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Journal of Integrative Medicine, 2011, 9(5): 546-552 doi: 10.3736/jcim20110513

实验论著

槐杞黄颗粒对阿霉素肾病大鼠肾组织nephrin和podocin表达的影响

孙雯1, 朱智1, 俞建,1, 汪永红1, 熊曼2, 高霞3, 赵仲华4, 刘学光4

1. 复旦大学儿科医院中医科,上海201102

2. 复旦大学儿科医院儿科研究所,上海201102

3. 复旦大学儿科医院肾脏科,上海201102

4. 复旦大学上海医学院病理学系,上海200032

Effects of Chinese herbal medicine Huaiqihuang Granule on nephrin and podocin expressions in renal tissues of rats with adriamycin-induced nephrosis

Wen Sun1, Zhi Zhu1, Jian Yu,1, Yong-hong Wang1, Man Xiong2, Xia Gao3, Zhong-hua Zhao4, Xue-guang Liu4

1. Department of Traditional Chinese Medicine, Children's Hospital, Fudan University, Shanghai 201102, China

2. Pediatric Institute, Children's Hospital, Fudan University, Shanghai 201102, China

3. Department of Nephrology, Children's Hospital, Fudan University, Shanghai 201102, China

4. Department of Pathology, Shanghai Medical College of Fudan University, Shanghai 200032, China

通讯作者: Jian Yu, MD, Professor; Tel: 021-64931219; E-mail: yuj@shmu.edu.cn

收稿日期: 2010-12-12   接受日期: 2011-01-9   网络出版日期: 2011-05-20

基金资助: 复旦大学附属儿科医院重点科研基金资助项目.  08ZD-01

Received: 2010-12-12   Accepted: 2011-01-9   Online: 2011-05-20

摘要

目的:通过观察槐杞黄颗粒对阿霉素肾病大鼠肾组织中nephrin与podocin基因及蛋白表达的影响,探讨槐杞黄颗粒治疗肾病综合征的作用机制。
方法:将Sprague-Dawley大鼠20只随机分为5组(即正常对照组、模型组、激素组、槐杞黄组和槐杞黄+激素组),除正常组大鼠外,各组大鼠尾静脉一次性注射阿霉素造模。分别在建模后第7、14、21及28天收集24 h尿液,检测大鼠24 h尿中的蛋白含量;第29天处死所有大鼠,取肾脏组织,光学显微镜和电子显微镜下观察大鼠肾脏病理改变,实时荧光定量聚合酶链式反应法检测肾组织nephrin与podocin mRNA的表达,蛋白质印迹法检测肾组织nephrin与podocin蛋白的表达。
结果:与正常对照组比较,从建模第14天开始模型组大鼠24 h尿液中蛋白量增多(P=0.005),并随造模时间延长逐渐增加(P=0.015,P=0.012);从第14天开始,槐杞黄组与正常对照组相比,其尿蛋白减少不明显(P<0.05);第21天时,激素组与正常对照组相比,其尿蛋白明显增加(P=0.045)。光学显微镜下发现:模型组肾间质有灶性炎症细胞浸润,伴少量纤维组织增生;药物治疗组炎症细胞浸润较少。电子显微镜下发现:模型组肾小球足突大部分融合、消失,或出现部分微绒毛;槐杞黄组基底膜基本光滑均匀,足突融合明显减轻;激素组及槐杞黄+激素组足突清晰,甚至接近正常对照组。与正常对照组相比,模型组肾组织中nephrin、podocin mRNA及蛋白表达均明显减少(P<0.05);与模型组比较,药物治疗组nephrin、podocin mRNA及蛋白表达均明显增加(P<0.05)。24 h尿蛋白量与nephrin mRNA以及nephrin和podocin蛋白的表达呈负相关。
结论:槐杞黄颗粒可通过上调裂孔隔膜上的nephrin及podocin的表达维持足细胞裂孔隔膜的完整性,减轻肾小球滤过屏障的损伤,以此降低尿蛋白漏出。

关键词: 中草药 ; 肾病 ; 足细胞 ; nephrin ; podocin ; 大鼠

Abstract

Object: To investigate the effects of Huaiqihuang Granule, a compound Chinese herbal medicine, on expressions of nephrin and podocin of slit diaphragm of glomerular podocytes in rats with adriamycin-induced nephrosis and to explore the mechanism in reducing the proteinuria.
Methods: Twenty SD rats were randomly divided into five groups: control group, model group, glucocorticoid group, Huaiqihuang Granule group and Huaiqihuang Granule plus glucocorticoid group. The 24-hour urine was collected 7, 14, 21 and 28 days after adriamycin injection respectively to measure 24-hour urinary protein, and all rats were sacrificed after 28-day treatment. Pathological changes in renal tissues were observed under a light microscope and an electron microscope. Expressions of nephrin and podocin mRNAs in renal cortex were determined by real-time polymerase chain reaction, and protein levels of nephrin and podocin were detected by Western blotting.
Results: (1) In the model group and the treatment groups, the level of urinary protein increased significantly from the 14th day. (2) Under the light microscope, inflammatory cells and slight fibroplasia were found in renal interstitium of the model group, but there were less inflammatory cells in renal interstitium in the intervention groups than in the model group. Under the electron microscope, 29 days after adriamycin injection, extensive fusion of foot processes was observed. (3) The expressions of nephrin and podocin were higher in treatment groups than in the model group. (4) Proteinuria level was negatively correlated with the expressions of nephrin mRNA and nephrin and podocin proteins.
Conclusion: The above results indicate that Huaiqihuang Granule can maintain the integrity of the slid diaphragram in podocyte, alleviate the lesion of glomerular filtration membrane, and decrease the proteinuria by up-regulating the expressions of nephrin and podocin. Huaiqihuang Granule plus glucocorticoid maybe has better effects than glucocorticoid alone.

Keywords: drugs,Chinese herbal ; nephrosis ; podocytes ; nephrin ; podocin ; rats

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Cite this article

孙雯, 朱智, 俞建, 汪永红, 熊曼, 高霞, 赵仲华, 刘学光. 槐杞黄颗粒对阿霉素肾病大鼠肾组织nephrin和podocin表达的影响[J]. 结合医学学报, 2011, 9(5): 546-552. Doi:10.3736/jcim20110513

Wen Sun, Zhi Zhu, Jian Yu, Yong-hong Wang, Man Xiong, Xia Gao, Zhong-hua Zhao, Xue-guang Liu. Effects of Chinese herbal medicine Huaiqihuang Granule on nephrin and podocin expressions in renal tissues of rats with adriamycin-induced nephrosis[J]. Journal of Integrative Medicine, 2011, 9(5): 546-552. Doi:10.3736/jcim20110513

原发性肾病综合征是儿童时期的常见病,虽然大部分患儿经肾上腺糖皮质激素治疗后的效果较好,但是仍有相当部分患儿对激素治疗抵抗或频繁复发。故而,临床上一直在寻求既能提高激素疗效,减轻激素副作用,又能减少感染,减少病情反复的新药物和方法。近年来,中医药在儿童肾病综合征治疗中取得了一定的进展[1],但其机制仍然不清楚。除汤剂外,许多中成药免疫调节剂应用于儿科临床,已取得了一定的疗效,但是对其作用机制仍然不明,槐杞黄颗粒剂就是其中之一。

本研究以微小病变性肾病综合征大鼠模型为研究对象,从蛋白和基因水平观察槐杞黄颗粒对大鼠肾组织中nephrin和podocin表达的影响,探讨该药物对阿霉素肾病大鼠肾小球滤过屏障的影响及其作用机制。

1 材料与方法

1.1 实验材料

20只雄性SD大鼠,体质量110~130 g,购自并饲养于复旦大学实验动物科学部,环境为清洁级,温度(20±2)℃,自由摄取饲料和水。动物使用符合上海市医学实验动物管理委员会管理条例,许可证号为SCXK(沪)2003-0002。阿霉素液射液,10 mg/支,批号为8NB002-A,购自意大利Pharmacia & Upjohn公司;强的松片,5 mg/片,批准文号为国药准字H31020675,购自上海医药有限公司信谊制药总厂;槐杞黄颗粒,10 g/包,由槐耳、枸杞子及黄精组成,每包含生药6.5 g,批号为HK12,购自启东盖天力药业有限公司;实时聚合酶链反应(polymerase chain reaction,PCR)Master Mix,批号为75660M3,购自日本TOYOBO公司;兔抗大鼠podocin抗体(批号为H2608),兔抗大鼠nephrin抗体(批号为I1208),购自美国Santa Cruz公司。

1.2 实验方法

1.2.1动物模型制备 利用随机数字表将大鼠随机分为正常对照组、模型组、激素组、槐杞黄组和槐杞黄+激素组,每组各4只大鼠,除正常对照组外,各组大鼠经尾静脉一次性注射盐酸阿霉素7.5 mg/kg,正常对照组经尾静脉一次性注射等量生理盐水。

1.2.2 药物干预方案及动物处理 从造模后第1天起连续灌胃28 d。(1)正常对照组:经胃灌服等量蒸馏水;(2)模型组:无任何干预,正常喂养;(3)激素组:经胃灌服强的松5 mg/(kg·d);(4)槐杞黄组:经胃灌服槐杞黄颗粒剂3.3 g/(kg·d);(5)槐杞黄+激素组:同时灌服槐杞黄颗粒和强的松,剂量如上。分别在阿霉素注射后当天和第7、14、21、28天收集24 h尿液;第29天处理大鼠,处死前经腹主动脉采血3 mL;取左肾皮质1 mm×1 mm×1 mm大小数块,一部分以10%中性甲醛固定用于光学显微镜检测,另一部分以3%戊二醛固定用于电子显微镜检测;取右肾分离皮质髓质后,用消毒锡箔纸包好速冻于液氮中,后转至–70 ℃保存,用于实时荧光定量PCR及Western blot的检测。

1.3 指标检测

1.3.1 尿蛋白定量 将24 h尿液离心,取上清液与磺基水杨酸-硫酸钠试剂作用产生沉淀,所形成之浊度用比色法比浊,与同样处理的标准液比较,测得其蛋白含量。

1.3.2 光学显微镜检查 大鼠肾脏常规石蜡包埋后,平行于肾脏短轴经肾门横断修块,4 μm切片。选取含有整个肾脏断面的切片,常规苏木精和伊红染色,光学显微镜下观察肾组织病理形态改变。

1.3.3 电子显微镜检查 肾皮质用含3%戊二醛、0.22 mmol/L蔗糖的磷酸盐缓冲液(pH 7.2)固定,1%锇酸后固定,逐级乙醇脱水,环氧树脂包埋,应用日立H-600型透射电子显微镜进行肾脏超微病理学检查。

1.3.4 实时荧光定量PCR检测 取肾皮质,TRIzol法抽提总RNA,以随机六核苷酸寡聚合物为引物,采用Promega公司的M-MLV逆转录酶将其逆转录为cDNA。实时PCR用SybrGreen法,引物采用Primer Premier 5.0辅助设计,由上海生工生物工程有限公司合成。检测基因的上下游引物序列见表1。实时荧光定量PCR反应体系:上下游引物各0.5 μL,样本cDNA 0.25 μL,SYBR Mix液12.5 μL,用灭菌注射用水补足25 μL。反应条件:95 ℃预变性20 min,95 ℃变性30 s,退火30 s,72 ℃延伸40 s,40个循环。所用实时PCR扩增仪为MX3000P型(美国Stratagene公司)。mRNA表达量采用管家基因β-actin校正。

Table 1   Sequence of primers and length of products

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1.3.5 蛋白质印迹法检测 冰上研磨肾皮质,并加入细胞裂解液和蛋白酶抑制剂,取上清,检测蛋白浓度。加入5×十二烷基硫酸钠聚丙烯酰胺凝胶(sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS-PAGE)蛋白上样缓冲液,100 ℃加热10 min变性,10% SDS-PAGE电泳,每孔上样总蛋白150 μg。电泳后用湿法将蛋白转移到硝酸纤维素膜上,室温下10%脱脂奶粉封闭2 h,分别加入podocin抗体(1︰250稀释)、nephrin抗体(1︰100),室温孵育3 h后,收入4 ℃冰箱内过夜。TBST洗膜后,加入辣根过氧化物酶标记的羊抗兔IgG和甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase, GAPDH)(均1︰5 000稀释),室温孵育1 h,洗膜后,加入电化学发光剂(美国Thermo公司),采用LAS-4000 Mini型发光成像分析仪(日本富士公司)对膜进行曝光时间叠加拍照,后应用仪器自带软件Multi Gauge Ver3.X 对检测蛋白和GAPDH蛋白条带的光密度进行分析。

1.4 统计学方法

实时PCR结果取对数后进行统计,蛋白质印迹目的蛋白光密度值与对应GAPDH蛋白光密度值的比值除以正常对照组比值后进行统计分析。应用SPSS 15.0统计软件进行分析,数据以x±s表示;对于24 h尿蛋白量的比较,采用一般线性模型的Repeated Measure过程实现重复测量资料的方差分析,并用Multivariate过程实现组间的两两比较;对于实时PCR和Western blot结果,采用析因设计的方差分析进行比较;同时采用Pearson法做线性相关性分析。P<0.05认为差异有统计学意义。

2 结果

2.1 各组大鼠24 h尿蛋白定量

各组大鼠造模前尿蛋白均在同一基线上。阿霉素注射后第7天,模型组及药物干预组大鼠24 h尿蛋白量开始增加,但与正常对照组比较无统计学意义。在第14天时,模型组24 h蛋白量较正常对照组增加(P=0.005),槐杞黄组同正常对照组比较差异亦有统计学意义(P=0.021)。在第21天时,与正常对照组相比,模型组、激素组、槐杞黄组24 h尿蛋白量显著增加(P=0.015,P=0.045,P=0.028);而槐杞黄+激素组尿蛋白含量与正常对照组比较差异无统计学意义(P=0.185)。在第28天时,与正常对照组相比,模型组和槐杞黄组24 h尿蛋白量显著增加(P=0.012,P=0.032);而激素组和槐杞黄+激素组与正常对照组间比较差异无统计学意义。此外,在各时间点,3个药物干预组与模型组相比,差异无统计学意义。见表2

Table 2   Content of 24-hour urinary protein of rats in different groups

*P<0.05, **P<0.01, vs control group.

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2.2 各组大鼠肾组织病理形态学改变

正常对照组大鼠肾组织无明显病变。模型组大鼠部分肾小球体积稍增大,系膜细胞及基质局灶节段性轻度增多,但未见坏死、新月体及硬化;肾小管上皮细胞可见部分灶性颗粒样、空泡样变性,小管未见明显萎缩,偶见小管有蛋白管型;肾间质有灶性炎症细胞浸润,伴少量纤维组织增生;部分小动脉内膜增厚。激素组、槐杞黄组及槐杞黄+激素组肾组织病理改变较模型组好转,肾间质炎症细胞浸润减少。见图1

图1

图1   光学显微镜下各组肾病大鼠肾组织(苏木精和伊红染色,×400)

Figure 1   Pathological changes of renal tissues of rats under the light microscope (Hematoxylin and eosin staining, ×400)

A: Control group; B: Model group; C: Huaiqihuang Granule group; D: Glucocorticoid group; E: Huaiqihuang Granule plus glucocorticoid group.


2.3 各组大鼠透射电子显微镜观察的肾脏超微病理改变

正常对照组肾小球基底膜光滑均匀,未见增厚,足突清晰完整、未见融合。模型组肾小球基底膜偶见增厚,但足突大部分融合、消失,或出现部分微绒毛,与正常对照组有明显差异,达到临床上微小病变型肾病病理诊断标准,证明模型成功建立。槐杞黄组大鼠肾小球基底膜基本光滑均匀,足突融合明显减轻,有少量新生绒毛长出;激素组及槐杞黄+激素组大鼠,足突清晰,甚至接近正常对照组,好转程度尤以槐杞黄+激素组最为明显。见图2

图2

图2   电子显微镜下各组大鼠肾病足突形态变化(×7 000)

Figure 2   Ultra-structural changes of foot processes of renal tissues of rats under the electron microscope (×7 000)

A: Control group; B: Model group; C: Huaiqihuang Granule group; D: Glucocorticoid group; E: Huaiqihuang Granule plus glucocorticoid group.


2.4 各组大鼠肾组织nephrin及podocin mRNA的表达

在建模后第29天,模型组大鼠肾组织nephrin mRNA表达明显低于正常对照组(P=0.014),激素及槐杞黄的应用和槐杞黄+激素的交互作用对提高nephrin mRNA的表达均有统计学意义,其中槐杞黄+激素的交互作用具有统计学意义(P=0.015,P=0.043,P=0.003)。模型组大鼠肾组织podocin mRNA表达明显低于正常对照组(P=0.007),激素的应用对上调podocin mRNA的表达具有统计学意义(P=0.031),其中,槐杞黄的应用和槐杞黄+激素的交互作用在此方面的统计学意义更为显著(P=0.001,P=0.006)。见表3

Table 3   Expressions of nephrin and podocin mRNAs in renal tissue of rats in different groups

*P<0.05, **P<0.01, vs control group; P<0.05, △△P<0.01, vs model group.

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2.5 各组大鼠肾组织nephrin及podocin蛋白的表达

在建模后第29天,模型组大鼠肾组织nephrin蛋白的表达明显低于正常对照组(P=0.001);激素组、槐杞黄组和槐杞黄+激素组在上调nephrin蛋白表达时具有显著的统计学意义(P=0.007,P=0.004,P<0.001)。模型组大鼠肾组织podocin蛋白的表达明显低于正常对照组(P<0.01),激素组、槐杞黄组和槐杞黄+激素组podocin蛋白的表达升高(P=0.041,P=0.010,P=0.003)。见表4图3

Table 4   Expressions of nephrin and podocin proteins in renal tissue of rats in different groups

*P<0.05, **P<0.01, vs control group; P<0.05, △△P<0.01, vs model group.

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图3

图3   Western blot法检测nephrin及podocin蛋白的表达

Figure 3   Expressions of nephrin and podocin proteins observed by Western blot method

A: Control group; B: Model group; C: Huaiqihuang Granule group; D: Glucocorticoid group; E: Huaiqihuang Granule plus glucocorticoid group.


2.6 肾组织nephrin和podocin表达与24 h尿蛋白排泄量的相关性分析

在建模后第29天,大鼠肾组织中nephrin mRNA、nephrin和podocin蛋白的表达与24 h尿蛋白排泄量呈负相关(r=-0.587, P=0.011; r=-0.611, P=0.007; r=-0.547, P=0.019);而podocin mRNA的表达与24 h尿蛋白排泄量无关(r=–0.282, P=0.257)。

3 讨论

槐耳为槐杞黄颗粒的君药,作为一种药用真菌[2],其性味“苦、辛、平,无毒”,能“治风,破血,益力”,偏于益气。枸杞子偏于滋阴,而黄精气阴并补。三药相合,补气而不致滞气,养阴而不过于滋腻。三药归经于心、肝、脾、肺、肾,益五脏之精气,滋脏腑之阴津,对整体之虚弱照顾全面,具有强壮补养之功效。研究发现,槐耳可以诱导正常小鼠分泌干扰素[3];对于化疗患者,其自然杀伤细胞和CD4+/CD8+细胞的提高率均高于对照组[4];对于癌症术后患者,其1型辅助性T细胞相关细胞因子γ干扰素和白细胞介素2显著增高[5]。临床证实,该药物在提高原发性肾病综合征儿童抵抗力、减少感染方面具有一定作用。

中医学认为,原发性肾病综合征多属“水肿”、“尿浊”范畴,其病理本质为本虚标实,以脾肾两虚为本,以血瘀、湿浊为辅。该病主要表现之一为大量蛋白尿的产生,其中脾肾两虚是蛋白尿产生的直接机制,此外,肺虚、湿热亦可引起蛋白尿。

目前研究认为,蛋白尿的产生与肾小球滤过屏障正常的结构遭到破坏和(或)其功能损伤有着十分密切的关系。肾小球滤过屏障由内皮细胞窗孔、肾小球基底膜和脏层细胞足突之间的裂孔隔膜组成,而影响蛋白滤过的主要屏障是足突裂孔隔膜,由“拉链样结构”组成,其中央为线状结构,两侧为横桥结构,横桥间为4 nm×14 nm的孔隙。该孔稍小于白蛋白分子的大小,起分子屏障作用,由podocin、nephrin、CD2AP、ACTN4、FAT、TRPC6和PLCE1等多种蛋白质分子组成[6]。Nephrin是一种跨膜蛋白,胞内部分含有8个酪氨酸残基,当nephrin与其配基结合时,可能导致胞内酪氨酸残基磷酸化,从而将信号传入细胞内以发挥其生物效应。Nephrin特异性表达在裂孔膜上,是裂孔膜蛋白复合体的主要成分[7]。研究发现,在阿霉素肾病大鼠中,nephrin在蛋白尿的产生和小球基膜延展方面具有重要作用;nephrin是该疾病中最敏感的分子[8]。Podocin也是一种跨膜蛋白,属stomatin家族。原位杂交显示编码podocin的基因NPHS2几乎全部表达在肾小球足细胞,而不在其他组织部位表达;podocin通过其C末端与nephrin及CD2AP的胞内段相互作用,可促进或放大nephrin诱导的信号传导[9]

本实验采用一次性尾静脉注射阿霉素7.5 mg/kg制备肾病微小病变型模型[10],并且每隔1周测定各组蛋白尿情况。我们发现,模型组在第2周后尿蛋白量明显增高(P=0.005),第4周时达高峰(P=0.012);同时,电子显微镜观察模型组肾小球足细胞足突融合明显,由此可以判断造模较为理想。此外,药物干预28 d后,蛋白尿排泄量均不同程度下降,尤以槐杞黄+激素组干预效果最为明显;但是在槐杞黄组中,其尿蛋白量控制不佳。肾组织电子显微镜观察显示,3个治疗组大鼠在阿霉素注射后28 d肾小球足细胞足突部分融合较模型组减轻,尤以槐杞黄+激素组最为明显,足突融合基本消失。以上实验结果表明,槐杞黄颗粒联合氢化可的松对阿霉素肾病大鼠模型有保护作用。

本研究发现,与正常对照组相比,模型组中的podocin、nephrin mRNA及蛋白水平的表达明显降低(均P<0.01),提示podocin、nephrin在肾病综合征的发生和蛋白尿的产生过程中起到了一定的作用。此外,与模型组相比,各治疗组podocin、nephrin mRNA及蛋白水平的表达都不同程度的增加,尤以槐杞黄+激素组增加明显(P<0.01)。以上结果提示,槐杞黄颗粒可能通过稳定裂孔隔膜上的podocin及nephrin蛋白,维持肾小球滤过屏障,以降低尿蛋白漏出,并且槐杞黄颗粒与激素合用干预效果更好,这也为临床上益气养阴方辅助激素治疗肾病综合征提供了理论依据。

本实验亦发现,大鼠肾组织中nephrin和podocin的表达与24 h尿蛋白排泄量呈负相关,不仅提示了蛋白尿的发生与nephrin和podocin蛋白的相关性,亦说明槐杞黄颗粒通过上调nephrin和podocin蛋白的表达,维持了滤过屏障上裂孔隔膜的完整性,减少了蛋白尿的产生,从而保护肾脏。

本实验从裂孔隔膜分子nephrin和podocin蛋白入手,初步探讨了槐杞黄颗粒对减少阿霉素肾病大鼠蛋白尿产生和保护肾小球滤过屏障的机制。进一步的研究方向,可以选择不同的干预时间点或选择肾小球滤过屏障上的其他分子,以求更深入地了解槐杞黄的作用机制。

4 利益冲突

本文作者声明不存在任何与本稿件相关的利益冲突。

The authors have declared that no competing interests exist.

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