糖尿病肾病瘀血阻络证大鼠肾皮质ACE2-Ang-(1-7)-Mas轴的变化及化瘀通络中药的干预作用
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摘要
目的：糖尿病肾病（diabetic nephropathy，DN）是糖尿病（diabetesmellitus，DM）最常见的微血管并发症之一，是目前我国慢性肾衰竭的主要原因之一，探寻DN发病机制及有效防治措施的意义不言而喻。中医药在防控DN方面，已显现出较大优势。现代中医肾病学者大多认为，DN除有DM“气阴两虚”的病机外，其主要病机是瘀血阻于肾络，DN治疗多遵循活血化瘀通络法则，已成为中医肾病领域的共识。现代医学证实，肾素-血管紧张素系统（renin-angiotensin system，RAS）的激活是参与DN发病机制的主要因素，基于对DN过程中RAS经典途径ACE-AngⅡ-AT1通路过度激活的认识，血管紧张素转换酶抑制剂（angiotensin convertingenzyme inhibitor，ACEI）、血管紧张素受体拮抗剂（angiotensin receptorblocker，ARB）已成为现代医学临床治疗和延缓DM肾损害的一线药物。然而，随着对RAS的深入研究发现，RAS的另一条功能轴——ACE2-Ang-(1-7)-Mas轴，可与ACE-AngⅡ-AT1轴相对抗。维持和调整RAS两条代谢通路的平衡成为治疗DM肾脏损伤的新理念。本研究拟以瘀血阻络证候和ACE2-Ang-(1-7)-Mas轴为主要切入点，通过动物实验及小鼠足细胞的离体培养，探索DN瘀血阻络证是否与RAS中ACE2-Ang-(1-7)-Mas轴功能失调存在关联，以及化瘀通络中药对DN瘀血阻络证的干预作用是否同时改善ACE2-Ang-(1-7)-Mas轴的功能失调。
方法：
1糖尿病肾病大鼠瘀血阻络证的确认
健康雄性SD大鼠32只，4w龄，80～100g，适应性喂养1w后，随机分为空白对照组（C组，n=8）、高糖高脂对照组（H组，n=10）和模型组（M组，n=14），C组大鼠予普通全价颗粒饲料喂养，其余大鼠均予高糖高脂饲料喂养。4w后，M组大鼠腹腔注射1%链脲佐菌素（streptozotocin，STZ）溶液（40mg/kg体重），72h后随机血糖≥16.7mmol/L为DM大鼠模型复制成功；C、H两组大鼠均腹腔注射相应体积的0.1M枸橼酸缓冲液。于注射STZ后第3d、4w、8w、16w末，测量各组大鼠的随机血糖。16w末收集大鼠24h尿液（检测尿蛋白定量）后，麻醉状态下腹主动脉取血，检测各组大鼠血液流变学指标、血脂和血小板参数，留取各组大鼠肾皮质，行光镜、透射电镜观察。
2化瘀通络中药对糖尿病肾病大鼠瘀血阻络证的干预作用
将实验大鼠40只，随机分为C组（n=10）和STZ注射组（n=30）。STZ注射组的模型复制方法同上。将模型复制成功的25只DM大鼠分为M组（n=12）和化瘀通络中药组（Z组，n=13）。用药剂量按照成人临床常用剂量的5.8倍计算，给予Z组大鼠中药颗粒剂混悬液灌胃（1.08g/kg体重，相当于饮片4.7g/kg体重），其余组大鼠给予相应体积饮用水灌胃，每日1次，连续16w。16w末，腹主动脉取血，检测各组大鼠血液流变学指标、血脂、血小板参数、血浆纤维连接蛋白（fibronectin，FN）含量（ELISA法），并冻存部分肾皮质，采用实时荧光定量PCR（real-time PCR）检测组织型纤溶酶原激活物（tissue plasminogen activator，t-PA）、纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor-1，PAI-1）mRNA的表达。
3糖尿病肾病瘀血阻络证大鼠肾皮质ACE2-Ang-(1-7)-Mas轴的变化及化瘀通络中药的干预作用
将实验大鼠93只，随机分为C组（n=22）和STZ注射组（n=71），模型复制方法同上，将成模DM大鼠62只随机分为M组（n=24）、厄贝沙坦组（I组，n=19）和Z组（n=19），给予相应药物灌胃干预。于注射STZ后4w末随机取C组、M组大鼠各6只处死，其余各组大鼠分别于8w、16w末分两次处死，取肾皮质组织，采用免疫组织化学法和蛋白印迹（western blot）检测肾皮质ACE2、Mas、ACE蛋白的表达，real-time PCR检测肾皮质ACE2、Mas、ACE mRNA的表达，ELISA法检测肾皮质Ang-(1-7)的含量。
4化瘀通络中药对糖尿病肾病瘀血阻络证肾损伤的干预作用
将实验大鼠57只，随机分为C组（n=10）和STZ注射组（n=47），模型复制、成模DM大鼠分组及干预方法同上。于注射STZ后第3d、4w、8w、16w末，动态计量各组大鼠体重、24h摄食量、饮水量、尿量和24h尿蛋白定量。16w末处死动物，腹主动脉取血，检测肾功能；称量肾重，计算肾脏指数（kidney index，KI）；留取肾皮质，通过光镜、透射电镜检测各组大鼠肾脏病理表现。
5化瘀通络中药对高糖刺激下足细胞的干预作用
将许可条件下培养的条件性永生化小鼠足细胞分为正常糖组（NG组）、高糖组（HG组）和12个药物浓度的高糖加药组分别给予相应干预24h、48h、72h，通过MTT试验观察不同药物浓度对高糖刺激下细胞增殖的影响，对比结果筛选最佳药物浓度。将非许可条件下分化成熟的小鼠足细胞分为NG组、HG组和最佳药物浓度的高糖加药组[(HG+Z)组]，分别给予相应干预24h、48h、72h，检测细胞上清液Ⅳ型胶原蛋白（collagen-Ⅳ，Col-Ⅳ）的含量；采用免疫细胞化学法和western blot检测足细胞ACE和ACE2蛋白的表达，real-time PCR检测足细胞ACE、ACE2mRNA的表达。
结果：
1糖尿病肾病大鼠瘀血阻络证的确认
1.1一般情况
与C、H两组大鼠比较，M组大鼠逐渐出现多饮、多食、多尿及体重降低的表现。C组大鼠爪心红润，尾部静脉暗红不显；H组与C组差异不明显；而M组大鼠爪心紫暗，尾部静脉深紫而清晰可见。
1.2随机血糖
C、H两组大鼠的血糖基本恒定，H组大鼠的血糖高于C组（P<0.01）；腹腔注射STZ后，M组血糖水平显著高于同时间点C、H两组（P<0.01）。
1.324h尿蛋白定量
16w末，C、H两组间UPE无统计学差异（P>0.05）；与C、H两组比较，M组大鼠尿蛋白显著增加（P<0.01）。
1.4病理形态学
C、H两组大鼠肾脏体积正常，表面光滑，肾小球结构正常、清晰，足突排列整齐、无融合；M组大鼠肾脏体积增大，表面不平滑且有苍白花斑，肾小球肿胀肥大，基底膜均质性增厚且结构杂乱，系膜基质增多，足突节段性融合。
1.5血液流变学指标
16w末，H组仅红细胞压积（hematocrit，HCT）低于C组（P<0.05）。与C组比较，M组大鼠各切变率全血粘度、血浆粘度和全血低切还原粘度均明显升高（P<0.01），HCT、红细胞变形指数（erythrocyte deformabilityindex，EDI）均明显降低（P<0.05或0.01）；其中，全血粘度、血浆粘度和EDI还同时与H组具有统计学差异（P<0.05或0.01）。
1.6血脂
16w末，H组血总胆固醇（total cholesterol，TC）、低密度脂蛋白（lowdensity lipid cholesterol， LDL）和高密度脂蛋白（high density lipidcholesterol，HDL）均高于C组（P<0.05或0.01），而甘油三酯（triglyceride，TG）和极低密度脂蛋白（very low density lipid cholesterol，VLDL）则与C组无明显差异（P>0.05）。与C、H两组比较，M组大鼠TC、TG、LDL、HDL和VLDL均明显升高（P<0.01）。
1.7血小板参数
16w末，H组仅平均血小板体积（mean platelet volume，MPV）高于C组（P<0.05）。与C组比较，M组血小板计数（platelet count，PLT）明显减少（P<0.01），MPV和血小板分布宽度（platelet distributing width，PDW）显著增高（P<0.01）；其中，PLT、MPV同时与H组具有显著差异（P<0.05或0.01）。各组间大型血小板比率（platelet-large cell ratio，P-LCR）无明显差异（P>0.05）。
2化瘀通络中药对糖尿病肾病大鼠瘀血阻络证的干预作用
2.1一般情况
C组大鼠爪心红润，尾部静脉暗红不显；M组大鼠爪心紫暗，尾部静脉深紫而清晰可见；I、Z两组大鼠爪心和尾部静脉较M组有所改善。
2.2血液流变学指标
16w末，与C组比较，M组大鼠各切变率全血粘度、血浆粘度和全血低切还原粘度均明显升高（P<0.01），HCT、红细胞变形指数（erythrocytedeformability index，EDI）均明显降低（P<0.01）；与M组比较，Z组切变率30S-1、200S-1全血粘度、血浆粘度和EDI均有显著改善（P<0.05或0.01）。
2.3血脂
16w末，与C组比较，M组大鼠TC、TG、LDL、HDL和VLDL均明显升高（P<0.01）。与M组比较，Z组以上各血脂指标均显著降低（P<0.05或0.01）。
2.4血小板参数
16w末，与C组比较，M组血小板计数（platelet count，PLT）明显减少（P<0.01），MPV和血小板分布宽度（platelet distributing width，PDW）显著增高（P<0.05或0.01）；与M组比较，Z组MPV、PDW均明显降低（P<0.01）。各组间大型血小板比率（platelet-large cell ratio，P-LCR）无明显差异（P>0.05）。
2.5血浆FN含量
16w末，M组血浆FN含量明显高于C组（P<0.01）；与M组比较，Z组含量明显降低（P<0.05）。
2.6肾皮质t-PA、PAI-1mRNA的表达
16w末，各组t-PA mRNA的表达无明显差异（P>0.05）；M组PAI-1mRNA的表达显著高于C组（P<0.05），而Z组的表达低于M组（P<0.05）。
3糖尿病肾病瘀血阻络证大鼠肾皮质ACE2-Ang-(1-7)-Mas轴的变化及化瘀通络中药的干预作用
3.1肾皮质ACE2、Mas、ACE的表达
ACE2和ACE主要表达在各组大鼠肾小球和肾小管的上皮细胞中，Mas主要表达在各组大鼠肾皮质组织的近端小管。
C组大鼠各时间点肾皮质ACE2、Mas和ACE蛋白的表达均无显著差异（P>0.05）。M组大鼠肾皮质ACE2、Mas蛋白的表达均随时间呈现降低趋势：16w末ACE2蛋白的表达低于4w末（P<0.05）；16w末Mas蛋白的表达分别低于4w末和8w末（P<0.05或0.01）。M组不同时间点ACE蛋白的表达无统计学差异（P>0.05）。
8w末，ACE2和Mas蛋白在各组大鼠肾皮质组织的表达水平，无统计学差异（P>0.05）；ACE蛋白的表达水平仅在M、I两组间存在差异（P<0.05）。
16w末，肾皮质ACE2蛋白的表达，M组低于C组（P<0.01），I、Z两组均高于M组（P<0.05），且Z组高于I组（P<0.05）；Mas蛋白的表达，M组低于C组（P<0.05）；I、Z两组均高于M组（P<0.01）。ACE蛋白的表达，M组显著高于C组（P<0.01）；Z组低于M、I两组（P<0.05或0.01）。
3.2肾皮质ACE2、Mas、ACE mRNA的表达
以4w末C组大鼠作为参照，进行相对表达定量。C组大鼠各时间点肾皮质ACE2、Mas、ACE mRNA的表达无显著差异（P>0.05）。M组各时间点ACE2mRNA的表达均低于同时间点C组（P<0.01），在4w至16w之间先上升（P<0.01）、后下降（P<0.05）；M组Mas mRNA的表达在4w高于C组（P<0.01），在4w至16w之间持续下降（P<0.01），16w低于C组（P<0.05）；M组ACE mRNA的表达在4w低于C组（P<0.01），在4w至16w之间先上升（P<0.01）、后下降（P<0.05），8w、16w均高于C组（P<0.05或0.01）。
8w末，各组Mas mRNA的表达无显著差异（P>0.05）。与C组比较，M组ACE2mRNA的表达显著降低（P<0.01），ACE mRNA的表达显著升高（P<0.01）；与M组比较，Z组ACE2mRNA的表达显著升高（P<0.01），且高于I组（P<0.05）；I、Z两组ACE mRNA的表达均低于M组（P<0.05）。16w末，与C组比较，M组ACE2mRNA的表达显著降低（P<0.01），ACE mRNA的表达显著升高（P<0.05），而Mas mRNA的表达无显著差异（P>0.05）；与M组比较，Z组ACE2和Mas mRNA的表达均显著升高（P<0.05或0.01）；其中， Z组Mas mRNA的表达还同时高于I组（P<0.05）；M、I、Z三组间ACE mRNA的表达差异不明显（P>0.05）。
3.3肾皮质Ang-(1-7)的含量
16w末，与C组比较，M组大鼠的肾皮质Ang-(1-7)含量明显降低（P<0.01）；而I、Z两组的Ang-(1-7)含量均高于M组（P<0.01）。
3.4M组大鼠血液粘度与ACE2、Mas和ACE mRNA及其蛋白表达的线性相关分析
16w末，M组大鼠血浆粘度和不同切变率下全血粘度与肾皮质ACE2mRNA及其蛋白的表达均呈负相关（P<0.05或0.01），与ACE mRNA及其蛋白的表达均呈正相关（P<0.05或0.01）；而与Mas mRNA及其蛋白的表达无显著线性相关关系（P>0.05）。
4化瘀通络中药对糖尿病肾病瘀血阻络证肾损伤的干预作用
4.1糖尿病症状性指标的动态变化
腹腔注射STZ4w后，M组大鼠各时间点体重均明显低于同时间点C组（P<0.01）；16w末，I、Z两组体重均明显高于M组（P<0.05或0.01）。与同时间点C组比较， M组各时间点RBG、24h摄食热量、饮水量和尿量均显著升高（P<0.01）；与同时间点M组比较，I、Z两组各时间点RBG、24h摄食热量和尿量均无明显差异（P>0.05），仅8w末I、Z两组和16w末Z组24h饮水量显著低于同时间点M组（P<0.05或0.01）。
4.224h尿蛋白定量的动态变化
M组大鼠各时间点尿蛋白均高于同时间点C组（P<0.01）；8w末，I、Z两组尿蛋白明显低于M组（P<0.05或0.01）；16w末，Z组显著低于M、I两组（P<0.05或0.01）。
4.3肾功能
16w末，M组血肌酐（serum creatinine，Scr）、尿素氮（blood ureanitrogen，BUN）、尿酸（uric acid，UA）均高于C组（P<0.01）；与M组比较，I、Z两组Scr、BUN均有显著改善（P<0.05或0.01），Z组UA也低于M组（P<0.05）。
4.4肾脏指数
16w末，M组KI值明显高于C组（P<0.01）；而I、Z两组KI值均显著低于M组（P<0.05）。
4.5光镜观察肾皮质结构
C组肾皮质结构正常；M组肾小球肾小球体积肥大，基底膜轻度增厚，系膜轻度增生，毛细血管管腔扩张，肾小囊腔狭窄，肾小管上皮细胞排列紊乱、肥大；I、Z两组的肾小囊腔较宽，系膜增生有所改善。
4.6透射电镜观察肾小球超微结构
C组肾小球超微结构基本正常；M组大鼠肾小球基底膜均质性增厚且结构欠清晰，系膜基质增多，足突节段性融合；I、Z两组结构较清晰，基底膜节段性增厚，足突少部分融合。
5化瘀通络中药对高糖刺激下足细胞的干预作用
5.1MTT试验
HG组各时间点光密度（optical density，OD）值均明显低于同时间点NG组（P<0.05或0.01）；多个药物浓度均对高糖刺激24h、48h、72h对足细胞的增殖抑制有不同程度的逆转作用，其中，各时间点均以药物浓度100ug/ml的含药高糖组的OD值最高，且高于HG组（P<0.01）。以100ug/ml作为最佳给药浓度。
5.2细胞上清液Col-Ⅳ的含量
给予足细胞不同干预24h后，各组细胞上清液Col-Ⅳ的含量无显著差异（P>0.05）；不同干预48h、72h后，HG组细胞上清液Col-Ⅳ的含量均高于同时间点NG组（P<0.01），而(HG+Z)组干预48h后Col-Ⅳ的含量显著低于HG组（P<0.05）。
5.3足细胞ACE、ACE2蛋白的表达
ACE和ACE2蛋白特异性表达于足细胞胞浆中。不同干预48h，与NG组比较，HG组ACE蛋白表达显著升高（P<0.05），ACE2蛋白表达显著降低（P<0.01）；与HG组比较，(HG+Z)组ACE蛋白表达较低，而ACE2蛋白表达较高（P<0.05）。而不同干预24h、72h后，各组ACE2、ACE蛋白的表达均无明显差异（P>0.05）。
5.4足细胞ACE、ACE2mRNA的表达
分别与同时间点NG组比较， HG组各时间点ACE mRNA表达均显著升高（P<0.05），HG组24h、48h ACE2mRNA表达显著降低（P<0.05或0.01）；分别与同时间点HG组比较，仅48h (HG+Z)组ACE mRNA表达明显降低，ACE2mRNA表达明显升高（P<0.01）。
结论：
1DN大鼠爪掌、尾静脉有紫暗瘀血表现，血液存在高粘、高凝状态，与瘀血阻络的中医证候特征相符，确认了DN瘀血阻络证的存在。
2化瘀通络中药对DN大鼠瘀血阻于肾络的证候具有良性调节作用。
3DN大鼠肾皮质ACE2-Ang-(1-7)-Mas轴功能下调，化瘀通络干预16w可不同程度上调其功能，且作用优于对照药物厄贝沙坦。
4化瘀通络中药能有效减轻DN瘀血阻络证大鼠的肾损伤，且作用略优于对照药物厄贝沙坦。
5高糖刺激对小鼠足细胞具有抑制增殖、促进Col-Ⅳ分泌、上调ACE、下调ACE2表达的效应；化瘀通络中药干预48h能不同程度逆转以上效应。
Objectives: Diabetic nephropathy (DN) is one of the most commonmicrovascular complications of diabetes mellitus (DM), which meanwhile isone of leading causes for chronic renal failure in our country. Therefore,searching for its pathogenesis and effective preventing and controllingmeasures is of great significance. Chinese medicine has shown greatadvantage in preventing and controlling DN. In the opinion of most modernChinese medical scholars in nephropathy, the primary pathogenesis of DN is“blood stasis blocking renal collaterals”, besides “Qi and Yin vacuity” of DM.The principle “activating blood, removing stasis and dredging collaterals” isfollowed in DN treatment, which has become a consensus in the Chinesemedical field of nephropathy. Modern medicine has confirmed that theactivation of renin-angiotensin system (RAS) is the main mechanism involvedin DN. Based on the cognition of over-activation of the classic pathway ofRAS, the angiotensin-converting enzyme-angiotensin Ⅱ-angiotensin type1receptor [ACE-AngⅡ-AT1] pathway, in the development of DN, angiotensinconverting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB)have been used as first-line agents in treating and delaying the renal injury ofDM in clinic. However, with the further study on RAS, another functional axis,angiotensin-converting enzyme-related carboxypeptidase2-angiotensin-(1-7)-Mas [ACE2-Ang-(1-7)-Mas] axis, was discovered to be against the function ofACE-AngⅡ-AT1axis. Maintaining and adjusting the balance between thesetwo pathways have become a new concept for diabetic renal injury therapy.Taking blood-stasis-blocking-collaterals syndrome and ACE2-Ang-(1-7)-Masaxis as two important entry points, this research is aimed to explore whether the blood-stasis-blocking-collaterals syndrome of DN is related to thedysfunction of ACE2-Ang-(1-7)-Mas axis, as well as whether the interventionof stasis-removing-and-collaterals-dredging medicine on blood-stasis-blocking-collaterals syndrome also improve the function of ACE2-Ang-(1-7)-Mas axis,by animal experiments and isolated culture of mouse podocytes.
Methods:
1Verification of stasis-blocking-collaterals syndrome in rats with diabeticnephropathy
A totle of32healthy male Sprague-Dawley rats of4weeks old, weighed80-100g were grouped randomly into blank control group (C group, n=8),high-glucose-high-fat control group (H group, n=10), and model group (Mgroup, n=14), after adaptive feeding for1week. Rats in C group were fed withregular rat chow, while others fed with high-glucose-high-fat diet. After4weeks, the rats in M group were injected intraperitoneailly withstreptozotocin (STZ) at the dose of40mg/kg body weight. The rats showingrandom blood glucose≥16.7mmol/L72hours after injection were consideredas successfully duplicated diabetic models. Rats in other two groups wereinjected with citrate buffer solution of equal volume. Random blood glucose(RBG) was measured on the3rd day and at the end of the4th,8th and16thweel after STZ injection. At the end of the16th week, after collection of urineof24hours for urine protein evaluation (UPE), blood samples were collectedby abdominal aortic method under anesthesia, which were used to test bloodviscosity measures, serum lipid profile and platelet parameters. Renal cortexof rats in different groups were examined by light microscope andtransmission electron microscope.
2Effect of stasis-removing-and-collaterals-dredging intervention upon stasis-blocking-collaterals syndrome in rats with diabetic nephropathy
A total of40experimental rats were divided into C group (n=10) andSTZ-injecting group (n=30). The establishment of models was same as above.Twenty-five diabetic rats were further divided into M group (n=12) andstasis-removing-and-collaterals-dredging group (Z group, n=13). Dosages of medicine in rats were calculated as5.8times as the clinical dosage in human.Rats in Z group were intragastrically fed with suspension of Chinese medicinegranules (1.08g/kg body weight, equal to crude drug4.7g/kg body weight),while rats in other groups with drinking water of equal volume, once daily for16weeks. At the end of the16th week, blood samples were collected byabdominal aortic method to test blood viscosity measures, serum lipid profile,platelet parameters and plasma fibronectin (FN) content. Renal cortex wascryopreserved to test the relative expression of tissue plasminogen activator(t-PA) and plasminogen activator inhibitor-1(PAI-1) mRNA by real-timePCR.
3The change of ACE2-Ang-(1-7)-Mas axis of renal cortex in DN rats ofstasis-blood-blocking-collaterals syndrome and effect of stasis-removing-and-collaterals-dredging intervention
A total of93experimental rats were divided into C group (n=22) andSTZ-injecting group (n=71). The establishment of models was same as above.Sixty-two diabetic rats were further divided into M group (n=24), irbesartangroup (I group, n=19), Z group (n=19). Interventions were given respectively.At the end of the4th week,6rats were choosed randomly from C and Mgroups respectively to be killed, and other rats were killed at the end of the8thand16th week separately. Renal cortex was cryopreserved to test theexpressions of ACE2, Mas, ACE proteins by immunohistochemical assay andwestern blot, relative expressions of ACE2, Mas, ACE mRNA by real-timePCR, and Ang-(1-7) content by ELISA.
4Effect of stasis-removing-and-collaterals-dredging intervention upon renalinjury in diabetic nephropathy of stasis-blood-blocking-collaterals syndrome
A total of57experimental rats were divided into C group (n=10) andSTZ-injecting group (n=47). The establishment of models, further division ofSTZ-injecting group and interventions were same as above. Body weight aswell as diet calorie, water intake, urine output and UPE of24hours, weremeasured on the3rd day and at the end of the4th,8th and16th week afterSTZ injection. Meanwhile, urine of24hours was collected to do the urine protein evaluation (UPE). At the sacrifice of rats at the end of the16th week,blood samples were collected to test kidney function; kidney indexes (KI)were calculated; and renal cortex was pathologically examined by lightmicroscope and transmission electron microscope.
5Effect of Chinese medicine of stasis-emoving-and-collaterals-dredgingintervention upon high glucose stimulating podocytes
The conditionally immortalized mouse podocytes cultured under growthpermissive condition were divided into normal glucose group (NG group),high glucose group (HG group) and medicated high glucose groups of12different concentrations, administered with corresponding culture mediainterventions for24,48and72hours. Effects of Chinese medicine of differentconcentrations upon cell proliferation under high glucose stimulation wereobserved by MTT test. Based on the comparison of data among differentconcentrations, the optimum concentration was screened for furtherexperiment. Differentiated and mature mouse podocytes cultured undergrowth restrictive condition were divided into NG group, HG group andmedicated high glucose group of the optimum concentration [(H+Z) group],administered with corresponding interventions for24,48and72hours.Collagen-Ⅳ (Col-Ⅳ) content in podocyte supernatant was tested by ELISA,and expressions of ACE and ACE2proteins in podocytes by immuno--cytochemical assay and western blot, relative expressions of ACE and ACE2mRNA by real-time PCR.
Results:
1Verification of stasis-blocking-collaterals syndrome in rats with diabeticnephropathy
1.1General condition
Compared with C and H groups, rats in M group showed polydipsia,polyphagia, polyuria and loss of weight gradually. Rats in C group had ruddypaws and unconspicuous dull-red tail veins, which was similar in H group,while M group had dark-purple paws and conspicuous dark-purple tail veins.1.2Random blood glucose
RBG of rats in C and H groups remained constant, with H group higherthan C group (P<0.01). After STZ injection, RBG in M group wassignificantly higher than C and H groups at same time points (P<0.01).
1.3UPE of24hours
At the end of16th week, there was no difference in UPE between C andH groups (P>0.05). Compared with C and H groups, UPE of M group washigher (P<0.01).
1.4Pathomorphology
Rats in C and H groups had normal sized kidneys, with smooth surface,from which glomeruli showed clear and well-distributed structures, with nofusion of foot processes arranged in order. Rats in M group had enlargedkidneys, with unsmooth surface and pale spots on them, from which enlargedswollen glomeruli revealed homogeneously thickened basement membranewith chaotic structure, increased mesangial matrix and segmental fusion ofpodocyte foot processes.
1.5Blood viscosity measures
At the end of16th week, only hematocrit (HCT) of rats in H group waslower than C group (P<0.05). Compared with C group, whole blood viscosity,plasma viscosity and whole blood low shear reductive viscosity of rats in Mgroup were all higher (P<0.01); while HCT and erythrocyte deformabilityindex (EDI) were lower (P<0.05or0.01). Among them, whole blood viscosity,plasma viscosity and EDI were also different from those in H group (P<0.05or0.01).
1.6Serum lipid profile
At the end of16th week, total cholesterol (TC), low density lipidcholesterol (LDL) and high density lipid cholesterol (HDL) of rats in H groupwere higher than C group (P<0.05or0.01); while triglyceride (TG) and verylow density lipid cholesterol (VLDL) were not different from C group(P>0.05). Compared with C and H groups, TC, TG, LDL, HDL and VLDL ofrats in M group were all significantly higher (P<0.01).
1.7Platelet parameters
At the end of16th week, only mean platelet volume (MPV) of rats in Hgroup was higher than C group (P<0.05). Compared with C group, plateletcount (PLT) of rats in M group decreased significantly (P<0.01); MPV andplatelet distributing width (PDW) of M group elevated significantly (P<0.01).And meanwhile, PLT and MPV of rats in M group were also different from Hgroup (P<0.05or0.01). There was no difference in platelet-large cell ratio(P-LCR) among different groups (P>0.05).
2Effect of stasis-removing-and-collaterals-dredging intervention upon stasis-blocking-collaterals syndrome in rats with diabetic nephropathy
2.1Blood viscosity measures
At the end of16th week, compared with C group, whole blood viscosity,plasma viscosity and whole blood low shear reductive viscosity of rats in Mgroup were all higher (P<0.01); while HCT and erythrocyte deformabilityindex (EDI) were lower (P<0.01). Compared with M group, whole bloodviscosity with30S-1and200S-1shear rates, plasma viscosity and EDI of Zgroup were improved significantly (P<0.05or0.01).
2.2Serum lipid profile
At the end of16th week, compared with C group, TC, TG, LDL, HDLand VLDL of rats in M group were all significantly higher (P<0.01).Compared with M group, all above indexes of Z group were lower (P<0.05or0.01).
2.3Platelet parameters
At the end of16th week, compared with C group, platelet count (PLT) ofrats in M group decreased significantly (P<0.01); MPV and plateletdistributing width (PDW) of M group elevated significantly (P<0.05or0.01).Compared with M group, MPV and PDW of Z group were lower (P<0.01).There was no difference in platelet-large cell ratio (P-LCR) among differentgroups (P>0.05).
2.4Plasma FN content
At the end of16th week, plasma FN content of rats in M group washigher significantly than C group (P<0.01). And compared with M group, Z group was lower (P<0.05).
2.5Relative expression of t-PA and PAI-1mRNA in renal cortex
At the end of16th week, there was no difference in the relativeexpression of t-PA mRNA among different groups (P>0.05). The relativeexpression of PAI-1mRNA in M group was significantly higher than C group(P<0.05). And its relative expression in Z group was lower than M group(P<0.05).
3The change of ACE2-Ang-(1-7)-Mas axis of renal cortex in DN rats ofstasis-blood-blocking-collaterals syndrome and effect of stasis-removing-and-collaterals-dredging intervention
3.1Expressions of ACE2, Mas, ACE proteins in renal cortex
ACE2and ACE expressed mainly in the epithelial cells of glomeruli andtubules of kidney, while Mas approximately expressed in the proximal tubules.There was no significant difference in expressions of ACE2, Mas and ACE inrenal cortex from rats in C group among different time points (P>0.05).Expressions of ACE2and Mas proteins in M group showed downtrend withtime. ACE2expression at the end of16th week was lower than4th week(P<0.05), while Mas expression at the end of16th week was lower than4thand8th week respectively (P<0.05or0.01). But there was no difference inACE expression in M group among different time points (P>0.05).
At the end of8th week, there was no significant difference in expressionsof ACE2and Mas among different groups (P>0.05). And there was statisticaldifference in ACE expression only between M and I groups (P<0.05).
At the end of16th week, ACE2expression in M group was lower than inC group (P<0.01); and that in I and Z groups were higher than in M group(P<0.05), with Z group higher than I group (P<0.05). Mas expression in Mgroup was lower than in C group (P<0.05); and that in I and Z groups washigher than in M group (P<0.01). ACE expression in M group was higher thanin C group (P<0.01); and that in Z group was lower than in M and I groups(P<0.05or0.01).
3.2Expressions of ACE2, Mas, ACE mRNA in renal cortex
Take rats in C group at the end of4th week as control to quantificate therelative expression. There was no difference in expressions of ACE2, Mas,ACE mRNA in C group among different time points (P>0.05). ACE2mRNAexpression in M group at each time point was lower than that in C group atsame time point (P<0.01), and experienced a course of first rising (P<0.01)then declining (P<0.05) from4th week to16th week. Mas mRNA expressionin M group at4th week was higher than that in C group at same time point(P<0.01), and experienced a declining course from4th week to16th week(P<0.01), which resulted in lower than C group at16th week (P<0.05). ACEmRNA expression in M group at4th week was lower than that in C group atsame time point (P<0.01), and experienced a course of first rising (P<0.01)then declining (P<0.05) from4th week to16th week, which resulted in higherthan C group at16th week (P<0.05).
At the end of8th week, there was no significant difference in Mas mRNAexpression among different groups (P>0.05). Compared with C group, ACE2mRNA expression in M group was lower (P<0.01), ACE mRNA expression inM group was higher (P<0.01). Compared with M group, ACE2mRNAexpression in Z group was higher (0.01), and higher than I group as well(P<0.05), while ACE mRNA expressions in both I and Z groups were lowerthan in M group (P<0.05).
At the end of16th week, compared with C group, ACE2mRNAexpression in M group was lower (P<0.01), ACE mRNA expression in Mgroup was higher (P<0.01), and Mas mRNA expression in M group showedno difference (P>0.05). Compared with M group, ACE2and Mas mRNAexpressions in Z group were both higher (P<0.05or0.01), with Mas mRNAexpression in Z group higher than I group (P<0.05). There was no statisticaldifference in ACE mRNA expressions among M, I and Z groups (P>0.05).
3.3Ang-(1-7) content in renal cortex
At the end of16th week, compared with C group, Ang-(1-7) content inrenal cortex from rats in M group was lower significantly (P<0.01). AndAng-(1-7) content in I and Z groups was higher than in M group (P<0.01).
3.4Linear correlation analysis between blood viscosity and expressions ofACE2, Mas, ACE mRNA and proteins of rats in M group
At the end of16th week, blood viscosity (including plasma viscosity andwhole blood viscosity under different shear rates) in M group was in negativecorrelation with ACE2mRNA and protein expressions (P<0.05or0.01), andin positive correlation with ACE mRNA and protein expressions (P<0.05or0.01). However, there was no linear correlation between blood viscosity andMas mRNA and protein expressions (P>0.05).
4Effect of stasis-removing-and-collaterals-dredging intervention upon renalinjury in diabetic nephropathy of stasis-blood-blocking-collaterals syndrome
4.1Dynamic trends of clinical indicators of diabetes
After4weeks, body weight of rats in M group at each time point waslower than C group at same time point (P<0.01). At the end of16th week,body weights in I and Z groups were higher than in M group (P<0.05or0.01).Compared with C group at same time point, RBG, diet calorie, water intakeand urine output of24hours of rats in M group at each time point were allsignificantly higher (P<0.01). Compared with M group at same time point,RBG, diet calorie and urine output of24hours of rats in I and Z groups ateach time point were not different significantly (P>0.05); while water intakeof24hours of rats in I and Z groups at the end of8th week and that in Z groupat the end of16th week were lower than M group (P<0.05or0.01).
4.2UPE of24hours
UPE in M group at each time point was significantly higher than C groupat same time point (P<0.01). At the end of8th week, UPE in I and Z groupswere lower than M group (P<0.05). And at the end of16th week, water intakein Z group was lower than M and I groups (P<0.05or0.01).
4.3Kidney function
At the end of16th week, serum creatinine (Scr), urea nitrogen (BUN) anduric acid (UA) of rats in M group were all higher than C group (P<0.01).Compared with M group, Scr and BUN of I and Z groups were lower (P<0.05or0.01), while UA of Z group was lower than M group as well (P<0.05).
4.4KI
At the end of16th week, KI of rats in M group was higher than C group(P<0.01). And KI in I and Z groups were lower than M group (P<0.05).
4.5Observation of renal cortical structures by light microscope
Renal cortex of rats in C group showed normal structures. Renal cortex ofM group revealed obvious hypertrophy of glomerular, slightly thickenedbasement membrane, expansion of mesangial matrix, expanded capillaries,narrow renal capsule and disarranged renal tubular epithelial cells. Comparedwith M group, renal capsules in I and Z groups were wider; and themesangial proliferations were both alleviated.
4.6Observation of ultrastructures of glomeruli by transmission electronmicroscope
Glomeruli from rats in C group showed normal ultrastructures. And thosefrom M group revealed homogeneously thickened basement membrane withnot clear structure, increased mesangial matrix and segmental fusion ofpodocyte foot processes. Compared with M group, glomeruli from rats in Iand Z groups showed clearer ultrastructures with segmentally thickenedbasement membrane and partially segmental fusion of podocyte footprocesses.
5Effect of Chinese medicine of stasis-emoving-and-collaterals-dredgingintervention upon high glucose stimulating podocytes
5.1MTT test
The optical density (OD) of HG group at each time point wassignificantly lower than NG group at same time point (P<0.05or0.01).Chinese medicine of several concentrations could reverse the suppressingproliferation effect of high glucose at each time point. Among differentconcentrations, the OD of100μg/ml medicated high glucose group was thehighest, and higher than HG group (P<0.01). The optimum concentration was100μg/ml.
5.2Col-Ⅳ content in podocyte supernatant
There was no significant difference in Col-Ⅳ content of podocyte supernatant among different groups podocyte at24hours (P>0.05). Col-Ⅳcontents of podocyte supernatant of HG group at48and72hours were bothhigher than NG group at same time points (P<0.01). And Col-Ⅳ content ofpodocyte supernatant of (HG+Z) group at48hours was significantly lowerthan HG group at same time point (P<0.05).
5.3Expressions of ACE and ACE2proteins in podocytes
The ACE and ACE2proteins expressed specificly in the cytoplasm ofpodocytes. Compared with NG group at48hours, the ACE protein expressionof HG group was higher (P<0.05), while the ACE2protein expression of HGgroup was lower (P<0.01). Compared with HG group at48hours, the ACEprotein expression of (HG+Z) group was higher (P<0.05), while the ACE2protein expression of (HG+Z) group was lower (P<0.05). However, there wasno difference in expressions of ACE and ACE2proteins among differentgroups at24and72hours (P>0.05).
5.4Relative expressions of ACE and ACE2mRNA in podocytes
Compared with NG group at same time point respectively, ACE mRNAexpression of HG group at each time point was higher significantly (P<0.05),and ACE2mRNA expression of HG group was lower significantly only at48and72hours time points (P<0.05or0.01). Compared with HG group at sametime point respectively, just at48hours time point, ACE mRNA expression of(HG+Z) group was lower (P<0.05), and ACE2mRNA expression of (HG+Z)group was higher (P<0.01).
Conclusions:
1DN rats had dark purple paws and tail veins; and their blood in highviscosity, hypercoagulable state. These were in accordance with the features ofblood-stasis-blocking-collaterals syndrome in Chinese medicine, whichaffirmed the existence of blood-stasis-blocking-collaterals syndrome.
2The stasis-removing-and-collaterals-dredging therapy played a benefitrole in regulating blood-stasis-blocking-renal-collaterals syndrome in DN.
3The function of ACE2-Ang-(1-7)-Mas axis was down-regulated in DMrats. The stasis-removing-and-collaterals-dredging intervention for16weeks could up-regulate its function, which effect was superior to thepositive control drug irbesartan.
4The stasis-removing-and-collaterals-dredging therapy alleviated therenal injury in diabetic nephropathy of stasis-blood-blocking-collateralssyndrome effectively, which effect was slightly superior to the positivecontrol drug irbesartan.
5High glucose stimulation had effects of suppressing proliferation,promoting Col-Ⅳ secretion, up-regulating ACE expression anddown-regulating ACE2expression upon mouse podocytes. Chinese medicineof stasis-removing-and-collaterals-dredging intervention for48h could reversethe negative effects of high glucose stimulating podocytes.
方法：
1糖尿病肾病大鼠瘀血阻络证的确认
健康雄性SD大鼠32只，4w龄，80～100g，适应性喂养1w后，随机分为空白对照组（C组，n=8）、高糖高脂对照组（H组，n=10）和模型组（M组，n=14），C组大鼠予普通全价颗粒饲料喂养，其余大鼠均予高糖高脂饲料喂养。4w后，M组大鼠腹腔注射1%链脲佐菌素（streptozotocin，STZ）溶液（40mg/kg体重），72h后随机血糖≥16.7mmol/L为DM大鼠模型复制成功；C、H两组大鼠均腹腔注射相应体积的0.1M枸橼酸缓冲液。于注射STZ后第3d、4w、8w、16w末，测量各组大鼠的随机血糖。16w末收集大鼠24h尿液（检测尿蛋白定量）后，麻醉状态下腹主动脉取血，检测各组大鼠血液流变学指标、血脂和血小板参数，留取各组大鼠肾皮质，行光镜、透射电镜观察。
2化瘀通络中药对糖尿病肾病大鼠瘀血阻络证的干预作用
将实验大鼠40只，随机分为C组（n=10）和STZ注射组（n=30）。STZ注射组的模型复制方法同上。将模型复制成功的25只DM大鼠分为M组（n=12）和化瘀通络中药组（Z组，n=13）。用药剂量按照成人临床常用剂量的5.8倍计算，给予Z组大鼠中药颗粒剂混悬液灌胃（1.08g/kg体重，相当于饮片4.7g/kg体重），其余组大鼠给予相应体积饮用水灌胃，每日1次，连续16w。16w末，腹主动脉取血，检测各组大鼠血液流变学指标、血脂、血小板参数、血浆纤维连接蛋白（fibronectin，FN）含量（ELISA法），并冻存部分肾皮质，采用实时荧光定量PCR（real-time PCR）检测组织型纤溶酶原激活物（tissue plasminogen activator，t-PA）、纤溶酶原激活物抑制剂-1（plasminogen activator inhibitor-1，PAI-1）mRNA的表达。
3糖尿病肾病瘀血阻络证大鼠肾皮质ACE2-Ang-(1-7)-Mas轴的变化及化瘀通络中药的干预作用
将实验大鼠93只，随机分为C组（n=22）和STZ注射组（n=71），模型复制方法同上，将成模DM大鼠62只随机分为M组（n=24）、厄贝沙坦组（I组，n=19）和Z组（n=19），给予相应药物灌胃干预。于注射STZ后4w末随机取C组、M组大鼠各6只处死，其余各组大鼠分别于8w、16w末分两次处死，取肾皮质组织，采用免疫组织化学法和蛋白印迹（western blot）检测肾皮质ACE2、Mas、ACE蛋白的表达，real-time PCR检测肾皮质ACE2、Mas、ACE mRNA的表达，ELISA法检测肾皮质Ang-(1-7)的含量。
4化瘀通络中药对糖尿病肾病瘀血阻络证肾损伤的干预作用
将实验大鼠57只，随机分为C组（n=10）和STZ注射组（n=47），模型复制、成模DM大鼠分组及干预方法同上。于注射STZ后第3d、4w、8w、16w末，动态计量各组大鼠体重、24h摄食量、饮水量、尿量和24h尿蛋白定量。16w末处死动物，腹主动脉取血，检测肾功能；称量肾重，计算肾脏指数（kidney index，KI）；留取肾皮质，通过光镜、透射电镜检测各组大鼠肾脏病理表现。
5化瘀通络中药对高糖刺激下足细胞的干预作用
将许可条件下培养的条件性永生化小鼠足细胞分为正常糖组（NG组）、高糖组（HG组）和12个药物浓度的高糖加药组分别给予相应干预24h、48h、72h，通过MTT试验观察不同药物浓度对高糖刺激下细胞增殖的影响，对比结果筛选最佳药物浓度。将非许可条件下分化成熟的小鼠足细胞分为NG组、HG组和最佳药物浓度的高糖加药组[(HG+Z)组]，分别给予相应干预24h、48h、72h，检测细胞上清液Ⅳ型胶原蛋白（collagen-Ⅳ，Col-Ⅳ）的含量；采用免疫细胞化学法和western blot检测足细胞ACE和ACE2蛋白的表达，real-time PCR检测足细胞ACE、ACE2mRNA的表达。
结果：
1糖尿病肾病大鼠瘀血阻络证的确认
1.1一般情况
与C、H两组大鼠比较，M组大鼠逐渐出现多饮、多食、多尿及体重降低的表现。C组大鼠爪心红润，尾部静脉暗红不显；H组与C组差异不明显；而M组大鼠爪心紫暗，尾部静脉深紫而清晰可见。
1.2随机血糖
C、H两组大鼠的血糖基本恒定，H组大鼠的血糖高于C组（P<0.01）；腹腔注射STZ后，M组血糖水平显著高于同时间点C、H两组（P<0.01）。
1.324h尿蛋白定量
16w末，C、H两组间UPE无统计学差异（P>0.05）；与C、H两组比较，M组大鼠尿蛋白显著增加（P<0.01）。
1.4病理形态学
C、H两组大鼠肾脏体积正常，表面光滑，肾小球结构正常、清晰，足突排列整齐、无融合；M组大鼠肾脏体积增大，表面不平滑且有苍白花斑，肾小球肿胀肥大，基底膜均质性增厚且结构杂乱，系膜基质增多，足突节段性融合。
1.5血液流变学指标
16w末，H组仅红细胞压积（hematocrit，HCT）低于C组（P<0.05）。与C组比较，M组大鼠各切变率全血粘度、血浆粘度和全血低切还原粘度均明显升高（P<0.01），HCT、红细胞变形指数（erythrocyte deformabilityindex，EDI）均明显降低（P<0.05或0.01）；其中，全血粘度、血浆粘度和EDI还同时与H组具有统计学差异（P<0.05或0.01）。
1.6血脂
16w末，H组血总胆固醇（total cholesterol，TC）、低密度脂蛋白（lowdensity lipid cholesterol， LDL）和高密度脂蛋白（high density lipidcholesterol，HDL）均高于C组（P<0.05或0.01），而甘油三酯（triglyceride，TG）和极低密度脂蛋白（very low density lipid cholesterol，VLDL）则与C组无明显差异（P>0.05）。与C、H两组比较，M组大鼠TC、TG、LDL、HDL和VLDL均明显升高（P<0.01）。
1.7血小板参数
16w末，H组仅平均血小板体积（mean platelet volume，MPV）高于C组（P<0.05）。与C组比较，M组血小板计数（platelet count，PLT）明显减少（P<0.01），MPV和血小板分布宽度（platelet distributing width，PDW）显著增高（P<0.01）；其中，PLT、MPV同时与H组具有显著差异（P<0.05或0.01）。各组间大型血小板比率（platelet-large cell ratio，P-LCR）无明显差异（P>0.05）。
2化瘀通络中药对糖尿病肾病大鼠瘀血阻络证的干预作用
2.1一般情况
C组大鼠爪心红润，尾部静脉暗红不显；M组大鼠爪心紫暗，尾部静脉深紫而清晰可见；I、Z两组大鼠爪心和尾部静脉较M组有所改善。
2.2血液流变学指标
16w末，与C组比较，M组大鼠各切变率全血粘度、血浆粘度和全血低切还原粘度均明显升高（P<0.01），HCT、红细胞变形指数（erythrocytedeformability index，EDI）均明显降低（P<0.01）；与M组比较，Z组切变率30S-1、200S-1全血粘度、血浆粘度和EDI均有显著改善（P<0.05或0.01）。
2.3血脂
16w末，与C组比较，M组大鼠TC、TG、LDL、HDL和VLDL均明显升高（P<0.01）。与M组比较，Z组以上各血脂指标均显著降低（P<0.05或0.01）。
2.4血小板参数
16w末，与C组比较，M组血小板计数（platelet count，PLT）明显减少（P<0.01），MPV和血小板分布宽度（platelet distributing width，PDW）显著增高（P<0.05或0.01）；与M组比较，Z组MPV、PDW均明显降低（P<0.01）。各组间大型血小板比率（platelet-large cell ratio，P-LCR）无明显差异（P>0.05）。
2.5血浆FN含量
16w末，M组血浆FN含量明显高于C组（P<0.01）；与M组比较，Z组含量明显降低（P<0.05）。
2.6肾皮质t-PA、PAI-1mRNA的表达
16w末，各组t-PA mRNA的表达无明显差异（P>0.05）；M组PAI-1mRNA的表达显著高于C组（P<0.05），而Z组的表达低于M组（P<0.05）。
3糖尿病肾病瘀血阻络证大鼠肾皮质ACE2-Ang-(1-7)-Mas轴的变化及化瘀通络中药的干预作用
3.1肾皮质ACE2、Mas、ACE的表达
ACE2和ACE主要表达在各组大鼠肾小球和肾小管的上皮细胞中，Mas主要表达在各组大鼠肾皮质组织的近端小管。
C组大鼠各时间点肾皮质ACE2、Mas和ACE蛋白的表达均无显著差异（P>0.05）。M组大鼠肾皮质ACE2、Mas蛋白的表达均随时间呈现降低趋势：16w末ACE2蛋白的表达低于4w末（P<0.05）；16w末Mas蛋白的表达分别低于4w末和8w末（P<0.05或0.01）。M组不同时间点ACE蛋白的表达无统计学差异（P>0.05）。
8w末，ACE2和Mas蛋白在各组大鼠肾皮质组织的表达水平，无统计学差异（P>0.05）；ACE蛋白的表达水平仅在M、I两组间存在差异（P<0.05）。
16w末，肾皮质ACE2蛋白的表达，M组低于C组（P<0.01），I、Z两组均高于M组（P<0.05），且Z组高于I组（P<0.05）；Mas蛋白的表达，M组低于C组（P<0.05）；I、Z两组均高于M组（P<0.01）。ACE蛋白的表达，M组显著高于C组（P<0.01）；Z组低于M、I两组（P<0.05或0.01）。
3.2肾皮质ACE2、Mas、ACE mRNA的表达
以4w末C组大鼠作为参照，进行相对表达定量。C组大鼠各时间点肾皮质ACE2、Mas、ACE mRNA的表达无显著差异（P>0.05）。M组各时间点ACE2mRNA的表达均低于同时间点C组（P<0.01），在4w至16w之间先上升（P<0.01）、后下降（P<0.05）；M组Mas mRNA的表达在4w高于C组（P<0.01），在4w至16w之间持续下降（P<0.01），16w低于C组（P<0.05）；M组ACE mRNA的表达在4w低于C组（P<0.01），在4w至16w之间先上升（P<0.01）、后下降（P<0.05），8w、16w均高于C组（P<0.05或0.01）。
8w末，各组Mas mRNA的表达无显著差异（P>0.05）。与C组比较，M组ACE2mRNA的表达显著降低（P<0.01），ACE mRNA的表达显著升高（P<0.01）；与M组比较，Z组ACE2mRNA的表达显著升高（P<0.01），且高于I组（P<0.05）；I、Z两组ACE mRNA的表达均低于M组（P<0.05）。16w末，与C组比较，M组ACE2mRNA的表达显著降低（P<0.01），ACE mRNA的表达显著升高（P<0.05），而Mas mRNA的表达无显著差异（P>0.05）；与M组比较，Z组ACE2和Mas mRNA的表达均显著升高（P<0.05或0.01）；其中， Z组Mas mRNA的表达还同时高于I组（P<0.05）；M、I、Z三组间ACE mRNA的表达差异不明显（P>0.05）。
3.3肾皮质Ang-(1-7)的含量
16w末，与C组比较，M组大鼠的肾皮质Ang-(1-7)含量明显降低（P<0.01）；而I、Z两组的Ang-(1-7)含量均高于M组（P<0.01）。
3.4M组大鼠血液粘度与ACE2、Mas和ACE mRNA及其蛋白表达的线性相关分析
16w末，M组大鼠血浆粘度和不同切变率下全血粘度与肾皮质ACE2mRNA及其蛋白的表达均呈负相关（P<0.05或0.01），与ACE mRNA及其蛋白的表达均呈正相关（P<0.05或0.01）；而与Mas mRNA及其蛋白的表达无显著线性相关关系（P>0.05）。
4化瘀通络中药对糖尿病肾病瘀血阻络证肾损伤的干预作用
4.1糖尿病症状性指标的动态变化
腹腔注射STZ4w后，M组大鼠各时间点体重均明显低于同时间点C组（P<0.01）；16w末，I、Z两组体重均明显高于M组（P<0.05或0.01）。与同时间点C组比较， M组各时间点RBG、24h摄食热量、饮水量和尿量均显著升高（P<0.01）；与同时间点M组比较，I、Z两组各时间点RBG、24h摄食热量和尿量均无明显差异（P>0.05），仅8w末I、Z两组和16w末Z组24h饮水量显著低于同时间点M组（P<0.05或0.01）。
4.224h尿蛋白定量的动态变化
M组大鼠各时间点尿蛋白均高于同时间点C组（P<0.01）；8w末，I、Z两组尿蛋白明显低于M组（P<0.05或0.01）；16w末，Z组显著低于M、I两组（P<0.05或0.01）。
4.3肾功能
16w末，M组血肌酐（serum creatinine，Scr）、尿素氮（blood ureanitrogen，BUN）、尿酸（uric acid，UA）均高于C组（P<0.01）；与M组比较，I、Z两组Scr、BUN均有显著改善（P<0.05或0.01），Z组UA也低于M组（P<0.05）。
4.4肾脏指数
16w末，M组KI值明显高于C组（P<0.01）；而I、Z两组KI值均显著低于M组（P<0.05）。
4.5光镜观察肾皮质结构
C组肾皮质结构正常；M组肾小球肾小球体积肥大，基底膜轻度增厚，系膜轻度增生，毛细血管管腔扩张，肾小囊腔狭窄，肾小管上皮细胞排列紊乱、肥大；I、Z两组的肾小囊腔较宽，系膜增生有所改善。
4.6透射电镜观察肾小球超微结构
C组肾小球超微结构基本正常；M组大鼠肾小球基底膜均质性增厚且结构欠清晰，系膜基质增多，足突节段性融合；I、Z两组结构较清晰，基底膜节段性增厚，足突少部分融合。
5化瘀通络中药对高糖刺激下足细胞的干预作用
5.1MTT试验
HG组各时间点光密度（optical density，OD）值均明显低于同时间点NG组（P<0.05或0.01）；多个药物浓度均对高糖刺激24h、48h、72h对足细胞的增殖抑制有不同程度的逆转作用，其中，各时间点均以药物浓度100ug/ml的含药高糖组的OD值最高，且高于HG组（P<0.01）。以100ug/ml作为最佳给药浓度。
5.2细胞上清液Col-Ⅳ的含量
给予足细胞不同干预24h后，各组细胞上清液Col-Ⅳ的含量无显著差异（P>0.05）；不同干预48h、72h后，HG组细胞上清液Col-Ⅳ的含量均高于同时间点NG组（P<0.01），而(HG+Z)组干预48h后Col-Ⅳ的含量显著低于HG组（P<0.05）。
5.3足细胞ACE、ACE2蛋白的表达
ACE和ACE2蛋白特异性表达于足细胞胞浆中。不同干预48h，与NG组比较，HG组ACE蛋白表达显著升高（P<0.05），ACE2蛋白表达显著降低（P<0.01）；与HG组比较，(HG+Z)组ACE蛋白表达较低，而ACE2蛋白表达较高（P<0.05）。而不同干预24h、72h后，各组ACE2、ACE蛋白的表达均无明显差异（P>0.05）。
5.4足细胞ACE、ACE2mRNA的表达
分别与同时间点NG组比较， HG组各时间点ACE mRNA表达均显著升高（P<0.05），HG组24h、48h ACE2mRNA表达显著降低（P<0.05或0.01）；分别与同时间点HG组比较，仅48h (HG+Z)组ACE mRNA表达明显降低，ACE2mRNA表达明显升高（P<0.01）。
结论：
1DN大鼠爪掌、尾静脉有紫暗瘀血表现，血液存在高粘、高凝状态，与瘀血阻络的中医证候特征相符，确认了DN瘀血阻络证的存在。
2化瘀通络中药对DN大鼠瘀血阻于肾络的证候具有良性调节作用。
3DN大鼠肾皮质ACE2-Ang-(1-7)-Mas轴功能下调，化瘀通络干预16w可不同程度上调其功能，且作用优于对照药物厄贝沙坦。
4化瘀通络中药能有效减轻DN瘀血阻络证大鼠的肾损伤，且作用略优于对照药物厄贝沙坦。
5高糖刺激对小鼠足细胞具有抑制增殖、促进Col-Ⅳ分泌、上调ACE、下调ACE2表达的效应；化瘀通络中药干预48h能不同程度逆转以上效应。
Objectives: Diabetic nephropathy (DN) is one of the most commonmicrovascular complications of diabetes mellitus (DM), which meanwhile isone of leading causes for chronic renal failure in our country. Therefore,searching for its pathogenesis and effective preventing and controllingmeasures is of great significance. Chinese medicine has shown greatadvantage in preventing and controlling DN. In the opinion of most modernChinese medical scholars in nephropathy, the primary pathogenesis of DN is“blood stasis blocking renal collaterals”, besides “Qi and Yin vacuity” of DM.The principle “activating blood, removing stasis and dredging collaterals” isfollowed in DN treatment, which has become a consensus in the Chinesemedical field of nephropathy. Modern medicine has confirmed that theactivation of renin-angiotensin system (RAS) is the main mechanism involvedin DN. Based on the cognition of over-activation of the classic pathway ofRAS, the angiotensin-converting enzyme-angiotensin Ⅱ-angiotensin type1receptor [ACE-AngⅡ-AT1] pathway, in the development of DN, angiotensinconverting enzyme inhibitor (ACEI) and angiotensin receptor blocker (ARB)have been used as first-line agents in treating and delaying the renal injury ofDM in clinic. However, with the further study on RAS, another functional axis,angiotensin-converting enzyme-related carboxypeptidase2-angiotensin-(1-7)-Mas [ACE2-Ang-(1-7)-Mas] axis, was discovered to be against the function ofACE-AngⅡ-AT1axis. Maintaining and adjusting the balance between thesetwo pathways have become a new concept for diabetic renal injury therapy.Taking blood-stasis-blocking-collaterals syndrome and ACE2-Ang-(1-7)-Masaxis as two important entry points, this research is aimed to explore whether the blood-stasis-blocking-collaterals syndrome of DN is related to thedysfunction of ACE2-Ang-(1-7)-Mas axis, as well as whether the interventionof stasis-removing-and-collaterals-dredging medicine on blood-stasis-blocking-collaterals syndrome also improve the function of ACE2-Ang-(1-7)-Mas axis,by animal experiments and isolated culture of mouse podocytes.
Methods:
1Verification of stasis-blocking-collaterals syndrome in rats with diabeticnephropathy
A totle of32healthy male Sprague-Dawley rats of4weeks old, weighed80-100g were grouped randomly into blank control group (C group, n=8),high-glucose-high-fat control group (H group, n=10), and model group (Mgroup, n=14), after adaptive feeding for1week. Rats in C group were fed withregular rat chow, while others fed with high-glucose-high-fat diet. After4weeks, the rats in M group were injected intraperitoneailly withstreptozotocin (STZ) at the dose of40mg/kg body weight. The rats showingrandom blood glucose≥16.7mmol/L72hours after injection were consideredas successfully duplicated diabetic models. Rats in other two groups wereinjected with citrate buffer solution of equal volume. Random blood glucose(RBG) was measured on the3rd day and at the end of the4th,8th and16thweel after STZ injection. At the end of the16th week, after collection of urineof24hours for urine protein evaluation (UPE), blood samples were collectedby abdominal aortic method under anesthesia, which were used to test bloodviscosity measures, serum lipid profile and platelet parameters. Renal cortexof rats in different groups were examined by light microscope andtransmission electron microscope.
2Effect of stasis-removing-and-collaterals-dredging intervention upon stasis-blocking-collaterals syndrome in rats with diabetic nephropathy
A total of40experimental rats were divided into C group (n=10) andSTZ-injecting group (n=30). The establishment of models was same as above.Twenty-five diabetic rats were further divided into M group (n=12) andstasis-removing-and-collaterals-dredging group (Z group, n=13). Dosages of medicine in rats were calculated as5.8times as the clinical dosage in human.Rats in Z group were intragastrically fed with suspension of Chinese medicinegranules (1.08g/kg body weight, equal to crude drug4.7g/kg body weight),while rats in other groups with drinking water of equal volume, once daily for16weeks. At the end of the16th week, blood samples were collected byabdominal aortic method to test blood viscosity measures, serum lipid profile,platelet parameters and plasma fibronectin (FN) content. Renal cortex wascryopreserved to test the relative expression of tissue plasminogen activator(t-PA) and plasminogen activator inhibitor-1(PAI-1) mRNA by real-timePCR.
3The change of ACE2-Ang-(1-7)-Mas axis of renal cortex in DN rats ofstasis-blood-blocking-collaterals syndrome and effect of stasis-removing-and-collaterals-dredging intervention
A total of93experimental rats were divided into C group (n=22) andSTZ-injecting group (n=71). The establishment of models was same as above.Sixty-two diabetic rats were further divided into M group (n=24), irbesartangroup (I group, n=19), Z group (n=19). Interventions were given respectively.At the end of the4th week,6rats were choosed randomly from C and Mgroups respectively to be killed, and other rats were killed at the end of the8thand16th week separately. Renal cortex was cryopreserved to test theexpressions of ACE2, Mas, ACE proteins by immunohistochemical assay andwestern blot, relative expressions of ACE2, Mas, ACE mRNA by real-timePCR, and Ang-(1-7) content by ELISA.
4Effect of stasis-removing-and-collaterals-dredging intervention upon renalinjury in diabetic nephropathy of stasis-blood-blocking-collaterals syndrome
A total of57experimental rats were divided into C group (n=10) andSTZ-injecting group (n=47). The establishment of models, further division ofSTZ-injecting group and interventions were same as above. Body weight aswell as diet calorie, water intake, urine output and UPE of24hours, weremeasured on the3rd day and at the end of the4th,8th and16th week afterSTZ injection. Meanwhile, urine of24hours was collected to do the urine protein evaluation (UPE). At the sacrifice of rats at the end of the16th week,blood samples were collected to test kidney function; kidney indexes (KI)were calculated; and renal cortex was pathologically examined by lightmicroscope and transmission electron microscope.
5Effect of Chinese medicine of stasis-emoving-and-collaterals-dredgingintervention upon high glucose stimulating podocytes
The conditionally immortalized mouse podocytes cultured under growthpermissive condition were divided into normal glucose group (NG group),high glucose group (HG group) and medicated high glucose groups of12different concentrations, administered with corresponding culture mediainterventions for24,48and72hours. Effects of Chinese medicine of differentconcentrations upon cell proliferation under high glucose stimulation wereobserved by MTT test. Based on the comparison of data among differentconcentrations, the optimum concentration was screened for furtherexperiment. Differentiated and mature mouse podocytes cultured undergrowth restrictive condition were divided into NG group, HG group andmedicated high glucose group of the optimum concentration [(H+Z) group],administered with corresponding interventions for24,48and72hours.Collagen-Ⅳ (Col-Ⅳ) content in podocyte supernatant was tested by ELISA,and expressions of ACE and ACE2proteins in podocytes by immuno--cytochemical assay and western blot, relative expressions of ACE and ACE2mRNA by real-time PCR.
Results:
1Verification of stasis-blocking-collaterals syndrome in rats with diabeticnephropathy
1.1General condition
Compared with C and H groups, rats in M group showed polydipsia,polyphagia, polyuria and loss of weight gradually. Rats in C group had ruddypaws and unconspicuous dull-red tail veins, which was similar in H group,while M group had dark-purple paws and conspicuous dark-purple tail veins.1.2Random blood glucose
RBG of rats in C and H groups remained constant, with H group higherthan C group (P<0.01). After STZ injection, RBG in M group wassignificantly higher than C and H groups at same time points (P<0.01).
1.3UPE of24hours
At the end of16th week, there was no difference in UPE between C andH groups (P>0.05). Compared with C and H groups, UPE of M group washigher (P<0.01).
1.4Pathomorphology
Rats in C and H groups had normal sized kidneys, with smooth surface,from which glomeruli showed clear and well-distributed structures, with nofusion of foot processes arranged in order. Rats in M group had enlargedkidneys, with unsmooth surface and pale spots on them, from which enlargedswollen glomeruli revealed homogeneously thickened basement membranewith chaotic structure, increased mesangial matrix and segmental fusion ofpodocyte foot processes.
1.5Blood viscosity measures
At the end of16th week, only hematocrit (HCT) of rats in H group waslower than C group (P<0.05). Compared with C group, whole blood viscosity,plasma viscosity and whole blood low shear reductive viscosity of rats in Mgroup were all higher (P<0.01); while HCT and erythrocyte deformabilityindex (EDI) were lower (P<0.05or0.01). Among them, whole blood viscosity,plasma viscosity and EDI were also different from those in H group (P<0.05or0.01).
1.6Serum lipid profile
At the end of16th week, total cholesterol (TC), low density lipidcholesterol (LDL) and high density lipid cholesterol (HDL) of rats in H groupwere higher than C group (P<0.05or0.01); while triglyceride (TG) and verylow density lipid cholesterol (VLDL) were not different from C group(P>0.05). Compared with C and H groups, TC, TG, LDL, HDL and VLDL ofrats in M group were all significantly higher (P<0.01).
1.7Platelet parameters
At the end of16th week, only mean platelet volume (MPV) of rats in Hgroup was higher than C group (P<0.05). Compared with C group, plateletcount (PLT) of rats in M group decreased significantly (P<0.01); MPV andplatelet distributing width (PDW) of M group elevated significantly (P<0.01).And meanwhile, PLT and MPV of rats in M group were also different from Hgroup (P<0.05or0.01). There was no difference in platelet-large cell ratio(P-LCR) among different groups (P>0.05).
2Effect of stasis-removing-and-collaterals-dredging intervention upon stasis-blocking-collaterals syndrome in rats with diabetic nephropathy
2.1Blood viscosity measures
At the end of16th week, compared with C group, whole blood viscosity,plasma viscosity and whole blood low shear reductive viscosity of rats in Mgroup were all higher (P<0.01); while HCT and erythrocyte deformabilityindex (EDI) were lower (P<0.01). Compared with M group, whole bloodviscosity with30S-1and200S-1shear rates, plasma viscosity and EDI of Zgroup were improved significantly (P<0.05or0.01).
2.2Serum lipid profile
At the end of16th week, compared with C group, TC, TG, LDL, HDLand VLDL of rats in M group were all significantly higher (P<0.01).Compared with M group, all above indexes of Z group were lower (P<0.05or0.01).
2.3Platelet parameters
At the end of16th week, compared with C group, platelet count (PLT) ofrats in M group decreased significantly (P<0.01); MPV and plateletdistributing width (PDW) of M group elevated significantly (P<0.05or0.01).Compared with M group, MPV and PDW of Z group were lower (P<0.01).There was no difference in platelet-large cell ratio (P-LCR) among differentgroups (P>0.05).
2.4Plasma FN content
At the end of16th week, plasma FN content of rats in M group washigher significantly than C group (P<0.01). And compared with M group, Z group was lower (P<0.05).
2.5Relative expression of t-PA and PAI-1mRNA in renal cortex
At the end of16th week, there was no difference in the relativeexpression of t-PA mRNA among different groups (P>0.05). The relativeexpression of PAI-1mRNA in M group was significantly higher than C group(P<0.05). And its relative expression in Z group was lower than M group(P<0.05).
3The change of ACE2-Ang-(1-7)-Mas axis of renal cortex in DN rats ofstasis-blood-blocking-collaterals syndrome and effect of stasis-removing-and-collaterals-dredging intervention
3.1Expressions of ACE2, Mas, ACE proteins in renal cortex
ACE2and ACE expressed mainly in the epithelial cells of glomeruli andtubules of kidney, while Mas approximately expressed in the proximal tubules.There was no significant difference in expressions of ACE2, Mas and ACE inrenal cortex from rats in C group among different time points (P>0.05).Expressions of ACE2and Mas proteins in M group showed downtrend withtime. ACE2expression at the end of16th week was lower than4th week(P<0.05), while Mas expression at the end of16th week was lower than4thand8th week respectively (P<0.05or0.01). But there was no difference inACE expression in M group among different time points (P>0.05).
At the end of8th week, there was no significant difference in expressionsof ACE2and Mas among different groups (P>0.05). And there was statisticaldifference in ACE expression only between M and I groups (P<0.05).
At the end of16th week, ACE2expression in M group was lower than inC group (P<0.01); and that in I and Z groups were higher than in M group(P<0.05), with Z group higher than I group (P<0.05). Mas expression in Mgroup was lower than in C group (P<0.05); and that in I and Z groups washigher than in M group (P<0.01). ACE expression in M group was higher thanin C group (P<0.01); and that in Z group was lower than in M and I groups(P<0.05or0.01).
3.2Expressions of ACE2, Mas, ACE mRNA in renal cortex
Take rats in C group at the end of4th week as control to quantificate therelative expression. There was no difference in expressions of ACE2, Mas,ACE mRNA in C group among different time points (P>0.05). ACE2mRNAexpression in M group at each time point was lower than that in C group atsame time point (P<0.01), and experienced a course of first rising (P<0.01)then declining (P<0.05) from4th week to16th week. Mas mRNA expressionin M group at4th week was higher than that in C group at same time point(P<0.01), and experienced a declining course from4th week to16th week(P<0.01), which resulted in lower than C group at16th week (P<0.05). ACEmRNA expression in M group at4th week was lower than that in C group atsame time point (P<0.01), and experienced a course of first rising (P<0.01)then declining (P<0.05) from4th week to16th week, which resulted in higherthan C group at16th week (P<0.05).
At the end of8th week, there was no significant difference in Mas mRNAexpression among different groups (P>0.05). Compared with C group, ACE2mRNA expression in M group was lower (P<0.01), ACE mRNA expression inM group was higher (P<0.01). Compared with M group, ACE2mRNAexpression in Z group was higher (0.01), and higher than I group as well(P<0.05), while ACE mRNA expressions in both I and Z groups were lowerthan in M group (P<0.05).
At the end of16th week, compared with C group, ACE2mRNAexpression in M group was lower (P<0.01), ACE mRNA expression in Mgroup was higher (P<0.01), and Mas mRNA expression in M group showedno difference (P>0.05). Compared with M group, ACE2and Mas mRNAexpressions in Z group were both higher (P<0.05or0.01), with Mas mRNAexpression in Z group higher than I group (P<0.05). There was no statisticaldifference in ACE mRNA expressions among M, I and Z groups (P>0.05).
3.3Ang-(1-7) content in renal cortex
At the end of16th week, compared with C group, Ang-(1-7) content inrenal cortex from rats in M group was lower significantly (P<0.01). AndAng-(1-7) content in I and Z groups was higher than in M group (P<0.01).
3.4Linear correlation analysis between blood viscosity and expressions ofACE2, Mas, ACE mRNA and proteins of rats in M group
At the end of16th week, blood viscosity (including plasma viscosity andwhole blood viscosity under different shear rates) in M group was in negativecorrelation with ACE2mRNA and protein expressions (P<0.05or0.01), andin positive correlation with ACE mRNA and protein expressions (P<0.05or0.01). However, there was no linear correlation between blood viscosity andMas mRNA and protein expressions (P>0.05).
4Effect of stasis-removing-and-collaterals-dredging intervention upon renalinjury in diabetic nephropathy of stasis-blood-blocking-collaterals syndrome
4.1Dynamic trends of clinical indicators of diabetes
After4weeks, body weight of rats in M group at each time point waslower than C group at same time point (P<0.01). At the end of16th week,body weights in I and Z groups were higher than in M group (P<0.05or0.01).Compared with C group at same time point, RBG, diet calorie, water intakeand urine output of24hours of rats in M group at each time point were allsignificantly higher (P<0.01). Compared with M group at same time point,RBG, diet calorie and urine output of24hours of rats in I and Z groups ateach time point were not different significantly (P>0.05); while water intakeof24hours of rats in I and Z groups at the end of8th week and that in Z groupat the end of16th week were lower than M group (P<0.05or0.01).
4.2UPE of24hours
UPE in M group at each time point was significantly higher than C groupat same time point (P<0.01). At the end of8th week, UPE in I and Z groupswere lower than M group (P<0.05). And at the end of16th week, water intakein Z group was lower than M and I groups (P<0.05or0.01).
4.3Kidney function
At the end of16th week, serum creatinine (Scr), urea nitrogen (BUN) anduric acid (UA) of rats in M group were all higher than C group (P<0.01).Compared with M group, Scr and BUN of I and Z groups were lower (P<0.05or0.01), while UA of Z group was lower than M group as well (P<0.05).
4.4KI
At the end of16th week, KI of rats in M group was higher than C group(P<0.01). And KI in I and Z groups were lower than M group (P<0.05).
4.5Observation of renal cortical structures by light microscope
Renal cortex of rats in C group showed normal structures. Renal cortex ofM group revealed obvious hypertrophy of glomerular, slightly thickenedbasement membrane, expansion of mesangial matrix, expanded capillaries,narrow renal capsule and disarranged renal tubular epithelial cells. Comparedwith M group, renal capsules in I and Z groups were wider; and themesangial proliferations were both alleviated.
4.6Observation of ultrastructures of glomeruli by transmission electronmicroscope
Glomeruli from rats in C group showed normal ultrastructures. And thosefrom M group revealed homogeneously thickened basement membrane withnot clear structure, increased mesangial matrix and segmental fusion ofpodocyte foot processes. Compared with M group, glomeruli from rats in Iand Z groups showed clearer ultrastructures with segmentally thickenedbasement membrane and partially segmental fusion of podocyte footprocesses.
5Effect of Chinese medicine of stasis-emoving-and-collaterals-dredgingintervention upon high glucose stimulating podocytes
5.1MTT test
The optical density (OD) of HG group at each time point wassignificantly lower than NG group at same time point (P<0.05or0.01).Chinese medicine of several concentrations could reverse the suppressingproliferation effect of high glucose at each time point. Among differentconcentrations, the OD of100μg/ml medicated high glucose group was thehighest, and higher than HG group (P<0.01). The optimum concentration was100μg/ml.
5.2Col-Ⅳ content in podocyte supernatant
There was no significant difference in Col-Ⅳ content of podocyte supernatant among different groups podocyte at24hours (P>0.05). Col-Ⅳcontents of podocyte supernatant of HG group at48and72hours were bothhigher than NG group at same time points (P<0.01). And Col-Ⅳ content ofpodocyte supernatant of (HG+Z) group at48hours was significantly lowerthan HG group at same time point (P<0.05).
5.3Expressions of ACE and ACE2proteins in podocytes
The ACE and ACE2proteins expressed specificly in the cytoplasm ofpodocytes. Compared with NG group at48hours, the ACE protein expressionof HG group was higher (P<0.05), while the ACE2protein expression of HGgroup was lower (P<0.01). Compared with HG group at48hours, the ACEprotein expression of (HG+Z) group was higher (P<0.05), while the ACE2protein expression of (HG+Z) group was lower (P<0.05). However, there wasno difference in expressions of ACE and ACE2proteins among differentgroups at24and72hours (P>0.05).
5.4Relative expressions of ACE and ACE2mRNA in podocytes
Compared with NG group at same time point respectively, ACE mRNAexpression of HG group at each time point was higher significantly (P<0.05),and ACE2mRNA expression of HG group was lower significantly only at48and72hours time points (P<0.05or0.01). Compared with HG group at sametime point respectively, just at48hours time point, ACE mRNA expression of(HG+Z) group was lower (P<0.05), and ACE2mRNA expression of (HG+Z)group was higher (P<0.01).
Conclusions:
1DN rats had dark purple paws and tail veins; and their blood in highviscosity, hypercoagulable state. These were in accordance with the features ofblood-stasis-blocking-collaterals syndrome in Chinese medicine, whichaffirmed the existence of blood-stasis-blocking-collaterals syndrome.
2The stasis-removing-and-collaterals-dredging therapy played a benefitrole in regulating blood-stasis-blocking-renal-collaterals syndrome in DN.
3The function of ACE2-Ang-(1-7)-Mas axis was down-regulated in DMrats. The stasis-removing-and-collaterals-dredging intervention for16weeks could up-regulate its function, which effect was superior to thepositive control drug irbesartan.
4The stasis-removing-and-collaterals-dredging therapy alleviated therenal injury in diabetic nephropathy of stasis-blood-blocking-collateralssyndrome effectively, which effect was slightly superior to the positivecontrol drug irbesartan.
5High glucose stimulation had effects of suppressing proliferation,promoting Col-Ⅳ secretion, up-regulating ACE expression anddown-regulating ACE2expression upon mouse podocytes. Chinese medicineof stasis-removing-and-collaterals-dredging intervention for48h could reversethe negative effects of high glucose stimulating podocytes.
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