止痛润肠浓煎饮防治混合痔术后便秘的临床及实验研究
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摘要
目的：便秘是混合痔术后常见的并发症之一，一旦发生将给患者带来严重的不良后果。本课题旨在通过临床研究观察止痛润肠浓煎饮防治混合痔术后便秘的疗效及不良反应；通过动物试验检测止痛润肠浓煎饮可能存在的毒性；探讨符合混合痔术后便秘临床特点的大鼠动物模型的制备方法；通过实验研究从基因及蛋白表达水平探讨止痛润肠浓煎饮防治混合痔术后便秘的可能作用机制，为更好地推广应用提供理论依据。
材料与方法：
第一部分：止痛润肠浓煎饮防治混合痔术后便秘的临床研究
将住院手术的混合痔患者随机分为两组。所有患者入院后完善各项相关检查，排除不符合研究标准的病例。两组患者均于骶管麻醉下行混合痔内扎外切创面开放术式（Milligan-Morgan），并根据需要加消痔灵注射术，术后在常规治疗的基础上，治疗组（n=41）术后给予止痛润肠浓煎饮口服，对照组（n=37）术后给予麻仁软胶囊口服。分别观察记录两组患者的术后首次排便时间，术后首次、第3-4天、6-7天及9-10天时间段内大便性状评分和排便时疼痛评分。
第二部分：止痛润肠浓煎饮防治混合痔术后便秘的实验研究
实验一：止痛润肠浓煎饮口服给药急性毒理试验研究
选用SPF级昆明种小鼠30只，随机分为A、B、C三组，每组10只，雌雄各半进行预实验。所有动物给药前禁食不禁水16小时。A组每天给药1次；B组每天给药2次，间隔6h；C组每天给药3次，间隔4h。小鼠每次灌胃给予40ml/kg的止痛润肠浓煎饮的浓缩液(3.5g生药／ml)。然后按常规饲养，观察小鼠外观、行为活动、呼吸、分泌物、排便、死亡等情况，共观察7天。结果未见小鼠死亡，未能测出小鼠灌胃给药的半数致死量（LD50），故进行小鼠灌胃给药的最大给药量试验研究。
选用SPF级昆明种小鼠40只，随机分为2组，分别为空白对照组及给药组，每组20只，雌雄各半。按止痛润肠浓煎饮的最大浓度(3.5g生药／ml)、小鼠一次灌胃最大容积(40ml／kg)给药，日3次，每次间隔4h；空白对照组给予等体积的纯净水，方法同给药组。第3次给药后两组小鼠均给予正常饮食，常规饲养14天。实验期间每天观察有无死亡、外观、行为、精神状态、分泌物、排泄物、饮食和中毒症状；给药前和给药后第7、14天分别称量小鼠的体重；在观察期结束后对存活动物进行大体解剖，记录相关情况，如有器官出现异常则对病变器官进行组织病理学检查。
实验二：混合痔术后便秘大鼠模型的制备
将24只SD大鼠随机分成3组：正常对照组、模型组和模型恢复组。每组8只，雌雄各半。造模方法根据文献报道及预实验结果进行。第1-3天模型组和模型恢复组大鼠禁水不禁食，给予洛哌丁胺悬浊液3mg/kg，灌胃日2次，间隔6h。正常对照组大鼠正常饮食，给予同体积纯净水灌胃，方法同模型组。第4天模型组和模型恢复组行肛门手术，正常对照组行假手术。模型组术后继续给予洛哌丁胺悬浊液3mg/kg，约2ml，灌胃日2次，间隔6h，并给予正常饮水量1/6（8ml）的纯净水，鼠粮不限。模型恢复组和正常对照组术后不限饮食。第5天以后各组大鼠给药及饮食情况同第4天。
肛门手术方法：乙醚麻醉成功后，将大鼠固定于解剖台上，碘伏消毒肛周皮肤，取肛管右后及左侧各做一放射状梭形切口，深达皮下，长约0.6cm，最宽处约0.2cm，内达直肠与肛管交界，外达肛缘。假手术方法，除不做切口外其它操作同上。
观察造模开始前一日及开始后每日进食量、排便量、饮水量及排便的难易程度，新排出粪便的质地，并测量计算新鲜粪便含水率。
实验三：止痛润肠浓煎饮防治混合痔术后便秘的实验研究
将48只SD大鼠随机分成6组：A组（正常对照组）、B组（模型组）、C（组麻仁软胶囊组）、D组（低剂量治疗组）、E组（中剂量治疗组）和F组（高剂量治疗组），每组8只，雌雄各半。
依照实验二方法造混合痔术后便秘模型，肛门手术后开始灌胃给予治疗药物，于洛哌丁胺灌胃后间隔2h给予。A、B组给予生理盐水（NS）2ml，日2次；C组给予麻仁软胶囊混悬液（0.0075g/ml）2ml，日2次，为人鼠等效剂量；D组给予止痛润肠浓煎饮（0.6g生药/ml）2ml，日2次，约为成人常用等效剂量1/2；E组给予止痛润肠浓煎饮（1.2g生药/ml）2ml，日2次，约为成人常用等效剂量；F组给予止痛润肠浓煎饮（2.4g生药/ml）2ml，日2次，约为成人常用等效剂量2倍。观察各组大鼠手术后排便情况，统计每24小时排便量及新鲜粪便含水率
各组大鼠在术后48h第5次给药后禁食水4h，各组大鼠均给予活性炭凝胶灌胃，剂量4ml/只。灌胃结束30min后，大鼠乙醚麻醉，取倒“T”形切口打开腹腔，腹主动脉采血至大鼠死亡。采血管室温静置20min，离心，3000rpm，20min，分别取血清0.5ml于EP管中，置入-80℃超低温冰箱保存。取出自幽门至盲肠全部小肠，拉直平铺于白纸上。取近端结肠组织，生理盐水冲洗肠腔内容物，分成两段，其一应用4%多聚甲醛固定，其二置于超低温冰箱-80℃冷藏。
测量并计算小肠活性炭凝胶推进长度和推进率；ELISA检测血清前列腺素E2（PGE2）水平；RT-PCR检测近端结肠水通道蛋白3（AQP3）mRNA表达温水平；免疫组化检测近端结肠粘膜AQP3、诱导型一氧化氮合酶（iNOS）蛋白表达情况。
结果：
第一部分：止痛润肠浓煎饮防治混合痔术后便秘的临床研究
术后首次排便时间，治疗组较对照组缩短，但无统计学差异（p>0.05）；与对照组相比，术后首次、第3-4天大便性状评分明显优于对照组（P＜0.01），第6-7天时亦有差异（P＜0.05），至第9-10天两者差异不显著（P>0.05）；与对照组相比，术后首次、3-4d大便疼痛评分明显低于对照组（P＜0.01），6-7d时亦有差异（P＜0.05），至9-10d两者差异不显著（P>0.05）。
第二部分：止痛润肠浓煎饮防治混合痔术后便秘的实验研究实验一：止痛润肠浓煎饮口服给药急性毒理试验研究
结果表明，小鼠在上述剂量灌胃给药后未见小鼠死亡，未能测出小鼠灌胃给药的半数致死量（LD50），故进行小鼠灌胃给药的做最大给药量实验研究。止痛润肠浓煎饮以最大给药量给予小鼠后，精神状况良好，毛色光亮，呼吸通畅、均匀，行为、分泌物、小便等均无异常。给药组给药后出现大便含水量增加，颜色变黑（中药颜色），个别呈稀糊状，1天后自动恢复正常，小鼠体重增长正常。给药组受试小鼠体重与空白对照组比无明显差异（P>0.05）。实验结束大体解剖各小鼠心、肺、肾、肝、脾、胃肠道等主要脏器均未见异常，故未进行组织病理学检查。
实验二：混合痔术后便秘大鼠模型的制备
1.在造模前测得正常情况下大鼠24h饮水量（48.25±4.15ml）、进食量（24.22±1.11g/d）、排便量（4.14±0.58g/d）及新鲜粪便含水率（58.16±2.95%），三组比较均无统计学差别（p>0.05）。
2.实验开始第2日上午9：00（即模型组禁水24h）模型组及模型恢复组进食量、排便量及新鲜粪便含水率与对照组相比有所下降，但无统计学差异（p>0.05）。
3.第3日上午9：00（即模型组禁水48h）模型组及模型恢复组进食量、排便量及新鲜粪便含水率与对照组相比明显下降，有统计学差异（p<0.01）。
4.第4日上午9：00（即模型组禁水72h）模型组及模型恢复组进食量、排便量及新鲜粪便含水率与对照组相比进一步明显下降，统计学差异显著（p<0.01）。
5.第5日上午9：00（即模型组手术后24h）模型组进食量、排便量及新鲜粪便含水率与对照组相比明显下降，统计学差异显著（p<0.01）。模型恢复组与对照相比饮水量明显增加（p<0.01），进食量、排便量及新鲜粪便含水率仍低于对照组（p<0.01），但均明显高于模型组（p<0.01）。
6.第6日上午9：00（即模型组手术后48h）模型组进食量、排便量及新鲜粪便含水率与对照组相比明显下降，统计学差异显著（p<0.01）。模型恢复组与对照相比饮水量、进食量、排便量及新鲜粪便含水率无显著差异（p>0.05）。
7.三组大鼠排便量及新鲜粪便含水率随时间变化结果：正常对照组无明显变化；模型组与模型恢复组大鼠自造模开始排便量及新鲜粪便含水率逐渐减少，模型组在造模结束后排便量及新鲜粪便含水率继续减少并维持在低水平，而模型恢复组在祛除造造模因素后，排便量及新鲜粪便含水率在48小内快速恢复至正常。
实验三：止痛润肠浓煎饮防治混合痔术后便秘的实验研究
1．排便量及新鲜粪便含水率
与A组（正常对照组）相比，其它各组大鼠术后24小时排便量及新鲜粪便含水率均明显降低（P<0.01）；与B组（模型组）相比术后24h排便量及新鲜粪便含水率C组（麻仁软胶囊组）、D组（低剂量治疗组）均增加，但无统计学差异（P>0.05），E组（中剂量治疗组）明显增多（P<0.05），F组（高剂量治疗组）增加更显著（P<0.01）；
与A组（正常对照组）相比，其它各组大鼠术后48小时排便量及新鲜粪便含水率均明显降低（P<0.01）；与B组相比，术后48h排便量C组、D组均明显增加（P<0.05），E、F组增加更显著（P<0.01）；术后48h新鲜粪便含水率C、D、E、F组显著升高（P<0.01）。与C组相比，48h排便量及新鲜粪便含水率D组无差别（P>0.05），E组明显增加（P<0.05），F组显著增加（P<0.01）；与D组相比，48h排便量及新鲜粪便含水率E组明显增加（P<0.05），F组显著增加（P<0.01）；E组与F组相比无差别。
止痛润肠浓煎饮对排便量及粪便含水率的影响呈量效依赖关系。
2．小肠活性炭凝胶推进长度和推进率
与A组（正常对照组）相比其它各组无论是小肠活性炭凝胶推进长度还是推进率均显著降低（P<0.01）；与B组（模型组）相比，各治疗组推进长度和推进率均显著改善（P<0.01）；与C组（麻仁软胶囊组）相比，D组（低剂量治疗组）无差别（P>0.05），E组（中剂量治疗组）增加明显（P<0.05），F组（高剂量治疗组）增加显著（P<0.01）；与D组相比，E组增加明显（P<0.05），F组增加显著（P<0.01）；E组与F组差别不显著（P>0.05）；止痛润肠浓煎饮对小肠活性炭凝胶推进长度和推进率的影响具有量效依赖关系。
3．各组大鼠血清PGE2水平
与A组（正常对照组）相比，模型组及各治疗组PGE2水平均明显升高（P<0.01）；而各治疗组PGE2水平与B组（模型组）相比都有下降，其中C组（中麻仁软胶囊组）及D组（低剂量治疗组）下降无统计学差异（P>0.05），E组（中剂量治疗组）、F组（高剂量治疗组）下降最为明显（P<0.01）；E、F组均明显低于C和D组（P<0.01）；
E组与F组间无统计学差异（P>0.05）。
4．各组大鼠结肠AQP3mRNA表达水平
与A组（正常对照组）相比，B组（模型组）近端结肠AQP3mRNA表达水平显著下调（P<0.01），C组（麻仁软胶囊组）、D组（低剂量治疗组）无统计学差异（P>0.05），E组（中剂量治疗组）、F组（高剂量治疗组）明显上调，统计学差异显著（P<0.01）；与B组相比，C、D、E、F组AQP3mRNA表达水平均显著上调（P<0.01）；C组与D组间无统计学差异（P>0.05）；E、F组均明显高于C、D组（P<0.01）；E组和F组间无统计学差异（P>0.05）。
5．各组大鼠AQP3、iNOS免疫组化结果比较
与A组（正常对照组）相比，B组（模型组）表达下调（P<0.01），C组（麻仁软胶囊组）、D组（低剂量治疗组）和E组（中剂量治疗组）无显著差别（P>0.05），F组（高剂量治疗组）表达上调（P<0.05）；与B组相比，C组、D组表达无差别，E组表达上调（P<0.01），F组上调更明显（P<0.01），但E、F组之间相比并无统计学差异（P>0.05）。
A组大鼠结肠iNOS呈低表达或不表达，而B组大鼠结肠iNOS表达比A组明显增加（P<0.01），各治疗组与A组相比iNOS表达也有不同程度的上调(p<0.01)。与B组相比，C组与D组表达无明显差别（P>0.05），E组表达水平降低（P<0.05），F组降低更为明显（P<0.01），但E、F组之间相比并无统计学差异（P>0.05）。
结论：
1．混合痔术后患者口服止痛润肠浓煎饮具有促进排便、软化大便、减轻疼痛的作用，且不易引起腹泻。应用止痛润肠浓煎饮是临床中防治混合痔患者术后便秘的有效方法。
2．止痛润肠浓煎饮短期应用无明显毒性作用，临床应用安全。
3．联合应用限水、灌服洛哌丁胺及肛门手术制作混合痔术后便秘大鼠模型是可行的，符合临床混合痔患者术后便秘的病理生理特点，稳定性好，可供进一步动物实验使用。
4．止痛润肠浓煎饮能增加混合痔术后便秘大鼠的排便量及新鲜粪便含水率，并呈量效依赖关系。
5．止痛润肠浓煎饮能增加混合痔术后便秘大鼠小肠活性炭凝胶推进长度及推进率，具有促进肠蠕动作用。
6．止痛润肠浓煎饮可降低混合痔术后便秘大鼠血清PGE2水平，减轻肛门疼痛程度。
7．混合痔术后便秘大鼠结肠AQP3mRNA及蛋白表达下调，应用止痛润肠浓煎饮治疗后表达上调，说明AQP3可能参与了止痛润肠浓煎饮对肠道水液代谢的调节作用。
8．正常大鼠结肠iNOS呈低表达或不表达，混合痔术后便秘大鼠结肠iNOS表达增强，应用止痛润肠浓煎饮可下调iNOS表达，增强肠动力。
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 hemorrhoids
Patients 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 intragastricadministration
30Kunming 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 hemorrhoids
24SD 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 hemorrhoids
48SD 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 measured
At48h 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 hemorrhoids
The 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 hemorrhoids
Experiment1Experimental study on acute toxicity of ALGCD by intragastricadministration
Preliminary 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 hemorrhoids
1. 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 hemorrhoids
1. 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 gel
The 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 colon
Compared 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.
材料与方法：
第一部分：止痛润肠浓煎饮防治混合痔术后便秘的临床研究
将住院手术的混合痔患者随机分为两组。所有患者入院后完善各项相关检查，排除不符合研究标准的病例。两组患者均于骶管麻醉下行混合痔内扎外切创面开放术式（Milligan-Morgan），并根据需要加消痔灵注射术，术后在常规治疗的基础上，治疗组（n=41）术后给予止痛润肠浓煎饮口服，对照组（n=37）术后给予麻仁软胶囊口服。分别观察记录两组患者的术后首次排便时间，术后首次、第3-4天、6-7天及9-10天时间段内大便性状评分和排便时疼痛评分。
第二部分：止痛润肠浓煎饮防治混合痔术后便秘的实验研究
实验一：止痛润肠浓煎饮口服给药急性毒理试验研究
选用SPF级昆明种小鼠30只，随机分为A、B、C三组，每组10只，雌雄各半进行预实验。所有动物给药前禁食不禁水16小时。A组每天给药1次；B组每天给药2次，间隔6h；C组每天给药3次，间隔4h。小鼠每次灌胃给予40ml/kg的止痛润肠浓煎饮的浓缩液(3.5g生药／ml)。然后按常规饲养，观察小鼠外观、行为活动、呼吸、分泌物、排便、死亡等情况，共观察7天。结果未见小鼠死亡，未能测出小鼠灌胃给药的半数致死量（LD50），故进行小鼠灌胃给药的最大给药量试验研究。
选用SPF级昆明种小鼠40只，随机分为2组，分别为空白对照组及给药组，每组20只，雌雄各半。按止痛润肠浓煎饮的最大浓度(3.5g生药／ml)、小鼠一次灌胃最大容积(40ml／kg)给药，日3次，每次间隔4h；空白对照组给予等体积的纯净水，方法同给药组。第3次给药后两组小鼠均给予正常饮食，常规饲养14天。实验期间每天观察有无死亡、外观、行为、精神状态、分泌物、排泄物、饮食和中毒症状；给药前和给药后第7、14天分别称量小鼠的体重；在观察期结束后对存活动物进行大体解剖，记录相关情况，如有器官出现异常则对病变器官进行组织病理学检查。
实验二：混合痔术后便秘大鼠模型的制备
将24只SD大鼠随机分成3组：正常对照组、模型组和模型恢复组。每组8只，雌雄各半。造模方法根据文献报道及预实验结果进行。第1-3天模型组和模型恢复组大鼠禁水不禁食，给予洛哌丁胺悬浊液3mg/kg，灌胃日2次，间隔6h。正常对照组大鼠正常饮食，给予同体积纯净水灌胃，方法同模型组。第4天模型组和模型恢复组行肛门手术，正常对照组行假手术。模型组术后继续给予洛哌丁胺悬浊液3mg/kg，约2ml，灌胃日2次，间隔6h，并给予正常饮水量1/6（8ml）的纯净水，鼠粮不限。模型恢复组和正常对照组术后不限饮食。第5天以后各组大鼠给药及饮食情况同第4天。
肛门手术方法：乙醚麻醉成功后，将大鼠固定于解剖台上，碘伏消毒肛周皮肤，取肛管右后及左侧各做一放射状梭形切口，深达皮下，长约0.6cm，最宽处约0.2cm，内达直肠与肛管交界，外达肛缘。假手术方法，除不做切口外其它操作同上。
观察造模开始前一日及开始后每日进食量、排便量、饮水量及排便的难易程度，新排出粪便的质地，并测量计算新鲜粪便含水率。
实验三：止痛润肠浓煎饮防治混合痔术后便秘的实验研究
将48只SD大鼠随机分成6组：A组（正常对照组）、B组（模型组）、C（组麻仁软胶囊组）、D组（低剂量治疗组）、E组（中剂量治疗组）和F组（高剂量治疗组），每组8只，雌雄各半。
依照实验二方法造混合痔术后便秘模型，肛门手术后开始灌胃给予治疗药物，于洛哌丁胺灌胃后间隔2h给予。A、B组给予生理盐水（NS）2ml，日2次；C组给予麻仁软胶囊混悬液（0.0075g/ml）2ml，日2次，为人鼠等效剂量；D组给予止痛润肠浓煎饮（0.6g生药/ml）2ml，日2次，约为成人常用等效剂量1/2；E组给予止痛润肠浓煎饮（1.2g生药/ml）2ml，日2次，约为成人常用等效剂量；F组给予止痛润肠浓煎饮（2.4g生药/ml）2ml，日2次，约为成人常用等效剂量2倍。观察各组大鼠手术后排便情况，统计每24小时排便量及新鲜粪便含水率
各组大鼠在术后48h第5次给药后禁食水4h，各组大鼠均给予活性炭凝胶灌胃，剂量4ml/只。灌胃结束30min后，大鼠乙醚麻醉，取倒“T”形切口打开腹腔，腹主动脉采血至大鼠死亡。采血管室温静置20min，离心，3000rpm，20min，分别取血清0.5ml于EP管中，置入-80℃超低温冰箱保存。取出自幽门至盲肠全部小肠，拉直平铺于白纸上。取近端结肠组织，生理盐水冲洗肠腔内容物，分成两段，其一应用4%多聚甲醛固定，其二置于超低温冰箱-80℃冷藏。
测量并计算小肠活性炭凝胶推进长度和推进率；ELISA检测血清前列腺素E2（PGE2）水平；RT-PCR检测近端结肠水通道蛋白3（AQP3）mRNA表达温水平；免疫组化检测近端结肠粘膜AQP3、诱导型一氧化氮合酶（iNOS）蛋白表达情况。
结果：
第一部分：止痛润肠浓煎饮防治混合痔术后便秘的临床研究
术后首次排便时间，治疗组较对照组缩短，但无统计学差异（p>0.05）；与对照组相比，术后首次、第3-4天大便性状评分明显优于对照组（P＜0.01），第6-7天时亦有差异（P＜0.05），至第9-10天两者差异不显著（P>0.05）；与对照组相比，术后首次、3-4d大便疼痛评分明显低于对照组（P＜0.01），6-7d时亦有差异（P＜0.05），至9-10d两者差异不显著（P>0.05）。
第二部分：止痛润肠浓煎饮防治混合痔术后便秘的实验研究实验一：止痛润肠浓煎饮口服给药急性毒理试验研究
结果表明，小鼠在上述剂量灌胃给药后未见小鼠死亡，未能测出小鼠灌胃给药的半数致死量（LD50），故进行小鼠灌胃给药的做最大给药量实验研究。止痛润肠浓煎饮以最大给药量给予小鼠后，精神状况良好，毛色光亮，呼吸通畅、均匀，行为、分泌物、小便等均无异常。给药组给药后出现大便含水量增加，颜色变黑（中药颜色），个别呈稀糊状，1天后自动恢复正常，小鼠体重增长正常。给药组受试小鼠体重与空白对照组比无明显差异（P>0.05）。实验结束大体解剖各小鼠心、肺、肾、肝、脾、胃肠道等主要脏器均未见异常，故未进行组织病理学检查。
实验二：混合痔术后便秘大鼠模型的制备
1.在造模前测得正常情况下大鼠24h饮水量（48.25±4.15ml）、进食量（24.22±1.11g/d）、排便量（4.14±0.58g/d）及新鲜粪便含水率（58.16±2.95%），三组比较均无统计学差别（p>0.05）。
2.实验开始第2日上午9：00（即模型组禁水24h）模型组及模型恢复组进食量、排便量及新鲜粪便含水率与对照组相比有所下降，但无统计学差异（p>0.05）。
3.第3日上午9：00（即模型组禁水48h）模型组及模型恢复组进食量、排便量及新鲜粪便含水率与对照组相比明显下降，有统计学差异（p<0.01）。
4.第4日上午9：00（即模型组禁水72h）模型组及模型恢复组进食量、排便量及新鲜粪便含水率与对照组相比进一步明显下降，统计学差异显著（p<0.01）。
5.第5日上午9：00（即模型组手术后24h）模型组进食量、排便量及新鲜粪便含水率与对照组相比明显下降，统计学差异显著（p<0.01）。模型恢复组与对照相比饮水量明显增加（p<0.01），进食量、排便量及新鲜粪便含水率仍低于对照组（p<0.01），但均明显高于模型组（p<0.01）。
6.第6日上午9：00（即模型组手术后48h）模型组进食量、排便量及新鲜粪便含水率与对照组相比明显下降，统计学差异显著（p<0.01）。模型恢复组与对照相比饮水量、进食量、排便量及新鲜粪便含水率无显著差异（p>0.05）。
7.三组大鼠排便量及新鲜粪便含水率随时间变化结果：正常对照组无明显变化；模型组与模型恢复组大鼠自造模开始排便量及新鲜粪便含水率逐渐减少，模型组在造模结束后排便量及新鲜粪便含水率继续减少并维持在低水平，而模型恢复组在祛除造造模因素后，排便量及新鲜粪便含水率在48小内快速恢复至正常。
实验三：止痛润肠浓煎饮防治混合痔术后便秘的实验研究
1．排便量及新鲜粪便含水率
与A组（正常对照组）相比，其它各组大鼠术后24小时排便量及新鲜粪便含水率均明显降低（P<0.01）；与B组（模型组）相比术后24h排便量及新鲜粪便含水率C组（麻仁软胶囊组）、D组（低剂量治疗组）均增加，但无统计学差异（P>0.05），E组（中剂量治疗组）明显增多（P<0.05），F组（高剂量治疗组）增加更显著（P<0.01）；
与A组（正常对照组）相比，其它各组大鼠术后48小时排便量及新鲜粪便含水率均明显降低（P<0.01）；与B组相比，术后48h排便量C组、D组均明显增加（P<0.05），E、F组增加更显著（P<0.01）；术后48h新鲜粪便含水率C、D、E、F组显著升高（P<0.01）。与C组相比，48h排便量及新鲜粪便含水率D组无差别（P>0.05），E组明显增加（P<0.05），F组显著增加（P<0.01）；与D组相比，48h排便量及新鲜粪便含水率E组明显增加（P<0.05），F组显著增加（P<0.01）；E组与F组相比无差别。
止痛润肠浓煎饮对排便量及粪便含水率的影响呈量效依赖关系。
2．小肠活性炭凝胶推进长度和推进率
与A组（正常对照组）相比其它各组无论是小肠活性炭凝胶推进长度还是推进率均显著降低（P<0.01）；与B组（模型组）相比，各治疗组推进长度和推进率均显著改善（P<0.01）；与C组（麻仁软胶囊组）相比，D组（低剂量治疗组）无差别（P>0.05），E组（中剂量治疗组）增加明显（P<0.05），F组（高剂量治疗组）增加显著（P<0.01）；与D组相比，E组增加明显（P<0.05），F组增加显著（P<0.01）；E组与F组差别不显著（P>0.05）；止痛润肠浓煎饮对小肠活性炭凝胶推进长度和推进率的影响具有量效依赖关系。
3．各组大鼠血清PGE2水平
与A组（正常对照组）相比，模型组及各治疗组PGE2水平均明显升高（P<0.01）；而各治疗组PGE2水平与B组（模型组）相比都有下降，其中C组（中麻仁软胶囊组）及D组（低剂量治疗组）下降无统计学差异（P>0.05），E组（中剂量治疗组）、F组（高剂量治疗组）下降最为明显（P<0.01）；E、F组均明显低于C和D组（P<0.01）；
E组与F组间无统计学差异（P>0.05）。
4．各组大鼠结肠AQP3mRNA表达水平
与A组（正常对照组）相比，B组（模型组）近端结肠AQP3mRNA表达水平显著下调（P<0.01），C组（麻仁软胶囊组）、D组（低剂量治疗组）无统计学差异（P>0.05），E组（中剂量治疗组）、F组（高剂量治疗组）明显上调，统计学差异显著（P<0.01）；与B组相比，C、D、E、F组AQP3mRNA表达水平均显著上调（P<0.01）；C组与D组间无统计学差异（P>0.05）；E、F组均明显高于C、D组（P<0.01）；E组和F组间无统计学差异（P>0.05）。
5．各组大鼠AQP3、iNOS免疫组化结果比较
与A组（正常对照组）相比，B组（模型组）表达下调（P<0.01），C组（麻仁软胶囊组）、D组（低剂量治疗组）和E组（中剂量治疗组）无显著差别（P>0.05），F组（高剂量治疗组）表达上调（P<0.05）；与B组相比，C组、D组表达无差别，E组表达上调（P<0.01），F组上调更明显（P<0.01），但E、F组之间相比并无统计学差异（P>0.05）。
A组大鼠结肠iNOS呈低表达或不表达，而B组大鼠结肠iNOS表达比A组明显增加（P<0.01），各治疗组与A组相比iNOS表达也有不同程度的上调(p<0.01)。与B组相比，C组与D组表达无明显差别（P>0.05），E组表达水平降低（P<0.05），F组降低更为明显（P<0.01），但E、F组之间相比并无统计学差异（P>0.05）。
结论：
1．混合痔术后患者口服止痛润肠浓煎饮具有促进排便、软化大便、减轻疼痛的作用，且不易引起腹泻。应用止痛润肠浓煎饮是临床中防治混合痔患者术后便秘的有效方法。
2．止痛润肠浓煎饮短期应用无明显毒性作用，临床应用安全。
3．联合应用限水、灌服洛哌丁胺及肛门手术制作混合痔术后便秘大鼠模型是可行的，符合临床混合痔患者术后便秘的病理生理特点，稳定性好，可供进一步动物实验使用。
4．止痛润肠浓煎饮能增加混合痔术后便秘大鼠的排便量及新鲜粪便含水率，并呈量效依赖关系。
5．止痛润肠浓煎饮能增加混合痔术后便秘大鼠小肠活性炭凝胶推进长度及推进率，具有促进肠蠕动作用。
6．止痛润肠浓煎饮可降低混合痔术后便秘大鼠血清PGE2水平，减轻肛门疼痛程度。
7．混合痔术后便秘大鼠结肠AQP3mRNA及蛋白表达下调，应用止痛润肠浓煎饮治疗后表达上调，说明AQP3可能参与了止痛润肠浓煎饮对肠道水液代谢的调节作用。
8．正常大鼠结肠iNOS呈低表达或不表达，混合痔术后便秘大鼠结肠iNOS表达增强，应用止痛润肠浓煎饮可下调iNOS表达，增强肠动力。
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 hemorrhoids
Patients 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 intragastricadministration
30Kunming 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 hemorrhoids
24SD 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 hemorrhoids
48SD 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 measured
At48h 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 hemorrhoids
The 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 hemorrhoids
Experiment1Experimental study on acute toxicity of ALGCD by intragastricadministration
Preliminary 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 hemorrhoids
1. 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 hemorrhoids
1. 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 gel
The 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 colon
Compared 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.
引文
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