【作者】 代春美；

【导师】 彭成；

【作者基本信息】 成都中医药大学 ， 中药学， 2011， 博士

【摘要】 目的：本课题拟以临床广泛应用,同时毒效双重作用客观存在的大黄为示范药物,主要以中药毒效相关性影响因素中量的因素及不同状态因素为研究内容,考察大黄蒽醌和鞣质类物质对HK-2细胞的毒效作用,毒效物质基础,影响因素及作用机制,同时为其它有毒中药的毒效相关性研究提供借鉴。方法：首先采用正常HK-2细胞,对大黄主要有效部位蒽醌及鞣质,蒽醌部位代表性单体大黄素,鞣质部位代表性类单体的聚合物EGCG的毒性进行研究,通过细胞形态学观察,细胞增殖活性测定,细胞LDH漏出量测定,细胞周期分析及凋亡率测定考察其毒性及量-毒关系。其次,建立细胞损伤模型即以过氧化氢损伤HK-2细胞,在此基础上探讨大黄蒽醌及鞣质、大黄素、EGCG对于细胞氧化应激损伤的保护作用,通过细胞形态学观察,细胞增殖活性测定,细胞LDH测定,细胞周期分析及凋亡率测定,考察其量-(毒/效)关系。最后综合分析大黄蒽醌、鞣质类物质对HK-2细胞的毒效物质基础,影响因素及作用机制。结果：(1)大黄蒽醌及大黄素总体上可使细胞形态发生皱缩,融合等形态学的变化；大黄蒽醌和大黄素使细胞生长受到抑制,阻滞了细胞周期的正常转化,并不同程度的引起了细胞的凋亡和坏死。表明大黄蒽醌和大黄素对HK-2细胞有潜在的肾毒性,能够导致细胞的损伤。大黄蒽醌对HK-2细胞增殖有直接的抑制作用,呈一定的量效关系,半抑制浓度约为20.62mg·L-1；大黄素显现的毒性作用较轻,半抑制浓度约为120μmol·L-1。大黄蒽醌和大黄素的毒性作用机制主要是阻滞了细胞周期从S期向G2/M期的转化,并引起细胞的凋亡和坏死。(2)大黄鞣质部位在考察剂量中对HK-2细胞的毒性作用轻微,对于细胞形态的影响只是轻微的使细胞形状变得狭长,抑制率在较高浓度200mg·L-1时仅达到20%,而大黄蒽醌的半抑制浓度20.62 mg·L-1,蒽醌类物质和鞣质类物质在大黄生药中所占的质量百分比约为1：2左右,因此,在应用大黄时鞣质的肾细胞毒性相对于蒽醌类物质有限。鞣质的单体的聚合物EGCG对HK-2细胞的毒性作用较轻,在80μmol·L-1以上的浓度可引起HK-2细胞早期凋亡。(3)HK-2细胞在过氧化氢损伤的状态下,一方面,大黄蒽醌主要表现出了对受损细胞的保护作用,如细胞形态上相对于损伤组细胞的形态有所恢复,细胞活性有所增强。尤其是在LDH的检测结果中,蒽醌组的光密度值明显减少。比较有意义的是,在凋亡率的检测中,蒽醌组的细胞凋亡率相对于损伤组有明显改善,近一步证实蒽醌类物质对受损细胞有保护作用。而另一方面,从剂量关系上看,15mg·L-1以下剂量范围内的大黄蒽醌对于受损细胞有一定的保护作用；当剂量达到30mg-L-1时,大黄蒽醌一方面发挥了细胞保护作用,同时也显示了一定的潜在毒性作用,但损伤细胞对大黄蒽醌的毒性耐受性比正常状态下增强。大黄素对过氧化氢损伤HK-2细胞的作用中,并未发现有明显的保护作用,尤其在对凋亡率的检测中,发现大黄素引起细胞晚期凋亡/坏死的作用显著,比大黄素对正常细胞的晚期凋亡/坏死的影响更为明显,提示在HK-2细胞损伤状态下,大黄素更多的体现了其毒性作用。(4)HK-2细胞在过氧化氢损伤的状态下,相同剂量大黄鞣质相对于对正常状态下细胞的毒性,其毒性作用变化不大,细胞形态改变不明显,细胞增殖活性没有显著下降；在细胞损伤状态下,大黄鞣质在100mg.L-1时对细胞的抑制率为27%,与该剂量对正常状态细胞的抑制率22%相差不大。尤其是LDH的检测中,相对于损伤组,LDH值下降明显,表明大黄鞣质对受损细胞具有保护作用,受损细胞对鞣质毒性的耐受性增加。在本实验中,EGCG对损伤的HK-2细胞表现了一定的保护作用,尤其对于凋亡率的影响上,早期凋亡和晚期凋亡/死亡的改善作用明显。结论：基于上述对大黄毒效辩证关系的认识,结合中药毒效相关性观点的科学内涵,提出对于有毒中药的毒效相关性研究应考察以下两个方面：剂量是有毒中药毒效相关性的影响因素；应在机体生理、病理不同状态下进行有毒中药毒效相关性研究。以大黄为例,蒽醌和鞣质类物质对HK-2细胞的毒效相关性主要体现在以下几个方面：(1)成分不同,毒性相异：蒽醌类物质(蒽醌有效部位和大黄素)的毒性作用强于鞣质类物质(鞣质有效部位和EGCG),主要影响了HK-2细胞形态、降低细胞增殖活性、增加细胞LDH的漏出、阻滞细胞周期、引起细胞的凋亡/坏死等。(2)成分不同,药效相似：HK-2细胞在过氧化氢损伤状态下,蒽醌类物质(主要是蒽醌有效部位)和鞣质类物质(主要是鞣质有效部位和EGCG)在一定剂量范围内都发挥了药效作用,如保护细胞形态,提高细胞增殖活性,降低细胞凋亡率等。(3)成分相同,机体状态有别,量-毒/效关系不同：相对于正常状态下,大黄蒽醌和鞣质对过氧化氢损伤的HK-2细胞的毒性作用降低,损伤细胞对药物毒性的耐受性增强,在相同剂量下主要体现了药效作用；相对于正常状态,大黄素对损伤的HK-2细胞毒性作用明显,EGCG则主要体现对损伤HK-2细胞的药效作用。(4)成分相同,用量有别,量-毒关系不同：如：对于正常细胞大黄蒽醌在30mg·L-1以下剂量时,其抑制细胞增殖作用和对细胞周期的阻滞作用相对较弱,在30mg·L-1增加到60mg·L-1时的过程中,抑制细胞增殖和细胞周期的阻滞作用增加明显,而且出现了明显的晚期凋亡/坏死,晚期凋亡/坏死程度在60mg·L-1时甚至大于了50%,毒性作用明显。(5)通过考察大黄蒽醌、鞣质类物质对HK-2细胞的毒效关系,为其它中药的毒效相关性研究可提供思路和方法借鉴。更多还原

【Abstract】 Objective The goal of this thesis is to explore correlation between toxicity and effect of anthraquinone and tannin extracted from Rhubarb, and the influencing factors of dose and different conditions of HK-2 cells are to be concerned about, what is more significant, this study may provide demonstration for researches on correlation between toxicity and effect of other toxic TCM.Methods The study on toxicity of anthraquinone and tannin extracted from Rhubarb, emodin and EGCG on HK-2 cells was carried out in different dose, and the change of cell morphology, OD of LDH, cell cycle and apoptosis were detected. Furthermore, HK-2 cells that were damaged at some level by exposing to H2O2 were pretreated with anthraquinone and tannin extracted from Rhubarb, emodin and EGCG,the effect of those drugs on impaired HK-2 cells were detected. By combinding with experimental data and traditional theory of TCM, the material basic, influencing factors and mechanism of correlation between toxicity and effct of Rhubarb were analyzed.Results (1) Anthraquinone extracted from Rhubarb and emodin possed some toxic on HK-2 cells, the toxic manifestation included:inhibition of cells proliferation, cell shrinkage and vacuolation, LDH leakage were increased, the cell cycle was blocked and induced apoptosis at different level. This result illustrated anthraquinone and emodin may be the materials that damage the kidney. The IC50 of anthraquinone extracted from Rhubarb is about 20.62mg·L-1 and the IC50 of emodin is about 120μmol·L-1. The main toxic mechanism of anthraquinone extracted from Rhubarb and emodin is to induce apoptosis and block the transformation of cell cycle from S period to G2/M period.(2) The toxicity of tannin extracted from Rhubarb on HK-2 cell was little, only leaded to lower grade change of cell shape, the inhibition ratio was 20% when the dose was 200mg·L-1.The percentage of anthraquinone and tannin extracted from Rhubarb is 1:2 approximately, contrast to the toxicity of anthraquinone, tannin is safe when the Rhubarb is administrated. The early apoptosis was induced by using EGCG when the dose exceed 80μmol·L-1.(3) In the condition of HK-2 cells were damaged by H2O2, anthraquinone extracted from Rhubarb protected the cells by improving the cell shape and activity, the OD was descended, especially, the apoptosis of cell was improved significantly. The effect of anthraquinone was concerned with the dose,15mg·L-1 anthraquinone mainly protected the cell, toxicity was detected when the dose was 30 mg·L-1, but the tolerance to drug toxicity of damaged cells was enhanced. Emodin represented the toxicity mainly by inducing advanced stage apoptosis and necrosis.(4) In the condition of HK-2 cell were damaged by H2O2, tannin extracted from Rhubarb protected the cells and the tolerance of damaged cells to drug toxicity was enhanced. The inhibitor ratio changed lighty and the OD of LDH was descended. EGCG protected the damaged cell by improving the early stage apoptosis and necrosis.Conclusions Base on the experiment on the toxicity and effct of Rhubarb, combind with the idea of correlation between toxicity and effect of toxic TCM, the thesis provide the strategy to the research on correlation between toxicity and effect of toxic TCM:The influencing factors of correlation between toxicity and effect are varied, but dose and different conditions play important roles. The correlation between toxicity and effect of anthraquinone and tannin extracted from Rhubarb include some aspects as follows:(1) Different materials may have different toxicity. Anthraquinone extracted from Rhubarb and emodin are more toxical than tannin extracted from Rhubarb and EGCG.. Anthraquinone extracted from Rhubarb and emodin change the cell shape, enhance the inhibition ratio and OD of LDH, block the cell cycle and induce the apoptosis and necrosis.(2) Different materials may have similar effect. Anthraquinone extracted from Rhubarb and emodin all protect the HK-2 cells damaged by H2O2. They all can maintain the normal cell shape, descend the inhibition ratio and cell apoptosis et al.(3) The same materials may have different toxicity and effect in different conditions. The tolerance of HK-2 cells to toxicity of anthraquinone and tannin extracted from Rhubarb are enhanced, anthraquinone and tannin extracted from Rhubarb mainly protect the damaged cells contrast to the normal condition at the same dose. The toxicity of emodin on damaged cells is more significant than the normal condition but EGCG protect the damaged cells.(4) The same materials may have different toxicity and effect in different dose. The toxicity of anthraquinone extracted from Rhubarb is little when the dose is lower than 30mg·L-1 and enhanced significantly when the dose is 60 mg·L-1, the inhibition ratio, the apoptosis and necrosis of HK-2 cells alter notablely.(5) The study may provide reference to explore correlation between toxicity and effect of other toxic TCM.更多还原