活血化瘀通络中药防治早期糖尿病肾病的实验研究
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摘要
目的：糖尿病肾病(diabetic nephropathy, DN)是糖尿病(diabetes mellitus,DM)最常见的慢性并发症之一，也是糖尿病致死的重要原因。糖尿病肾病现已成为发达国家慢性肾衰竭的第一位原发病；在我国，随着糖尿病发病率的快速增加，由DN导致的终末期肾功能衰竭也逐年上升，预计在未来的20年中DN将成为终末期肾衰竭最重要的原因。但到目前为止尚无有效的方法能阻止DN的发生和发展，故防治DN的发生发展已经成为当前急需解决的问题，且DN严重影响患者的生存质量及预后，增加患者的痛苦，因此探讨DN的发病机制，寻求防治DN的有效方法具有重要的意义。
DN主要的病理表现是肾小球毛细血管基底膜弥漫增厚，系膜基质增生，进而造成结节性肾小球硬化。临床表现则以蛋白尿为主，早期的表现为微量白蛋白尿，临床期的表现为大量蛋白尿。DN发生发展的病理机制非常复杂，包括了蛋白的非酶糖基化、氧化应激以及足细胞损伤等等，是多种因素、多条通路协同作用的结果，因而针对DN的治疗也必须是多因素、多靶点的。目前，现代医学防治DN的措施主要有以下几方面：严格控制血糖、血压，纠正血脂紊乱，血管紧张素转化酶抑制剂(angiotensionconverting enzyme inhibitors, ACEI)或血管紧张素受体拮抗剂(angiotensinreceptor blockers, ARBs)的应用等，尚无控制DN的特异性药物，且西药副作用较多，在控制临床症状方面难使患者满意。而中医药以其独特的整体调节与辨证论治体系，通过三因制宜的个性化治疗方案，可能对DN发生发展的多个环节起到干预作用，在临床防治DN中显示出巨大的潜力和广阔的应用前景。
课题组结合多年临床治疗DN的经验和中医学病因病机理论，尤其是络病理论，认为DN主要的病机是瘀血阻于肾络，治疗当活血化瘀通络，采用丹参、川芎、地龙、水蛭、全蝎等中药配伍组成活血化瘀通络方，临床观察和前期实验表明该方对DN有较好疗效。本研究采用一次性大剂量链脲佐菌素(Streptozocin, STZ)腹腔注射复制DN大鼠模型，以活血化瘀通络中药进行干预，进行整体研究，观察其对DN模型大鼠肾脏的保护作用；并且采用小鼠条件永生系足细胞进行细胞实验，观察活血化瘀通络中药含药血清及药物单体丹参酮IIA对足细胞增殖的影响，进一步验证该复方中药的疗效，探讨其对DN的作用机制，并以效测证，为临床应用提供参考。
方法：
1活血化瘀通络中药对糖尿病肾病大鼠肾脏的保护作用
选取健康、雄性、4～5周龄，体重120g～150g，SD大鼠48只，适应性饲养1周后，检测血糖和尿蛋白，均为阴性者用于实验。随机分为正常组(normal group, NG)和造模组。造模组大鼠一次性腹腔注射STZ60mg·kg~(-1)，NG注射相应体积的枸橼酸缓冲液。72h后检测造模组大鼠的空腹血糖(fasting blood glucose, FBG)，三次连续随机尾静脉血糖≥16.7mmol·L~(-1)为DM模型成功。成模大鼠随机分为模型组(model group,MG)、厄贝沙坦组(Irbesartan group, XG)及活血化瘀通络中药组(huoxuehuayutongluo Chinese medicine group, ZG)。ZG给予活血化瘀通络中药免煎颗粒进行灌胃，给药量按照体型系数换算给药剂量的基本公式进行换算。分别按川芎0.96g·kg~(-1)·d~(-1)、丹参1.2g·kg~(-1)·d~(-1)、地龙0.8g·kg~(-1)·d~(-1)、水蛭0.48g·kg~(-1)·d~(-1)、全蝎0.48g·kg~(-1)·d~(-1)进行灌胃。厄贝沙坦组大鼠给予厄贝沙坦15mg·kg~(-1)·d~(-1)灌胃。每日给药一次，连续16周。于给药后的第四周末(4W)、第八周末(8W)、第十二周末(12W)、第十六周末(16W)，代谢笼收集24小时尿液，并测量大鼠的饮水量(water intake)、进食量(foodintake)、尿量(urine output)等。
给药16周末杀检动物，大鼠称体重(body weight, BW)，尾静脉取血检测FBG和糖化血红蛋白(HbA1c)。之后，水合氯醛麻醉大鼠，腹主动脉取血，分离血清，检测肝功能(总蛋白TP、白蛋白ALB)、肾功能(血肌酐SCr、尿素氮BUN)、血脂(总胆固醇TC、甘油三酯TG、高密度脂蛋白胆固醇HDL、极低密度脂蛋白胆固醇VLDL)。留取双肾，称重，计算肾脏指数(kidney index, KI)。分离皮质，FAA固定液固定，行光镜检查；4%戊二醛固定，行透射电镜检查，从透射电镜图片上测量肾小球基底膜(glomerular basement membrane, GBM)厚度。
2活血化瘀通络中药对糖尿病肾病大鼠尿微量白蛋白及足细胞裂孔膜蛋白p-cadherin的影响
2.1活血化瘀通络中药对糖尿病肾病大鼠尿微量白蛋白的影响
于给药后的第四周末(4W)、第八周末(8W)、第十二周末(12W)、第十六周末(16W)，代谢笼收集24小时尿液，记录24h尿量，尿液经3000转/分,离心10分钟，取上清，-20℃保存。采用Beckman cx7全自动生化分析仪检测尿微量白蛋白(urinary microalbumin, U-Alb)含量。
2.2活血化瘀通络中药对糖尿病肾病大鼠p-cadherin的影响
取材时，快速留取肾皮质，-80℃保存。提取肾组织总RNA，采用反转录酶聚合酶链式反应(RT-PCR)和实时定量聚合酶链式反应(Real-TimePCR)检测肾组织p-cadherin mRNA的表达。采用免疫组织化学法(Immunohistochemistry，IHC)检测肾组织p-cadherin蛋白的表达。
3活血化瘀通络中药对糖尿病肾病大鼠24小时尿蛋白及肾脏纤维化相关因子的影响
3.1活血化瘀通络中药对糖尿病肾病大鼠24小时尿蛋白的影响
于给药后的第四周末(4W)、第八周末(8W)、第十二周末(12W)、第十六周末(16W)，代谢笼收集24小时尿液，记录24h尿量，尿液经3000转/分,离心10分钟，取上清，-20℃保存。采用Beckman cx7全自动生化分析仪测定24小时尿蛋白量(24h Upro)。
3.2活血化瘀通络中药对糖尿病肾病大鼠肾脏纤维化相关因子的影响
取材时，快速留取肾皮质，-80℃保存。检测与肾脏纤维化相关的细胞因子的表达。采用蛋白印迹法(Western blot)、IHC法及Real-Time PCR法检测肾组织BMP-7及TGF-β1蛋白和mRNA的表达；采用Real-TimePCR法检测肾组织OPN mRNA的表达；采用Western blot法检测肾组织Smad7蛋白的表达；并分析BMP-7及TGF-β1之间的相关性。
4活血化瘀通络中药含药血清及药物单体——丹参酮II A对小鼠条件永生系足细胞增殖的影响
体外细胞培养技术培养小鼠条件永生系足细胞，给予大鼠的活血化瘀通络中药含药血清及各浓度的药物单体——丹参酮II A干预后，以MTT法检测足细胞增殖情况。
5统计学方法
所有资料以±s表示，各组间不同观察点空腹血糖、体重、饮水量、进食量、尿量、24小时尿蛋白和尿微量白蛋白采用重复测量设计资料的单变量方差分析进行比较，各观察点进行组间两两比较；其他数据多组间比较采用One-Way ANOVA进行分析，进一步组间两两比较采用SNK法进行；对于不符合正态分布的资料先进行秩和检验，再对秩次进行One-Way ANOVA；变量间的相关性采用Person直线相关分析。所有数据分析应用SAS V8软件进行，以P<0.05为差异有显著性。
结果：
1活血化瘀通络中药对糖尿病肾病大鼠肾脏的保护作用
1.1各组大鼠不同观察点空腹血糖(FBG)的比较
与NG比较，MG、ZG和XG大鼠FBG明显升高(P<0.05)；而与MG比较，ZG和XG大鼠FBG无统计学差异(P>0.05)；且在4个月内MG、ZG和XG大鼠FBG水平一直稳定，没有明显变化(P>0.05)。
1.2各组大鼠不同观察点体重(BW)的比较
NG大鼠体重逐渐增加；与NG比较，MG、ZG、XG大鼠BW明显减轻(P<0.05)，且MG大鼠BW呈现逐渐下降趋势。而与MG比较，ZG和XG大鼠BW下降有所减轻，且ZG和XG大鼠从8W开始体重明显高于MG，差异有显著性(P<0.05)；治疗组间比较，在各观察点大鼠体重无统计学差异(P>0.05)。
1.3各组大鼠不同观察点饮水量(water intake)的比较
与NG比较，MG、XG和ZG大鼠water intake显著增加(P<0.05)；且MG大鼠water intake随时间呈逐渐增加趋势。与MG比较，ZG大鼠water intake从12W开始显著减少(P<0.05)；XG大鼠water intake无显著性差异(P>0.05)。
1.4各组大鼠不同观察点进食量(food intake)的比较
与NG比较，MG、ZG和XG大鼠food intake显著增加(P<0.05)；与MG比较，ZG和XG大鼠food intake增加有所改善，但显效缓慢，从12W开始food intake增加显著改善(P<0.05)。治疗组间比较，在各观察点foodintake无统计学差异(P>0.05)。
1.5各组大鼠不同观察点尿量(urine output)的比较
与NG比较，MG、ZG和XG大鼠urine output显著增加(P<0.05)；MG urine output呈逐步增加趋势。与MG比较，ZG大鼠urine output增加趋势趋缓，且在16W时显示出明显改善(P<0.05)；XG urine output呈逐步增加趋势，且于8W时高于MG。两治疗组间比较，第8W时ZG urine output显著少于XG，有统计学差异(P<0.05)。
1.6各组大鼠HbA1c、KI、BUN、SCr、TP和ALB的比较
与NG相比，MG、ZG和XG大鼠HbA1c、KI显著升高(P<0.05)；与MG相比，ZG和XG大鼠HbA1c、KI无统计学差异(P>0.05)。与NG比较，MG、ZG和XG大鼠BUN均显著升高(P<0.05)，而与MG比较，ZG和XG大鼠BUN显著降低(P<0.05)，两治疗组间比较，无统计学差异(P>0.05)。与NG比较，MG、ZG和XG大鼠SCr均显著降低(P<0.05)，而与MG比较，ZG和XG大鼠SCr显著升高(P<0.05)，两治疗组间比较，XG大鼠SCr显著升高(P<0.05)。与NG相比，MG大鼠TP和ALB显著降低(P<0.05)；与MG相比，ZG和XG大鼠TP和ALB显著升高(P<0.05)；两治疗组间比较，无统计学差异(P>0.05)。
1.7各组大鼠血脂(TC、TG、HDL、VLDL)的比较
TC和HDL：各组大鼠之间无显著性差异(P>0.05)。与NG比较，MG大鼠TG显著升高(P<0.05)，而与MG比较，ZG和XG大鼠TG显著降低(P<0.05)，两治疗组间比较，无统计学差异(P>0.05)。与NG比较，MG大鼠VLDL显著升高(P<0.05)，而与MG比较，ZG和XG大鼠VLDL显著降低(P<0.05)，两治疗组间比较，无统计学差异(P>0.05)。
1.8各组大鼠肾组织病理形态学改变的比较
光镜观察：NG大鼠肾脏肾小球结构清晰完整，未见明显肥大及萎缩，基底膜无增厚，系膜无增生。MG大鼠可见肾小球明显肥大，肾小囊腔呈裂隙状，肾小球基底膜明显增厚，系膜基质增多，系膜区增宽，部分肾小管上皮细胞空泡样变；与MG比较，ZG和XG大鼠肾脏病变有一定程度的改善，两治疗组间差异不明显。
电镜观察：NG大鼠肾组织结构正常，肾小球毛细血管基底膜(GBM)结构清晰，均匀无增厚，上皮足突分布均匀，足突排列整齐、系膜细胞无增殖、系膜基质无增生。MG可见GBM结构欠清晰、均匀弥漫增厚，足细胞足突增宽或大片融合，内皮细胞高度融合，窗口大部分消失。与MG比较，ZG和XG病理改变均有所减轻，GBM结构较清晰，足突节段融合。两治疗组间差异不明显。
1.9各组大鼠GBM厚度的比较
与NG比较，MG、ZG和XG大鼠GBM明显增厚(P<0.05)；与MG比较，ZG和XG大鼠GBM明显减薄(P<0.05)；与XG比较，ZG大鼠GBM明显减薄(P<0.05)。
2活血化瘀通络中药对糖尿病肾病大鼠尿微量白蛋白及足细胞裂孔膜蛋白p-cadherin的影响
2.1各组大鼠不同观察点尿微量白蛋白(U-Alb)的比较
与NG比较，MG、ZG和XG大鼠U-Alb显著增加(P<0.05)。与MG比较，ZG和XG大鼠U-Alb增加趋势趋缓；且从8W至16W各时间点均显示出明显改善(P<0.05)。两治疗组间比较，在各观察点U-Alb无统计学差异(P>0.05)。
2.2各组大鼠肾组织中p-cadherin mRNA表达的比较
与NG比较，MG p-cadherin mRNA表达水平明显降低(P<0.05)；与MG比较，ZG和XG p-cadherin mRNA表达水平均明显升高(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
2.3各组大鼠肾组织中p-cadherin蛋白表达的比较
IHC染色显示：NG p-cadherin蛋白表达较强，主要呈线性分布于肾小球基底膜处，棕黄色颗粒呈深染。与NG比较，MG棕黄色颗粒明显减少。与MG比较，ZG和XG棕黄色颗粒不同程度地增多。
3活血化瘀通络中药对糖尿病肾病大鼠24小时尿蛋白及肾脏纤维化相关因子的影响
3.1各组大鼠不同观察点24h Upro的比较
与NG比较，MG、ZG和XG大鼠24h Upro显著增加(P<0.05)。与MG比较，ZG和XG大鼠24h Upro增加趋势趋缓，且在12W、16W时显示出明显改善(P<0.05)。两治疗组间比较，在各观察点24h Upro无统计学差异(P>0.05)。
3.2各组大鼠肾组织中BMP-7蛋白及mRNA表达的比较
IHC染色显示：在正常及DN大鼠肾脏均有BMP-7蛋白表达，而在NG组呈强阳性表达，主要位于肾小管和集合管，呈胞浆表达阳性，胞浆呈棕黄色颗粒状。IHC、Western-blot及Real-Time PCR法检测结果均显示：与NG大鼠比较，MG、ZG和XG BMP-7蛋白和mRNA表达水平明显降低(P<0.05)；与MG比较，ZG和XG BMP-7蛋白和mRNA表达水平均明显增加(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.3各组大鼠肾组织中TGF-β1蛋白及mRNA的表达
IHC染色显示大鼠肾脏TGF-β1蛋白在肾小球及肾小管间质均有表达。NG大鼠肾脏TGF-β1表达量很少，着色浅。MG大鼠肾脏TGF-β1蛋白表达明显增加，呈强阳性表达，着色深。ZG和XG大鼠肾脏TGF-β1蛋白呈弱阳性表达，介于NG和MG之间。Western-blot及Real-Time PCR法检测结果显示：与NG比较，模型组TGF-β1蛋白和mRNA表达水平明显升高(P<0.05)；与MG比较，TGF-β1蛋白和mRNA表达水平均明显降低(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.4各组大鼠肾组织中Smad7蛋白的表达
Western-blot结果显示：与NG比较，MG、ZG和XG Smad7蛋白的表达水平明显降低(P<0.05)；与MG比较，ZG和XG Smad7蛋白的表达水平明显升高(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.5各组大鼠肾组织OPN mRNA的表达
与NG比较，MG、ZG和XG OPN mRNA表达明显增加(P<0.05)；与MG比较，ZG和XG OPN mRNA表达均明显减少(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.6MG BMP-7和TGF-β1表达的相关性分析
分析结果显示：肾组织BMP-7和TGF-β1表达的关系：两者在核酸和蛋白表达的相关系数r分别为-0.9269和-0.9055，说明两者的表达呈负相关(P<0.05)。
4活血化瘀通络中药含药血清及药物单体——丹参酮II A对小鼠条件永生系足细胞增殖的影响
4.1活血化瘀通络中药含药血清对足细胞增殖的影响
与低糖对照组相比，活血化瘀通络中药含药血清组在任何时间段均没有统计学差异(P>0.05)；高糖对照组在36h出现了增殖抑制作用，有统计学差异(P<0.05)；与高糖对照组相比，活血化瘀通络中药含药血清组只在48h出现了明显的促进增殖作用(P<0.05)。
4.2丹参酮II A对足细胞增殖的影响
与低糖对照组相比，足细胞在含有不同浓度(1×10~(-8)mol/L～1×10-4mol/L)Tanshinone II A的培养基中：培养12h后，各浓度未出现明显的促增殖作用(P>0.05)；培养24h后，1×10~(-7)mol/L、1×10~(-8)mol/L浓度出现了明显的促进增殖的作用(P<0.05)；培养36h后，1×10~(-6)mol/L、1×10~(-7)mol/L、1×10~(-8)mol/L浓度出现了明显的促进增殖的作用(P<0.05)；培养48h后，各浓度均有明显的促进增殖作用(P<0.05)。
与高糖对照组相比，足细胞在含有不同浓度(1×10~(-8)mol/L～1×10-4mol/L)Tanshinone II A的培养基中：培养12h后，各浓度未出现明显的促增殖作用(P>0.05)；培养24h后，1×10~(-7)mol/L、1×10~(-8)mol/L浓度出现了明显的促进增殖的作用(P<0.05)；培养36h后，1×10-5mol/L、1×10~(-6)mol/L、1×10~(-7)mol/L、1×10~(-8)mol/L各浓度均出现了明显的促进增殖的作用(P<0.05)；培养48h后，各浓度均有明显的促进增殖作用(P<0.05)。
结论：
1一次性大剂量STZ腹腔注射可成功复制DN大鼠模型。
2活血化瘀通络中药能显著改善DN大鼠一般状况，“三多一少”表现及脂代谢紊乱，减少尿蛋白排泄，保护肾功能，减轻肾脏病理损害及肾小球肥大和肾小球基底膜增厚，具有肾保护作用。
3活血化瘀通络中药能明显上调p-cadherin蛋白和mRNA的表达，从而减少蛋白尿的产生，进而保护肾功能，延缓肾小球硬化的发生和发展。
4活血化瘀通络中药能明显减少DN大鼠24小时尿蛋白的产生，并能上调BMP-7和Smad7的表达、明显下调TGF-β1和OPN的表达，从而减少细胞外基质堆积，进而延缓肾脏纤维化和肾小球硬化的发展。
5活血化瘀通络中药含药血清对足细胞具有促进增殖作用；丹参酮IIA各浓度对于足细胞具有明显的促进增殖的作用，从而增强足细胞的自我修复能力，进而减少蛋白尿的产生，保护肾功能。这一结果与本实验中整体研究结果一致，从而从细胞生物学上对活血化瘀通络中药治疗DN的可能作用机制予以了进一步的佐证，并对中药单体的应用提供了依据。
6以效测证，从本研究结果可以反证DN的证候属于瘀血阻络证。
7活血化瘀通络中药与西药对照药厄贝沙坦的疗效相当。
Objective: Diabetic nephropathy (DN) is one of the most commonchronic complications of diabetes mellitus (DM), and also one of main causesleading to death. Diabetic nephropathy has become the first primary diseasedeveloped chronic renal failure. In our country, with the rapid increase of theincidence of diabetes, end-stage renal failure is increasing caused by DN.Expected in the next20years DN will become the most important cause ofend-stage renal failure, but so far there is no effective method to prevent theoccurrence and development of DN, which has become an urgent problem.Furthermore, DN has great impact on life quality and prognosis of the patientsand increases the suffering of patients. So, it has important significance toexplore the pathogenesis of DN and effective methods for prevention of DN.
The main pathological manifestation of DN is glomerular capillarybasement membrane thickening, mesangial matrix proliferation and nodularglomerulosclerosis. The main clinical manifestation of DN is proteinuria,which is microalbuminuria at early manifestations and proteinuria at clinicalperformance. Pathogenesis of diabetic nephropathy is very complex, includingprotein non-enzymatic glycation, oxidative stress and podocyte injury and soon. DN is the result of multiple factors and multiple paths synergy. Therefore,treatment for diabetic nephropathy must also be multiple factor, multipletargets. At present, the modern medical prevention and control measures ofDN are mainly in the following aspects: strict control of blood glucose, bloodpressure, lipid disorders correction and application of ACEI and ARB. There isno specific drug for controlling DN. And western medicine has more sideeffects, which can not satisfy the patients in the control of clinical symptoms.However, Chinese medicine may develop effect on many aspects of DN andshow great potential and broad application prospect in the clinical prevention and treatment of DN with its unique overall adjustment, syndromedifferentiation system and three for individualized treatment plan system.
Our research group arrival at a conclusion that the pathogenesis of DN ismainly blood stasis in the kidney collateral combined with years of clinicaltreatment experience of DN and TCM etiology and pathogenesis theory,especially the collateral disease theory. And the treatment should be adoptedhuoxue-huayu-tongluo therapy. We compose huoxue-huayu-tongluo Chineserecipe using salvia, chuanxiong, earthworm, leech, Scorpio and so on, whichadopting compatibility of traditional Chinese medicine. Clinical observationand preliminary experiments show that the recipe has a good effect on DN.This study adopts the model of DN rats with a single high dose streptozotocinintraperitoneal injection. First of all, we make an overall study on theprotective effect on the DN model in kidney of rats of huoxue-huayu-tongluoChinese medicine. Secondly, we develop a cell experiment using mice livingsystem condition of podocyte. And we observe the effect ofhuoxue-huayu-tongluo Chinese medicine decoction drug-containing serum ofrats and drug monomer-tanshinone II A on podocyte, in order to furthervalidate the efficacy of the party, to investigate the mechanism of DN, and theeffect of measurement, and provide reference for clinical application.
Methods:
1Renal protective effects of huoxue-huayu-tongluo Chinese medicine on DNrats
Healthy male4～5weeks old50SD rats with body weight120g～150gafter adaptive feeding for1week were grouped randomly. And they were fedfor one week after making sure the rats’ urine protein negative. Ten rats werenormal control group. Other40rats were fed with high fat diet throughout thestudy. The animals were randomly divided into normal group (NG) for ten ratsand DM model group for forty rats. The DM model group was given a singledose of60mg·kg~(-1)streptozotocin intraperitonally and rats in NG were ip vehic.Blood glucose levels were measured for three times continuously (≥16.7mmol·L~(-1)) on the third day after streptozotocin injection to confirm the development of diabetes. We divide the model rats randomly into three groups:model group (MG), irbesartan group (XG) and huoxue-huayu-tongluo Chinesemedicine group (ZG). Rats in XG were orally administered with15mg·kg~(-1)·d~(-1)irbesartan, while the ZY group with decoction-free herbal granules of salvia,chuanxiong, earthworm, leech and Scorpio by1.0g·kg~(-1)·d~(-1), and the left groupswith the same dose of Sodium Chloride everyday for sixteen weeks.
At the end of4Weeks,8Weeks,12Weeks and16Weeks, rats wereplaced into metabolic cages for24hours to collected24h urine. Rats′waterintake, diet intake, urine output, and body weight were measured.
FBG and HbA1c were measured with sampling from tail vein. Then ratswere anesthesiaed with chloraldurate and blood sample was collected fromaortaventralis and serum was separated. Serum creatinine, urinary creatinine,serum lipid (including total choleaterol, triglyceride, high density lipidcholeaterol and very low density lipid choleaterol) were measured byfull-automatic biochemistry checker. Renal weight was weighed. Kidneyindexes (KI) were calculated. Renal cortex samples were stored for rulemicroscopy or transmission electron microscopy and glomerular basementmenbrane thickness was measured by transmission electron microscopes.
2Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin andthe expression of p-cadherin in DN rats
2.1Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin inDN rats
At the end of4Weeks,8Weeks,12Weeks and16Weeks, rats wereplaced into metabolic cages for24hours to collected24h urine. Urine sampleswere centrifuged at speed of3500rpm for10minutes, and supernate ofsamples were stored at-20℃. Urinary albumin concentration was measuredby Beckman cx7full automatic biochemical analyzer.
2.2Effects of huoxue-huayu-tongluo Chinese medicine on the expression ofp-cadherin in DN rats
Renal cortex samples were cut fast and stored at-80℃. We extract totalRNA in renal tissue and use reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (Real-TimePCR) to detect expression of p-cadherin mRNA in renal tissue. Also, theexpression of p-cadherin protein was detected by Western blot.
3Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein andfactors associated with renal fibrosis in DN rats
3.1Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein inDN rats
According to the special instrument manual, urinary protein in24hoursin rats in each group was determined by using Beckman cx7automaticbiochemical analyzer.
3.2Effects of huoxue-huayu-tongluo Chinese medicine on factors associatedwith renal fibrosis in DN rats
Renal cortex samples were cut fast and expression of BMP-7and TGF-β1protein and mRNA were detected using immunohistochemistry, Western blot,reverse transcriptase polymerase chain reaction and fluorescence quantitativereal-time polymerase chain reaction. We also detected the expression of OPNmRNA by RT-PCR and Real-Time PCR and the expression of Smad7proteinby Western-blot. Correlation of BMP-7and TGF-β1also analyzed.
4Effects of huoxue-huayu-tongluo serum containing medicine and drugmonomer-tanshinone II A on the proliferation of podocyte
Mice living system condition of podocyte were cultured in vitro using thetechnique of cell culture and observing the effect on the proliferation ofpodocyte of huoxue-huayu-tongluo serum containing medicine and theconcentration of the monomer-tanshinone II A, using MTT method.
5Statistical analyses
All results were expressed as±s. Repeated measures and multivariateanalysis of variance (ANOVA) process of the general linear model was used toidentify comparison among different groups and different measure timepairwise; One-Way ANOVA was used to compare mean values among groups,and SNK to compare mean values between each two group. And the datawhich was not conforming to the normal distribution was analyzed by rank sum test, then One-Way ANOVA to rank. Correlations between variables usePerson linear correlation analysis. Analysis was carried out using SAS V8.P-value of <0.05was considered to be statistically significant.
Results:
1Renal protective effects of huoxue-huayu-tongluo Chinese medicine on DNrats
1.1Comparison of FBG in each group at different time
Compared with NG, FBG in MG, ZG and XG increased notably (P<0.05)at different time. Compared with MG, there was no significant difference ofFBG in ZG and XG (P>0.05). Furthermore, within4months of MG, ZG andXG in rat FBG levels have been stable, no obvious change.
1.2Comparison of body weight in each group at different time
Body weight of NG rats gradually increased. Compared with NG, bodyweight of MG, ZG and XG rats decreased notably (P<0.05) at different time.Compared with MG, body weight of rats in ZG and XG decline eased. Andbody weight of ZG and XG rats was significantly higher than that of MG from8W, there was significant difference (P<0.05). Furthermore, there was nosignificant difference of body weight in ZG and XG at different time (P>0.05).
1.3Comparison of water intake in each group at different time
Compared with NG, water-intake in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG, water-intake of ZG rats wassignificantly lower than that of MG from12W (P<0.05), however, there wasno significant difference of water-intake between ZG and XG rats (P>0.05).
1.4Comparison of food intake in each group at different time
Compared with NG, diet-intake in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG, diet-intake in ZG and XGincreased less, but the effect is slow. Until12W the increase of diet-intake inZG and XG began to improve significantly (P<0.05). However, there was nosignificant difference of water-intake between ZG and XG rats (P>0.05).
1.5Comparison of urine output in each group at different time
Compared with NG, urine-output in MG, ZG and XG increased notably (P<0.05) at different time. Compared with MG, urine-output in ZG increasedless, but the effect is slow. Until16W the increase of urine-output in ZG beganto improve significantly (P<0.05). And there was significant difference ofurine-output between ZG and XG rats (P<0.05).
1.6Comparison of HbA1c, KI, BUN, SCR, TP and ALB in each group
Compared with NG, HbA1c and KI in MG, ZG and XG increased notably(P<0.05). Compared with MG, there was no significant difference of FBG inZG and XG (P>0.05). Compared with NG, BUN in MG, ZG and XG ratsincreased notably (P<0.05), and compared with MG, BUN in ZG and XG ratsdecreased notably (P<0.05), and there was no significant difference of BUNbetween ZG and XG (P>0.05). Compared with NG, SCr in MG, ZG and XGrats decreased notably (P<0.05), and compared with MG, SCr in ZG and XGrats increased notably (P<0.05), and SCr in XG rats increased notablycompared with ZG (P<0.05).
1.7Comparison of serum lipid (including TC, TG, HDL, and VLDL) in eachgroup
There was no significant difference of TC and HDL in all groups(P>0.05). Compared with NG, TG and VLDL in MG, ZG and XG ratsincreased notably (P<0.05), and compared with MG, TG and VLDL in ZG andXG rats decreased notably (P<0.05), and there was no significant difference ofTG and VLDL between ZG and XG (P>0.05).
1.8Comparison of changes of pathomorphology observations in kidney ineach group
Observations under light microscope: in the normal group, there were noobvious hypertrophy of renal glomerulus, thickening of GBM and the changeof the MCs quantity. In model group, there were obvious hypertrophy of renalglomerulus, thickening of GBM, expansion of mesangial matrix andvacuolar/granular degeneration of renal tubular cell. In irbesartan group andZY groups, the pathomorphology changes above were improved comparedwith that of the model group. There was no obvious difference between twotreated groups.
Observations under transmission electron microscopy: In the normalgroup, the structure of glomerular capillary basement membrane was clear andcomplete, microcirculatary endothelial cell, foot processes was normal. Inmodel group, there were obvious thickening of GBM, the fusion of the footprocesses was extensive, microcirculatary endothelial cell confluence andwindow structure disappeared. In irbesartan group and ZY groups, thepathomorphology changes above were improved compared with that of themodel group. There was no obvious difference between two treated groups.
1.9Comparison of GBM thickness in each group
Compared with NG, GBM in MG, ZG and XG thickened notably(P<0.05). Compared with MG, the thickness of GBM in ZG and XG reducednotably (P<0.05), however, which in ZG reduced notably compared with XG(P<0.05).
2Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin andthe expression of p-cadherin in DN rats
2.1Comparison of urinary albumin in each group at different time
Compared with NG, U-Alb in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG, U-Alb in ZG and XGincreased less, but the effect is slow. Until12W to16W the increase of U-Albbegan to improve significantly (P<0.05). However, there was no significantdifference of U-Alb between ZG and XG rats (P>0.05).
2.2Comparison of expression of p-cadherin mRNA in renal tissue in eachgroup
Compared with NG, expression of p-cadherin mRNA in MG, ZG and XGdecreased notably (P<0.05). Compared with MG, expression of p-cadherinmRNA in ZG and XG induced notably (P<0.05) and There was no obviousdifference between two treated groups (P>0.05).
2.3Comparison of expression of p-cadherin protein in renal tissue in eachgroup
IHC staining showed: in NG, brown yellow particles took on a lineardistribution along glomerular basement membrane. Compared with NG, the brown yellow particles in MG significantly reduced. However, compared withMG, the brown yellow particles increased significantly in ZG and XG.
3Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein andand factors associated with renal fibrosis in DN rats
3.1Comparison of24h urine protein in each group at different time
Compared with NG,24h Upro in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG,24h Upro in ZG and XGincreased less, but the effect is slow. Until12W to16W the increase of24hUpro began to improve significantly (P<0.05). However, there was nosignificant difference of24h Upro between ZG and XG rats (P>0.05).
3.2Comparison of the expression of BMP-7protein and mRNA in each group
After detection with IHC, Western blot and Real-Time PCR, comparedwith NG, expression of BMP-7protein and mRNA in MG, ZG and XGdecreased notably (P<0.05). Compared with MG, expression of BMP-7protein and mRNA in ZG and XG increased notably (P<0.05). There was noobvious difference between two treated groups (P>0.05).
3.3Comparison of the expression of TGF-β1protein and mRNA in each group
After detection with IHC, Western-blot and Real-Time PCR, comparedwith NG, expression of TGF-β1protein and mRNA in MG, ZG and XGincreased notably (P<0.05). Compared with MG, expression of TGF-β1protein and mRNA in ZG and XG decreased notably (P<0.05). There was noobvious difference between two treated groups (P>0.05).
3.4Comparison of the expression of Smad7protein in each group
After detection with Western-blot, compared with NG, expression ofSmad7protein in MG, ZG and XG decreased notably (P<0.05). Comparedwith MG, expression of Smad7protein in ZG and XG increased notably(P<0.05). There was no obvious difference between two treated groups(P>0.05).
3.5Comparison of the expression of OPN mRNA in each group
After detection with Real-Time PCR, compared with NG, expression ofOPN mRNA in MG, ZG and XG increased notably (P<0.05). Compared with MG, expression of OPN mRNA in ZG and XG decreased notably (P<0.05).There was no obvious difference between two treated groups (P>0.05).
3.6Analysis of correlation of BMP-7and TGF-β1in MG
Analysis results showed that relationship between the expression ofBMP-7and TGF-β1in renal tissue was as follows: the correlation coefficientbetween the expression of BMP-7and TGF-β1in nucleic acid and proteinwere-0.9269and-0.9055. That means that there was a negative correlationbetween the expression of BMP-7and TGF-β1(P<0.01).
4Effects of huoxue-huayu-tongluo serum containing medicine and drugmonomer-tanshinone II A on the proliferation of podocyte
4.1Effects of huoxue-huayu-tongluo serum containing medicine on theproliferation of podocyte
There was no obvious difference between huoxue-huayu-tongluo serumcontaining medicine group and low glucose control group (P>0.05).Compared with high glucose control group, the proliferation of podocyteobviously promote in huoxue-huayu-tongluo serum containing medicine group(P<0.05).
4.2Effects of drug monomer-tanshinone II A on the proliferation of podocyte
There was no obvious difference between tanshinone II A group and lowglucose control group after incubation for12hours (P>0.05). Compared withlow glucose control group, tanshinone II A with1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed an obvious role in promoting proliferationafter incubation for24hours (P<0.05) and tanshinone II A with1×10~(-6)mol/L,1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed an obvious role inpromoting proliferation after incubation for36hours (P<0.05) and tanshinoneII A with all concentration developed an obvious role in promotingproliferation after incubation for48hours (P<0.05).
There was no obvious difference between tanshinone II A group and highglucose control group after incubation for12hours (P>0.05). Compared withhigh glucose control group, tanshinone II A with1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed an obvious role in promoting proliferation after incubation for24hours (P<0.05) and tanshinone II A with1×10-5mol/L,1×10~(-6)mol/L,1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed anobvious role in promoting proliferation after incubation for36hours (P<0.05)and tanshinone II A with all concentration developed an obvious role inpromoting proliferation after incubation for48hours (P<0.05).
Conclusions:
1A single high dose of STZ ip can repulate DN rat model succesfully.
2Huoxue-huayu-tongluo Chinese medicines could attenuate polydipsia,polyphagia, polyuria, and body weight reduction notably, as well as metabolicdisorder of lipid. It also could decrease U-Alb and24h Upro extenuate thepathological lesion attenuate glomerular hyrerfiltration and glomerularhypertrophy, thickened GBM, so, it has a renal protective role.
3Huoxue-huayu-tongluo Chinese medicines could up-regulate theexpression of p-cadherin protein and mRNA significantly, so as to decreasethe proteinuria, make further efforts to protect the kidney function and delaythe occurrence and development of glomerulosclerosis.
4Huoxue-huayu-tongluo Chinese medicines could up-regulate theexpression of BMP-7and Smad7, and could down-regulate the expression ofTGF-β1and OPN significantly, so as to decrease the accumulation ofextracellular matrix, make further efforts to delay the occurrence anddevelopment of glomerulosclerosis.
5Huoxue-huayu-tongluo serum containing medicine developed anobvious role in promoting proliferation of podocyte. Drug monomer-tanshinone II A also developed an obvious role in promoting proliferation ofpodocyte, which could enhance the ability to repair itself of podocyte, so as todecrease the proteinuria and make further efforts to protect the kidney function.This result was consistent with the overall experiments. Thus it gave furtherevidence from the cell biological mechanism of huoxue-huayu-tongluo herbsin the treatment of DN. And provide a basis for the application of traditionalChinese medicine monomer.
6To postulate the essence of DN according to the results of present study, it is blood stasis syndrome.
7Huoxue-huayu-tongluo Chinese medicines have a therapeuticequivalence compared with Western medicine.
DN主要的病理表现是肾小球毛细血管基底膜弥漫增厚，系膜基质增生，进而造成结节性肾小球硬化。临床表现则以蛋白尿为主，早期的表现为微量白蛋白尿，临床期的表现为大量蛋白尿。DN发生发展的病理机制非常复杂，包括了蛋白的非酶糖基化、氧化应激以及足细胞损伤等等，是多种因素、多条通路协同作用的结果，因而针对DN的治疗也必须是多因素、多靶点的。目前，现代医学防治DN的措施主要有以下几方面：严格控制血糖、血压，纠正血脂紊乱，血管紧张素转化酶抑制剂(angiotensionconverting enzyme inhibitors, ACEI)或血管紧张素受体拮抗剂(angiotensinreceptor blockers, ARBs)的应用等，尚无控制DN的特异性药物，且西药副作用较多，在控制临床症状方面难使患者满意。而中医药以其独特的整体调节与辨证论治体系，通过三因制宜的个性化治疗方案，可能对DN发生发展的多个环节起到干预作用，在临床防治DN中显示出巨大的潜力和广阔的应用前景。
课题组结合多年临床治疗DN的经验和中医学病因病机理论，尤其是络病理论，认为DN主要的病机是瘀血阻于肾络，治疗当活血化瘀通络，采用丹参、川芎、地龙、水蛭、全蝎等中药配伍组成活血化瘀通络方，临床观察和前期实验表明该方对DN有较好疗效。本研究采用一次性大剂量链脲佐菌素(Streptozocin, STZ)腹腔注射复制DN大鼠模型，以活血化瘀通络中药进行干预，进行整体研究，观察其对DN模型大鼠肾脏的保护作用；并且采用小鼠条件永生系足细胞进行细胞实验，观察活血化瘀通络中药含药血清及药物单体丹参酮IIA对足细胞增殖的影响，进一步验证该复方中药的疗效，探讨其对DN的作用机制，并以效测证，为临床应用提供参考。
方法：
1活血化瘀通络中药对糖尿病肾病大鼠肾脏的保护作用
选取健康、雄性、4～5周龄，体重120g～150g，SD大鼠48只，适应性饲养1周后，检测血糖和尿蛋白，均为阴性者用于实验。随机分为正常组(normal group, NG)和造模组。造模组大鼠一次性腹腔注射STZ60mg·kg~(-1)，NG注射相应体积的枸橼酸缓冲液。72h后检测造模组大鼠的空腹血糖(fasting blood glucose, FBG)，三次连续随机尾静脉血糖≥16.7mmol·L~(-1)为DM模型成功。成模大鼠随机分为模型组(model group,MG)、厄贝沙坦组(Irbesartan group, XG)及活血化瘀通络中药组(huoxuehuayutongluo Chinese medicine group, ZG)。ZG给予活血化瘀通络中药免煎颗粒进行灌胃，给药量按照体型系数换算给药剂量的基本公式进行换算。分别按川芎0.96g·kg~(-1)·d~(-1)、丹参1.2g·kg~(-1)·d~(-1)、地龙0.8g·kg~(-1)·d~(-1)、水蛭0.48g·kg~(-1)·d~(-1)、全蝎0.48g·kg~(-1)·d~(-1)进行灌胃。厄贝沙坦组大鼠给予厄贝沙坦15mg·kg~(-1)·d~(-1)灌胃。每日给药一次，连续16周。于给药后的第四周末(4W)、第八周末(8W)、第十二周末(12W)、第十六周末(16W)，代谢笼收集24小时尿液，并测量大鼠的饮水量(water intake)、进食量(foodintake)、尿量(urine output)等。
给药16周末杀检动物，大鼠称体重(body weight, BW)，尾静脉取血检测FBG和糖化血红蛋白(HbA1c)。之后，水合氯醛麻醉大鼠，腹主动脉取血，分离血清，检测肝功能(总蛋白TP、白蛋白ALB)、肾功能(血肌酐SCr、尿素氮BUN)、血脂(总胆固醇TC、甘油三酯TG、高密度脂蛋白胆固醇HDL、极低密度脂蛋白胆固醇VLDL)。留取双肾，称重，计算肾脏指数(kidney index, KI)。分离皮质，FAA固定液固定，行光镜检查；4%戊二醛固定，行透射电镜检查，从透射电镜图片上测量肾小球基底膜(glomerular basement membrane, GBM)厚度。
2活血化瘀通络中药对糖尿病肾病大鼠尿微量白蛋白及足细胞裂孔膜蛋白p-cadherin的影响
2.1活血化瘀通络中药对糖尿病肾病大鼠尿微量白蛋白的影响
于给药后的第四周末(4W)、第八周末(8W)、第十二周末(12W)、第十六周末(16W)，代谢笼收集24小时尿液，记录24h尿量，尿液经3000转/分,离心10分钟，取上清，-20℃保存。采用Beckman cx7全自动生化分析仪检测尿微量白蛋白(urinary microalbumin, U-Alb)含量。
2.2活血化瘀通络中药对糖尿病肾病大鼠p-cadherin的影响
取材时，快速留取肾皮质，-80℃保存。提取肾组织总RNA，采用反转录酶聚合酶链式反应(RT-PCR)和实时定量聚合酶链式反应(Real-TimePCR)检测肾组织p-cadherin mRNA的表达。采用免疫组织化学法(Immunohistochemistry，IHC)检测肾组织p-cadherin蛋白的表达。
3活血化瘀通络中药对糖尿病肾病大鼠24小时尿蛋白及肾脏纤维化相关因子的影响
3.1活血化瘀通络中药对糖尿病肾病大鼠24小时尿蛋白的影响
于给药后的第四周末(4W)、第八周末(8W)、第十二周末(12W)、第十六周末(16W)，代谢笼收集24小时尿液，记录24h尿量，尿液经3000转/分,离心10分钟，取上清，-20℃保存。采用Beckman cx7全自动生化分析仪测定24小时尿蛋白量(24h Upro)。
3.2活血化瘀通络中药对糖尿病肾病大鼠肾脏纤维化相关因子的影响
取材时，快速留取肾皮质，-80℃保存。检测与肾脏纤维化相关的细胞因子的表达。采用蛋白印迹法(Western blot)、IHC法及Real-Time PCR法检测肾组织BMP-7及TGF-β1蛋白和mRNA的表达；采用Real-TimePCR法检测肾组织OPN mRNA的表达；采用Western blot法检测肾组织Smad7蛋白的表达；并分析BMP-7及TGF-β1之间的相关性。
4活血化瘀通络中药含药血清及药物单体——丹参酮II A对小鼠条件永生系足细胞增殖的影响
体外细胞培养技术培养小鼠条件永生系足细胞，给予大鼠的活血化瘀通络中药含药血清及各浓度的药物单体——丹参酮II A干预后，以MTT法检测足细胞增殖情况。
5统计学方法
所有资料以±s表示，各组间不同观察点空腹血糖、体重、饮水量、进食量、尿量、24小时尿蛋白和尿微量白蛋白采用重复测量设计资料的单变量方差分析进行比较，各观察点进行组间两两比较；其他数据多组间比较采用One-Way ANOVA进行分析，进一步组间两两比较采用SNK法进行；对于不符合正态分布的资料先进行秩和检验，再对秩次进行One-Way ANOVA；变量间的相关性采用Person直线相关分析。所有数据分析应用SAS V8软件进行，以P<0.05为差异有显著性。
结果：
1活血化瘀通络中药对糖尿病肾病大鼠肾脏的保护作用
1.1各组大鼠不同观察点空腹血糖(FBG)的比较
与NG比较，MG、ZG和XG大鼠FBG明显升高(P<0.05)；而与MG比较，ZG和XG大鼠FBG无统计学差异(P>0.05)；且在4个月内MG、ZG和XG大鼠FBG水平一直稳定，没有明显变化(P>0.05)。
1.2各组大鼠不同观察点体重(BW)的比较
NG大鼠体重逐渐增加；与NG比较，MG、ZG、XG大鼠BW明显减轻(P<0.05)，且MG大鼠BW呈现逐渐下降趋势。而与MG比较，ZG和XG大鼠BW下降有所减轻，且ZG和XG大鼠从8W开始体重明显高于MG，差异有显著性(P<0.05)；治疗组间比较，在各观察点大鼠体重无统计学差异(P>0.05)。
1.3各组大鼠不同观察点饮水量(water intake)的比较
与NG比较，MG、XG和ZG大鼠water intake显著增加(P<0.05)；且MG大鼠water intake随时间呈逐渐增加趋势。与MG比较，ZG大鼠water intake从12W开始显著减少(P<0.05)；XG大鼠water intake无显著性差异(P>0.05)。
1.4各组大鼠不同观察点进食量(food intake)的比较
与NG比较，MG、ZG和XG大鼠food intake显著增加(P<0.05)；与MG比较，ZG和XG大鼠food intake增加有所改善，但显效缓慢，从12W开始food intake增加显著改善(P<0.05)。治疗组间比较，在各观察点foodintake无统计学差异(P>0.05)。
1.5各组大鼠不同观察点尿量(urine output)的比较
与NG比较，MG、ZG和XG大鼠urine output显著增加(P<0.05)；MG urine output呈逐步增加趋势。与MG比较，ZG大鼠urine output增加趋势趋缓，且在16W时显示出明显改善(P<0.05)；XG urine output呈逐步增加趋势，且于8W时高于MG。两治疗组间比较，第8W时ZG urine output显著少于XG，有统计学差异(P<0.05)。
1.6各组大鼠HbA1c、KI、BUN、SCr、TP和ALB的比较
与NG相比，MG、ZG和XG大鼠HbA1c、KI显著升高(P<0.05)；与MG相比，ZG和XG大鼠HbA1c、KI无统计学差异(P>0.05)。与NG比较，MG、ZG和XG大鼠BUN均显著升高(P<0.05)，而与MG比较，ZG和XG大鼠BUN显著降低(P<0.05)，两治疗组间比较，无统计学差异(P>0.05)。与NG比较，MG、ZG和XG大鼠SCr均显著降低(P<0.05)，而与MG比较，ZG和XG大鼠SCr显著升高(P<0.05)，两治疗组间比较，XG大鼠SCr显著升高(P<0.05)。与NG相比，MG大鼠TP和ALB显著降低(P<0.05)；与MG相比，ZG和XG大鼠TP和ALB显著升高(P<0.05)；两治疗组间比较，无统计学差异(P>0.05)。
1.7各组大鼠血脂(TC、TG、HDL、VLDL)的比较
TC和HDL：各组大鼠之间无显著性差异(P>0.05)。与NG比较，MG大鼠TG显著升高(P<0.05)，而与MG比较，ZG和XG大鼠TG显著降低(P<0.05)，两治疗组间比较，无统计学差异(P>0.05)。与NG比较，MG大鼠VLDL显著升高(P<0.05)，而与MG比较，ZG和XG大鼠VLDL显著降低(P<0.05)，两治疗组间比较，无统计学差异(P>0.05)。
1.8各组大鼠肾组织病理形态学改变的比较
光镜观察：NG大鼠肾脏肾小球结构清晰完整，未见明显肥大及萎缩，基底膜无增厚，系膜无增生。MG大鼠可见肾小球明显肥大，肾小囊腔呈裂隙状，肾小球基底膜明显增厚，系膜基质增多，系膜区增宽，部分肾小管上皮细胞空泡样变；与MG比较，ZG和XG大鼠肾脏病变有一定程度的改善，两治疗组间差异不明显。
电镜观察：NG大鼠肾组织结构正常，肾小球毛细血管基底膜(GBM)结构清晰，均匀无增厚，上皮足突分布均匀，足突排列整齐、系膜细胞无增殖、系膜基质无增生。MG可见GBM结构欠清晰、均匀弥漫增厚，足细胞足突增宽或大片融合，内皮细胞高度融合，窗口大部分消失。与MG比较，ZG和XG病理改变均有所减轻，GBM结构较清晰，足突节段融合。两治疗组间差异不明显。
1.9各组大鼠GBM厚度的比较
与NG比较，MG、ZG和XG大鼠GBM明显增厚(P<0.05)；与MG比较，ZG和XG大鼠GBM明显减薄(P<0.05)；与XG比较，ZG大鼠GBM明显减薄(P<0.05)。
2活血化瘀通络中药对糖尿病肾病大鼠尿微量白蛋白及足细胞裂孔膜蛋白p-cadherin的影响
2.1各组大鼠不同观察点尿微量白蛋白(U-Alb)的比较
与NG比较，MG、ZG和XG大鼠U-Alb显著增加(P<0.05)。与MG比较，ZG和XG大鼠U-Alb增加趋势趋缓；且从8W至16W各时间点均显示出明显改善(P<0.05)。两治疗组间比较，在各观察点U-Alb无统计学差异(P>0.05)。
2.2各组大鼠肾组织中p-cadherin mRNA表达的比较
与NG比较，MG p-cadherin mRNA表达水平明显降低(P<0.05)；与MG比较，ZG和XG p-cadherin mRNA表达水平均明显升高(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
2.3各组大鼠肾组织中p-cadherin蛋白表达的比较
IHC染色显示：NG p-cadherin蛋白表达较强，主要呈线性分布于肾小球基底膜处，棕黄色颗粒呈深染。与NG比较，MG棕黄色颗粒明显减少。与MG比较，ZG和XG棕黄色颗粒不同程度地增多。
3活血化瘀通络中药对糖尿病肾病大鼠24小时尿蛋白及肾脏纤维化相关因子的影响
3.1各组大鼠不同观察点24h Upro的比较
与NG比较，MG、ZG和XG大鼠24h Upro显著增加(P<0.05)。与MG比较，ZG和XG大鼠24h Upro增加趋势趋缓，且在12W、16W时显示出明显改善(P<0.05)。两治疗组间比较，在各观察点24h Upro无统计学差异(P>0.05)。
3.2各组大鼠肾组织中BMP-7蛋白及mRNA表达的比较
IHC染色显示：在正常及DN大鼠肾脏均有BMP-7蛋白表达，而在NG组呈强阳性表达，主要位于肾小管和集合管，呈胞浆表达阳性，胞浆呈棕黄色颗粒状。IHC、Western-blot及Real-Time PCR法检测结果均显示：与NG大鼠比较，MG、ZG和XG BMP-7蛋白和mRNA表达水平明显降低(P<0.05)；与MG比较，ZG和XG BMP-7蛋白和mRNA表达水平均明显增加(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.3各组大鼠肾组织中TGF-β1蛋白及mRNA的表达
IHC染色显示大鼠肾脏TGF-β1蛋白在肾小球及肾小管间质均有表达。NG大鼠肾脏TGF-β1表达量很少，着色浅。MG大鼠肾脏TGF-β1蛋白表达明显增加，呈强阳性表达，着色深。ZG和XG大鼠肾脏TGF-β1蛋白呈弱阳性表达，介于NG和MG之间。Western-blot及Real-Time PCR法检测结果显示：与NG比较，模型组TGF-β1蛋白和mRNA表达水平明显升高(P<0.05)；与MG比较，TGF-β1蛋白和mRNA表达水平均明显降低(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.4各组大鼠肾组织中Smad7蛋白的表达
Western-blot结果显示：与NG比较，MG、ZG和XG Smad7蛋白的表达水平明显降低(P<0.05)；与MG比较，ZG和XG Smad7蛋白的表达水平明显升高(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.5各组大鼠肾组织OPN mRNA的表达
与NG比较，MG、ZG和XG OPN mRNA表达明显增加(P<0.05)；与MG比较，ZG和XG OPN mRNA表达均明显减少(P<0.05)，两治疗组之间无统计学差异(P>0.05)。
3.6MG BMP-7和TGF-β1表达的相关性分析
分析结果显示：肾组织BMP-7和TGF-β1表达的关系：两者在核酸和蛋白表达的相关系数r分别为-0.9269和-0.9055，说明两者的表达呈负相关(P<0.05)。
4活血化瘀通络中药含药血清及药物单体——丹参酮II A对小鼠条件永生系足细胞增殖的影响
4.1活血化瘀通络中药含药血清对足细胞增殖的影响
与低糖对照组相比，活血化瘀通络中药含药血清组在任何时间段均没有统计学差异(P>0.05)；高糖对照组在36h出现了增殖抑制作用，有统计学差异(P<0.05)；与高糖对照组相比，活血化瘀通络中药含药血清组只在48h出现了明显的促进增殖作用(P<0.05)。
4.2丹参酮II A对足细胞增殖的影响
与低糖对照组相比，足细胞在含有不同浓度(1×10~(-8)mol/L～1×10-4mol/L)Tanshinone II A的培养基中：培养12h后，各浓度未出现明显的促增殖作用(P>0.05)；培养24h后，1×10~(-7)mol/L、1×10~(-8)mol/L浓度出现了明显的促进增殖的作用(P<0.05)；培养36h后，1×10~(-6)mol/L、1×10~(-7)mol/L、1×10~(-8)mol/L浓度出现了明显的促进增殖的作用(P<0.05)；培养48h后，各浓度均有明显的促进增殖作用(P<0.05)。
与高糖对照组相比，足细胞在含有不同浓度(1×10~(-8)mol/L～1×10-4mol/L)Tanshinone II A的培养基中：培养12h后，各浓度未出现明显的促增殖作用(P>0.05)；培养24h后，1×10~(-7)mol/L、1×10~(-8)mol/L浓度出现了明显的促进增殖的作用(P<0.05)；培养36h后，1×10-5mol/L、1×10~(-6)mol/L、1×10~(-7)mol/L、1×10~(-8)mol/L各浓度均出现了明显的促进增殖的作用(P<0.05)；培养48h后，各浓度均有明显的促进增殖作用(P<0.05)。
结论：
1一次性大剂量STZ腹腔注射可成功复制DN大鼠模型。
2活血化瘀通络中药能显著改善DN大鼠一般状况，“三多一少”表现及脂代谢紊乱，减少尿蛋白排泄，保护肾功能，减轻肾脏病理损害及肾小球肥大和肾小球基底膜增厚，具有肾保护作用。
3活血化瘀通络中药能明显上调p-cadherin蛋白和mRNA的表达，从而减少蛋白尿的产生，进而保护肾功能，延缓肾小球硬化的发生和发展。
4活血化瘀通络中药能明显减少DN大鼠24小时尿蛋白的产生，并能上调BMP-7和Smad7的表达、明显下调TGF-β1和OPN的表达，从而减少细胞外基质堆积，进而延缓肾脏纤维化和肾小球硬化的发展。
5活血化瘀通络中药含药血清对足细胞具有促进增殖作用；丹参酮IIA各浓度对于足细胞具有明显的促进增殖的作用，从而增强足细胞的自我修复能力，进而减少蛋白尿的产生，保护肾功能。这一结果与本实验中整体研究结果一致，从而从细胞生物学上对活血化瘀通络中药治疗DN的可能作用机制予以了进一步的佐证，并对中药单体的应用提供了依据。
6以效测证，从本研究结果可以反证DN的证候属于瘀血阻络证。
7活血化瘀通络中药与西药对照药厄贝沙坦的疗效相当。
Objective: Diabetic nephropathy (DN) is one of the most commonchronic complications of diabetes mellitus (DM), and also one of main causesleading to death. Diabetic nephropathy has become the first primary diseasedeveloped chronic renal failure. In our country, with the rapid increase of theincidence of diabetes, end-stage renal failure is increasing caused by DN.Expected in the next20years DN will become the most important cause ofend-stage renal failure, but so far there is no effective method to prevent theoccurrence and development of DN, which has become an urgent problem.Furthermore, DN has great impact on life quality and prognosis of the patientsand increases the suffering of patients. So, it has important significance toexplore the pathogenesis of DN and effective methods for prevention of DN.
The main pathological manifestation of DN is glomerular capillarybasement membrane thickening, mesangial matrix proliferation and nodularglomerulosclerosis. The main clinical manifestation of DN is proteinuria,which is microalbuminuria at early manifestations and proteinuria at clinicalperformance. Pathogenesis of diabetic nephropathy is very complex, includingprotein non-enzymatic glycation, oxidative stress and podocyte injury and soon. DN is the result of multiple factors and multiple paths synergy. Therefore,treatment for diabetic nephropathy must also be multiple factor, multipletargets. At present, the modern medical prevention and control measures ofDN are mainly in the following aspects: strict control of blood glucose, bloodpressure, lipid disorders correction and application of ACEI and ARB. There isno specific drug for controlling DN. And western medicine has more sideeffects, which can not satisfy the patients in the control of clinical symptoms.However, Chinese medicine may develop effect on many aspects of DN andshow great potential and broad application prospect in the clinical prevention and treatment of DN with its unique overall adjustment, syndromedifferentiation system and three for individualized treatment plan system.
Our research group arrival at a conclusion that the pathogenesis of DN ismainly blood stasis in the kidney collateral combined with years of clinicaltreatment experience of DN and TCM etiology and pathogenesis theory,especially the collateral disease theory. And the treatment should be adoptedhuoxue-huayu-tongluo therapy. We compose huoxue-huayu-tongluo Chineserecipe using salvia, chuanxiong, earthworm, leech, Scorpio and so on, whichadopting compatibility of traditional Chinese medicine. Clinical observationand preliminary experiments show that the recipe has a good effect on DN.This study adopts the model of DN rats with a single high dose streptozotocinintraperitoneal injection. First of all, we make an overall study on theprotective effect on the DN model in kidney of rats of huoxue-huayu-tongluoChinese medicine. Secondly, we develop a cell experiment using mice livingsystem condition of podocyte. And we observe the effect ofhuoxue-huayu-tongluo Chinese medicine decoction drug-containing serum ofrats and drug monomer-tanshinone II A on podocyte, in order to furthervalidate the efficacy of the party, to investigate the mechanism of DN, and theeffect of measurement, and provide reference for clinical application.
Methods:
1Renal protective effects of huoxue-huayu-tongluo Chinese medicine on DNrats
Healthy male4～5weeks old50SD rats with body weight120g～150gafter adaptive feeding for1week were grouped randomly. And they were fedfor one week after making sure the rats’ urine protein negative. Ten rats werenormal control group. Other40rats were fed with high fat diet throughout thestudy. The animals were randomly divided into normal group (NG) for ten ratsand DM model group for forty rats. The DM model group was given a singledose of60mg·kg~(-1)streptozotocin intraperitonally and rats in NG were ip vehic.Blood glucose levels were measured for three times continuously (≥16.7mmol·L~(-1)) on the third day after streptozotocin injection to confirm the development of diabetes. We divide the model rats randomly into three groups:model group (MG), irbesartan group (XG) and huoxue-huayu-tongluo Chinesemedicine group (ZG). Rats in XG were orally administered with15mg·kg~(-1)·d~(-1)irbesartan, while the ZY group with decoction-free herbal granules of salvia,chuanxiong, earthworm, leech and Scorpio by1.0g·kg~(-1)·d~(-1), and the left groupswith the same dose of Sodium Chloride everyday for sixteen weeks.
At the end of4Weeks,8Weeks,12Weeks and16Weeks, rats wereplaced into metabolic cages for24hours to collected24h urine. Rats′waterintake, diet intake, urine output, and body weight were measured.
FBG and HbA1c were measured with sampling from tail vein. Then ratswere anesthesiaed with chloraldurate and blood sample was collected fromaortaventralis and serum was separated. Serum creatinine, urinary creatinine,serum lipid (including total choleaterol, triglyceride, high density lipidcholeaterol and very low density lipid choleaterol) were measured byfull-automatic biochemistry checker. Renal weight was weighed. Kidneyindexes (KI) were calculated. Renal cortex samples were stored for rulemicroscopy or transmission electron microscopy and glomerular basementmenbrane thickness was measured by transmission electron microscopes.
2Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin andthe expression of p-cadherin in DN rats
2.1Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin inDN rats
At the end of4Weeks,8Weeks,12Weeks and16Weeks, rats wereplaced into metabolic cages for24hours to collected24h urine. Urine sampleswere centrifuged at speed of3500rpm for10minutes, and supernate ofsamples were stored at-20℃. Urinary albumin concentration was measuredby Beckman cx7full automatic biochemical analyzer.
2.2Effects of huoxue-huayu-tongluo Chinese medicine on the expression ofp-cadherin in DN rats
Renal cortex samples were cut fast and stored at-80℃. We extract totalRNA in renal tissue and use reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence quantitative polymerase chain reaction (Real-TimePCR) to detect expression of p-cadherin mRNA in renal tissue. Also, theexpression of p-cadherin protein was detected by Western blot.
3Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein andfactors associated with renal fibrosis in DN rats
3.1Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein inDN rats
According to the special instrument manual, urinary protein in24hoursin rats in each group was determined by using Beckman cx7automaticbiochemical analyzer.
3.2Effects of huoxue-huayu-tongluo Chinese medicine on factors associatedwith renal fibrosis in DN rats
Renal cortex samples were cut fast and expression of BMP-7and TGF-β1protein and mRNA were detected using immunohistochemistry, Western blot,reverse transcriptase polymerase chain reaction and fluorescence quantitativereal-time polymerase chain reaction. We also detected the expression of OPNmRNA by RT-PCR and Real-Time PCR and the expression of Smad7proteinby Western-blot. Correlation of BMP-7and TGF-β1also analyzed.
4Effects of huoxue-huayu-tongluo serum containing medicine and drugmonomer-tanshinone II A on the proliferation of podocyte
Mice living system condition of podocyte were cultured in vitro using thetechnique of cell culture and observing the effect on the proliferation ofpodocyte of huoxue-huayu-tongluo serum containing medicine and theconcentration of the monomer-tanshinone II A, using MTT method.
5Statistical analyses
All results were expressed as±s. Repeated measures and multivariateanalysis of variance (ANOVA) process of the general linear model was used toidentify comparison among different groups and different measure timepairwise; One-Way ANOVA was used to compare mean values among groups,and SNK to compare mean values between each two group. And the datawhich was not conforming to the normal distribution was analyzed by rank sum test, then One-Way ANOVA to rank. Correlations between variables usePerson linear correlation analysis. Analysis was carried out using SAS V8.P-value of <0.05was considered to be statistically significant.
Results:
1Renal protective effects of huoxue-huayu-tongluo Chinese medicine on DNrats
1.1Comparison of FBG in each group at different time
Compared with NG, FBG in MG, ZG and XG increased notably (P<0.05)at different time. Compared with MG, there was no significant difference ofFBG in ZG and XG (P>0.05). Furthermore, within4months of MG, ZG andXG in rat FBG levels have been stable, no obvious change.
1.2Comparison of body weight in each group at different time
Body weight of NG rats gradually increased. Compared with NG, bodyweight of MG, ZG and XG rats decreased notably (P<0.05) at different time.Compared with MG, body weight of rats in ZG and XG decline eased. Andbody weight of ZG and XG rats was significantly higher than that of MG from8W, there was significant difference (P<0.05). Furthermore, there was nosignificant difference of body weight in ZG and XG at different time (P>0.05).
1.3Comparison of water intake in each group at different time
Compared with NG, water-intake in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG, water-intake of ZG rats wassignificantly lower than that of MG from12W (P<0.05), however, there wasno significant difference of water-intake between ZG and XG rats (P>0.05).
1.4Comparison of food intake in each group at different time
Compared with NG, diet-intake in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG, diet-intake in ZG and XGincreased less, but the effect is slow. Until12W the increase of diet-intake inZG and XG began to improve significantly (P<0.05). However, there was nosignificant difference of water-intake between ZG and XG rats (P>0.05).
1.5Comparison of urine output in each group at different time
Compared with NG, urine-output in MG, ZG and XG increased notably (P<0.05) at different time. Compared with MG, urine-output in ZG increasedless, but the effect is slow. Until16W the increase of urine-output in ZG beganto improve significantly (P<0.05). And there was significant difference ofurine-output between ZG and XG rats (P<0.05).
1.6Comparison of HbA1c, KI, BUN, SCR, TP and ALB in each group
Compared with NG, HbA1c and KI in MG, ZG and XG increased notably(P<0.05). Compared with MG, there was no significant difference of FBG inZG and XG (P>0.05). Compared with NG, BUN in MG, ZG and XG ratsincreased notably (P<0.05), and compared with MG, BUN in ZG and XG ratsdecreased notably (P<0.05), and there was no significant difference of BUNbetween ZG and XG (P>0.05). Compared with NG, SCr in MG, ZG and XGrats decreased notably (P<0.05), and compared with MG, SCr in ZG and XGrats increased notably (P<0.05), and SCr in XG rats increased notablycompared with ZG (P<0.05).
1.7Comparison of serum lipid (including TC, TG, HDL, and VLDL) in eachgroup
There was no significant difference of TC and HDL in all groups(P>0.05). Compared with NG, TG and VLDL in MG, ZG and XG ratsincreased notably (P<0.05), and compared with MG, TG and VLDL in ZG andXG rats decreased notably (P<0.05), and there was no significant difference ofTG and VLDL between ZG and XG (P>0.05).
1.8Comparison of changes of pathomorphology observations in kidney ineach group
Observations under light microscope: in the normal group, there were noobvious hypertrophy of renal glomerulus, thickening of GBM and the changeof the MCs quantity. In model group, there were obvious hypertrophy of renalglomerulus, thickening of GBM, expansion of mesangial matrix andvacuolar/granular degeneration of renal tubular cell. In irbesartan group andZY groups, the pathomorphology changes above were improved comparedwith that of the model group. There was no obvious difference between twotreated groups.
Observations under transmission electron microscopy: In the normalgroup, the structure of glomerular capillary basement membrane was clear andcomplete, microcirculatary endothelial cell, foot processes was normal. Inmodel group, there were obvious thickening of GBM, the fusion of the footprocesses was extensive, microcirculatary endothelial cell confluence andwindow structure disappeared. In irbesartan group and ZY groups, thepathomorphology changes above were improved compared with that of themodel group. There was no obvious difference between two treated groups.
1.9Comparison of GBM thickness in each group
Compared with NG, GBM in MG, ZG and XG thickened notably(P<0.05). Compared with MG, the thickness of GBM in ZG and XG reducednotably (P<0.05), however, which in ZG reduced notably compared with XG(P<0.05).
2Effects of huoxue-huayu-tongluo Chinese medicine on urinary albumin andthe expression of p-cadherin in DN rats
2.1Comparison of urinary albumin in each group at different time
Compared with NG, U-Alb in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG, U-Alb in ZG and XGincreased less, but the effect is slow. Until12W to16W the increase of U-Albbegan to improve significantly (P<0.05). However, there was no significantdifference of U-Alb between ZG and XG rats (P>0.05).
2.2Comparison of expression of p-cadherin mRNA in renal tissue in eachgroup
Compared with NG, expression of p-cadherin mRNA in MG, ZG and XGdecreased notably (P<0.05). Compared with MG, expression of p-cadherinmRNA in ZG and XG induced notably (P<0.05) and There was no obviousdifference between two treated groups (P>0.05).
2.3Comparison of expression of p-cadherin protein in renal tissue in eachgroup
IHC staining showed: in NG, brown yellow particles took on a lineardistribution along glomerular basement membrane. Compared with NG, the brown yellow particles in MG significantly reduced. However, compared withMG, the brown yellow particles increased significantly in ZG and XG.
3Effects of huoxue-huayu-tongluo Chinese medicine on24h urine protein andand factors associated with renal fibrosis in DN rats
3.1Comparison of24h urine protein in each group at different time
Compared with NG,24h Upro in MG, ZG and XG increased notably(P<0.05) at different time. Compared with MG,24h Upro in ZG and XGincreased less, but the effect is slow. Until12W to16W the increase of24hUpro began to improve significantly (P<0.05). However, there was nosignificant difference of24h Upro between ZG and XG rats (P>0.05).
3.2Comparison of the expression of BMP-7protein and mRNA in each group
After detection with IHC, Western blot and Real-Time PCR, comparedwith NG, expression of BMP-7protein and mRNA in MG, ZG and XGdecreased notably (P<0.05). Compared with MG, expression of BMP-7protein and mRNA in ZG and XG increased notably (P<0.05). There was noobvious difference between two treated groups (P>0.05).
3.3Comparison of the expression of TGF-β1protein and mRNA in each group
After detection with IHC, Western-blot and Real-Time PCR, comparedwith NG, expression of TGF-β1protein and mRNA in MG, ZG and XGincreased notably (P<0.05). Compared with MG, expression of TGF-β1protein and mRNA in ZG and XG decreased notably (P<0.05). There was noobvious difference between two treated groups (P>0.05).
3.4Comparison of the expression of Smad7protein in each group
After detection with Western-blot, compared with NG, expression ofSmad7protein in MG, ZG and XG decreased notably (P<0.05). Comparedwith MG, expression of Smad7protein in ZG and XG increased notably(P<0.05). There was no obvious difference between two treated groups(P>0.05).
3.5Comparison of the expression of OPN mRNA in each group
After detection with Real-Time PCR, compared with NG, expression ofOPN mRNA in MG, ZG and XG increased notably (P<0.05). Compared with MG, expression of OPN mRNA in ZG and XG decreased notably (P<0.05).There was no obvious difference between two treated groups (P>0.05).
3.6Analysis of correlation of BMP-7and TGF-β1in MG
Analysis results showed that relationship between the expression ofBMP-7and TGF-β1in renal tissue was as follows: the correlation coefficientbetween the expression of BMP-7and TGF-β1in nucleic acid and proteinwere-0.9269and-0.9055. That means that there was a negative correlationbetween the expression of BMP-7and TGF-β1(P<0.01).
4Effects of huoxue-huayu-tongluo serum containing medicine and drugmonomer-tanshinone II A on the proliferation of podocyte
4.1Effects of huoxue-huayu-tongluo serum containing medicine on theproliferation of podocyte
There was no obvious difference between huoxue-huayu-tongluo serumcontaining medicine group and low glucose control group (P>0.05).Compared with high glucose control group, the proliferation of podocyteobviously promote in huoxue-huayu-tongluo serum containing medicine group(P<0.05).
4.2Effects of drug monomer-tanshinone II A on the proliferation of podocyte
There was no obvious difference between tanshinone II A group and lowglucose control group after incubation for12hours (P>0.05). Compared withlow glucose control group, tanshinone II A with1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed an obvious role in promoting proliferationafter incubation for24hours (P<0.05) and tanshinone II A with1×10~(-6)mol/L,1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed an obvious role inpromoting proliferation after incubation for36hours (P<0.05) and tanshinoneII A with all concentration developed an obvious role in promotingproliferation after incubation for48hours (P<0.05).
There was no obvious difference between tanshinone II A group and highglucose control group after incubation for12hours (P>0.05). Compared withhigh glucose control group, tanshinone II A with1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed an obvious role in promoting proliferation after incubation for24hours (P<0.05) and tanshinone II A with1×10-5mol/L,1×10~(-6)mol/L,1×10~(-7)mol/L and1×10~(-8)mol/L concentration developed anobvious role in promoting proliferation after incubation for36hours (P<0.05)and tanshinone II A with all concentration developed an obvious role inpromoting proliferation after incubation for48hours (P<0.05).
Conclusions:
1A single high dose of STZ ip can repulate DN rat model succesfully.
2Huoxue-huayu-tongluo Chinese medicines could attenuate polydipsia,polyphagia, polyuria, and body weight reduction notably, as well as metabolicdisorder of lipid. It also could decrease U-Alb and24h Upro extenuate thepathological lesion attenuate glomerular hyrerfiltration and glomerularhypertrophy, thickened GBM, so, it has a renal protective role.
3Huoxue-huayu-tongluo Chinese medicines could up-regulate theexpression of p-cadherin protein and mRNA significantly, so as to decreasethe proteinuria, make further efforts to protect the kidney function and delaythe occurrence and development of glomerulosclerosis.
4Huoxue-huayu-tongluo Chinese medicines could up-regulate theexpression of BMP-7and Smad7, and could down-regulate the expression ofTGF-β1and OPN significantly, so as to decrease the accumulation ofextracellular matrix, make further efforts to delay the occurrence anddevelopment of glomerulosclerosis.
5Huoxue-huayu-tongluo serum containing medicine developed anobvious role in promoting proliferation of podocyte. Drug monomer-tanshinone II A also developed an obvious role in promoting proliferation ofpodocyte, which could enhance the ability to repair itself of podocyte, so as todecrease the proteinuria and make further efforts to protect the kidney function.This result was consistent with the overall experiments. Thus it gave furtherevidence from the cell biological mechanism of huoxue-huayu-tongluo herbsin the treatment of DN. And provide a basis for the application of traditionalChinese medicine monomer.
6To postulate the essence of DN according to the results of present study, it is blood stasis syndrome.
7Huoxue-huayu-tongluo Chinese medicines have a therapeuticequivalence compared with Western medicine.
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12Yoshikawa M, Hishikawa K, Marumo T, et al. Inhibition of histonedeacetylase activity suppresses epithelial-to-mesenchymal transitioninduced by TGF-beta1in human renal epithelial cells. J Am Soc Nephrol,2007,18(1):58-65
13Zeisberg M, Shah AA, Kalluri R. Bone morphogenic protein-7inducesmesenchymal to epithelial transition in adult renal fibroblasts andfacilitates regeneration of injured kidney. J Biol Chem,2005,280(9):8094-8100
14Veerasamy M, Nguyen TQ, Motazed R, et al. Differential regulation ofE-cadherin and alpha-smooth muscle actin by BMP7in human renalproximal tubule epithelial cells and its implication in renal fibrosis．Am JPhysiol Renal Physiol,2009,297(5):1238-1248
15肖炜,马云,魏连波. TGF-β/Smads信号通路与糖尿病肾病.国外医学泌尿系统分册,2005,25(5):676-679
16Nakao A，Afrakhte M，Moren A，et al．Identification of Smad7, aTGF-beta-inducible antagonist of TGF-beta signaling．Nature，1997，389:631-635
17Ka SM, Yeh YC, Huang XR, et al. Kidney-targeting Smad7gene transferinhibits renal TGF-β/MAD homologue (SMAD) and nuclear factorκB(NF-κB) signalling pathways, and improves diabetic nephropathy in mice.Diabetologia,2012,55:509-519
18Chen HY，Huang XR，Wang W，et al． The protective role of Smad7indiabetic kidney disease: mechanism and therapeutic potential．Diabetes,2011,60:590-601
1陈立平,周巧玲等.足细胞超微结构及其相关分子表达变化在糖尿病肾病发病中的作用.中南大学学报(医学版).2007,32(4):620-625
2Jalako H. Pathogenesis of proteinuria: lessons learned from nephrin andpodocin. Pediatr Nephrol,2003,18(6):487-491
3李仪奎，吴健宇.血清药理实验中采血时间的通法方案.中国药理学通报,1999,15(6):569-570
4李仪奎.中药血清药理学实验方法的若干问题.中药新药与临床药理.1999,10(2):95-98
5White KE. Research into the glomerular podocyte-is it relevant to diabeticnephropathy. Diabet Med.2006,23(7):715-719
6Sawai K, Mukoyama M, Mori K, et al. Redistirbution of connexin43expression in glomerular podocytes predicts poor renal prognosis inpatients with type2diabetes and overt nephropathy. Nephrol DialTransplant,2006,21(9):2472-2477
7Izzedine H, Brocheriou I, Eymard B, et al. Loss of podocyte dysferlinexpression is associated with minimal change nephropathy. Am J KidneyDis.2006,48(1):143-150
8余椿生,宋忠花.丹参.食品与药品,2005,7(12):72-74
9柴瑞震.丹参的药理研究近况.中国中医药科技,2003,10(6):390-392
10潘雪莲,周青山,杜大萍.丹参酮II A对糖尿病大鼠神经病理性疼痛的影响.中华麻醉学杂志.2006,26(8):692
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3Nicole SH, David AA. Regulation of tight junctions and loss of barrierfunction in pathophysiology. Biochemistry and Cell Biology Int,2004,36:1206-1237
4王瑞英，姚敏，王绵，等.2型糖尿病模型大鼠肾小管上皮细胞中TGF-β1的表达及与肾功能的关系.中国老年学杂志,2005,25:549-551
5Verzola D, Gandolfo MT，Ferrario F，et al．Apoptosis in the kidneys ofpatients with type Ⅱd iabetic nephyopathy． Kidney Int,2007,72:1262-1272
6Lan HY, Chung AC. Transforming growth factor-beta and Smads. ContribNephrol,2011,170:75-82
7Lan HY. Diverse roles of TGF-beta/Smads in renal fibrosis andinflammation. Int J Biol Sci,2011,7:1056-1067
8张芸,周蓉,沈杰等.糖尿病肾病蛋白尿与尿TGF-β1的关系.上海交通大学学报（医学版）.2009,3(29):358-359
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10Junnan Zeng, Yannong Dou, Jiaomei Guo, et al. Paeoniflorin of Paeonialactiflora prevents renal interstitial fibrosis induced by unilateral ureteralobstruction in mice. Phytomedicine,2013(20):753-759
11SAN-CHER CHEN, JINN-YUH GUH, TAI-DU LIN, et al. Gefitinibattenuates transforming growth factor-β1–activated mitogen-activatedprotein kinases and mitogenesis in NRK-49F cells. Translational Research,2011,11(158):214-224
12Ming-Ming Pan, Ming-Hui Zhang, Hai-Feng Ni, et al. Inhibition ofTGF-β1/Smad signal pathway is involved in the effect of Cordycepssinensis against renal fibrosis in5/6nephrectomy rats. Food and ChemicalToxicology,2013,4:1-8
13Xin Xin, Xian-HuiLi, Jian-ZhaoWu, et al. Pentamethylquercetinameliorates fibrosisindiabetic Goto-Kakizaki rat kidneys and mesangialcells with suppression of TGF-β/Smads signaling. European Journal ofPharmacology,2013,(713):6-15
14Fraser D, Bruskill N, Ito T, et al. Long-term exposure of proximal tubularepithelial cells to glucose induces transforming growth factor-β1synthesisvia an autocrine PDGF loop. American Journal of Pathology,2003,163:2565-2574
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1Reiser J, Kriz W, Kretzler M, et al. The glomerular slit diaphragm is amodified adherens junction. J Am Soc Nephrol,2000,11(1):1-8
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1Yang J, Dai C, Liu Y. Hepatocyte growth factor gene therapy andangiotensin Ⅱ blockade synergistically attenuate renal interstitial fibrosisin mice. J Am Soc Nephrol,2002,(13):2464-2477
2Klahr S, Morrissey J, Hruska K, et al. New approaches to delay theprogression of chronic renal failure. Kidney Int,2002, Suppl (80):23-26
3Kelynack KJ, Hewitson TD, Martic M, et al. Lovastatin downregulatesrenal myofibroblast function in vitro. Nephron,2002,91(4):701-707
4KanwarY S, Wada J, Sun L, et al. Diabetic nephropathy: mechanisms ofrenaldisease progression. Exp BiolMed,2008,233(1):4-11
5Sharma K, Ziyadeh FN. Biochemical events and cytokine interactionslinking glucose metabolism to the development of diabetic nephropathy.Semin Nephrol,1997,17(2):80-92
6Takahashi F, Takahashi K, Okazaki T, et a1. Role of osteopontin in thepathogenesis of bleomycin-induced pulmonary fibrosis. Am J Respir CellMol Biol,2001,(24):264-271
7Yanling Zhang, Qingxian Zhang. Bone morphogenetic protein-7andGremlin: New emerging therapeutic targets for diabetic nephropathy.Biochemical and Biophysical Research Communications.2009,(383):1-3
8杜月光,杨明华,柴可夫等.骨形成蛋白-7在糖尿病大鼠肾脏中的表达及意义.浙江中医药大学学报,2011,(35):559-562
9J. Massague, The transforming growth factor-beta family, Annu. Rev. CellBiol.1990,(6):597-641
10EITNER A, FRANK B, FlLOEGE A. Jurgen novel insights into renalfibrosis. Current Opinion in Nephrology. Hypertension,2003,12(3):227-252
11J. Morrissey, K. Hruska, G. Guo, S. et, al. Bone morphogenetic protein-7improves renal fibrosis and accelerates the return of renal function, J. Am.Soc. Nephrol.2002,13(Suppl.1):14-21
12Yoshikawa M, Hishikawa K, Marumo T, et al. Inhibition of histonedeacetylase activity suppresses epithelial-to-mesenchymal transitioninduced by TGF-beta1in human renal epithelial cells. J Am Soc Nephrol,2007,18(1):58-65
13Zeisberg M, Shah AA, Kalluri R. Bone morphogenic protein-7inducesmesenchymal to epithelial transition in adult renal fibroblasts andfacilitates regeneration of injured kidney. J Biol Chem,2005,280(9):8094-8100
14Veerasamy M, Nguyen TQ, Motazed R, et al. Differential regulation ofE-cadherin and alpha-smooth muscle actin by BMP7in human renalproximal tubule epithelial cells and its implication in renal fibrosis．Am JPhysiol Renal Physiol,2009,297(5):1238-1248
15肖炜,马云,魏连波. TGF-β/Smads信号通路与糖尿病肾病.国外医学泌尿系统分册,2005,25(5):676-679
16Nakao A，Afrakhte M，Moren A，et al．Identification of Smad7, aTGF-beta-inducible antagonist of TGF-beta signaling．Nature，1997，389:631-635
17Ka SM, Yeh YC, Huang XR, et al. Kidney-targeting Smad7gene transferinhibits renal TGF-β/MAD homologue (SMAD) and nuclear factorκB(NF-κB) signalling pathways, and improves diabetic nephropathy in mice.Diabetologia,2012,55:509-519
18Chen HY，Huang XR，Wang W，et al． The protective role of Smad7indiabetic kidney disease: mechanism and therapeutic potential．Diabetes,2011,60:590-601
1陈立平,周巧玲等.足细胞超微结构及其相关分子表达变化在糖尿病肾病发病中的作用.中南大学学报(医学版).2007,32(4):620-625
2Jalako H. Pathogenesis of proteinuria: lessons learned from nephrin andpodocin. Pediatr Nephrol,2003,18(6):487-491
3李仪奎，吴健宇.血清药理实验中采血时间的通法方案.中国药理学通报,1999,15(6):569-570
4李仪奎.中药血清药理学实验方法的若干问题.中药新药与临床药理.1999,10(2):95-98
5White KE. Research into the glomerular podocyte-is it relevant to diabeticnephropathy. Diabet Med.2006,23(7):715-719
6Sawai K, Mukoyama M, Mori K, et al. Redistirbution of connexin43expression in glomerular podocytes predicts poor renal prognosis inpatients with type2diabetes and overt nephropathy. Nephrol DialTransplant,2006,21(9):2472-2477
7Izzedine H, Brocheriou I, Eymard B, et al. Loss of podocyte dysferlinexpression is associated with minimal change nephropathy. Am J KidneyDis.2006,48(1):143-150
8余椿生,宋忠花.丹参.食品与药品,2005,7(12):72-74
9柴瑞震.丹参的药理研究近况.中国中医药科技,2003,10(6):390-392
10潘雪莲,周青山,杜大萍.丹参酮II A对糖尿病大鼠神经病理性疼痛的影响.中华麻醉学杂志.2006,26(8):692
11唐锦辉,占成业.丹参酮II A对大鼠肾间质纤维化的抑制作用.内科急危重症杂志,2008,14(1):1
1Yang J, Dai C, Liu Y. Hepatocyte growth factor gene therapy andangiotensin Ⅱ blockade synergisticallyattenuate renal interstitial fibrosisin mice. J Am Soc Nephrol,2002,13:2464-2477
2Nangaku M. Mechanisms of tubulointerstitial injury in the kidney:final-common pathways to end stage renal failure. Intern Med,2004,43:9-17
3Nicole SH, David AA. Regulation of tight junctions and loss of barrierfunction in pathophysiology. Biochemistry and Cell Biology Int,2004,36:1206-1237
4王瑞英，姚敏，王绵，等.2型糖尿病模型大鼠肾小管上皮细胞中TGF-β1的表达及与肾功能的关系.中国老年学杂志,2005,25:549-551
5Verzola D, Gandolfo MT，Ferrario F，et al．Apoptosis in the kidneys ofpatients with type Ⅱd iabetic nephyopathy． Kidney Int,2007,72:1262-1272
6Lan HY, Chung AC. Transforming growth factor-beta and Smads. ContribNephrol,2011,170:75-82
7Lan HY. Diverse roles of TGF-beta/Smads in renal fibrosis andinflammation. Int J Biol Sci,2011,7:1056-1067
8张芸,周蓉,沈杰等.糖尿病肾病蛋白尿与尿TGF-β1的关系.上海交通大学学报（医学版）.2009,3(29):358-359
9Liu YH. Epithelial to mesenchymal transition in renal fibrogenesis:pathologic significance, molecular mechanism, and therapeuticintervention. J Am Soc Nephrol,2004,15:1-12
10Junnan Zeng, Yannong Dou, Jiaomei Guo, et al. Paeoniflorin of Paeonialactiflora prevents renal interstitial fibrosis induced by unilateral ureteralobstruction in mice. Phytomedicine,2013(20):753-759
11SAN-CHER CHEN, JINN-YUH GUH, TAI-DU LIN, et al. Gefitinibattenuates transforming growth factor-β1–activated mitogen-activatedprotein kinases and mitogenesis in NRK-49F cells. Translational Research,2011,11(158):214-224
12Ming-Ming Pan, Ming-Hui Zhang, Hai-Feng Ni, et al. Inhibition ofTGF-β1/Smad signal pathway is involved in the effect of Cordycepssinensis against renal fibrosis in5/6nephrectomy rats. Food and ChemicalToxicology,2013,4:1-8
13Xin Xin, Xian-HuiLi, Jian-ZhaoWu, et al. Pentamethylquercetinameliorates fibrosisindiabetic Goto-Kakizaki rat kidneys and mesangialcells with suppression of TGF-β/Smads signaling. European Journal ofPharmacology,2013,(713):6-15
14Fraser D, Bruskill N, Ito T, et al. Long-term exposure of proximal tubularepithelial cells to glucose induces transforming growth factor-β1synthesisvia an autocrine PDGF loop. American Journal of Pathology,2003,163:2565-2574
15De Winter P, Leoni P, Abraham D. Connective tissue growth factor:structure-function relationships of a mosaic, multifunctional protein.Growth Factors,2008,26(2):80-91
16Black SA Jr, Trackman PC. Transforming growth factor-beta1(TGF-beta1)stimulates connective tissue growth factor (CCN2/CTGF) expression inhuman gingival fibroblasts through aRhoA-independent,Rac1/Cdc42-dependent mechanism: statins with forskolin blockTGF-beta1-induced CCN2/CTGF expression. J Biol Chem,2008,283(16):10835-10847
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