商陆皂苷甲急性毒性与通利二便药效学及其作用机制研究
发布时间：2018-07-21 15:02
【摘要】：2010版《中国药典》一部记载商陆有毒，具有逐水消肿、通利二便等功能。商陆皂苷甲（Esculentoside A，以下简称EsA）是从商陆中提取的一种含量最高的三萜类皂苷。本研究是国家自然基金面上项目——《基于峻泻逐水及毒性与商陆皂苷甲变化相关的商陆炮制科学内涵的构建》（编号：81173555）任务的一部分，主要围绕EsA的急性毒性和通利二便作用以及从水通道蛋白角度揭示EsA利尿作用机制展开一系列研究。 目的：初步了解EsA对小鼠急性毒性的强弱及特点，并测定相关参数；观察EsA对水负荷大鼠的利尿作用，研究其对肾脏水通道蛋白2（AQP2）和水通道蛋白4（AQP4）的蛋白及mRNA表达调节作用，探讨EsA利尿作用机制；通过复方地芬诺酯致便秘小鼠小肠推进试验及排便试验，研究EsA的泻下作用。 方法：向小鼠腹腔注射不同浓度的EsA，观察给药后小鼠毒性反应并记录各组小鼠死亡情况；向水负荷状态大鼠腹腔注射不同浓度EsA，每2h收集一次尿液，连续收集6h，分别测定各时段尿量并计算6h总尿量。之后将大鼠麻醉，解剖取出肾脏，运用免疫组化及实时荧光定量PCR方法检测肾脏AQP2、AQP4蛋白及mRNA表达的变化；向复方地芬诺酯致便秘小鼠腹腔注射不同浓度EsA并灌服墨汁，每2h收集一次粪便，，连续收集10h，记录每只小鼠首粒黑便出现时间，粪便阴干后称量每一时段内的排便量并计算总排便量；向复方地芬诺酯致便秘小鼠腹腔注射不同浓度EsA并灌服墨汁，给药25min后脱椎处死，测量并计算小肠推进率； 结果： ①Bliss法测得EsA的LD50为26.19mg kg-1，95%的可信区间为23.11mg kg-1~29.85mg kg-1；随着EsA浓度的增加，小鼠的毒性反应表现越明显，且小鼠的死亡率随之增加，空白对照组小鼠无异常反应；由病理切片的检查可知，EsA对小鼠肝脏、肾脏造成了不同程度的损伤。②利尿实验中，高剂量组大鼠尿量与阴性对照组比较有显著性差异（P0.05），中、低剂量与阴性对照组比较无显著性差异。高剂量组EsA利尿作用主要发挥在给药后4h内，而阳性对照药氢氯噻嗪主要在给药后2h内发挥药效，说明高剂量EsA有一定的利尿作用且作用时间较氢氯噻嗪长。 ③运用免疫组化及实时荧光定量PCR技术检测发现，高剂量组大鼠肾脏AQP2、AQP4的蛋白及mRNA表达均较阴性对照组下调（P0.05）,差异具有统计学意义；中、低剂量组大鼠肾脏AQP2、AQP4的蛋白及mRNA表达与阴性对照组相比无显著性差异。 ④在排便试验中，与模型组比较，EsA高、中剂量组小鼠首粒黑便出现时间缩短且总排便量增加，差异具有统计学意义（P0.05）。小肠推进试验中EsA高、中、低剂量组小鼠小肠推进率与模型组比较均无显著性差异。 结论：EsA有一定的毒性，但对于该成分是否是商陆中的唯一或主要毒性成分，尚需深入研究；EsA在给药量为5.2mg kg-1时对大鼠有明显的利尿作用，按体表面积折算大鼠与人的等效剂量，这一剂量在临床有效剂量范围之内。这表明，EsA可能是商陆中起利尿作用的主要成分之一；高剂量EsA能降低肾脏AQP2、AQP4的蛋白及mRNA的表达，增加尿量，提示通过降低肾脏AQP2、AQP4的蛋白及mRNA的表达量可能是其产生利尿作用的机制之一。排便试验结果表明高、中剂量的EsA对复方地芬诺酯致便秘小鼠有一定的泻下作用。小肠推进试验结果表明EsA对复方地芬诺酯致便秘小鼠没有促进小肠推进的作用。结合小肠推进试验及排便试验可知，EsA对复方地芬诺酯致便秘小鼠有一定的泻下作用，但其作用机制可能不是促进小肠蠕动，其具体的泻下机制还需要进一步研究。
[Abstract]:The 2010 edition of the Chinese Pharmacopoeia (China Pharmacopoeia), one of the records of the Chinese Pharmacopoeia (Esculentoside A) is the highest content three terpenoid Saponins Extracted from the Phytolacca Phytolacca (EsA). This study is a national natural fund project - based on the change of water and toxicity and phytolaccin a. The construction of scientific connotation of Phytolacca arecurus (No. 81173555) is part of the task, which mainly focuses on the acute toxicity of EsA and the two defecation and the mechanism of EsA diuretic action from the angle of aquaporin.
Objective: To investigate the acute toxicity and characteristics of EsA in mice and determine the related parameters, observe the diuretic effect of EsA on water load rats, study the regulation of the protein and mRNA expression of renal aquaporin 2 (AQP2) and aquaporin 4 (AQP4), explore the mechanism of EsA diuretic action, and reduce the constipation by compound diphenol. Rat intestinal propulsion test and defecation test were performed to study the purging effect of EsA.
Methods: the mice were intraperitoneally injected with different concentrations of EsA, observed the toxicity of the mice after the administration and recorded the mortality of mice in each group. The rats were injected with different concentrations of EsA in the abdominal cavity and collected a urine per 2H per 2H. The urine volume was collected and the total urine volume was measured at each time period and the total amount of urine was calculated respectively. Then the rats were anaesthetized and dissected to remove the kidney. Immunohistochemistry and real time fluorescence quantitative PCR were used to detect the changes in the expression of AQP2, AQP4 protein and mRNA in the kidney. Intraperitoneal injection of EsA in the constipation mice with compound dip was injected into the abdominal cavity, and the excrement was collected every 2h, 10h was collected continuously, and the appearance time of the first black stool in each mouse was recorded. The amount of defecation and the amount of total defecation were calculated; the constipated mice were injected with different concentrations of EsA in the constipation compound and filled with ink. After the drug was given 25min, the devertebrate was executed, and the propulsive rate of the small intestine was measured and calculated.
Result:
(1) the Bliss method showed that the LD50 of LD50 of EsA was 23.11mg kg-1~29.85mg kg-1. With the increase of EsA concentration, the toxicity of mice was more obvious, and the mortality of mice increased, and there was no abnormal reaction in the blank control group. It was found that EsA was different in the liver and kidney of mice by pathological section examination. In the diuresis test, there was a significant difference in the urine volume between the high dose group and the negative control group (P0.05). In the high dose group, there was no significant difference between the low dose and the negative control group. The EsA diuretic effect of the high dose group was mainly in the 4h after the administration, while the positive control hydrochlorothiazide was mainly used in the 2h after the administration, indicating that the high dose of hydrochlorothiazide was high. The dose of EsA has a certain diuretic effect and the time of action is longer than that of hydrochlorothiazide.
(3) using immunohistochemistry and real-time fluorescence quantitative PCR technique, it was found that the expression of AQP2, AQP4 and mRNA in the kidney of the high dose group were lower than those of the negative control group (P0.05), and the difference was statistically significant. In the low dose group, there was no significant difference between the AQP2, the AQP4 protein and the mRNA table in the kidney of the low dose group.
(4) in the test of defecation, compared with the model group, EsA was high, the time of the first black stool in the middle dose group shortened and the total defecation increased, the difference was statistically significant (P0.05). The small intestinal propulsion test was high in EsA, and there was no significant difference in the small intestinal propulsion rate in the low dose group and the model group.
Conclusion: EsA has certain toxicity, but it is still necessary to study whether this component is the only or major toxic ingredient in Phytolacca; EsA has an obvious diuretic effect on rats when the dose is 5.2mg kg-1, and the equivalent dose of the rat is converted to the human body according to the body surface area. This dose is within the range of clinical effective dose. This indicates that EsA may be in the range of effective dose. It is one of the main components of diuretic effect in Phytolacca; high dose EsA can reduce the expression of AQP2, AQP4 protein and mRNA, and increase urine volume. It suggests that the expression of protein and mRNA in AQP4 may be one of the mechanisms of diuresis by lowering the AQP2 of the kidney. The results of defecation test show that high, medium dose EsA to compound diuronl The results of small intestine propulsion test showed that EsA had no effect on promoting small intestinal propulsion in constipation mice induced by compound ground fluid. The combination of small intestine propulsion test and defecation test showed that EsA had a certain diarrhea effect on constipation induced constipation mice, but the mechanism may not promote small intestinal creep. Its specific mechanism should be further studied.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285
本文编号：2135905
[Abstract]:The 2010 edition of the Chinese Pharmacopoeia (China Pharmacopoeia), one of the records of the Chinese Pharmacopoeia (Esculentoside A) is the highest content three terpenoid Saponins Extracted from the Phytolacca Phytolacca (EsA). This study is a national natural fund project - based on the change of water and toxicity and phytolaccin a. The construction of scientific connotation of Phytolacca arecurus (No. 81173555) is part of the task, which mainly focuses on the acute toxicity of EsA and the two defecation and the mechanism of EsA diuretic action from the angle of aquaporin.
Objective: To investigate the acute toxicity and characteristics of EsA in mice and determine the related parameters, observe the diuretic effect of EsA on water load rats, study the regulation of the protein and mRNA expression of renal aquaporin 2 (AQP2) and aquaporin 4 (AQP4), explore the mechanism of EsA diuretic action, and reduce the constipation by compound diphenol. Rat intestinal propulsion test and defecation test were performed to study the purging effect of EsA.
Methods: the mice were intraperitoneally injected with different concentrations of EsA, observed the toxicity of the mice after the administration and recorded the mortality of mice in each group. The rats were injected with different concentrations of EsA in the abdominal cavity and collected a urine per 2H per 2H. The urine volume was collected and the total urine volume was measured at each time period and the total amount of urine was calculated respectively. Then the rats were anaesthetized and dissected to remove the kidney. Immunohistochemistry and real time fluorescence quantitative PCR were used to detect the changes in the expression of AQP2, AQP4 protein and mRNA in the kidney. Intraperitoneal injection of EsA in the constipation mice with compound dip was injected into the abdominal cavity, and the excrement was collected every 2h, 10h was collected continuously, and the appearance time of the first black stool in each mouse was recorded. The amount of defecation and the amount of total defecation were calculated; the constipated mice were injected with different concentrations of EsA in the constipation compound and filled with ink. After the drug was given 25min, the devertebrate was executed, and the propulsive rate of the small intestine was measured and calculated.
Result:
(1) the Bliss method showed that the LD50 of LD50 of EsA was 23.11mg kg-1~29.85mg kg-1. With the increase of EsA concentration, the toxicity of mice was more obvious, and the mortality of mice increased, and there was no abnormal reaction in the blank control group. It was found that EsA was different in the liver and kidney of mice by pathological section examination. In the diuresis test, there was a significant difference in the urine volume between the high dose group and the negative control group (P0.05). In the high dose group, there was no significant difference between the low dose and the negative control group. The EsA diuretic effect of the high dose group was mainly in the 4h after the administration, while the positive control hydrochlorothiazide was mainly used in the 2h after the administration, indicating that the high dose of hydrochlorothiazide was high. The dose of EsA has a certain diuretic effect and the time of action is longer than that of hydrochlorothiazide.
(3) using immunohistochemistry and real-time fluorescence quantitative PCR technique, it was found that the expression of AQP2, AQP4 and mRNA in the kidney of the high dose group were lower than those of the negative control group (P0.05), and the difference was statistically significant. In the low dose group, there was no significant difference between the AQP2, the AQP4 protein and the mRNA table in the kidney of the low dose group.
(4) in the test of defecation, compared with the model group, EsA was high, the time of the first black stool in the middle dose group shortened and the total defecation increased, the difference was statistically significant (P0.05). The small intestinal propulsion test was high in EsA, and there was no significant difference in the small intestinal propulsion rate in the low dose group and the model group.
Conclusion: EsA has certain toxicity, but it is still necessary to study whether this component is the only or major toxic ingredient in Phytolacca; EsA has an obvious diuretic effect on rats when the dose is 5.2mg kg-1, and the equivalent dose of the rat is converted to the human body according to the body surface area. This dose is within the range of clinical effective dose. This indicates that EsA may be in the range of effective dose. It is one of the main components of diuretic effect in Phytolacca; high dose EsA can reduce the expression of AQP2, AQP4 protein and mRNA, and increase urine volume. It suggests that the expression of protein and mRNA in AQP4 may be one of the mechanisms of diuresis by lowering the AQP2 of the kidney. The results of defecation test show that high, medium dose EsA to compound diuronl The results of small intestine propulsion test showed that EsA had no effect on promoting small intestinal propulsion in constipation mice induced by compound ground fluid. The combination of small intestine propulsion test and defecation test showed that EsA had a certain diarrhea effect on constipation induced constipation mice, but the mechanism may not promote small intestinal creep. Its specific mechanism should be further studied.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R285
【参考文献】
相关期刊论文 前10条
1 郑钦岳,王慧峰,郑向民,肖振宇,易杨华;商陆皂苷甲对兔滑膜细胞产生IL-1和TNF的影响[J];第二军医大学学报;2001年05期
2 肖振宇,郑钦岳,郑向民,程明和,易杨华;陆皂苷甲对小鼠胸腺细胞凋亡的影响[J];第二军医大学学报;2002年06期
3 肖振宇,郑钦岳,张俊平,陆峰,张大志;商陆皂苷甲对自身免疫综合征模型小鼠的疗效[J];第二军医大学学报;2003年10期
4 郑钦岳,方军,王洪斌;商陆皂甙甲对人外周血单核细胞产生肿瘤坏死因子的抑制作用[J];第二军医大学学报;1997年05期
5 陈春玲;李涛平;朱丽华;;MAPKs信号阻断剂U0126对油酸致急性肺损伤大鼠肺泡Ⅱ型上皮细胞中AQP4表达的影响[J];南方医科大学学报;2009年08期
6 张克非,张亮,吴雄飞,张祥贵,于泓,易杨华,赵士启;商陆皂甙甲治疗大鼠抗-Thy1.1系膜增生性肾炎的实验研究[J];四川大学学报(医学版);2004年05期
7 邓俐,张堂德,杜江;商陆皂苷甲对银屑病患者外周血单个核细胞产生α肿瘤坏死因子和可溶性白介素2受体的影响[J];临床皮肤科杂志;2004年07期
8 师忠芳;赵焕英;袁芳;韩明;路杨;;MAPKs信号通路干预对体外培养大鼠星形胶质细胞划痕损伤后水通道蛋白4表达的影响[J];首都医科大学学报;2010年02期
9 太史春;邰东梅;邹晓明;王德山;;肾主水液与水通道蛋白内在关系探讨[J];实用中医内科杂志;2007年08期
10 张祥贵;汤杰印;;商陆皂苷甲对肾小球系膜细胞增殖的影响[J];陕西中医;2013年08期
相关博士学位论文 前1条
1 黄飞;人参皂甙Rb1调节AQP4表达在大鼠脊髓缺血再灌注损伤中作用的实验研究[D];吉林大学;2012年
本文编号：2135905
本文链接：https://www.wllwen.com/yixuelunwen/mazuiyixuelunwen/2135905.html
最近更新
教材专著
