小柴胡汤干预化疗药物伊立替康所致迟发性腹泻的实验研究

发布时间：2018-05-11 08:46

本文选题：CPT-11 + 迟发性腹泻　；参考：《贵州医科大学》2017年硕士论文

【摘要】：目的:伊立替康(Irinotecan,CPT-11)是临床大肠癌常用的化疗药,其剂量限制性毒性反应为迟发性腹泻,严重影响了患者的生活质量和对化疗的耐受。本研究目的:1.建立CPT-11所致小鼠迟发性腹泻模型;2.探讨预先灌胃小柴胡汤对CPT-11所致小鼠迟发性腹泻是否有预防作用;3.通过超高效液相色谱串联三重四级杆质谱法(Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry,UPLC-MS/MS)测定CPT-11及其代谢产物以及小柴胡汤中有效成分含量,探寻小柴胡汤作用于迟发性腹泻的物质基础,为机制研究奠定基础。方法:1.建立小鼠迟发性腹泻模型:健康雄性C57BL6小鼠20只,适应性饲喂高脂饲料三周后,随机分为模型组(n=10)和正常组(n=10),连续腹腔注射CPT-11 7天,每天一次,剂量为50 mg·kg-1,正常组腹腔注射等量的右旋糖苷40葡萄糖注射液,每天观察小鼠的肛门,拍照记录,收集粪便,用尿粪便隐血试剂盒测定血便情况。2.小柴胡汤干预研究:健康雄性C57BL6小鼠40只,适应性饲喂高脂饲料三周后,随机分为4组,每组10只:空白对照组(等容生理盐水),迟发性腹泻模型组(等容生理盐水),小柴胡汤组(1500 mg·kg-1·d-1),洛哌丁胺组(0.5333 mg·kg-1·d-1),连续灌胃17天,每天1次。于实验第4~10天,除空白对照组外,其余各组小鼠均腹腔注射CPT-11 7天诱导迟发性腹泻。在注射CPT-11的第0、2、4、6、8、10天取小鼠尾尖血,置预涂肝素的微量离心管(eppendorf tube,EP管)中,取血时间为注射后2 h。观察小鼠体重变化,收集粪便用尿粪便隐血试剂盒测定血便,实验第18天所有小鼠乙醚麻醉后取心脏血,置预涂肝素的EP管中,为寻找CPT-11代谢产物,测定小柴胡汤有效成分含量,然后处死小鼠取的肝脏、肾脏、大肠、小肠组织,分为两部分保存,一部分冻存于-80℃冰箱,用于测量组织中药物含量;另一部分存于4%甲醛溶液,用于制作病理切片,了解组织损伤。3.采用UPLC-MS/MS方法,测定组织及血液样品中CPT-11及其代谢产物和小柴胡汤中有效成分的含量。结果:1.迟发性腹泻模型建立:(1)与正常组相比,模型组小鼠皮毛污秽、潮湿、结块,身体发抖,抓拿时不挣扎,肛门乌黑红肿;(2)正常组小鼠体重上升了0.57±0.41g,模型组小鼠下降了5.98±2.08g;(3)正常组小鼠血便率为0,模型组于连续腹腔注射cpt-11后第7天血便率达100%(p0.01)。2.小柴胡汤干预研究:(1)体重比较:第1天和第4天各组小鼠体重无明显差异(p0.05),第10天空白对照组小鼠体重高于其他组小鼠(p0.05),说明迟发性腹泻模型能导致小鼠体重显著降低;第17天空白对照组小鼠体重仍高于其他组(p0.05),但小柴胡汤组小鼠体重高于模型对照组(p0.05),说明小柴胡汤能一定程度地减轻小鼠体重降低的症状;(2)血便率:正常对照组、模型对照组、小柴胡汤组、洛哌丁胺组血便率分别为0%、100%、50%、60%;实验第8~17天,小柴胡汤组和洛哌丁胺组小鼠血便率均显著低于模型对照组(p0.01),且小柴胡汤组小鼠血便率低于洛哌丁胺组(p0.05);(3)病理组织学观察:空白对照组小鼠大肠和小肠结构完整,模型对照组小鼠大肠和小肠黏膜上皮细胞伴明显弥漫性坏死脱落和炎症细胞浸润,小柴胡汤组和洛哌丁胺组小鼠肠黏膜组织均趋于正常;连续注射cpt-117天对肝脏和肾脏无明显损伤。3.药物浓度含量测定:(1)cpt-11及其代谢产物:小柴胡汤组小鼠注射cpt-11第6天时cpt-11血药浓度最高,与模型对照组第4天的最高浓度没有显著性差异(p0.05);模型对照组sn-38最高浓度是实验组的7倍;模型对照组注射cpt-11第4天时sn-38g血药浓度最高,而小柴胡汤组是第6天,显著高于模型对照组(p0.05),除了第4天实验组血药浓度比对照组低外,其他天数实验组血药浓度均高于对照组;模型对照组cpt-11浓度在各组织中排序为小肠肝肾大肠,sn-38和sn-38g浓度在各组织中排序为小肠肾大肠肝,模型对照组小鼠cpt-11、sn-38、sn-38g均在小肠中分布最多(p0.05),小柴胡汤组cpt-11浓度在各组织中排序为肝肾小肠大肠,sn-38和sn-38g浓度在各组织中排序为肝小肠肾大肠,小柴胡汤组小鼠cpt-11、sn-38、sn-38g这三种物质均在肝中分布最多(p0.05),小柴胡汤组各成分在各组织的含量均显著小于模型对照组;(2)小柴胡汤中17种有效成分:注射cpt-11后0、2、4、6天血液中只能测到14种成分,黄芩苷、槲皮素和黄芩苷元这三种成分在检测限以下,而各组织中均可以测到这3种成分,提示这3种成分具有器官靶向性;甘草酸的血药浓度远远高于其他成分,在各组织中只少量存在,可能是甘草酸主要在血液中代谢。在肝中汉黄芩素的浓度最高(21700.7430ng/ml),而柴胡皂苷a的浓度最低(12.4930 ng/mL);在肾脏中人参皂苷Rd的浓度最高(21563.0884ng/mL),柴胡皂苷a的含量最低(20.7965 ng/mL);在大肠中汉黄芩素的浓度最高(26370.0697 ng/mL),柴胡皂苷a的含量最低(3.7625 ng/mL);在小肠中人参皂苷Rd的浓度最高(22622.3167 ng/mL),人参皂苷Rg1的含量最低(45.0834 ng/mL)。结论:1.首次使用C57BL6小鼠,适应性饲喂高脂饲料三周后腹腔注射CPT-11 50 mg/kg,连续7天,每天一次,能成功建立CPT-11所致迟发性腹泻小鼠模型;2.小柴胡汤能预防CPT-11所致小鼠血便,使迟发性腹泻小鼠体重正常增长、降低血便率、改善肠组织损伤;3.小柴胡汤中含有17种活性成分,主要干预SN-38的代谢,能抑制CPT-11、SN-38、SN-38G的胆汁外排并加速这三种物质的消除;CPT-11的肠肝循环能引起迟发性腹泻,小柴胡汤预防CPT-11所致迟发性腹泻是多个靶器官共同作用的,可能与抑制肠肝循环有关,可为后续作用机制研究提供参考。
[Abstract]:Objective: Irinotecan (CPT-11) is a commonly used chemotherapeutic agent in clinical colorectal cancer. The dose restrictive toxicity is delayed diarrhea, which seriously affects the patient's quality of life and tolerance to chemotherapy. 1. the purpose of this study was to establish a model of delayed diarrhea caused by CPT-11 in mice, and 2. to explore the effect of small Bupleurum Decoction on CPT-11 induced mice in advance. Does delayed diarrhea have a preventive effect; 3. the content of CPT-11, its metabolites and the effective components in small Bupleurum soup are determined by super high performance liquid chromatography tandem mass spectrometry (Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry, UPLC-MS/MS), and the substance base of small Bupleurum Decoction on delayed diarrhea. Foundation. Methods: 1. a model of delayed diarrhea in mice was established: 20 healthy male C57BL6 mice were randomly divided into model group (n=10) and normal group (n=10) after adaptive feeding of high fat diet for three weeks. A continuous intraperitoneal injection of CPT-11 for 7 days, once a day, was 50 mg. Kg-1, and the normal group was intraperitoneally injected with the equal dextran 40 glucoside. Glucose injection, daily observation of the mice's anus, photographing records, collecting feces, using urine fecal occult kit to determine the blood stool.2. small Bupleurum Decoction intervention study: 40 healthy male C57BL6 mice, adaptive feeding high fat feed for three weeks, randomly divided into 4 groups, each group of 10: blank control group (equal volume of saline), delayed diarrhea model group (equal volume saline), small Chaihu Decoction group (1500 mg. Kg-1. D-1), luperidine group (0.5333 mg. Kg-1. D-1), continuously gavage for 17 days, 1 times a day. On day 4~10 of the experiment, except for the blank control group, the rest of the mice were intraperitoneally injected with CPT-11 for 7 days to induce delayed diarrhea. In the micro centrifuge tube (Eppendorf tube, EP tube), the time of taking blood was 2 h. after injection to observe the body weight change of mice. The excrement was collected by urine fecal occult blood reagent box for blood stool. All mice were taken heart blood after eighteenth days of ether anaesthesia, and the EP tube precoated with heparin was used to find the metabolite of CPT-11 and the content of the effective component of Xiao Chai Hu soup was determined. Then the liver, kidney, large intestine and small intestine tissue were put to death in mice, and two parts were stored in two parts. Some were frozen in the refrigerator to measure the drug content in the tissue. The other was stored in 4% Formaldehyde Solution to make pathological sections. The tissue damage.3. was used to determine CPT-11 and its metabolism in tissue and blood samples. Results: the content of the effective components in the material and small Bupleurum soup. Results: 1. the model of delayed diarrhea was established: (1) compared with the normal group, the mice in the model group were filthy, wet, caking, trembling, holding and holding no struggles, and anus black and red swelling; (2) the weight of the normal group increased by 0.57 + 0.41g, and the model mice decreased by 5.98 + 2.08g; (3) normal group mice blood The rate of stool was 0. The rate of blood stool in the model group was 100% (P0.01).2. small Chaihu Decoction after seventh days of continuous intraperitoneal injection of CPT-11. (1) weight comparison: there was no significant difference in weight between the first days and fourth days (P0.05), and the weight of the blank control group was higher than that of the other group (P0.05) on the tenth day, indicating that the delayed diarrhea model could lead to the weight of the mice. The weight of the mice in the seventeenth day blank control group was still higher than that of the other groups (P0.05), but the weight of the mice in the small Chaihu soup group was higher than that of the model control group (P0.05), indicating that the small Bupleurum soup could reduce the symptoms of the weight loss of the mice to a certain extent; (2) the blood stool rate: the normal control group, the model control group, the small Bupleurum soup group and the luperidine group were 0%, 10 respectively. 0%, 50%, 60%; on day 8~17, the blood stool rate of mice in the small Chaihu soup group and the loperbuamine group were significantly lower than that in the model control group (P0.01), and the blood stool rate of the mice in the small Chaihu Decoction group was lower than that of the loperbuchine group (P0.05); (3) the histopathological observation: the large intestine and small intestine structure of the blank control group was complete, and the mice in the model control group of large intestine and small intestinal mucosa epithelium were in the control group. The intestinal mucosal tissue in the small Chaihu soup group and the roperamine group tended to be normal, and the concentration of.3. in the liver and kidney was not significantly damaged by the continuous injection of cpt-117 days. (1) CPT-11 and its metabolites: the mice of the small Chaihu soup group were injected with CPT-11 at sixth days after the injection of CPT-11. The highest concentration was no significant difference between the model control group and the control group at fourth days (P0.05); the highest concentration of SN-38 in the model control group was 7 times of the experimental group; the model control group had the highest concentration of sn-38g blood at fourth days of CPT-11 injection, and the small Bupleurum soup group was sixth days, significantly higher than the model control group (P0.05), except for the blood concentration ratio of the fourth day experimental group. The concentration of blood drug in the experimental group was higher than that of the control group. The concentration of CPT-11 in the model control group was arranged in the small intestine liver and kidney large intestine, and the concentration of SN-38 and sn-38g was arranged in the small intestine and kidney large intestine in the tissues, and the mice in the model control group, CPT-11, SN-38, and sn-38g were all distributed most in the small intestine (P0.05), and the small Bupleurum soup group was CPT-11 concentrated. The degree of SN-38 and sn-38g concentration in the tissues of the liver and kidney of the small intestine, the concentration of SN-38 and sn-38g in the tissues of the liver and kidney large intestine, CPT-11, SN-38 and sn-38g in the small Bupleurum soup group were most distributed in the liver (P0.05), and the content of each component in the small Chaihu soup group was significantly smaller than that in the model control group; (2) 17 species in the small Bupleurum soup were found in the small Bupleurum soup. Effective components: 14 components can be detected only in the blood 0,2,4,6 days after CPT-11, baicalin, quercetin and baicalin, and the three components are below the detection limit, and all of the 3 components can be detected in each tissue, suggesting that these 3 components have organ targeting, and the concentration of glycyrrhizic acid is far higher than that of other components, and only a small amount in each tissue is stored. In the liver, the concentration of Radix scutellarin is the highest (21700.7430ng/ml), and the concentration of Bupleurum saponin A is the lowest (12.4930 ng/mL); the concentration of ginsenoside Rd in the kidney is the highest (21563.0884ng/mL), and the content of Bupleurum saponins A is the lowest (20.7965 ng/mL), and the concentration of baicalein in the large intestine is the highest (26370.06 97 ng/mL), the content of saponins a was the lowest (3.7625 ng/mL); the concentration of ginsenoside Rd in the small intestine was the highest (22622.3167 ng/mL) and the content of ginsenoside Rg1 was the lowest (45.0834 ng/mL). Conclusion: 1., C57BL6 mice were first used, and three weeks after adaptive feeding of high fat feed, the peritoneal injection of CPT-11 50 mg/kg, for 7 days, once a day, could successfully establish CPT. The mice model of delayed diarrhea caused by -11, 2. small Chaihu Decoction can prevent the blood stool of mice caused by CPT-11, make the mice of delayed diarrhea normal weight increase, reduce the rate of blood stool and improve the injury of intestinal tissue. 3. small Bupleurum soup contains 17 kinds of active ingredients, which mainly interfere with the metabolism of SN-38, and can inhibit the bile excretion of CPT-11, SN-38, SN-38G and accelerate these three substances. CPT-11's intestinal hepatic circulation can cause delayed diarrhea. The prevention of delayed diarrhea caused by the small Bupleurum soup is the common effect of multiple target organs, which may be related to the inhibition of the intestinal liver circulation, and can provide reference for the follow-up mechanism.

【学位授予单位】：贵州医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R965

【参考文献】