商陸皂苷甲急性毒性與通利二便藥效學(xué)及其作用機(jī)制研究
發(fā)布時(shí)間：2018-07-21 15:02
【摘要】：2010版《中國藥典》一部記載商陸有毒，具有逐水消腫、通利二便等功能。商陸皂苷甲（Esculentoside A，以下簡稱EsA）是從商陸中提取的一種含量最高的三萜類皂苷。本研究是國家自然基金面上項(xiàng)目——《基于峻瀉逐水及毒性與商陸皂苷甲變化相關(guān)的商陸炮制科學(xué)內(nèi)涵的構(gòu)建》（編號：81173555）任務(wù)的一部分，主要圍繞EsA的急性毒性和通利二便作用以及從水通道蛋白角度揭示EsA利尿作用機(jī)制展開一系列研究。 目的：初步了解EsA對小鼠急性毒性的強(qiáng)弱及特點(diǎn)，并測定相關(guān)參數(shù)；觀察EsA對水負(fù)荷大鼠的利尿作用，研究其對腎臟水通道蛋白2（AQP2）和水通道蛋白4（AQP4）的蛋白及mRNA表達(dá)調(diào)節(jié)作用，探討EsA利尿作用機(jī)制；通過復(fù)方地芬諾酯致便秘小鼠小腸推進(jìn)試驗(yàn)及排便試驗(yàn)，研究EsA的瀉下作用。 方法：向小鼠腹腔注射不同濃度的EsA，觀察給藥后小鼠毒性反應(yīng)并記錄各組小鼠死亡情況；向水負(fù)荷狀態(tài)大鼠腹腔注射不同濃度EsA，每2h收集一次尿液，連續(xù)收集6h，分別測定各時(shí)段尿量并計(jì)算6h總尿量。之后將大鼠麻醉，解剖取出腎臟，運(yùn)用免疫組化及實(shí)時(shí)熒光定量PCR方法檢測腎臟AQP2、AQP4蛋白及mRNA表達(dá)的變化；向復(fù)方地芬諾酯致便秘小鼠腹腔注射不同濃度EsA并灌服墨汁，每2h收集一次糞便，，連續(xù)收集10h，記錄每只小鼠首粒黑便出現(xiàn)時(shí)間，糞便陰干后稱量每一時(shí)段內(nèi)的排便量并計(jì)算總排便量；向復(fù)方地芬諾酯致便秘小鼠腹腔注射不同濃度EsA并灌服墨汁，給藥25min后脫椎處死，測量并計(jì)算小腸推進(jìn)率； 結(jié)果： ①Bliss法測得EsA的LD50為26.19mg kg-1，95%的可信區(qū)間為23.11mg kg-1~29.85mg kg-1；隨著EsA濃度的增加，小鼠的毒性反應(yīng)表現(xiàn)越明顯，且小鼠的死亡率隨之增加，空白對照組小鼠無異常反應(yīng)；由病理切片的檢查可知，EsA對小鼠肝臟、腎臟造成了不同程度的損傷。②利尿?qū)嶒?yàn)中，高劑量組大鼠尿量與陰性對照組比較有顯著性差異（P0.05），中、低劑量與陰性對照組比較無顯著性差異。高劑量組EsA利尿作用主要發(fā)揮在給藥后4h內(nèi)，而陽性對照藥氫氯噻嗪主要在給藥后2h內(nèi)發(fā)揮藥效，說明高劑量EsA有一定的利尿作用且作用時(shí)間較氫氯噻嗪長。 ③運(yùn)用免疫組化及實(shí)時(shí)熒光定量PCR技術(shù)檢測發(fā)現(xiàn)，高劑量組大鼠腎臟AQP2、AQP4的蛋白及mRNA表達(dá)均較陰性對照組下調(diào)（P0.05）,差異具有統(tǒng)計(jì)學(xué)意義；中、低劑量組大鼠腎臟AQP2、AQP4的蛋白及mRNA表達(dá)與陰性對照組相比無顯著性差異。 ④在排便試驗(yàn)中，與模型組比較，EsA高、中劑量組小鼠首粒黑便出現(xiàn)時(shí)間縮短且總排便量增加，差異具有統(tǒng)計(jì)學(xué)意義（P0.05）。小腸推進(jìn)試驗(yàn)中EsA高、中、低劑量組小鼠小腸推進(jìn)率與模型組比較均無顯著性差異。 結(jié)論：EsA有一定的毒性，但對于該成分是否是商陸中的唯一或主要毒性成分，尚需深入研究；EsA在給藥量為5.2mg kg-1時(shí)對大鼠有明顯的利尿作用，按體表面積折算大鼠與人的等效劑量，這一劑量在臨床有效劑量范圍之內(nèi)。這表明，EsA可能是商陸中起利尿作用的主要成分之一；高劑量EsA能降低腎臟AQP2、AQP4的蛋白及mRNA的表達(dá)，增加尿量，提示通過降低腎臟AQP2、AQP4的蛋白及mRNA的表達(dá)量可能是其產(chǎn)生利尿作用的機(jī)制之一。排便試驗(yàn)結(jié)果表明高、中劑量的EsA對復(fù)方地芬諾酯致便秘小鼠有一定的瀉下作用。小腸推進(jìn)試驗(yàn)結(jié)果表明EsA對復(fù)方地芬諾酯致便秘小鼠沒有促進(jìn)小腸推進(jìn)的作用。結(jié)合小腸推進(jìn)試驗(yàn)及排便試驗(yàn)可知，EsA對復(fù)方地芬諾酯致便秘小鼠有一定的瀉下作用，但其作用機(jī)制可能不是促進(jìn)小腸蠕動(dòng)，其具體的瀉下機(jī)制還需要進(jìn)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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R285
本文編號：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.
【學(xué)位授予單位】：湖北中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R285
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