form pylorus to caecum, thenstraightened and spread it on the white paper. We took out the proximal colonspecimens with saline washed the lumen content, then cut into two pieces, onewas fixed in4%polyformaldehyde, and the other was placed into ultra lowtemperature freezer.The length of Small intestinal transiting activated carbon gel was measured,and then the rate of transiting was calculated, the level of serum prostaglandinE2(PGE2) was tested by ELISA, Aquaporin3(AQP3) mRNA in proximal colon was testedby RT-PCR, The expression of AQP3and inducible nitric oxide synthase (iNOS)in proximal colon was detected by immunohistochemistry.Results：The first part: Clinical research on ALGCD in prevention and treatment ofconstipation after operation of mixed hemorrhoidsThe time of first defecation after operation in the treatment group was shorter than that in control group, but no significant difference in statistics(P>0.05), The stool form scale of defecation at first time,3-4th day in thetreatment group was better than that in the control group (P<0.01), and alsobetter At6-7th day (P<0.05), until9-10th day there was no significantdifference between the two groups (P>0.05). The pain scale of defecation at firsttime,3-4th day in the treatment group was better than that in the control group(P<0.01), and also better at6-7th day (P<0.05), until9-10th day there was nosignificant difference between the two groups (P>0.05).The second part: experimental research on ALGCD in prevention and treatment ofconstipation after operation of mixed hemorrhoidsExperiment1Experimental study on acute toxicity of ALGCD by intragastricadministrationPreliminary test indicated there were no dead mouse was found when abovedoses administered, and the lethal median dose (LD50) of ALGCD orallyadministered to mice can’t be found. So we do the experimental research of miceorally administered ALGCD with maximum amount. After maximum amount of ALGCDadministration to the mice, we found that the mice had good spirits, shiny hair,smooth breathing, normal behavior, secretions and urine. In the ALGCDadministration group the water content of feces increased, and the color changedinto black (the color of ALGCD), even some feces became liking paste. The changesof feces recovered automatically after one day. The body weight of ALGCDadministration group increased normally, there are no significant differencecompared with blank normal group (P>0.05). Anatomy at the end of experimentshowed that each mouse’s major organ such as heart, lung, kidney, liver, spleen,and gastrointestinal tract were not be found abnormal, so the histopathologicalexamination was not be done.Experiment2Setting up rat model of constipation after operation of hemorrhoids1. Before making model, we obtained the volume of drink(48.25±4.15ml), weightof food intake(24.22±1.11g/d), weight of dried feces(4.14±0.58g/d) during24h and the rate of water content in fresh feces(58.16±2.95%) of rats in normal condition. There was no significant difference in statistics between the threegroups.2. At9:00AM of the2ndday since experiment began (model group rats withoutdrinking for24h), model group and model recovery group compared with normalcontrol group, the weight of food intake, weight of dried feces and the rateof water content in fresh feces decreased, but there were no significantdifference in statistics(p>0.05).3. At9:00AM of the3rdday since experiment began (model group rats withoutdrinking for48h), model group and model recovery group compared with normalcontrol group, the weight of food intake, weight of dried feces and the rateof water content in fresh feces decreased significantly (p<0.05).4.At9:00AM of the4thday since experiment began (model group rats withoutdrinking for72h), model group and model recovery group compared with normalcontrol group, the weight of food intake, weight of dried feces and the rateof water content in fresh feces decreased more significantly (p<0.01).5At9:00AM of the5thday since experiment began (model group rats24h afteranus operation), model group compared with normal control group, the weightof food intake, weight of dried feces and the rate of water content in freshfeces decreased significantly (p<0.01).model recovery group compared withnormal control group, the volume of drinking increased significantly (p<0.01),but the weight of food intake, weight of dried feces and the rate of watercontent in fresh feces still decreased (p<0.01), but increased compared withmodel group (p<0.01).6. At9:00AM of the6thday since experiment began (model group rats48h afteranal operation), model group compared with normal control group, the weightof food intake, weight of dried feces and the rate of water content in freshfeces decreased significantly (p<0.01). Model recovery group compared withnormal control group, there were no difference in the weight of food intake,weight of dried feces and the rate of water content in fresh feces.7. The change with time of weight of dried feces and the rate of water content in fresh feces in three groups. There was no obviously change in normal controlgroup. The weight of dried feces and the rate of water content in fresh fecesin the model and model recovery group decreased gradually since experimentbegan. These data of model group continuous decreased after model made and keptat a low level. But the weight of dried feces and the rate of water contentin fresh feces in model recovery group recovered rapidly in48h when makingmodel factors dispelled.Experiment3: experimental research on ALGCD in prevention and treatment ofconstipation after operation of mixed hemorrhoids1. The weight of defecation and the rate of water content in fresh fecesThe rats of other groups compared with group A(normal control group),theweight of defecation and the rate of water content in fresh feces of24h afteroperation all significantly decreased (p<0.01). Compared with group B(modelgroup), the weight of defecation and the rate of water content in fresh fecesof24h after operation in group C（Maren Soft Gelatin Capsule group） and D(lowdose therapy group) increased, but there were no difference in statistics. Thosein Group E (middle dose therapy group) increased significantly (p<0.05), andin group F (high dose therapy group) increased even more significantly (p<0.01).The rats of other groups Compared with group A(normal control group),theweight of defecation and the rate of water content in fresh feces of48h afteroperation all significantly decreased (p<0.01). The weight of defecation of48hafter operation in group C and D increased compared with that in group B increasedsignificantly (p<0.05), and group E and F increased even more significantly(p<0.01). The rate of water content in fresh feces in group C, D, E, F increasedsignificantly compared with group B(p<0.01). Compared with group C, the weightof defecation and the rate of water content in fresh feces of48h after operation,there were no difference in group D(p>0.05),and those in group E increasedsignificantly (p<0.05), in group F even more significantly (p<0.01). Comparedwith group D, the weight of defecation and the rate of water content in freshfeces of48h after operation in group E increased significantly (p<0.05), in group F even more significantly (p<0.01), but there were no difference betweengroup E and F (p>0.05).The effect of ALGCD on the weight of defecation and the rate of water contentin fresh feces showed a Dose-response relationship.2. The length and rate of small intestinal transiting activated carbon gelThe length and rate of small intestinal transiting activated carbon gel inother groups all decreased compared with those in group A(normal control group)(p<0.01), Those of each therapy groups all improved compared with group B(modelgroup)(p<0.01), Compared with those in group C（Maren Soft Gelatin Capsule group）,there were no difference in group D(low dose therapy group)(p>0.05),and thosein group E(middle dose therapy group) increased significantly(p<0.05), in groupF(high dose therapy group) even more significantly (p<0.01). Compared with groupD, those in group E increased significantly (p<0.05), and in group F increasedmore significantly (p<0.01). There were no difference in group E and F. The effectof ALGCD on the length and rate of small intestinal transiting activated carbongel showed a Dose-response relationship.3. The level of serum prostaglandin E2(PGE2)Compared with group A, the level of serum PGE2in group B (model group, andgroup C（Maren Soft Gelatin Capsule group）, D(low dose therapy group), E(middledose therapy group), F ((high dose therapy group)) all increased significantly(p<0.01), Compared with group B, the level of serum PGE2in group C and groupD both decreased, but there were no difference in statistics. The level of serumPGE2in group E and F decreased significantly compared with that in group B(p<0.01). The level of serum PGE2in group E and F decreased significantlycompared with that in group C and D. There were no difference between group Eand group F in statistics.4. Aquaporin3(AQP3) mRNA in proximal colonCompared with group A, the level of proximal colon AQP3mRNA expression ingroup B down regulated significantly (p<0.01), there were no statisticaldifference in group C and D, and that in group E, F up regulated significantly in statistics (p<0.01). AQP3mRNA expression in Group C, D, E, F all up regulatedcompared with group B (p<0.01), There were no difference between group C andD (p>0.05), The level of AQP3mRNA expression in group E and F is higher thanthat in group C and D (p<0.01), There were no difference between group E andF in statistics.5. Immunohistochemical results of the expression of AQP3and inducible nitricoxide synthase (iNOS) in proximal colon.Compared with group A, the expression of AQP3down regulated in group B(p<0.01), no difference was found in group C, D, E contrast to group A (p>0.05),and the expression of AQP3in group F up regulated compared with that in groupA (p<0.05). Compared with group B, there were no difference of expression ofAQP3in group C and D (p>0.05), and the expression of AQP3in group E up regulated(p<0.01), more significantly in group F (p<0.01). No statistical difference wasfound between group E and F (p>0.05).There was no or little expression of iNOS in colon of group A rats. Theexpression of iNOS in colon of group B rats increased significantly contrastedto group A rats. And the expression of iNOS in colon of in each therapy groupup regulated in difference degree compared with that in group A (p<0.01). Therewere no difference of group C and D contrasted to group B (p>0.05). The expressionof iNOS in colon of group E rats down regulated compared with that in group B(p<0.05), and more significantly in the colon of group F rats (p<0.01). Therewere no difference between group E and F in statistics.Conclusion：1. ALGCD can stimulate bowel movement, intenerate the stool and alleviate thepain, and can’t cause diarrhea easily. Application of ALGCD is an effectivemethod in prevention and treatment of constipation after operation of mixedhemorrhoids.2. Application of ALGCD for a short period has no obvious toxicity, so it issafe in clinical application. 3.Using of limiting water, orally administered loperamide and anus operationtogether is a possible method of making rat model of constipation after operationof hemorrhoids。And the model is consistent with pathophysiologic character ofconstipation after operation of hemorrhoids in clinic, stable, can be used forfurther animal experiment.4. ALGCD implicated to the rats with constipation after operation of hemorrhoidscan increase the weight of defecation and the rate of water content in freshfeces, and showed a Dose-response relationship.5. Application ALGCD to the rats with constipation after operation of hemorrhoidscan improve the length and rate of small intestinal transiting activated carbongel, ALGCD can stimulate bowel movement.6. Oral administration of ALGCD to the rats with constipation after operationof hemorrhoids can reduced the level of serum PEG2, and alleviates anal pain.7. The expression of AQP3mRNA and protein in the proximal colon of the ratswith constipation after operation of hemorrhoids down regulated, afterapplication of ALGCD the expression up regulated, this illustrates that AQP3participate the effect of ALGCD on regulating liquid metabolism of intestinaltract.8. There was no or little expression of iNOS in colon of normal rat. The expressionof iNOS in colon of the rats with constipation after operation of hemorrhoidsincreased, application of ALGCD can down regulate the expression of iNOS; enhancethe motivity of intestinal tract. |
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