【作者】 胡秀芳；

【导师】 杨贤强；

【作者基本信息】 浙江大学 ， 茶学， 2001， 博士

【摘要】 本论文用动物模型和细胞模型研究了茶儿茶素对肾病的药效与药理。首先，研究了茶儿茶素对家兔C-BSA和Masugi两种肾炎模型的药效，及对水负荷家兔肾功能的影响。继而，研究了茶儿茶素对小鼠免疫和炎症的作用，及对氧化应激(H₂O₂、As³⁺和Cr⁶⁺)细胞（正常肾细胞Vero和肿瘤肾细胞786-0）凋亡模型的影响。结果分述如下：1.茶几茶素对家兔水负荷和肾炎模型的作用 药效实验表明，茶儿茶素对水负荷家兔和肾炎家兔有明显的疗效。对于水负荷家兔，茶儿茶素在大（137mg/kg）、中（68mg/kg）、小（34mg/kg）剂量下均有显著的利尿作用，且大、小剂量间差异显著，有明显的量效关系（p＜0.01），利尿峰时为用药后2～2.5h。利尿剂——氢氯噻嗪（5.7mg/kg）的利尿作用弱于大剂量茶儿茶素（p＜0.05），峰时为0.5～1.0h。可见，大剂量茶儿茶素显著增强水负荷家兔肾脏的滤过功能，利尿效果强于氢氯噻嗪，且药效更持久。 茶儿茶素对家兔C-BSA和Masugi两种肾炎模型均有良好的疗效。大（306mg/kg）、中（102mg/kg）、小（34mg/kg）剂量茶儿茶素明显降低尿蛋白、血清肌酐及尿素氮含量（P＜0.05或P＜0.01）；抑制C-BSA肾炎的血清免疫复合物形成（CIC，P＜0.05）；减轻肾小球肿胀、细胞增生、血栓形成及白细胞浸润，修复肾小球病理改变；大、小剂量间差异显著，具有剂量-效应关系。大剂量茶儿茶素的效果强于地塞米松。可见，茶儿茶素明显减轻肾炎的症状。 上述结果表明茶儿茶素在肾病防治中有潜在价值。继而，根据目前关于肾炎的发病机理，研究茶儿茶素对肾病的药理。2.茶儿茶素对免疫和炎症的作用 免疫机理是肾炎的始发机理，控制免疫水平是预防肾炎的重要措施。本课题组以前的研究表明，粗儿茶素或茶提取物对免疫低下的动物（如荷瘤小鼠），表现增强免疫的作用；对于正常小鼠，则增强细胞免疫，而抑制体液免疫。本研究 中茶儿茶素对正常小鼠的兔疫均表现抑制作用。茶儿茶素剂量为 31 omghg、 155mg／kg、77．4mgdig时，明显抑制小鼠血清溶菌酶及腹腔巨噬细胞的活性D O＜00)；抑制小鼠红细胞＊3b受体花环和IC花环的形成O＜0刀5及P＜0刀1)；抑DD 制小鼠总T细胞花环和h花环的形成（P＜0刀1＼大、小剂量间差异显著，存在明DD 显的量效关系。大剂量茶儿茶素明显抑制小鼠脾细胞产生抗体的能力。地塞米松D ①．3＜ghg）对上述免疫指标的抑制效果不及茶儿茶素…功刀5)可见，茶儿茶D 素显著抑制小鼠的非特异性免疫和特异性免疫；且其抑制效果优于地塞米松。DD 用角叉菜胶致大鼠足路炎性肿胀法和纸片肉芽肿法研究大、中、小剂量D 份omghg、180mghg、90mg／kg)茶儿茶素的抗炎作用。结果，茶儿茶素剂量依D 赖性地抑制角叉菜胶诱发的大鼠急性炎症，大剂量茶儿茶素效果优于阿司匹林。DD 对于纸片所致的肉芽肿亚急性炎症，只有大剂量茶儿茶素才有抑制作用，且效果DD 不及地塞米松。可见，茶儿茶素显著抑制急性炎症模型，而对亚急性炎症模型的Dl 以比几、As‘“和 Cr’”应激建立细胞凋亡模型。MTT测定结果显示：H，0，、As‘“和 lICr”呈时间和剂量依赖性抑制细胞活力，三者对 Vero细胞的半抑制浓度（IC。。）l 分别为178．spM、155．6pM和9．SUM；对786－0细胞的IC。。分别为125．6pM、77．spM 和 8．6pM。H＊（50pM／24h）、As‘”（80pM／24h）、Cr’”（ZUM／24h或 400pM／Zh）分l 别应激处理 Ve。o和 786－0细胞，24h后，荧光显微镜、流式细胞仪和 DNA电泳 ll 检测，发现细胞 DNA和 RNA凝聚浓缩，进而产生凋亡小体，出现典型的 G－1亚峰 ll 及 DNA梯形条带。可见，以上氧化应激诱导细胞了凋亡，借此氧化应激细胞凋亡 lD 模型进行以下研究。Dl 茶儿茶素及其单体对VerO细胞和786－0细胞的毒性实验表明，低浓度促进细l胞生长，高浓度抑制细胞活力，茶儿茶素、EGCG、ECG、EGC对Vr细胞n。nD 分别为：233卜合1、17工lpg／nil、136．3pg／nil和112．spg／ffil：对旭6－0的1Q。分 别为：Zm．7卜咖二134．印咖L 11O．sll咖1和98．叫咖1。茶儿茶素及其单体对D 两种细胞的毒性大小为：茶儿茶素＜＊＊＊＊、＊＊G＜＊＊G，786－0细胞的毒性敏感性Dl 大十VeO细M。l121l 根据 IC。。，在非毒性浓度范围研究茶儿茶素及其单体对应激细胞凋亡模型的 l影响。结果：叨V钉ml茶儿茶素显著抑制几A和k’”应激诱导的W＊细胞凋亡，l 但对同样应激引起的 786—0细胞凋亡没有保护效果。另外，茶儿茶素对 AS‘”诱导更多还原

【Abstract】 The Phannacodyndrics and Phimacology of Tea catechins on Nwttis werestUdied in Anmal and ce1l systems. First, the theraPy of tea catechins on C-BSANePhritis, Masugi NePhritis and overwatered model rabbits were investigated. Next,the effects of tea catechins on immwhty and inf[ammation in animals, as well as onaPoptosis of cells stressed by H,O,, As3+ and Cr6+ were stUdied. The results aredescribed as follows:l. Effect of tea catechins on over--watered and Nephritis mode1 rabbitsTea catedris at differen concentrations (higher dose 137 mg/Kg, middle dose 68mg/Kg, 1ower dose 34mg/Kg) signficanly increased the ndne excretion of overwatered rabbits, which did in a dose-dePendent manner (P< 0.01). The urine reachedmaxbo between 2 h and 2.5 h. The cbloAnalidone (5.7 mg/Kg, a kind ofhydragogUe, had weaker effect than higher dose of tea catechins. It is thus eviden thathigher do se o f tea c atechins remarkably enhanced filtrating c aPability ofkidne y inoverwatered rabbits, with more effeCtive than chiorthalidone.Tea catechins (higher dose 306 mg/Kg, middle dose l02 mghg, lower dose34mg/Kg) aPparenly relieved the srwtom of nePhritis model rabbits. It reduced theurine protein, serum creatinine and serum nitrOgen (P<0.05 or P<0.01), and inhibitedthe formation of serum circling nie comPlex (CIC) in C-BSA nePdritis(P<0.05). Italso alleVated glomeru1us swelling and cell proliferation, decreased thIombosis andleukocyte infiltraion in rena1 tubule, and so rePaired the pathological change in thenePdritic tubule. All the effects were concforation-dePendent.From these resuts, we can see tea catechins he1P to the theraPy ofnchtis.2. Effect of tea catechins on irnunity and inf1armationNormally, immune system can eliminate some foreign substances such aspathogens and toxin, and protects the body against the risk of free radicals. Bot if theimmune system becomes abnormal, Whether StrOnger or weakeq the body is suscePtthleto disease. Our former results showed that tea catechins imProVed body immnity inlow-immedty Afals (mice with tUmor). hi normal mice, tea catechins increased cell-mediated imInwhty but decreased humoral immnityhi ths research, tea catechins showed idriitory effeCt on immune system innorma1 mice. Tea catechins (3l0 mg/Kg, 155 mg/Kg and 77.4 mg/Kg) distinctlyinhibited the activities of serum bacteriolysin and celiac macroPhago cyte(P<0. 0 l ), andreduced the formation of C3b and IC wreath in red blood cell, as well as total T celland Th w reath in m ice. Sighfican difference e xisted a mong ditheen do ses o ft eacatechins. The results also indicated that higher dose of tea catedris remarkablyichbited the formation of anibody in spleen cell. These results demonstrated that teacatechins could remarkably inhibit specific immunity and nonspecific immboty, andthe effects were bcher than that of Dexamethasone.FurthCr We stUdied the effect of tea catechins on acute and drinic inflaxnmationin fats. Results indcated that 90--360 mg/Kg tea catechns suPpressed the swelling ofrat’s hind paws induced by caxrageenan. The effects lasted for more than 4 hours. After360mgiKg tea catechins were administered intraperitonealy once a day for 7consecutive days, the proliferation of granuloma induced by a piece of paPer wasevidently inhibited only by higher dose of tea catechins, and the effeCt was weaker thanthat of Dexamethasone.3. Effect of tea catechins on the oxidant--stressed ce11sThe oxidam-stfessed cell model was founded with H,O,, As3" and Cr6+. Accordingto the toxicity checked by MTT thIee oxidans inhiliited cell viabilities in time- anddose-dePendent manners. The IC,, of H,O,, As3+ and Cr6+ were l78.5 UM, l55.6UMand 9.8llM in Ver cel1s, and 125.6pM, 77.8pM and 8.6UM in 786-0 cells reSPectivelyJudged by fluoromicrograPhs, flow cytOmeter and DNA electrophoresis, H,O,(50llM/24h), As’+(80llM/24h) and Cr6+(2llM/24h or 400llM/2h) induced aPoptosis ofVer cells and 786-0 cells, and so wer更多还原