益气活血中药复方通脉口服液干预DN的疗效观察及肾保护作用研究
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摘要
目的：
探讨益气活血中药复方通脉口服液干预糖尿病肾病的疗效及肾保护作用。
方法：
一、临床部分：
病例来源：广东省中医院本部及二沙、芳村分院的住院及门诊病人。
诊断标准：
①西医诊断标准：
(1)糖尿病诊断标准：参照1999年WHO标准。
(2)糖尿病肾病分期标准：参照Mogensen标准及王海燕《肾脏病学》。
②中医证型诊断标准：
参照2002年版《中药新药临床研究指导原则》中关于气虚证及血瘀证的辨证标准。
③症状积分标准：
症状按无、轻、中、重记0、1、2、3分，舌脉记“有”为1分，“无”为0分。
监测项目：每二周监测症状、舌脉、血压、餐后2h毛细血管全血血糖，试验前后各监测尿24h尿蛋白定量，血清尿素氮(BUN)、血肌酐(SCr)、总胆固醇(TCh)、甘油三酯(TG)。
疗程：8周。
治疗方案：
①基础治疗：
(1)糖尿病健康教育、体育锻炼及低盐优质蛋白糖尿病饮食：每日食盐摄入量≤6克，精蛋白摄入量：0.8g／Kg·d；
(12)乃控制血压：目标：＜130／80mmHg：可应用非ACEI及ARB外的所有降压药；
(3)控制血糖：目标：FPG≤7.0mmol／L 2HPG≤10.0mmol／L全部应用糖适平和／或胰岛素治疗；
(4)对症处理。
②分组治疗：
(1)T组：通脉口服液：1支／次，3次／日，口服；
(2)L组：洛汀新片：10mg／次，1次／日，口服。
调查方法：
①药物洗脱：共2周，停用所有ACEI、ARB、益气活血类中药等制剂；给予受试者DM教育，低盐低脂优质低蛋白饮食；血压控制于＜130／80mmHg水平；血糖控制在FPG≤7.0mmol／L，2HPG≤10.0mmol／L水平；血脂控制在TCH≤5.98mmol／L，TG≤1.7mmol／L水平。结束复查24h尿蛋白定量：
②分组：将合格病例分为洛汀新组(L组)及通脉组(T组)：
③试验过程依从性：采用药物记量的方法了解患者依从性。研究者应耐心向患者做好解释工作，提前电话通知，使其充分理解，配合研究。免除挂号费以保证按期就诊率。
④干扰和沾染的控制：所有病人受试期间不得接受试验措施以外的相关药物或治疗，尽量保证病人按照治疗方案规定用药，而不受其他药物等治疗措施的影响。
二、实验部分：
实验一：通脉口服液对实验性糖尿病模型大鼠肾脏保护作用的研究
选鼠分组：选用SD雄性大鼠，设立正常对照组(C组)；余动物采用STZ单次腹腔注射方法制备糖尿病(DM)动物模型，DM大鼠模型成功后随机分为模型组(M组)、通脉组(T组)及洛汀新组(L组)。
干预起始点：DM模型成功，分组后即开始干预。
干预方式：①饲料：T、L、M组给予隔日高脂饲料，C组给予普通饲料；实验期间均自由进食和饮水；②药物：T组给予通脉口服液灌胃，L组给予洛汀新灌胃，C及M组给予生理盐水灌胃。
干预时限：6周。
大鼠淘汰标准：①干预过程中，测定血糖值＜16.8mmol／L者，予以淘汰；②6周后处死大鼠，光镜示肾组织无明显病变者，予以淘汰。
观察项目：大鼠一般情况、体重、尿量、24h尿蛋白定量、总胆固醇(TCh)、甘油三脂(TG)、血糖(BG)、血尿素氮(BUN)、血肌酐(SCr)、肾重／体重比值(K／W)；光镜、电镜下进行肾脏组织病理形态学观察。
实验二：通脉口服液对实验性糖尿病肾病模型大鼠的肾脏保护作用研究
选鼠、C组设立、DM动物模型制备：同实验一。
DN模型制备及分组：DM大鼠模型成功后，给予隔日高脂饲料饲养4周，DN大鼠模型成功，随机分为M组、T组及L组。
干预起始点：DN模型成功，分组后即开始干预。
干预方式、干预时限、大鼠淘汰标准：同实验一。
观察项目：测量肾小球截面积(MGA)、细胞外基质截面积(MMA)、肾小球基底膜厚度(GBM)，计算细胞外基质截面积／肾小球截面积(MMA／MGA)比值；
余项目同实验一。
实验三：通脉口服液对实验性糖尿病肾病模型大鼠肾组织MMP-9及其作用底物、调控因子表达影响的研究
标本来源：选择实验二干预后C组、M组、T组大鼠肾组织，每组随机选择6例标本。
实验方法：免疫组化法。
观察项目：测定肾组织Col-Ⅳ、RMP-9、TIMP-1、TGF-β_1等的免疫组化染色面积、面密度及积分光密度，计算MMP-9／TIMP-1免疫组化染色面积比值。
结果：
一、临床部分：
①疗程前两组患者一般情况、24h尿蛋白定量、肾功能等，差异无统计学意义(P＞0.05)；疗程中，两组患者血压、血糖、血脂等差异无统计学意义(P＞0.05)；
②疗程后T组DN患者24h尿蛋白定量、症状积分较治疗前明显降低，差异有显著性统计学意义(P＜0.01)；
③疗程后T组与L组比较：24h尿蛋白定量前后差值，差异无统计学意义(P＞0.05)，T组症状积分前后差值高于L组，差异有显著性统计学意义(P＜0.01)。
二、实验部分：
实验一：通脉口服液对实验性糖尿病模型大鼠肾脏保护作用的研究
体重、尿量：
①T、L、M三组较C组大鼠尿量增加，体重降低，差异有显著性统计学意义(P＜0.01)；
②T组大鼠尿量较L、M组有减少的趋势，体重较L、M组有增加的趋势，但T、L、M三组间尿量及体重差异无统计学意义(P＞0.05)。
24h尿蛋白定量：
①T、L、M三组24h尿蛋白定量较C组升高，差异有显著性统计学意义(P＜0.01)；
②T组24h尿蛋白定量较M组降低，差异有显著性统计学意义(P＜0.01)；与L组比较差异无统计学意义(P＞0.05)。
血糖：
①T、L、M三组与C组比较血糖持续升高，差异有显著性统计学意义(P＜0.01)；
②T、L、M三组组间血糖比较差异无统计学意义(P＞0.05)。
血尿素氮、肌酐：
C、T、L、M四组组间比较差异无统计学意义(P＞0.05)。
总胆固醇、甘油三脂：
①T、L、M三组TCh、TG较C组均升高，差异有显著性统计学意义(P＜0.01)；
②T组TCh、TG较M组、L组均降低，差异有统计学意义(P＜0.05或P＜0.01)。
肾重／体重比值；
①T、L、M三组K／W比值较C组均升高，差异有显著性统计学意义(P＜0.01)；
②T组K／W比值较M组降低，差异有显著性统计学意义(P＜0.01)，与L组比较差异无统计学意义(P＞0.05)。
肾组织病理形态学：
①M组肾小球肥大、系膜区增宽等病变较C组明显加重；
②T组肾小球肥大、系膜区增宽等病变较M组减轻，和L组无明显差异。
实验二：通脉口服液对实验性糖尿病肾病模型大鼠的肾脏保护作用研究
体重、尿量：
T组大鼠尿量较M组、L组明显减少，体重较M组、L组增加，差异有统计学意义(P＜0.01或P＜0.05)。
尿素氮：
T组较M组、L组均降低，差异有统计学意义(P＜0.01或P＜0.05)。
24h尿蛋白定量、总胆固醇、甘油三脂、血糖、血肌酐、肾重／体重比值：
结果同实验一。
肾组织病理形态学：
①M组系膜区增宽、基底膜增厚及肾小管上皮细胞空泡变性等，较C组明显加重；
②T组系膜区增宽、基底膜增厚及肾小管上皮细胞空泡变性等，较M组明显减轻，和L组无明显差异。
细胞外基质截面积／肾小球截面积比值：
①M组MMA／MGA比值较C组增加，差异有显著性统计学意义(P＜0.01)；
②T组MMA／MGA比值较M组、L组降低，差异有统计学意义(P＜0.01或P＜0.05)。
肾小球基底膜厚度：
①M组GBM较C组增厚，差异有显著性统计学意义(P＜0.01)；
②T组GBM较M组变薄，差异有显著性统计学意义(P＜0.01)，与L组比较差异无统计学意义(P＞0.05)。
实验三：通脉口服液对实验性糖尿病肾病模型大鼠肾组织MMP-9及其作用底物、调控因子表达影响的研究
Ⅳ型胶原：主要表达定位于肾小球，和C组比较，M组呈强阳性表达；T组免疫组化染色面密度及积分光密度均较M组降低，差异有显著性，差异有显著性统计学意义(P＜0.01)；
MMP-9：主要表达定位于肾小球，肾间质内有少量表达，和C组比较，M组较弱表达；T组免疫组化染色面密度及积分光密度均较M组升高，差异有显著性，差异有显著性统计学意义(P＜0.01)；
TIMP-1：主要表达定位于肾小球，肾小管及间质区内表达较弱，和C组比较，M组呈强阳性表达；T组免疫组化染色面密度及积分光密度均较M组降低，差异有统计学意义(P＜0.05)；
TGF-β_1：主要定位在肾小管及间质处，肾小球表达较弱；T组免疫组化染色面密度及积分光密度均较M组降低，差异有显著性，差异有显著性统计学意义(P＜0.01)。
MMP-9／TIMP-1：M组MMP-9／TIMP-1比值较C组降低，差异有显著性，差异有显著性统计学意义(P＜0.01)；T组MMP-9／TIMP-1比值较M组升高，差异有显著性，差异有显著性统计学意义(P＜0.01)；MMP-9／TIMP-1比值与Col-Ⅳ、TGF-β_1免疫组化染色面积均存在负相关(r=-0.919或r=-0.877，P＜0.01)；Col-Ⅳ免疫组化染色面积与TGF-β_1免疫组化染色面积存在正相关(r=0.820，P＜0.01)。
结论：
1．益气活血中药复方通脉口服液临床观察，结果显示：该中药可以有效改善DN患者的临床症状，减轻尿蛋白，保护肾功能；动物实验结果显示：该中药可以有效减轻实验性DN模型大鼠尿蛋白、调节血脂及改善肾脏病理损害。
2．益气活血中药复方通脉口服液对DN肾脏保护作用的机制可能是：改善脂质代谢，减轻尿蛋白，抑制肾组织TGF-β_1表达，调节MMP-9／TIMP-1比例失衡，增加Ⅳ型胶原降解，减轻ECM积聚，改善肾脏肥大，从而调节细胞、细胞外基质、细胞因子和生长因子间的网络调控，延缓DN肾小球硬化的进展。
3．益气活血中药复方通脉口服液，在预防实验性DM模型大鼠尿蛋白出现，及预防实验性DN模型大鼠尿蛋白加重两方面，均可获得较为满意的疗效，和西药比较，其优势体现在改善症状，调节血脂。结合临床，提示持续蛋白尿可以作为本药干预糖尿病肾病的起始点。
Background
Recently, the prevalence rate of diabetes is rising unceasingly in ourcountry. Along with insulin widespread application, the mortality rate ofDiabetes' acute complication,such as ketoacidosis, are obviouslydropped, and the chronic complication such as diabetic nephropathy has becomeone of most common complications with diabetic life extension. In clinicalpractice, diabetes nephrosis is often divided into 3 stages, namely: morningstage, intermediate stage and advanced stage. In different stage, the emphasisof treatment is also different.
Professor Yang consider that the Deficiency of Qi and the Blood Stasis isthe mainly etiopathogenesis of diabetic nephropathy, especially when diseaseis morning or intermediate stage, so the therapeutic rule-"benefiting vitalenergy and promoting blood flow" can applied in the treatment of diabeticnephropathy; when their stage are morning or intermediate, the effectivetreatment is the key of pathogenetic condition reverse. So we study themechanism of action of Tongmai Oral Solution to the diabetic nephropathy bythe methods of pharmacodynamics and morphology, in order to provide thesubstantial theory and plenitudinous scientic evidence for Tongmai OralSolution which is composed according to benefiting vital energy and promotingblood flow method cure diabetic nephropathy.
Objective.
To explore the effect and mechanism of TongMai Oral Solution in the defence and treatment of diabetic nephropathy, and found Tongmai Oral Solution' sfunction superiority stage.
Methods:
1). Clinical study:
This part of the study is controlled. According to degree of proteinuriaand general condition, 63 patients suffered from morning or intermediate stagediabetic nephropathy were divided into treatment and control group. There wasno statistically significant difference between two groups in sex, age,severity of the disease and laboratory items. The patients of the treatmentgroup received management of rongMai Oral Solution while the patients of thecontrol group took the drug of Lotensin. The course of treatment lasted for8 weeks, during which the changes of clinical findings and lab index weremonitored and recorded, including renal function, proteinuria, blood fat,blood pressure, blood glucose, and clinical symptoms and signs. Thedifferences between the symptom index before and after treatment were thencalculated and tested, as well as the quantities of the urine protein of 24hours.
2). Experimental Study: (1). PartⅠ. Study of protective effects of TongMai oral solution on thekidney of Diabetes Mellitus rats.
a. preparation of animal model. 42 SD male adult rats of specified-pathogenfree were injected with streptozotocin (STZ) intraperitoneally to prepare forDiabetes Mellitus animal model and then randomly divided into 3 groupsincluding model group(M group), TongMai Oral Solution group(T group)andLotensin group(L group) after diabetes was induced successfully. Another groupwas set for experiment control, consisted of normal SD rats.
b. Methods of intervention. The rats of T, L, M groups were raised withhigh fat forage every other day while the rats of normal group were raisedwith normal one. Different drugs were perfused into stomach of rats includingTongMai Oral Solution for T group, Lotensin for L group, and sodium chloridefor C and M groups. All the rats were observed closely for 6 weeks. Duringthis period, the rats with values of blood glucose less than 16.8 mmol/L orslight renal pathogenic abnormalities under light microscope were excludedfrom the experimental studies. Several items were recorded to assess renalprotective effects of TongMai oral solution, including general condition, body mass, urine volume, quantities of urinary protein of 24 hours, serum bloodcholesterol total, serum glycerol, blood glucose, blood urea nitrogen, serumcreatinine, and ratio of kidney mass to body mass (K/W).
(2) PartⅡ. Study of renal protective effects of TongMai oral solutionon the rats of diabetes nephropathy animal model.
After diabetes was induced successfully, the rats of diabetes animal modelwere continually raised with high fat food every other day for another 4 weeksin order to induce the diabetes nephropathy in them. The rats, in which diabetesmellitus were induced successfully, were then grouped and treated in a mannerthe same as that of PartⅠ, controlled by normal SD rats. After 6 weeks ofdrugs treatments or placebo managements, all the animals were put to deathand their kidneys were removed for the pathological study including sectionarea of glomerulus (MGA), section area of extracel]ular matrix (MMA),thickness of glemerular basement menbrance (GBM), and the ratio of thesection area of MMA to MGA.
PatrⅢ. Study of effects of the TongMai oral solution on the expressionof MMP-9 and its substrates, as well as regulation factor
6 renal samples were randomly obtained from the rats of group C, M, andT in PartⅡstudy, respectively. Expression of typeⅣcollagen, MMP-9, TIMP-1,and TGF-β1 were detected by immunohistochemistry. The stained area, areadensity, and photodensity were then observed and recorded. The ratio of stainedarea of MMP-9 to TIMP-1 was also determined.
Results:
1). Clinical study.
(a). there's no statistical significant difference in the generalcondition of the patients, the quantities of urinary protein of 24 hours, andrenal function before treatment. No statistical significance was found in thedifference of blood pressure, values of blood glucose, and values of the bloodfat during the course of treatment.
(b).As to the patients of T group, the symptomatic scores and quantity ofurinary protein of 24 hours after treatment decreased obviously compared tothat before the treatment, with statistical significance (P＜0.01).
(c).There were no statistical significance found in the difference ofchanges of quantity of urinary protein of 24 hours after treatment betweenthe T group and L group (P＞0.05). However, the changes of symptomatic scores in T group was higher than that of L group after treatment with statisticalsignificance (P＜0.01).
2) Experimental Study:
(1). PartⅠ. Study of protective effects of TongMai oral solution on thekidney of Diabetes Mellitus rats.
a. The changes of body mass and urine volume. The mean values of urine volumein the T, L, and M groups increased concomitant with loss of body mass withstatistical significance (p＜0.01), in contrast to that of C group. Althoughthere' s descent trend in the urine volume and increasing trend in the bodymass of T group compare to that of L and M group, no statistical significancewas demonstrated in the difference of the values of both items among T, L,and M group (P＞0.05).
b. Quantity of urinary protein of 24 hours. Quantity of urinary proteinin T, L, and M group increased with statistical significance, compared to thatof C group (P＜0.01). Excretion of urinary protein in 24 hours in T groupdecreased statistically significantly, in contrast to that of M group (P＜0.01).No statistical significance was demonstrated in the difference of dischargeof urinary protein in 24 hours between the L and T group (P＞0.05).
c. Changes of blood glucose level. The mean value of blood glucose of T,L, and M group increased with statistical significance, compare to that ofC group (P＜0.01). No statistical significance was demonstrated in thedifferences of values of blood glucose among T, L, and M group (P＞0.05).
d. Changes of values of blood urea nitrogen and serum creatinine. There' sno statistical significant difference demonstrated between C, T, L, and M group
e. The cholesterol total(TCh) and glycerol total (TG) fluctuation. The meanvalues of ZCh and TG in T, L, and M group increased statisticallysignificantly, compared that of C group (P＜0.01). In T group, the values ofboth TCh and TG decreased with statistical significance, compared to that ofM and L group (P＜0.01 or P＜0.05).
f. Ratio of kidney mass to body mass (K/W). The ratios of K/W of T, L, andM group increased with statistical significance, in contrast to that of C group(P＜0.01). The ratio of K/W of T group was less than that of M group withstatistical significance (P＜0.01). However, no statistical significance wasdemonstrated in the difference of ratio of K/W between the T and L group
g. Pathological changes of kidney. Renal pathohistology changes in M group,such as glomerular hypertrophy and widening of mesangial region, were foundworse than that of C group. The similar pathohistology abnormalities were alsodetected in both T and L group but to a less degree. No difference was determinedto be statistically significant as to the severity of renal pathohistoloyabnormality between T and L group.
(2) PartⅡ. Study of renal protective effects of TongMai oral solutionon the rats of diabetes nephropathy animal model.
a. changes of body mass and urine volume. The mean value of urine volumein T group decreased while the body mass in T group increased, compared tothat of M and L group. Both the changes of body mass and urine volume werestatistically significantly (P＜0.05 or P＜0.01).
b. The blood urea nitrogen. The mean value of blood urea nitrogen of T groupdecreased with statistical significance (P＜0.01 or P＜0.05).
c. other items. Outcomes of quantity of urinary protein in 24 hours, TCh,TG, Blood glucose, serum creatinine, and ratio of K/W were referred to thatof PartⅠof the experimental study.
d. The renal pathohistology. Microscopic abnormalities of the kidney formrats of M group included widening of mesangial region, thickness of renalbasement membrane, and vacuolar degeneration of renal tubular epithelia. Theserenal changes in M group were worse than that seen in the C group.Although similar renal abnormalities could also be found in T group, it' sobvious that the abnormalities became less serious in T group and L group.No difference was demonstrated between T and L group.
e. Ratio of section area of extracellular matrix (MMA) and glomerulus(MGA). it' s higher of ratio of MMA/MGA in M group than that of C group withstatistical significance (P＜0.01). However, in T group the ratio of MMA/MGAwas less than that of both the M and L group (P＜0.01 or P＜0.05).
f. The thickness of renal glomerulus basement membrane. The renal basementmembrane got thicker than that of C group with statistical significance(P＜0.01). Compared to that of M group, the GBM of T group was thinner withstatistical significance (P＜0.01).No difference of the same index was foundto be statistical between T and L group (P＞0.05).
(3) PartⅢ. Study of effects of the TongMai oral solution on the expression of MMP-9 and its substrates, as well as regulation factor.
a. Expression of typeⅣcollagen. The typeⅣcollagen was located in theglomerulus. Strong positive expression was found in group M. According to theoutcomes of immunohistology, the values of area density and photodensity ofpositively stained area were less than that of M group with statisticalsignificance (P＜0.01).
b. Expression of MMP-9. The expression of MMP-9 was located mainly inglomerulus and sparse expression could be seen in renal interstitium. It'smore weakly expressed in group M than in C group. The value of area densityand photodensity in MMP-9 positively stained area were higher than that ofM group (P＜0.01).
c. Expression of TIMP-1. Expression of TIMP-1 was mainly located inglomerulus with weakly expression located in renal tubule and interstitium.It's stronger positively expressed in M group than in C. The values of areadensity and photodensity of TIMP positively stained area in group T were lowerthan that of group M with statistical significance (P＜0.05).
d. Ratio of MMP-9/TIMP-1. It's higher of ratio of MMP-9/TIMP-1 in groupC than that of group M with statistical significance (P＜0.01). However, thisratio in group T was higher than that of group M with statistical significance(P＜0.01). The ratio of MMP-9/TIMP-1 and photodensity of typeⅣcollagenchanged in reverse direction.
e. Expression of TGF-β1. Expression of TGF-β1 was located in the renaltubule and interstitium. It's weakly expressed in the glomerulus. It's lowerfor the value of area density and photodensity in group T than that of groupM with statistical significance (P＜0.01).
Conclusion
(1). Tongmai Oral Solution can improve the patients' clinical symptomsand renal function, decrease the urine protein.
(2). The mechanism that Tongmai Oral Solution ameliorated the structureand function damage of DN model rats may be due to enhancement of metabolismof blood fat, up-regulation of the expression of MMP-9, down-regulation ofthe expression of TGF-β1, rebuilding of balance between MMP-9 and TIMP-1,inhibition of thickening of the glomerular basement membrane, increase ofdegeneration of typeⅣcollagen, reduction of extracellular matrixaccumulation, and amelioration of renal pathological lesions. These therapeutic effects may improve renal hypertrophy, regulation of cells, ECM,cytokines, and growth factors resulting in delayed progress of DN.
(3). In the course of intervention of DN model rats, excellent renalprotective effects could be obtained not only at the beginning of initiatestage of model induction but at the time when urinary protein appears in theurine of DN model rats. This reveals that the point that the urinary proteinappearances could be regarded as initiate point when Tongmai Oral Solutionis given.
探讨益气活血中药复方通脉口服液干预糖尿病肾病的疗效及肾保护作用。
方法：
一、临床部分：
病例来源：广东省中医院本部及二沙、芳村分院的住院及门诊病人。
诊断标准：
①西医诊断标准：
(1)糖尿病诊断标准：参照1999年WHO标准。
(2)糖尿病肾病分期标准：参照Mogensen标准及王海燕《肾脏病学》。
②中医证型诊断标准：
参照2002年版《中药新药临床研究指导原则》中关于气虚证及血瘀证的辨证标准。
③症状积分标准：
症状按无、轻、中、重记0、1、2、3分，舌脉记“有”为1分，“无”为0分。
监测项目：每二周监测症状、舌脉、血压、餐后2h毛细血管全血血糖，试验前后各监测尿24h尿蛋白定量，血清尿素氮(BUN)、血肌酐(SCr)、总胆固醇(TCh)、甘油三酯(TG)。
疗程：8周。
治疗方案：
①基础治疗：
(1)糖尿病健康教育、体育锻炼及低盐优质蛋白糖尿病饮食：每日食盐摄入量≤6克，精蛋白摄入量：0.8g／Kg·d；
(12)乃控制血压：目标：＜130／80mmHg：可应用非ACEI及ARB外的所有降压药；
(3)控制血糖：目标：FPG≤7.0mmol／L 2HPG≤10.0mmol／L全部应用糖适平和／或胰岛素治疗；
(4)对症处理。
②分组治疗：
(1)T组：通脉口服液：1支／次，3次／日，口服；
(2)L组：洛汀新片：10mg／次，1次／日，口服。
调查方法：
①药物洗脱：共2周，停用所有ACEI、ARB、益气活血类中药等制剂；给予受试者DM教育，低盐低脂优质低蛋白饮食；血压控制于＜130／80mmHg水平；血糖控制在FPG≤7.0mmol／L，2HPG≤10.0mmol／L水平；血脂控制在TCH≤5.98mmol／L，TG≤1.7mmol／L水平。结束复查24h尿蛋白定量：
②分组：将合格病例分为洛汀新组(L组)及通脉组(T组)：
③试验过程依从性：采用药物记量的方法了解患者依从性。研究者应耐心向患者做好解释工作，提前电话通知，使其充分理解，配合研究。免除挂号费以保证按期就诊率。
④干扰和沾染的控制：所有病人受试期间不得接受试验措施以外的相关药物或治疗，尽量保证病人按照治疗方案规定用药，而不受其他药物等治疗措施的影响。
二、实验部分：
实验一：通脉口服液对实验性糖尿病模型大鼠肾脏保护作用的研究
选鼠分组：选用SD雄性大鼠，设立正常对照组(C组)；余动物采用STZ单次腹腔注射方法制备糖尿病(DM)动物模型，DM大鼠模型成功后随机分为模型组(M组)、通脉组(T组)及洛汀新组(L组)。
干预起始点：DM模型成功，分组后即开始干预。
干预方式：①饲料：T、L、M组给予隔日高脂饲料，C组给予普通饲料；实验期间均自由进食和饮水；②药物：T组给予通脉口服液灌胃，L组给予洛汀新灌胃，C及M组给予生理盐水灌胃。
干预时限：6周。
大鼠淘汰标准：①干预过程中，测定血糖值＜16.8mmol／L者，予以淘汰；②6周后处死大鼠，光镜示肾组织无明显病变者，予以淘汰。
观察项目：大鼠一般情况、体重、尿量、24h尿蛋白定量、总胆固醇(TCh)、甘油三脂(TG)、血糖(BG)、血尿素氮(BUN)、血肌酐(SCr)、肾重／体重比值(K／W)；光镜、电镜下进行肾脏组织病理形态学观察。
实验二：通脉口服液对实验性糖尿病肾病模型大鼠的肾脏保护作用研究
选鼠、C组设立、DM动物模型制备：同实验一。
DN模型制备及分组：DM大鼠模型成功后，给予隔日高脂饲料饲养4周，DN大鼠模型成功，随机分为M组、T组及L组。
干预起始点：DN模型成功，分组后即开始干预。
干预方式、干预时限、大鼠淘汰标准：同实验一。
观察项目：测量肾小球截面积(MGA)、细胞外基质截面积(MMA)、肾小球基底膜厚度(GBM)，计算细胞外基质截面积／肾小球截面积(MMA／MGA)比值；
余项目同实验一。
实验三：通脉口服液对实验性糖尿病肾病模型大鼠肾组织MMP-9及其作用底物、调控因子表达影响的研究
标本来源：选择实验二干预后C组、M组、T组大鼠肾组织，每组随机选择6例标本。
实验方法：免疫组化法。
观察项目：测定肾组织Col-Ⅳ、RMP-9、TIMP-1、TGF-β_1等的免疫组化染色面积、面密度及积分光密度，计算MMP-9／TIMP-1免疫组化染色面积比值。
结果：
一、临床部分：
①疗程前两组患者一般情况、24h尿蛋白定量、肾功能等，差异无统计学意义(P＞0.05)；疗程中，两组患者血压、血糖、血脂等差异无统计学意义(P＞0.05)；
②疗程后T组DN患者24h尿蛋白定量、症状积分较治疗前明显降低，差异有显著性统计学意义(P＜0.01)；
③疗程后T组与L组比较：24h尿蛋白定量前后差值，差异无统计学意义(P＞0.05)，T组症状积分前后差值高于L组，差异有显著性统计学意义(P＜0.01)。
二、实验部分：
实验一：通脉口服液对实验性糖尿病模型大鼠肾脏保护作用的研究
体重、尿量：
①T、L、M三组较C组大鼠尿量增加，体重降低，差异有显著性统计学意义(P＜0.01)；
②T组大鼠尿量较L、M组有减少的趋势，体重较L、M组有增加的趋势，但T、L、M三组间尿量及体重差异无统计学意义(P＞0.05)。
24h尿蛋白定量：
①T、L、M三组24h尿蛋白定量较C组升高，差异有显著性统计学意义(P＜0.01)；
②T组24h尿蛋白定量较M组降低，差异有显著性统计学意义(P＜0.01)；与L组比较差异无统计学意义(P＞0.05)。
血糖：
①T、L、M三组与C组比较血糖持续升高，差异有显著性统计学意义(P＜0.01)；
②T、L、M三组组间血糖比较差异无统计学意义(P＞0.05)。
血尿素氮、肌酐：
C、T、L、M四组组间比较差异无统计学意义(P＞0.05)。
总胆固醇、甘油三脂：
①T、L、M三组TCh、TG较C组均升高，差异有显著性统计学意义(P＜0.01)；
②T组TCh、TG较M组、L组均降低，差异有统计学意义(P＜0.05或P＜0.01)。
肾重／体重比值；
①T、L、M三组K／W比值较C组均升高，差异有显著性统计学意义(P＜0.01)；
②T组K／W比值较M组降低，差异有显著性统计学意义(P＜0.01)，与L组比较差异无统计学意义(P＞0.05)。
肾组织病理形态学：
①M组肾小球肥大、系膜区增宽等病变较C组明显加重；
②T组肾小球肥大、系膜区增宽等病变较M组减轻，和L组无明显差异。
实验二：通脉口服液对实验性糖尿病肾病模型大鼠的肾脏保护作用研究
体重、尿量：
T组大鼠尿量较M组、L组明显减少，体重较M组、L组增加，差异有统计学意义(P＜0.01或P＜0.05)。
尿素氮：
T组较M组、L组均降低，差异有统计学意义(P＜0.01或P＜0.05)。
24h尿蛋白定量、总胆固醇、甘油三脂、血糖、血肌酐、肾重／体重比值：
结果同实验一。
肾组织病理形态学：
①M组系膜区增宽、基底膜增厚及肾小管上皮细胞空泡变性等，较C组明显加重；
②T组系膜区增宽、基底膜增厚及肾小管上皮细胞空泡变性等，较M组明显减轻，和L组无明显差异。
细胞外基质截面积／肾小球截面积比值：
①M组MMA／MGA比值较C组增加，差异有显著性统计学意义(P＜0.01)；
②T组MMA／MGA比值较M组、L组降低，差异有统计学意义(P＜0.01或P＜0.05)。
肾小球基底膜厚度：
①M组GBM较C组增厚，差异有显著性统计学意义(P＜0.01)；
②T组GBM较M组变薄，差异有显著性统计学意义(P＜0.01)，与L组比较差异无统计学意义(P＞0.05)。
实验三：通脉口服液对实验性糖尿病肾病模型大鼠肾组织MMP-9及其作用底物、调控因子表达影响的研究
Ⅳ型胶原：主要表达定位于肾小球，和C组比较，M组呈强阳性表达；T组免疫组化染色面密度及积分光密度均较M组降低，差异有显著性，差异有显著性统计学意义(P＜0.01)；
MMP-9：主要表达定位于肾小球，肾间质内有少量表达，和C组比较，M组较弱表达；T组免疫组化染色面密度及积分光密度均较M组升高，差异有显著性，差异有显著性统计学意义(P＜0.01)；
TIMP-1：主要表达定位于肾小球，肾小管及间质区内表达较弱，和C组比较，M组呈强阳性表达；T组免疫组化染色面密度及积分光密度均较M组降低，差异有统计学意义(P＜0.05)；
TGF-β_1：主要定位在肾小管及间质处，肾小球表达较弱；T组免疫组化染色面密度及积分光密度均较M组降低，差异有显著性，差异有显著性统计学意义(P＜0.01)。
MMP-9／TIMP-1：M组MMP-9／TIMP-1比值较C组降低，差异有显著性，差异有显著性统计学意义(P＜0.01)；T组MMP-9／TIMP-1比值较M组升高，差异有显著性，差异有显著性统计学意义(P＜0.01)；MMP-9／TIMP-1比值与Col-Ⅳ、TGF-β_1免疫组化染色面积均存在负相关(r=-0.919或r=-0.877，P＜0.01)；Col-Ⅳ免疫组化染色面积与TGF-β_1免疫组化染色面积存在正相关(r=0.820，P＜0.01)。
结论：
1．益气活血中药复方通脉口服液临床观察，结果显示：该中药可以有效改善DN患者的临床症状，减轻尿蛋白，保护肾功能；动物实验结果显示：该中药可以有效减轻实验性DN模型大鼠尿蛋白、调节血脂及改善肾脏病理损害。
2．益气活血中药复方通脉口服液对DN肾脏保护作用的机制可能是：改善脂质代谢，减轻尿蛋白，抑制肾组织TGF-β_1表达，调节MMP-9／TIMP-1比例失衡，增加Ⅳ型胶原降解，减轻ECM积聚，改善肾脏肥大，从而调节细胞、细胞外基质、细胞因子和生长因子间的网络调控，延缓DN肾小球硬化的进展。
3．益气活血中药复方通脉口服液，在预防实验性DM模型大鼠尿蛋白出现，及预防实验性DN模型大鼠尿蛋白加重两方面，均可获得较为满意的疗效，和西药比较，其优势体现在改善症状，调节血脂。结合临床，提示持续蛋白尿可以作为本药干预糖尿病肾病的起始点。
Background
Recently, the prevalence rate of diabetes is rising unceasingly in ourcountry. Along with insulin widespread application, the mortality rate ofDiabetes' acute complication,such as ketoacidosis, are obviouslydropped, and the chronic complication such as diabetic nephropathy has becomeone of most common complications with diabetic life extension. In clinicalpractice, diabetes nephrosis is often divided into 3 stages, namely: morningstage, intermediate stage and advanced stage. In different stage, the emphasisof treatment is also different.
Professor Yang consider that the Deficiency of Qi and the Blood Stasis isthe mainly etiopathogenesis of diabetic nephropathy, especially when diseaseis morning or intermediate stage, so the therapeutic rule-"benefiting vitalenergy and promoting blood flow" can applied in the treatment of diabeticnephropathy; when their stage are morning or intermediate, the effectivetreatment is the key of pathogenetic condition reverse. So we study themechanism of action of Tongmai Oral Solution to the diabetic nephropathy bythe methods of pharmacodynamics and morphology, in order to provide thesubstantial theory and plenitudinous scientic evidence for Tongmai OralSolution which is composed according to benefiting vital energy and promotingblood flow method cure diabetic nephropathy.
Objective.
To explore the effect and mechanism of TongMai Oral Solution in the defence and treatment of diabetic nephropathy, and found Tongmai Oral Solution' sfunction superiority stage.
Methods:
1). Clinical study:
This part of the study is controlled. According to degree of proteinuriaand general condition, 63 patients suffered from morning or intermediate stagediabetic nephropathy were divided into treatment and control group. There wasno statistically significant difference between two groups in sex, age,severity of the disease and laboratory items. The patients of the treatmentgroup received management of rongMai Oral Solution while the patients of thecontrol group took the drug of Lotensin. The course of treatment lasted for8 weeks, during which the changes of clinical findings and lab index weremonitored and recorded, including renal function, proteinuria, blood fat,blood pressure, blood glucose, and clinical symptoms and signs. Thedifferences between the symptom index before and after treatment were thencalculated and tested, as well as the quantities of the urine protein of 24hours.
2). Experimental Study: (1). PartⅠ. Study of protective effects of TongMai oral solution on thekidney of Diabetes Mellitus rats.
a. preparation of animal model. 42 SD male adult rats of specified-pathogenfree were injected with streptozotocin (STZ) intraperitoneally to prepare forDiabetes Mellitus animal model and then randomly divided into 3 groupsincluding model group(M group), TongMai Oral Solution group(T group)andLotensin group(L group) after diabetes was induced successfully. Another groupwas set for experiment control, consisted of normal SD rats.
b. Methods of intervention. The rats of T, L, M groups were raised withhigh fat forage every other day while the rats of normal group were raisedwith normal one. Different drugs were perfused into stomach of rats includingTongMai Oral Solution for T group, Lotensin for L group, and sodium chloridefor C and M groups. All the rats were observed closely for 6 weeks. Duringthis period, the rats with values of blood glucose less than 16.8 mmol/L orslight renal pathogenic abnormalities under light microscope were excludedfrom the experimental studies. Several items were recorded to assess renalprotective effects of TongMai oral solution, including general condition, body mass, urine volume, quantities of urinary protein of 24 hours, serum bloodcholesterol total, serum glycerol, blood glucose, blood urea nitrogen, serumcreatinine, and ratio of kidney mass to body mass (K/W).
(2) PartⅡ. Study of renal protective effects of TongMai oral solutionon the rats of diabetes nephropathy animal model.
After diabetes was induced successfully, the rats of diabetes animal modelwere continually raised with high fat food every other day for another 4 weeksin order to induce the diabetes nephropathy in them. The rats, in which diabetesmellitus were induced successfully, were then grouped and treated in a mannerthe same as that of PartⅠ, controlled by normal SD rats. After 6 weeks ofdrugs treatments or placebo managements, all the animals were put to deathand their kidneys were removed for the pathological study including sectionarea of glomerulus (MGA), section area of extracel]ular matrix (MMA),thickness of glemerular basement menbrance (GBM), and the ratio of thesection area of MMA to MGA.
PatrⅢ. Study of effects of the TongMai oral solution on the expressionof MMP-9 and its substrates, as well as regulation factor
6 renal samples were randomly obtained from the rats of group C, M, andT in PartⅡstudy, respectively. Expression of typeⅣcollagen, MMP-9, TIMP-1,and TGF-β1 were detected by immunohistochemistry. The stained area, areadensity, and photodensity were then observed and recorded. The ratio of stainedarea of MMP-9 to TIMP-1 was also determined.
Results:
1). Clinical study.
(a). there's no statistical significant difference in the generalcondition of the patients, the quantities of urinary protein of 24 hours, andrenal function before treatment. No statistical significance was found in thedifference of blood pressure, values of blood glucose, and values of the bloodfat during the course of treatment.
(b).As to the patients of T group, the symptomatic scores and quantity ofurinary protein of 24 hours after treatment decreased obviously compared tothat before the treatment, with statistical significance (P＜0.01).
(c).There were no statistical significance found in the difference ofchanges of quantity of urinary protein of 24 hours after treatment betweenthe T group and L group (P＞0.05). However, the changes of symptomatic scores in T group was higher than that of L group after treatment with statisticalsignificance (P＜0.01).
2) Experimental Study:
(1). PartⅠ. Study of protective effects of TongMai oral solution on thekidney of Diabetes Mellitus rats.
a. The changes of body mass and urine volume. The mean values of urine volumein the T, L, and M groups increased concomitant with loss of body mass withstatistical significance (p＜0.01), in contrast to that of C group. Althoughthere' s descent trend in the urine volume and increasing trend in the bodymass of T group compare to that of L and M group, no statistical significancewas demonstrated in the difference of the values of both items among T, L,and M group (P＞0.05).
b. Quantity of urinary protein of 24 hours. Quantity of urinary proteinin T, L, and M group increased with statistical significance, compared to thatof C group (P＜0.01). Excretion of urinary protein in 24 hours in T groupdecreased statistically significantly, in contrast to that of M group (P＜0.01).No statistical significance was demonstrated in the difference of dischargeof urinary protein in 24 hours between the L and T group (P＞0.05).
c. Changes of blood glucose level. The mean value of blood glucose of T,L, and M group increased with statistical significance, compare to that ofC group (P＜0.01). No statistical significance was demonstrated in thedifferences of values of blood glucose among T, L, and M group (P＞0.05).
d. Changes of values of blood urea nitrogen and serum creatinine. There' sno statistical significant difference demonstrated between C, T, L, and M group
e. The cholesterol total(TCh) and glycerol total (TG) fluctuation. The meanvalues of ZCh and TG in T, L, and M group increased statisticallysignificantly, compared that of C group (P＜0.01). In T group, the values ofboth TCh and TG decreased with statistical significance, compared to that ofM and L group (P＜0.01 or P＜0.05).
f. Ratio of kidney mass to body mass (K/W). The ratios of K/W of T, L, andM group increased with statistical significance, in contrast to that of C group(P＜0.01). The ratio of K/W of T group was less than that of M group withstatistical significance (P＜0.01). However, no statistical significance wasdemonstrated in the difference of ratio of K/W between the T and L group
g. Pathological changes of kidney. Renal pathohistology changes in M group,such as glomerular hypertrophy and widening of mesangial region, were foundworse than that of C group. The similar pathohistology abnormalities were alsodetected in both T and L group but to a less degree. No difference was determinedto be statistically significant as to the severity of renal pathohistoloyabnormality between T and L group.
(2) PartⅡ. Study of renal protective effects of TongMai oral solutionon the rats of diabetes nephropathy animal model.
a. changes of body mass and urine volume. The mean value of urine volumein T group decreased while the body mass in T group increased, compared tothat of M and L group. Both the changes of body mass and urine volume werestatistically significantly (P＜0.05 or P＜0.01).
b. The blood urea nitrogen. The mean value of blood urea nitrogen of T groupdecreased with statistical significance (P＜0.01 or P＜0.05).
c. other items. Outcomes of quantity of urinary protein in 24 hours, TCh,TG, Blood glucose, serum creatinine, and ratio of K/W were referred to thatof PartⅠof the experimental study.
d. The renal pathohistology. Microscopic abnormalities of the kidney formrats of M group included widening of mesangial region, thickness of renalbasement membrane, and vacuolar degeneration of renal tubular epithelia. Theserenal changes in M group were worse than that seen in the C group.Although similar renal abnormalities could also be found in T group, it' sobvious that the abnormalities became less serious in T group and L group.No difference was demonstrated between T and L group.
e. Ratio of section area of extracellular matrix (MMA) and glomerulus(MGA). it' s higher of ratio of MMA/MGA in M group than that of C group withstatistical significance (P＜0.01). However, in T group the ratio of MMA/MGAwas less than that of both the M and L group (P＜0.01 or P＜0.05).
f. The thickness of renal glomerulus basement membrane. The renal basementmembrane got thicker than that of C group with statistical significance(P＜0.01). Compared to that of M group, the GBM of T group was thinner withstatistical significance (P＜0.01).No difference of the same index was foundto be statistical between T and L group (P＞0.05).
(3) PartⅢ. Study of effects of the TongMai oral solution on the expression of MMP-9 and its substrates, as well as regulation factor.
a. Expression of typeⅣcollagen. The typeⅣcollagen was located in theglomerulus. Strong positive expression was found in group M. According to theoutcomes of immunohistology, the values of area density and photodensity ofpositively stained area were less than that of M group with statisticalsignificance (P＜0.01).
b. Expression of MMP-9. The expression of MMP-9 was located mainly inglomerulus and sparse expression could be seen in renal interstitium. It'smore weakly expressed in group M than in C group. The value of area densityand photodensity in MMP-9 positively stained area were higher than that ofM group (P＜0.01).
c. Expression of TIMP-1. Expression of TIMP-1 was mainly located inglomerulus with weakly expression located in renal tubule and interstitium.It's stronger positively expressed in M group than in C. The values of areadensity and photodensity of TIMP positively stained area in group T were lowerthan that of group M with statistical significance (P＜0.05).
d. Ratio of MMP-9/TIMP-1. It's higher of ratio of MMP-9/TIMP-1 in groupC than that of group M with statistical significance (P＜0.01). However, thisratio in group T was higher than that of group M with statistical significance(P＜0.01). The ratio of MMP-9/TIMP-1 and photodensity of typeⅣcollagenchanged in reverse direction.
e. Expression of TGF-β1. Expression of TGF-β1 was located in the renaltubule and interstitium. It's weakly expressed in the glomerulus. It's lowerfor the value of area density and photodensity in group T than that of groupM with statistical significance (P＜0.01).
Conclusion
(1). Tongmai Oral Solution can improve the patients' clinical symptomsand renal function, decrease the urine protein.
(2). The mechanism that Tongmai Oral Solution ameliorated the structureand function damage of DN model rats may be due to enhancement of metabolismof blood fat, up-regulation of the expression of MMP-9, down-regulation ofthe expression of TGF-β1, rebuilding of balance between MMP-9 and TIMP-1,inhibition of thickening of the glomerular basement membrane, increase ofdegeneration of typeⅣcollagen, reduction of extracellular matrixaccumulation, and amelioration of renal pathological lesions. These therapeutic effects may improve renal hypertrophy, regulation of cells, ECM,cytokines, and growth factors resulting in delayed progress of DN.
(3). In the course of intervention of DN model rats, excellent renalprotective effects could be obtained not only at the beginning of initiatestage of model induction but at the time when urinary protein appears in theurine of DN model rats. This reveals that the point that the urinary proteinappearances could be regarded as initiate point when Tongmai Oral Solutionis given.
引文
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