补肾滋阴方药对实验性糖尿病肾病大鼠治疗作用及机制的研究
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摘要
目的:糖尿病肾病是糖尿病常见而难治的微血管并发症,已成为糖尿病患者的主要死因之一。其发病机制主要与高血糖、肾脏血流动力学异常、家族遗传性等有关。
肾素-血管紧张素系统在糖尿病肾病发病机制中占有重要地位,血管紧张素Ⅱ不仅是一种血管活性因子,影响肾脏血流动力学,还是一种生长因子及促纤维化因子,影响肾脏固有细胞的生物学功能。转化生长因子-β1在糖尿病肾病相关的生长因子网络中处于中心地位,可通过多种机制促进肾小球肥大、刺激细胞外基质合,参与肾小球硬化和肾小管间质纤维化的发生。
糖尿病肾病与中医“消渴”之“下消”关系密切,“肾阴亏虚”在其发生发展的过程中具有重要作用,也是早期糖尿病肾病(Ⅰ、Ⅱ、Ⅲ期)的基本病机之一,补肾滋阴方药常用于早期糖尿病肾病治疗。六味地黄丸是补肾滋阴的经典方剂,很多临床研究均证实其对糖尿病肾病具有良好的治疗作用,但其作用机制尚不清楚。本实验采用腹腔注射链尿佐菌素的方法先复制大鼠糖尿病模型,进而发展为糖尿病肾病,观察六味地黄汤对其血糖、肾功能、24小时尿蛋白定量、肾组织病理形态学及肾组织肾素活性、血管紧张素Ⅱ和转化生长因子-β1表达的影响,初步探讨本方的作用机理,为临床应用补肾滋阴方药治疗早期糖尿病肾病提供实验依据。
第一部分六味地黄汤对糖尿病肾病大鼠肾功能及肾组织病理形态学的影响
方法:选用清洁级雄性健康SD大鼠30只体重200±10 g,普通饲料适应性喂养一周,随机抽6只为正常组(NC),其余大鼠一次性腹腔注射链脲佐菌素(STZ) 60 mg/kg,正常组注射相应量生理盐水,72小时后,测血糖以≥16.65mmol/L,尿糖阳性为DM模型成模标准。成模大鼠被随机分为:模型组(DM)、苯那普利组(BN)、六味地黄汤(LD)组,成模后一周按成人剂量的20倍给药,每日一次。六味地黄汤组按25g /kg予六味地黄汤,苯那普利组按6.67mg/kg灌胃,各组共给药8周;正常组和模型组均灌服相应量的生理盐水。于DM成模后第9周末禁食不禁水留取各组大鼠24小时尿液检测24小时尿蛋白定量、尿肌酐(Ucr);各组大鼠禁食8小时,称重,麻醉动物,股动脉取血,分离血清,检测空腹血糖(FPG),血尿素氮(BUN ),血肌酐(Scr ),计算肌酐清除率(Ccr )。无菌操作取双肾,剥离被膜称重,计算肾重/体重指数(KW/BW);取右肾组织4%多聚甲醛初步固定,用于观察肾脏病理形态学变化;血、尿生化指标均用全自动生化分析仪测定。
结果:1一般情况及空腹血糖、肾重/体重指数、24h尿蛋白定量:大鼠注射STZ后体重增长缓慢,毛色差,实验结束时,出现肾脏体积增大,尿蛋白增多;空腹血糖模型组与正常组比较明显增高(P<0.01 ),苯那普利组空腹血糖与模型组比较无明显变化;六味地黄汤组与模型组比较明显降低(P<0.05);24h尿蛋白定量模型组与正常组比较明显升高(P<0.01 ),苯那普利组、六味地黄汤组与模型组比较明显降低(P<0.05),六味地黄汤组与苯那普利组比较明显降低(P<0.05);肾重/体重指数模型组与正常组比较明显增高(P<0.01 ),六味地黄汤组、苯那普利组与模型组比较均有显著降低(P<0.05),其中六味地黄汤组优于苯那普利组但无显著性差异。
2血尿素氮、肌酐清除率的测定结果:血尿素氮模型组与正常组比较明显增高(P<0.01 ),苯那普利组与模型组比较均有明显降低(P<0.05),六味地黄汤组与苯那普利组比较明显降低(P<0.01),仍高于正常组但无显著差异;肌酐清除率模型组与正常组比较有显著性增高(P<0.01),苯那普利组、六味地黄汤组与模型组比较均有显著性降低(P<0.05),六味地黄汤组优于苯那普利组但无显著性差异。
3肾组织病理形态学观察:HE、PAS染色光镜下可见正常组肾小球结构完整,未见肾小球肥大,肾小球毛细血管基底膜、系膜、基质未见明显异常改变;模型组肾小球系膜区弥漫性增宽,系膜细胞轻、中度增生,肾小球基底膜增厚,PAS染色物质明显增多,部分毛细血管受压塌陷等表现。苯那普利组、六味地黄汤组的肾小球结构也有类似的病理改变,但明显较模型组减轻(P<0.05)。
第二部分六味地黄汤对糖尿病肾病大鼠肾组织肾素-血管紧张素系统、转化生长因子-β_1的影响
方法:动物分组、造模方法和给药方法同第一部分,用放射免疫法测定肾组织匀浆肾素活性和血管紧张素Ⅱ水平,结果用肾组织总蛋白校正,计算出肾组织每克蛋白中的肾素活性和血管紧张素Ⅱ含量。TGF-β_1蛋白表达采用免疫组织化学PV法检测,病理图像分析系统测积分光密度和相对阳性面积。
结果:1肾组织肾素活性和血管紧张素Ⅱ含量:肾素活性模型组显著高于正常组(P<0.05),苯那普利组比模型组显著增高(P<0.05),六味地黄汤组显著低于苯那普利组(P<0.05),六味地黄汤组略低于模型组但无明显差异。血管紧张素Ⅱ含量,模型组与正常组比较有显著增加(P<0.01);苯那普利组与模型组比较显著降低(P<0.05),六味地黄汤组低于苯那普利组但无显著性差异。2肾组织TGF-β_1的表达:光镜下观察,模型组TGF-β_1表达主要表达于肾小球系膜区,肾小管上皮细胞也有不同程度表达,阳性颗粒主要位于胞浆中。病理图象半定量分析:模型组肾小球TGF-β_1表达的相对面积及积分光密度均明显高于正常组(P<0.01);苯那普利组、六味地黄汤组均明显低于模型组(P<0.05);六味地黄汤组低于苯那普利组但无显著性差异。
结论:1补肾滋阴之六味地黄汤具有改善DN大鼠一般状态,降低血糖、BUN和Ccr,改善肾功能,对早期DN有确切的疗效。
2补肾滋阴之六味地黄汤具有减少DN大鼠尿蛋白的排出,抑制早期肾脏肥大,减轻肾脏病理损害和延缓肾脏病理进展的作用。
3补肾滋阴之六味地黄汤对DN大鼠肾组织AngⅡ有一定的抑制作用,这可能是其具有改善肾功能、减轻肾脏病理损害和延缓肾脏病理进展的作用机制之一。
4补肾滋阴之六味地黄汤能够降低DN大鼠肾组织TGF-β_1蛋白表达,这可能是其具有改善肾功能、减轻肾脏病理损害和延缓肾脏病理进展的作用机制之一。
Objective: Diabetic nephropathy is a microvascular complication of diabetic mellitus, and it has been the most common cause of death for diabetic patients.
Its pathologic mechanism has the close relation to high blood sugar, the excrescent of kidney blood dynamics and the household hereditary, etc. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of diabetic nephropathy, In addition to its ability to induce systemic and intraglomerular hypertension and to increase glomerular capillary permeability, AngII is a renal growth factor and causes hypertrophy of mesangial cells and tubular epithelial cells and promotes production of the prosclerotic cytokine transforming growth factor-β1. Transforming growth factor-beta1 (TGF-β1) plays a critical role in the pathogenesis of diabetic nephropathy(DN) through inducing cell hypertrophy of glomerulus and renal tubule, accelerating the accumulation of extracellular matrix(ECM), and promoting glomerulosclerosis and tubulointerstitial fibrosis, etc。
According to traditional Chinese medicine theory, diabetic nephropathy is included in categories of "wasting-thirsting" and "lower-wasting", Deficience of kidney-yin has been thought as the importent pathogeny of diabetic nephropathy. At the same time, Liuweidihuang Decoction has been proved to have satisfactory therapeutic effect on DN although the mechanism of its action is still unkown. In this experiment, Diabetes mellitus (DM) animal model was first established, then developed for diabetic nephropathy (DN), and carried on the treatment usingLiuweidihuang Decoction. we observe the change of its blood sugar, renal function, the appearance of kidney and expressions of transforming growth factor-β1、renal tissue rennin activity、the content of angiotensin II,and inquiry the prevention and cure mechanism of Liuweidihuang Decoction, so as to provid scientific experimental foundation for reinforcing kidney toreplenish Yin method of clinical application.
PartⅠ:Effect of reinforcing kidney toreplenish Yin method on the renal function and pathomorphology of the diabetic nephropathy rats
Methods: 6 rats were selected for normal control group (NC) randomly in the 30 clean healthy male Sprague-Dawley rats. Others were injected the streptozocin (STZ) intraperitoneally after one week according to 60 mg/kg.We regarded the rats as the model rats whose blood sugar≥16.65mmol/L, urine glucose was masculine.The model rats were randomly divided into: model group(DM), Benazepril group (BN), Liuweidihuang Decoction group (LD),n=8. After one week of DM models made successfully, the drugs started be given to the rats according to adult dosage 20 times, daily one time. The rats of Liuweidihuang Decoction group were given the Liuweidihuang Decoction by gavage, 25g/kg, the benazepril group were administrated with benazepril by gavage, 6.67mg/kg, the normal group and the model group were given physiological saline by gavage. Each group was given the medicine for 8 weeks. After 9 weeks of DM models made successfully, We took rats 24h urine in metabolic cages to measure urinary protein and urinary creatinine (Ucr). Animals were sacrificed to measure fasting plasma glucose (FPG), blood urea nitrogen (BUN), serum creatinine(Scr) and to calculate creatinine clearance rate(Ccr). Some renal tissue was obtained for the pathomorphology.
Results: 1. Fasting plasma glucose (FPG)、Kidney weight / body weigh(tKW/BW)and the data of urinary protein:The body weight of model groups increased slowly after the injection . The worse hair color, high gloucose and the expanded kidney were showed at the end of test. Five rats died of higher glucose. The FPG of Model group was higher than that of NC group significantly (p<0.01); there was no difference between the BN group and DM group ; LD group was lower than that of DM group significantly (p<0.05). The 24h total urinary protein of DM group was higher than that of NC group significantly (p<0.01); BN group were decreased remarkably compared with that of DM group (p<0.05), LD group was superior to BN group (p<0.05). The KW/BW of DM group was higher than that of NC group significantly (p<0.01); BN group were lower than that of DM group significantly (p<0.05); LD group was lower than that of BN group,but there was nosignificant difference between them. 2. The results of blood urea nitrogen(BUN), creatirine clearance rate (Ccr):The BUN of DM group was higher than that of NC group significantly (p<0.01); BN group was decreased remarkably compared with that of DM group (p<0.05), and that of LD group was superior to BN group (p<0.01), LD group was higher than that of NC group, but there was nosignificant difference between them.The Ccr of DM group were higher than NC group significantly (p<0.01); BN group was lower than that of DM group significantly (p<0.05); The effect of LD group superior to that of BN group, but that had no nosignificant difference. 3.Renal tissue pathomorphology: The staining of HE and PAS of renal tissues,In the NC group, the light microscope observation result showed that: the structure of renal glomerulus was complete, glomerular capillary basement membrane, mesangial and matrix did not show abnormal change. In the DM group,mesangial area of renal glomerulus broadening, even protein substances with PAS positive markebly increasing, extracellular matrix of glomerulus enlarging in number, glomerular basement membrance (GBM) thickening and the blood capillary is compressd、narrowed and vanished, so on. But in the LD group, the changes were better than those in the DM group. In BN group, renal tissue pathomorphology was similar to the LD group. PartⅡ:Effect of Liuweidihuang Decoction on the renal renin angiotensin system and TGF-β1 in diabetic nephropathy rats
Methods: The methods for test were as the same as those in PartⅠ. The levels of renin activity and angiotensinⅡof renal tissue homogenate were tested with radioimmunoassay method. The results were adjusted with renal tissue total protein to correct the renin activity and the content of angiotensinⅡin each sample. Some samples of renal tissue were fixed in 4% paraform, dehydration, paraffin embedding, and by the means of immunohistochemistry method to observe the expression of TGF-β1. The protein expression of TGF-β1 adopted SP method, The integral optical density and relative areal were measured with pathological image analysis system.
Results: 1.The renin activity and the content of angiotensionⅡin renal tissue, which could be founded in the experimental animals’kidney samples, were assayed by radiative immune method. The results showed: It was obviously that the renin activity strengthened in DM group against NC group (P<0.05), The data of BN group decreased comparing with that of DM group (P<0.05). The data of LN group was lower than that of BN group (P<0.05);There was no statistic sense between LD group with DM group. AngⅡ’s content of renal tissue in DM group increased markely,which refered to a significant difference in comparing with group NC(P<0.01). The data of BN group and LD group were decreased comparing with that of DM group(P<0.05). There was no statistic sense between BN group with LD group. 2.The semi-quantitative analysis of TGF-β_1: It was seen that the TGF-β_1 protein expressed hardly in the renal tissue in NC group observed by light microscope; The expression of TGF-β_1mainly localized in the cell cytoplasm of the renal Mesangial area. The expression of TGF-β_1in BN group and LD group were both lower than that in Model group significantly. Pathologic image semi-quantitative analysis shows: Compared with group NC, the expression of TGF-β_1 in DM group increased separately (P<0.01). Compared with group DM, the expression of TGF-β_1 in BN group and LD group decreased (P<0.05).There was no statistic sense between BN group with LD group.
Conclusions:
1 The results indicated that Liuweidihuang Decoction was benefit for regulating glucose metabolism, improving renal funition.
2 The results indicated that Liuweidihuang Decoction was benefit for reducing the urinary protein excretion, inhibiting renal hypertrophy and delaying the advancement of renal phathematology.
3 It indicated that Liuweidihuang Decoction had a certain effect on reducing the content of AngⅡin renal tissue, which probaboly had correlation with its ability to treat early DN.
4 It indicated that Liuweidihuang Decoction had the ability to inhibit extra express of TGF-β_1 in drabetic rats’renal tissue, which probaboly had correlation with its ability to treat early DN.
肾素-血管紧张素系统在糖尿病肾病发病机制中占有重要地位,血管紧张素Ⅱ不仅是一种血管活性因子,影响肾脏血流动力学,还是一种生长因子及促纤维化因子,影响肾脏固有细胞的生物学功能。转化生长因子-β1在糖尿病肾病相关的生长因子网络中处于中心地位,可通过多种机制促进肾小球肥大、刺激细胞外基质合,参与肾小球硬化和肾小管间质纤维化的发生。
糖尿病肾病与中医“消渴”之“下消”关系密切,“肾阴亏虚”在其发生发展的过程中具有重要作用,也是早期糖尿病肾病(Ⅰ、Ⅱ、Ⅲ期)的基本病机之一,补肾滋阴方药常用于早期糖尿病肾病治疗。六味地黄丸是补肾滋阴的经典方剂,很多临床研究均证实其对糖尿病肾病具有良好的治疗作用,但其作用机制尚不清楚。本实验采用腹腔注射链尿佐菌素的方法先复制大鼠糖尿病模型,进而发展为糖尿病肾病,观察六味地黄汤对其血糖、肾功能、24小时尿蛋白定量、肾组织病理形态学及肾组织肾素活性、血管紧张素Ⅱ和转化生长因子-β1表达的影响,初步探讨本方的作用机理,为临床应用补肾滋阴方药治疗早期糖尿病肾病提供实验依据。
第一部分六味地黄汤对糖尿病肾病大鼠肾功能及肾组织病理形态学的影响
方法:选用清洁级雄性健康SD大鼠30只体重200±10 g,普通饲料适应性喂养一周,随机抽6只为正常组(NC),其余大鼠一次性腹腔注射链脲佐菌素(STZ) 60 mg/kg,正常组注射相应量生理盐水,72小时后,测血糖以≥16.65mmol/L,尿糖阳性为DM模型成模标准。成模大鼠被随机分为:模型组(DM)、苯那普利组(BN)、六味地黄汤(LD)组,成模后一周按成人剂量的20倍给药,每日一次。六味地黄汤组按25g /kg予六味地黄汤,苯那普利组按6.67mg/kg灌胃,各组共给药8周;正常组和模型组均灌服相应量的生理盐水。于DM成模后第9周末禁食不禁水留取各组大鼠24小时尿液检测24小时尿蛋白定量、尿肌酐(Ucr);各组大鼠禁食8小时,称重,麻醉动物,股动脉取血,分离血清,检测空腹血糖(FPG),血尿素氮(BUN ),血肌酐(Scr ),计算肌酐清除率(Ccr )。无菌操作取双肾,剥离被膜称重,计算肾重/体重指数(KW/BW);取右肾组织4%多聚甲醛初步固定,用于观察肾脏病理形态学变化;血、尿生化指标均用全自动生化分析仪测定。
结果:1一般情况及空腹血糖、肾重/体重指数、24h尿蛋白定量:大鼠注射STZ后体重增长缓慢,毛色差,实验结束时,出现肾脏体积增大,尿蛋白增多;空腹血糖模型组与正常组比较明显增高(P<0.01 ),苯那普利组空腹血糖与模型组比较无明显变化;六味地黄汤组与模型组比较明显降低(P<0.05);24h尿蛋白定量模型组与正常组比较明显升高(P<0.01 ),苯那普利组、六味地黄汤组与模型组比较明显降低(P<0.05),六味地黄汤组与苯那普利组比较明显降低(P<0.05);肾重/体重指数模型组与正常组比较明显增高(P<0.01 ),六味地黄汤组、苯那普利组与模型组比较均有显著降低(P<0.05),其中六味地黄汤组优于苯那普利组但无显著性差异。
2血尿素氮、肌酐清除率的测定结果:血尿素氮模型组与正常组比较明显增高(P<0.01 ),苯那普利组与模型组比较均有明显降低(P<0.05),六味地黄汤组与苯那普利组比较明显降低(P<0.01),仍高于正常组但无显著差异;肌酐清除率模型组与正常组比较有显著性增高(P<0.01),苯那普利组、六味地黄汤组与模型组比较均有显著性降低(P<0.05),六味地黄汤组优于苯那普利组但无显著性差异。
3肾组织病理形态学观察:HE、PAS染色光镜下可见正常组肾小球结构完整,未见肾小球肥大,肾小球毛细血管基底膜、系膜、基质未见明显异常改变;模型组肾小球系膜区弥漫性增宽,系膜细胞轻、中度增生,肾小球基底膜增厚,PAS染色物质明显增多,部分毛细血管受压塌陷等表现。苯那普利组、六味地黄汤组的肾小球结构也有类似的病理改变,但明显较模型组减轻(P<0.05)。
第二部分六味地黄汤对糖尿病肾病大鼠肾组织肾素-血管紧张素系统、转化生长因子-β_1的影响
方法:动物分组、造模方法和给药方法同第一部分,用放射免疫法测定肾组织匀浆肾素活性和血管紧张素Ⅱ水平,结果用肾组织总蛋白校正,计算出肾组织每克蛋白中的肾素活性和血管紧张素Ⅱ含量。TGF-β_1蛋白表达采用免疫组织化学PV法检测,病理图像分析系统测积分光密度和相对阳性面积。
结果:1肾组织肾素活性和血管紧张素Ⅱ含量:肾素活性模型组显著高于正常组(P<0.05),苯那普利组比模型组显著增高(P<0.05),六味地黄汤组显著低于苯那普利组(P<0.05),六味地黄汤组略低于模型组但无明显差异。血管紧张素Ⅱ含量,模型组与正常组比较有显著增加(P<0.01);苯那普利组与模型组比较显著降低(P<0.05),六味地黄汤组低于苯那普利组但无显著性差异。2肾组织TGF-β_1的表达:光镜下观察,模型组TGF-β_1表达主要表达于肾小球系膜区,肾小管上皮细胞也有不同程度表达,阳性颗粒主要位于胞浆中。病理图象半定量分析:模型组肾小球TGF-β_1表达的相对面积及积分光密度均明显高于正常组(P<0.01);苯那普利组、六味地黄汤组均明显低于模型组(P<0.05);六味地黄汤组低于苯那普利组但无显著性差异。
结论:1补肾滋阴之六味地黄汤具有改善DN大鼠一般状态,降低血糖、BUN和Ccr,改善肾功能,对早期DN有确切的疗效。
2补肾滋阴之六味地黄汤具有减少DN大鼠尿蛋白的排出,抑制早期肾脏肥大,减轻肾脏病理损害和延缓肾脏病理进展的作用。
3补肾滋阴之六味地黄汤对DN大鼠肾组织AngⅡ有一定的抑制作用,这可能是其具有改善肾功能、减轻肾脏病理损害和延缓肾脏病理进展的作用机制之一。
4补肾滋阴之六味地黄汤能够降低DN大鼠肾组织TGF-β_1蛋白表达,这可能是其具有改善肾功能、减轻肾脏病理损害和延缓肾脏病理进展的作用机制之一。
Objective: Diabetic nephropathy is a microvascular complication of diabetic mellitus, and it has been the most common cause of death for diabetic patients.
Its pathologic mechanism has the close relation to high blood sugar, the excrescent of kidney blood dynamics and the household hereditary, etc. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of diabetic nephropathy, In addition to its ability to induce systemic and intraglomerular hypertension and to increase glomerular capillary permeability, AngII is a renal growth factor and causes hypertrophy of mesangial cells and tubular epithelial cells and promotes production of the prosclerotic cytokine transforming growth factor-β1. Transforming growth factor-beta1 (TGF-β1) plays a critical role in the pathogenesis of diabetic nephropathy(DN) through inducing cell hypertrophy of glomerulus and renal tubule, accelerating the accumulation of extracellular matrix(ECM), and promoting glomerulosclerosis and tubulointerstitial fibrosis, etc。
According to traditional Chinese medicine theory, diabetic nephropathy is included in categories of "wasting-thirsting" and "lower-wasting", Deficience of kidney-yin has been thought as the importent pathogeny of diabetic nephropathy. At the same time, Liuweidihuang Decoction has been proved to have satisfactory therapeutic effect on DN although the mechanism of its action is still unkown. In this experiment, Diabetes mellitus (DM) animal model was first established, then developed for diabetic nephropathy (DN), and carried on the treatment usingLiuweidihuang Decoction. we observe the change of its blood sugar, renal function, the appearance of kidney and expressions of transforming growth factor-β1、renal tissue rennin activity、the content of angiotensin II,and inquiry the prevention and cure mechanism of Liuweidihuang Decoction, so as to provid scientific experimental foundation for reinforcing kidney toreplenish Yin method of clinical application.
PartⅠ:Effect of reinforcing kidney toreplenish Yin method on the renal function and pathomorphology of the diabetic nephropathy rats
Methods: 6 rats were selected for normal control group (NC) randomly in the 30 clean healthy male Sprague-Dawley rats. Others were injected the streptozocin (STZ) intraperitoneally after one week according to 60 mg/kg.We regarded the rats as the model rats whose blood sugar≥16.65mmol/L, urine glucose was masculine.The model rats were randomly divided into: model group(DM), Benazepril group (BN), Liuweidihuang Decoction group (LD),n=8. After one week of DM models made successfully, the drugs started be given to the rats according to adult dosage 20 times, daily one time. The rats of Liuweidihuang Decoction group were given the Liuweidihuang Decoction by gavage, 25g/kg, the benazepril group were administrated with benazepril by gavage, 6.67mg/kg, the normal group and the model group were given physiological saline by gavage. Each group was given the medicine for 8 weeks. After 9 weeks of DM models made successfully, We took rats 24h urine in metabolic cages to measure urinary protein and urinary creatinine (Ucr). Animals were sacrificed to measure fasting plasma glucose (FPG), blood urea nitrogen (BUN), serum creatinine(Scr) and to calculate creatinine clearance rate(Ccr). Some renal tissue was obtained for the pathomorphology.
Results: 1. Fasting plasma glucose (FPG)、Kidney weight / body weigh(tKW/BW)and the data of urinary protein:The body weight of model groups increased slowly after the injection . The worse hair color, high gloucose and the expanded kidney were showed at the end of test. Five rats died of higher glucose. The FPG of Model group was higher than that of NC group significantly (p<0.01); there was no difference between the BN group and DM group ; LD group was lower than that of DM group significantly (p<0.05). The 24h total urinary protein of DM group was higher than that of NC group significantly (p<0.01); BN group were decreased remarkably compared with that of DM group (p<0.05), LD group was superior to BN group (p<0.05). The KW/BW of DM group was higher than that of NC group significantly (p<0.01); BN group were lower than that of DM group significantly (p<0.05); LD group was lower than that of BN group,but there was nosignificant difference between them. 2. The results of blood urea nitrogen(BUN), creatirine clearance rate (Ccr):The BUN of DM group was higher than that of NC group significantly (p<0.01); BN group was decreased remarkably compared with that of DM group (p<0.05), and that of LD group was superior to BN group (p<0.01), LD group was higher than that of NC group, but there was nosignificant difference between them.The Ccr of DM group were higher than NC group significantly (p<0.01); BN group was lower than that of DM group significantly (p<0.05); The effect of LD group superior to that of BN group, but that had no nosignificant difference. 3.Renal tissue pathomorphology: The staining of HE and PAS of renal tissues,In the NC group, the light microscope observation result showed that: the structure of renal glomerulus was complete, glomerular capillary basement membrane, mesangial and matrix did not show abnormal change. In the DM group,mesangial area of renal glomerulus broadening, even protein substances with PAS positive markebly increasing, extracellular matrix of glomerulus enlarging in number, glomerular basement membrance (GBM) thickening and the blood capillary is compressd、narrowed and vanished, so on. But in the LD group, the changes were better than those in the DM group. In BN group, renal tissue pathomorphology was similar to the LD group. PartⅡ:Effect of Liuweidihuang Decoction on the renal renin angiotensin system and TGF-β1 in diabetic nephropathy rats
Methods: The methods for test were as the same as those in PartⅠ. The levels of renin activity and angiotensinⅡof renal tissue homogenate were tested with radioimmunoassay method. The results were adjusted with renal tissue total protein to correct the renin activity and the content of angiotensinⅡin each sample. Some samples of renal tissue were fixed in 4% paraform, dehydration, paraffin embedding, and by the means of immunohistochemistry method to observe the expression of TGF-β1. The protein expression of TGF-β1 adopted SP method, The integral optical density and relative areal were measured with pathological image analysis system.
Results: 1.The renin activity and the content of angiotensionⅡin renal tissue, which could be founded in the experimental animals’kidney samples, were assayed by radiative immune method. The results showed: It was obviously that the renin activity strengthened in DM group against NC group (P<0.05), The data of BN group decreased comparing with that of DM group (P<0.05). The data of LN group was lower than that of BN group (P<0.05);There was no statistic sense between LD group with DM group. AngⅡ’s content of renal tissue in DM group increased markely,which refered to a significant difference in comparing with group NC(P<0.01). The data of BN group and LD group were decreased comparing with that of DM group(P<0.05). There was no statistic sense between BN group with LD group. 2.The semi-quantitative analysis of TGF-β_1: It was seen that the TGF-β_1 protein expressed hardly in the renal tissue in NC group observed by light microscope; The expression of TGF-β_1mainly localized in the cell cytoplasm of the renal Mesangial area. The expression of TGF-β_1in BN group and LD group were both lower than that in Model group significantly. Pathologic image semi-quantitative analysis shows: Compared with group NC, the expression of TGF-β_1 in DM group increased separately (P<0.01). Compared with group DM, the expression of TGF-β_1 in BN group and LD group decreased (P<0.05).There was no statistic sense between BN group with LD group.
Conclusions:
1 The results indicated that Liuweidihuang Decoction was benefit for regulating glucose metabolism, improving renal funition.
2 The results indicated that Liuweidihuang Decoction was benefit for reducing the urinary protein excretion, inhibiting renal hypertrophy and delaying the advancement of renal phathematology.
3 It indicated that Liuweidihuang Decoction had a certain effect on reducing the content of AngⅡin renal tissue, which probaboly had correlation with its ability to treat early DN.
4 It indicated that Liuweidihuang Decoction had the ability to inhibit extra express of TGF-β_1 in drabetic rats’renal tissue, which probaboly had correlation with its ability to treat early DN.
引文
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