摘要
前言
中药关木通可以引起人类严重肾小管损伤、间质纤维化和肾功能衰竭。有实验证实，该药对大鼠肾脏的损伤以小管间质为主。目前其发病机制有缺血学说、转分化学说等，缺血学说认为马兜铃酸能导致肾小动脉壁增厚，管腔狭窄，引起缺血肾损害。近年来的研究表明，缺血缺氧可促进肾小管上皮系胞、间质细胞活化，分泌致纤维化因子，而且可直接调控纤维化相关基因，促进细胞外基质(extra cellularmatrix，ECM)的积聚，而且越来越多的证据表明，肾小管周围毛细血管丢失与肾小管间质的缺氧性损伤及间质纤维化有关，且肾脏缺血，引起肾脏局部氧自由基生成增加，清除减少。因其机制尚有许多未明之处，故其治疗仍处于探讨阶段。本实验拟制定马兜铃酸肾病模型，试用银杏叶、凯时，探讨应用改变微循环药物在马兜铃酸肾病纤维化中治疗的价值。银杏叶主要含有银杏苦内脂、银杏叶总黄酮等成分，银杏苦内脂是一种特异性的血小板激活因子(Plateletactivating factor PAF)受体拮抗剂，多种动物实验证实，PAF受体拮抗剂能使肾血流动力学改变，银杏叶总黄酮具有抗自由基和扩血管作用，且银杏叶制剂可使肾小管-肾间质中增殖细胞核抗原(proliferating cell nuclear antigen，PCNA~+)细胞数明显减少，降低肾间质纤维化程度。凯时为前列腺素E_1(prostaglandin E_1，PGE_1)脂微球载体制剂，具有改善肾血流量、抑制肾小球固有细胞增生ECM积聚作用。
材料与方法
一、实验材料
1．实验动物
雌性Wistar大鼠35只，随机分为四组，其中对照组5只，未治疗组、治疗组(1)(2)均为10只。
2．主要试剂及药品
关木通、银杏叶、凯时、转化生长因子(TGF一p，)、结缔组织生长因子
(CTGF)、纤连蛋白(FN)免疫组化试剂盒。
3.主要仪器
光学显微镜、显微照相仪、显微图象分析仪、分光光度计、全自动生化分
析仪、离心机
二、实验方法
1.关木通水煎剂取关木通生药材2000克，加6倍饮用水，浸泡30分
钟，加热至沸，煎煮30分钟，倾出药液，残渣再加人4倍饮用水，煎煮30分
钟，合并两次药液，过滤、浓缩后制成r000nil水煎剂(相当于29生药/而)，
4℃放置备用。
2.动物分组及处理将大鼠随机分为四组，均经口灌胃，各组(除对照
组外)均给大鼠予关木通煎剂20群(kg·d)(相当于ron1F(kg·d))灌胃
12周，即(l)对照组(n二5)，饮用水灌胃，剂量10rnl/(kg·d)，分二次灌胃;
(2)未治疗组(n二10)，给关木通剂量为20克/(kg·d)，，分二次灌胃;(3)
治疗组1(n二10)，关木通煎剂灌胃(剂量和方法同未治疗组)同时予银杏
叶按300mg/(kg·d)(相当于7.smF(kg·d))灌胃，一日一次，连续用药
12周;(4)治疗组2(n二10)，关木通煎剂灌胃(剂量和方法同未治疗组)始
予凯时10林扩(kg·d)一日一次，尾静脉注射，连续用药10日。实验期间大
鼠均自由饮食、饮水。
3.实验12周将大鼠放入代谢笼收集24小时尿液，记24小时尿量，采
用黄柳酸硫酸钠比浊法检测尿蛋白;收集尿液后10%水合氯醛腹腔麻醉大
鼠，剖开腹腔，下腔静脉采血，进行血红蛋白、Scr、BUN测定，血红蛋白采用
光电比色法测定，scr、BUN采用Keysys全自动生化分析仪测定。
4.肾脏病理检查:摘除肾脏后，切取适当大小组织块于4%多聚甲醛溶
液(0.IM PBS配制，PH7.0一7.4，经DEPC处理)中固定2一4小时。常规
石蜡包埋，切取3林m石蜡切片，行HE染色。镜下观察病理变化并拍照。
5.用免疫组化学采用ABC法检测肾组织TGF一p;、 CTGF、FN，使用显微
图像分析系统进行定量分析及显徽照相，定量分析采用光密度测定:每组切
片随机选取5张，随机采集10个含阳性着色肾小管的视野(x400)，测定光
密度，取平均值。
6.统计学分析
所有结果均用又土s表示，采用SPSS10.O软件，多组间用单因素(完全
随机设计)方差分析，P<0.05为显著性差异。
结果
实验12周时体重未治疗组明显低于正常对照组及治疗组(l)、(2)(P
<0.05)，治疗组(1)、(2)之间差异不明显(P>0.05);血红蛋白未治疗组
与对照组、治疗组(l)、(2)比较明显下降(P<0.05)，治疗组(1)、(2)差异
不明显(P>0.05);24小时尿蛋白定量未治疗组明显增加与对照组比较有
显著差异(p<0.01)，与治疗组(1)、(2)比较有明显差异(p<0.05)，治疗
组(1)明显低于治疗组(2)(P<0.05)。肾功能未治疗组BUN、Scr显著增
高，与对照组、治疗组(1)、(2)比较均有显著差异(p<0.01)，治疗组(2)
BUN较治疗组(1)明显增高(p<0 .05)，治疗组(1)、(2)间Ser无明显差异
(P>0.05);肾脏病理未治疗组较治疗组损伤明显;免疫组化测定TGF一p，、
CTGF、FN光密度，未治疗组TGF一p，、CTGF、刚光密度值较对照组显著增加
(p 治疗组明显降低(P<0.05)，治疗组(1) CTGF、FN光密度值较未治疗组显
著降低(P<0.01)，治疗组(1) TGF一pl、FN光密度值与治疗组(2)无明显差
异(P>0.05)，治疗组(1)CTGF光密度值较治疗组(2)明显降低(P<
0 .05)。
讨论
近年来，关木通等含马兜铃酸的中草
Manchurian dutchmanspipe stem can cause serious injuries of renal tubules, and renal failure in human. The harm to kidneys has been observed and many investigations have proceeded. There are experimental proof that Manchurian dutchmanspipe stem mainly causes interstitial fibrosis in renal tubules. The machanism is not very clear. Depierreux etal foud that the patients with aristolo-chic acid nephropathy (AAN) had dramatical lesion in small arteries, such as thickened walls and constricted cava. The lesion causes ischemia and interstitial fibrosis of the kidneys. Chen Yipu etal thout that aristolochic acid accumulated in epithelial cells of renal tubules, persistantly stimulating and poisoning the cells. The results were the activation, transdifferentiation and necrosis of the cells. Stimulated by all kinds of cytokines, the fibroblast cells in the interstitial synthesized and exreted many extracellular matrix ( ECM) , which caused interstitial fibrosis. At present, the therapy is in studies. The main contain
s of folium ginkgo are bitter intralipid, omniflavone. The bitter intralipid is a specif!tial antagonist of platelet activating factor ( PAF) receptor. Many animal experiments conformed that the antagonist of PAF could therapy the ischemic renal injuries by the alteration of renal hemodynamics. The flavone could obviously clean-hy-droxy free radicals. In vitro experiments conformed that flavone could inhibit the activity of angiotensin converting enzyme( ACE). In vivo experiments conformed flavone obviously inhibited endothelin (ET) after injected in rats'abdomy. The effects of expanding vessels was not inhibited by all kinds of antagonist of neuro-
transmitters or hormome receptor. Moreover, folium ginkgo reduces the proliferating cell nuclear antigen (PCNA + ) in the renal tubules and renal interstitial and decrease the level of interstitial fibrosis. Hormone can inhibit the releation of inflammatory mediators, such as cytokines, adhesivemolecules, chemotactic factor, etal. Besides that, it can block the activation of inflammatory cells and reduce the inflammatory reaction. As the carrier of Prostaglandin E1 ( PGE, ) , PGE1 can alter the renal blood flow ( RBF) , inhibiting the proliferation of intrinsic cells in glomerulus and the accumulation of extracellular matrix. The purpose of this experiment is to find out the function of the folium ginkgo and Prostaglandin E1 ( PGE1) to the aristolochic acid nephropathy ( AAN) and the mechanism.
Materials and Methods
1. Materials
1. 1 Animal ; 35 female Wistar rats whose body weight are from 200 to 250g are divided into four groups , the control group has 5 rats; each of non -therapy and therapy (1) (2)group has 10 rats. AAN groups and therapy groups (1) (2) contrast groups.
1.2 Major reagent and physic: Manchurian Dutchman pipe Ginkgo leaf Prostaglandin E1TGF- CTGF FN test kits.
1.3 Apparatus: light microscope image analyzer spectro-light meter cen-trifugation machine.
2. Methods
2. 1 Biochemical detection
Sulfosalicylic acid-natrium sulfuricum turbidimetry is used to detect urine protein 24h; hemoglobin is detected by light-electricity chromatometry; BUN and Scr is detected with auto-biochemical-instrument.
2.2 Pathology detection
with cut paraffin sample into 3 m slide, which are stained with HE moth-
od.
2. 3 Immunohistochemical staining is performed by peroxidase-labeled stretavidin-biotin technique to detect TGF CTGF and FN on renal tissue .
2.4 Statistics
Quantity data is appeared as X S, analyse the variance with F - test and T -test,linear regression is used to correlative analysis. Above all are performed with SPSS10.0 software,the difference is significant if p <0. 05.
Result
After three months mean Hb level was found significantly lower in MD group than in FG group and PGE, group (p <0. 05) ; Mean urinary protein excretion level was found significantly higher in MD group than in FG group and PGE1 group , Mean BUN and Scr level was found significantly higher in MD group than in FG grop and PGE1 grop.
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