糖克煎剂对2型糖尿病大鼠肾功能保护作用的实验研究
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摘要
糖尿病肾病(DN)是糖尿病(DM)主要的慢性微血管并发症之一,是导致慢性肾衰的常见原因,据统计,在终末期肾病病人中约三分之一是由DN引起的。本论文通过理论研究对中西医防治DN的进展进行了较为全面的概述,并通过动物实验,从整体、器官、细胞分子等不同水平对中药复方糖克煎剂防治DN的机理进行了较为深入的探讨。
1.理论研究 由五部分组成,具体包括DN中医药研究概况、祛痰活血法治疗DN、DN现代医学研究进展、转化生长因子β(TGF-β)及其受体与DN、细胞凋亡与DN。在理论研究及文献综述中,参照古、今中医有关文献对糖尿病肾病的中医病名、病因病机、辨证论治方法、临床及实验研究进展和中医研究中存在的问题及对策进行了探讨,总结了导师李敬林教授辨治糖尿病肾病的方法和学术观点,强调从痰瘀论治糖尿病肾病,并对现代医学在糖尿病肾病研究中所取得的进展进行了总结,进一步明确了糖尿病肾病的病理特征、发病机制、分期和治疗,并具体探讨了现代医学关于细胞凋亡和TGF-β及其受体在DN发病机制中的作用。
2.实验研究 采用高糖高脂饲料喂养结合尾静脉注射链脲佐菌素(STZ)的造模方法复制出糖尿病模型,此模型首先采用高糖高脂饲料喂养一个月,诱发动物肥胖,引起胰岛素抵抗,再以小剂量STZ35mg/kg尾静脉注射造成胰岛素代偿性分泌障碍,从而引发高血糖,并出现微量白蛋白尿,系膜基质增生、基底膜增厚等糖尿病肾病典型的早期病理形态学改变,符合2型DN的发病机制。
本实验分正常对照组、中药糖克煎剂预防组、高低剂量组、模型组和西药洛汀新对照组,预防组大鼠灌胃给药12周,其余各治疗组均给药8周。观察糖克煎剂对DN大鼠血糖、糖化血红蛋白、血脂、体重、肾重、尿蛋白排泄、肾功能的影响;通过光镜、电镜观察各组实验性DM大鼠肾组织病理形念学变化;采用逆转录聚合酶链式反应(RT-PCR法)、蛋白质印迹杂交(Western Blot)及免疫组化法,进一步观察糖克煎剂对DN大鼠肾小球细胞外基质(ECM)的主要成分IV型胶原(ColⅣ)和纤维连接蛋白(FN)、转化生长因子(TGF-β_1)及其受体(TGF-BR)mRNA及蛋白表达的影响;采用原位缺口标记技术(TUNEL)以及免疫组化法检测糖尿病大鼠肾组织中细胞凋亡及BCL-2及BAX等凋亡相关基因的表达,以进一步探讨DN发病机制,了解bcl-2、bax在糖尿病肾病发生、发展中的作用,以及中药糖克煎剂对DN细胞凋亡及其相关基因bcl-2/bax表达的影响。
Diabetic nephropathy(DN) is one of the chronic complications of diabetes mellitus(DM) and the common reason leading to chronic renal failure ,one third of which is caused by DN. Systematic research on prevention and treatment of DN by TangKeJianJi (TangKe decoction) has been performed from the whole ,organ, cell and molecule levels in this essay which was composed of the reference review and experimental study.1. Theory reviewThe theoretical review including five sections mainly discussed the recent research progresses in prevention and treatment of DN both from the aspects of traditional Chinese medicine (TCM) and modern medicine. In detail, the five sections were made up of the skeleton review of TCM and modern medicine on DN, the relationship between transforming growth factor-P (TGF- β ) , transforming growth factor- P receptor (TGF- βR) and DN , the relationship between apoptosis and DN. In the theoretical research, we have referred to the related ancient and modern TCM literature in order to explore the source of disease name of TCM for DN, etiology and pathogenesis of TCM, selection of therapeutic methods by different stages or according to the differential syndrome diagnosis, advance on the clinical and experimental research as well as the problems lying in the research of TCM and corresponding strategies. In the meanwhile ,we also have summarized the tutor's ( professor Li Jingling) academic point of view on the differentiation and treatment of DN, moreover further exploited the pathologic characteristics ,pathogenesis , pathologic stages and treatment of DN as well as the function of TGF- P and apoptosis on DN.2. Experimental studyThe experimental rat models at first were fed by a diet enriched with sucrose ,cholesterol and cholic acid for a month in order to induce the models' obesity and result in insulin resistance, and then injected the low dosage of 35mg/kg streptozotocin(STZ) through vein of the rat's tail to develop hyperglycemia. This kind of model closely simulated the metabolic abnormalities of the human type 2 diabetes mellitus and its renal structural lesion with microalbuminuria, mild mesangial matrix expansion, thickening of glomerular basement membrane and so on. The experimental rats were randomly divided into 6 groups: normal contrast group, pattern group, prevention group, high dosage group, low dosage group and Lotension group. Drug was delivered daily by gavage for 8 weeks in the therapeutic groups except for the prevention group for 12 weeks. We have detected, BUN, Scr, hemoglobin Aic (HbAic), body weight, kidney weight, urinary albumin excretion (UAE) and renal pathologic lesion through light microscopy and electron microscopy and further observed mRNA and protein expression of TGF- β 1, TGF- β R, Collagen IV and fibronection through RT-PCR, western blot and immunohistochemistry methods to investigate the effects of TangKe decoction on the experimental rat models of DN. The results showed that TangKe decoction possessed the following functions: (1)decreasing blood glucose (BG), ameliorating glucose and lipid metabolic disorders, improving nonenzymatic glycation, attenuating urinary albumin excretion and protecting renal function. (2)ameliorating glomerular hypertrophy, mesangial matrix expansion and thickening of glomerular basement membrane.(3)inhibiting
mRNA and protein expression of TGF-3 i, TGF-P R, Collagen IV and fibronection and synthesis of extracellular matrix(ECM),decreasing abnomal accumulation of ECM and preventing the glomerular sclerosis.It has been reported that the number of apoptotic cells increased obviously in the renal cortex of the DN rats and glomerular apoptosis was concerned with deterioration of renal function. Apdptotic cells in renal cortex were evaluated by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) and the protein expression of BAX and BCL-2 relating to apoptosis was detected by means of immunohistochemistry to investigate the effects of bax and bcl-2 on DN as well as the influence of them on apoptosis. The e
1.理论研究 由五部分组成,具体包括DN中医药研究概况、祛痰活血法治疗DN、DN现代医学研究进展、转化生长因子β(TGF-β)及其受体与DN、细胞凋亡与DN。在理论研究及文献综述中,参照古、今中医有关文献对糖尿病肾病的中医病名、病因病机、辨证论治方法、临床及实验研究进展和中医研究中存在的问题及对策进行了探讨,总结了导师李敬林教授辨治糖尿病肾病的方法和学术观点,强调从痰瘀论治糖尿病肾病,并对现代医学在糖尿病肾病研究中所取得的进展进行了总结,进一步明确了糖尿病肾病的病理特征、发病机制、分期和治疗,并具体探讨了现代医学关于细胞凋亡和TGF-β及其受体在DN发病机制中的作用。
2.实验研究 采用高糖高脂饲料喂养结合尾静脉注射链脲佐菌素(STZ)的造模方法复制出糖尿病模型,此模型首先采用高糖高脂饲料喂养一个月,诱发动物肥胖,引起胰岛素抵抗,再以小剂量STZ35mg/kg尾静脉注射造成胰岛素代偿性分泌障碍,从而引发高血糖,并出现微量白蛋白尿,系膜基质增生、基底膜增厚等糖尿病肾病典型的早期病理形态学改变,符合2型DN的发病机制。
本实验分正常对照组、中药糖克煎剂预防组、高低剂量组、模型组和西药洛汀新对照组,预防组大鼠灌胃给药12周,其余各治疗组均给药8周。观察糖克煎剂对DN大鼠血糖、糖化血红蛋白、血脂、体重、肾重、尿蛋白排泄、肾功能的影响;通过光镜、电镜观察各组实验性DM大鼠肾组织病理形念学变化;采用逆转录聚合酶链式反应(RT-PCR法)、蛋白质印迹杂交(Western Blot)及免疫组化法,进一步观察糖克煎剂对DN大鼠肾小球细胞外基质(ECM)的主要成分IV型胶原(ColⅣ)和纤维连接蛋白(FN)、转化生长因子(TGF-β_1)及其受体(TGF-BR)mRNA及蛋白表达的影响;采用原位缺口标记技术(TUNEL)以及免疫组化法检测糖尿病大鼠肾组织中细胞凋亡及BCL-2及BAX等凋亡相关基因的表达,以进一步探讨DN发病机制,了解bcl-2、bax在糖尿病肾病发生、发展中的作用,以及中药糖克煎剂对DN细胞凋亡及其相关基因bcl-2/bax表达的影响。
Diabetic nephropathy(DN) is one of the chronic complications of diabetes mellitus(DM) and the common reason leading to chronic renal failure ,one third of which is caused by DN. Systematic research on prevention and treatment of DN by TangKeJianJi (TangKe decoction) has been performed from the whole ,organ, cell and molecule levels in this essay which was composed of the reference review and experimental study.1. Theory reviewThe theoretical review including five sections mainly discussed the recent research progresses in prevention and treatment of DN both from the aspects of traditional Chinese medicine (TCM) and modern medicine. In detail, the five sections were made up of the skeleton review of TCM and modern medicine on DN, the relationship between transforming growth factor-P (TGF- β ) , transforming growth factor- P receptor (TGF- βR) and DN , the relationship between apoptosis and DN. In the theoretical research, we have referred to the related ancient and modern TCM literature in order to explore the source of disease name of TCM for DN, etiology and pathogenesis of TCM, selection of therapeutic methods by different stages or according to the differential syndrome diagnosis, advance on the clinical and experimental research as well as the problems lying in the research of TCM and corresponding strategies. In the meanwhile ,we also have summarized the tutor's ( professor Li Jingling) academic point of view on the differentiation and treatment of DN, moreover further exploited the pathologic characteristics ,pathogenesis , pathologic stages and treatment of DN as well as the function of TGF- P and apoptosis on DN.2. Experimental studyThe experimental rat models at first were fed by a diet enriched with sucrose ,cholesterol and cholic acid for a month in order to induce the models' obesity and result in insulin resistance, and then injected the low dosage of 35mg/kg streptozotocin(STZ) through vein of the rat's tail to develop hyperglycemia. This kind of model closely simulated the metabolic abnormalities of the human type 2 diabetes mellitus and its renal structural lesion with microalbuminuria, mild mesangial matrix expansion, thickening of glomerular basement membrane and so on. The experimental rats were randomly divided into 6 groups: normal contrast group, pattern group, prevention group, high dosage group, low dosage group and Lotension group. Drug was delivered daily by gavage for 8 weeks in the therapeutic groups except for the prevention group for 12 weeks. We have detected, BUN, Scr, hemoglobin Aic (HbAic), body weight, kidney weight, urinary albumin excretion (UAE) and renal pathologic lesion through light microscopy and electron microscopy and further observed mRNA and protein expression of TGF- β 1, TGF- β R, Collagen IV and fibronection through RT-PCR, western blot and immunohistochemistry methods to investigate the effects of TangKe decoction on the experimental rat models of DN. The results showed that TangKe decoction possessed the following functions: (1)decreasing blood glucose (BG), ameliorating glucose and lipid metabolic disorders, improving nonenzymatic glycation, attenuating urinary albumin excretion and protecting renal function. (2)ameliorating glomerular hypertrophy, mesangial matrix expansion and thickening of glomerular basement membrane.(3)inhibiting
mRNA and protein expression of TGF-3 i, TGF-P R, Collagen IV and fibronection and synthesis of extracellular matrix(ECM),decreasing abnomal accumulation of ECM and preventing the glomerular sclerosis.It has been reported that the number of apoptotic cells increased obviously in the renal cortex of the DN rats and glomerular apoptosis was concerned with deterioration of renal function. Apdptotic cells in renal cortex were evaluated by terminal-deoxynucleotidyl transferase mediated d-UTP nick end labeling (TUNEL) and the protein expression of BAX and BCL-2 relating to apoptosis was detected by means of immunohistochemistry to investigate the effects of bax and bcl-2 on DN as well as the influence of them on apoptosis. The e
引文
1.郭啸华,刘志红,李恒,等.高糖高脂饮食诱导的2型糖尿病大鼠模型及其肾病特点.中国糖尿病杂志,2002:10(5):290-294
2.王艳菊,李敬林,汤连玲,等.糖克煎剂对大鼠糖尿病肾病早期滤过率影响的实验研究.辽宁中医杂志,2002;29(2):120-122
3. Like AA, Rossini AA. Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science 1979,193(425): 415
4.左文静,王忠山,许宗革,等.青春期糖尿病模型复制方法研究.白求恩医科大学学报,1995;21(2):117-119
5. Jounod A, Lambert AE, Orci L, et al. Studies of the diabetogenic action of streptozotocin. Proc Soc Exp Biol Med 1967,126:201
6. Anderson S. Renal effect of angiotensin-converting enzyme inhibitors in hypertension and diabetes. J Pharmacol, 1990,151(Suppl 3):11
7. Φsterby R. Fast accumulation of basement membrane material and the rate morphological changes in acute experimental diabetic glomerular hypertrophy. Diabetologia, 1980,18:493-500
8. 张喆,池光盛.糖尿病高血压药物的选择.国外医学《内分泌学分册》,1997;17(1):19.
9.陆菊明,潘长玉,郭爱岩.巯甲丙脯酸治疗糖尿病伴蛋白尿.中华肾脏病杂志,1991:7(5):284
10. Yosida H, Kon V, Ichikaua I. Polgmophism of the rennin-angiotens in system genes in progressive renal diseases. Kidney .Inet, 1996;50:732
11.郭汉城,陈孝文,孔令泉等.苯那普利对糖尿病肾病患者尿液内皮素一1排泄的影响及其意义.中国糖尿病杂志,2001:9(1):32
12.田文,刘志发.血管紧张素转换酶抑制剂洛汀新对早期糖尿病肾病的保护作用.实用医药杂志,2001:14(3):5
13.胡伟新.血管紧张素受体拮抗剂的保肾作用是否优于血管紧张素转换酶抑制剂.肾脏病与透析肾移植杂志,1997:6(4):338-340
14. Gilbert BE, Cox A, wull, et al. Expression of transforming growth factor β and type Ⅳ collagen in the renal tubulionterstitium in experimental diabetes: effects of ACE inhibition. Diabetes, 1998;47:414-422
15. Remuzzi A, et al. Effect of low dose Enalapril on glomerular size selectivity in human diabetic nephropathy. T Nephrol, 1993;6:36
16.江清林,李延君,辛华,等.黄芪甲苷对胰岛素G肽分泌作用研究.黑龙江医药科学,1999;22(3):24-27
17.汪德清,沈文梅,田亚平等.黄芪总黄酮对羟自由基作用的所致哺乳动物细胞损伤的保护作用.中国中药杂志,1995;20(4):240.
18.祁忠华,林善锬,黄宇峰.黄芪改善糖尿病早期肾血流动力异常的研究.中国糖尿病杂志,1999;7(3):147-148
19.赵洪伍.党参黄芪注射液对冠状动脉作用的研究.中国医院药学杂志,1989;9(6):162
20.蒋斗发,殷昌好,俞能高,等.黄芪总皂甙对老年大鼠血液流变性的影响.基层中药杂志,2002;16(5):15-16
21.顾文珍.尿囊素的作用及其临床应用.新药与临床,1990;9(4):232
22.郝志奇.山药水煎剂对实验性小鼠的降血糖作用.中国药科大学学报,1991;22(3):15
23.何书英.山药水溶性多糖的化学及体外抗氧化活性.中国药科大学学报,1994;25(6):369
24.曹凯.四种中药对小鼠组织谷胱甘肽过氧化物酶活性和脂质过氧化物含量的影响.中国中西医结合杂志,1993;13:82
25.舒思洁,洪爱蓉,胡宗礼等.山药对糖尿病小鼠血糖、血脂、肝糖元和心肌糖原含量的影响.咸宁医学院学报,1998;12(4):223-226
26.蔡缨,沈忠松.茯苓对2型糖尿病患者的降糖作用.解放军医学杂志,2001;26(5):371—372
27.高应斗.木通、茯苓、旋复花的利尿作用.中华医学杂志,1955;41(10):963
28.韩桂秋,白光清,王夕红.牛蒡子中血小板活化因子(PAF)受体拮抗剂的分离和结构鉴定.中草药,1992,23(11):563
29.吴涛.牛蒡子治疗Ⅱ型糖尿病.中医杂志,1997,38(10):581
30.王克勤.牛蒡子治疗肾性蛋白尿.中医杂志,1997,38(10):581
31.崔佰瑛.牛蒡子治疗糖尿病。肾病.中医杂志,1997,38(10):582
32. Parving HH, Osterby M, Ritz E. Diabetic nephropathy. In:The Kidney.Brenner BM ed. 10th ed. Philadelphia: WB Saunders Company, 2000,1731-1754
33.郭春燕,李振良.川芎嗪的药理作用与临床应用进展.医学综述,2001;7(7):436-437
2.王艳菊,李敬林,汤连玲,等.糖克煎剂对大鼠糖尿病肾病早期滤过率影响的实验研究.辽宁中医杂志,2002;29(2):120-122
3. Like AA, Rossini AA. Streptozotocin-induced pancreatic insulitis: new model of diabetes mellitus. Science 1979,193(425): 415
4.左文静,王忠山,许宗革,等.青春期糖尿病模型复制方法研究.白求恩医科大学学报,1995;21(2):117-119
5. Jounod A, Lambert AE, Orci L, et al. Studies of the diabetogenic action of streptozotocin. Proc Soc Exp Biol Med 1967,126:201
6. Anderson S. Renal effect of angiotensin-converting enzyme inhibitors in hypertension and diabetes. J Pharmacol, 1990,151(Suppl 3):11
7. Φsterby R. Fast accumulation of basement membrane material and the rate morphological changes in acute experimental diabetic glomerular hypertrophy. Diabetologia, 1980,18:493-500
8. 张喆,池光盛.糖尿病高血压药物的选择.国外医学《内分泌学分册》,1997;17(1):19.
9.陆菊明,潘长玉,郭爱岩.巯甲丙脯酸治疗糖尿病伴蛋白尿.中华肾脏病杂志,1991:7(5):284
10. Yosida H, Kon V, Ichikaua I. Polgmophism of the rennin-angiotens in system genes in progressive renal diseases. Kidney .Inet, 1996;50:732
11.郭汉城,陈孝文,孔令泉等.苯那普利对糖尿病肾病患者尿液内皮素一1排泄的影响及其意义.中国糖尿病杂志,2001:9(1):32
12.田文,刘志发.血管紧张素转换酶抑制剂洛汀新对早期糖尿病肾病的保护作用.实用医药杂志,2001:14(3):5
13.胡伟新.血管紧张素受体拮抗剂的保肾作用是否优于血管紧张素转换酶抑制剂.肾脏病与透析肾移植杂志,1997:6(4):338-340
14. Gilbert BE, Cox A, wull, et al. Expression of transforming growth factor β and type Ⅳ collagen in the renal tubulionterstitium in experimental diabetes: effects of ACE inhibition. Diabetes, 1998;47:414-422
15. Remuzzi A, et al. Effect of low dose Enalapril on glomerular size selectivity in human diabetic nephropathy. T Nephrol, 1993;6:36
16.江清林,李延君,辛华,等.黄芪甲苷对胰岛素G肽分泌作用研究.黑龙江医药科学,1999;22(3):24-27
17.汪德清,沈文梅,田亚平等.黄芪总黄酮对羟自由基作用的所致哺乳动物细胞损伤的保护作用.中国中药杂志,1995;20(4):240.
18.祁忠华,林善锬,黄宇峰.黄芪改善糖尿病早期肾血流动力异常的研究.中国糖尿病杂志,1999;7(3):147-148
19.赵洪伍.党参黄芪注射液对冠状动脉作用的研究.中国医院药学杂志,1989;9(6):162
20.蒋斗发,殷昌好,俞能高,等.黄芪总皂甙对老年大鼠血液流变性的影响.基层中药杂志,2002;16(5):15-16
21.顾文珍.尿囊素的作用及其临床应用.新药与临床,1990;9(4):232
22.郝志奇.山药水煎剂对实验性小鼠的降血糖作用.中国药科大学学报,1991;22(3):15
23.何书英.山药水溶性多糖的化学及体外抗氧化活性.中国药科大学学报,1994;25(6):369
24.曹凯.四种中药对小鼠组织谷胱甘肽过氧化物酶活性和脂质过氧化物含量的影响.中国中西医结合杂志,1993;13:82
25.舒思洁,洪爱蓉,胡宗礼等.山药对糖尿病小鼠血糖、血脂、肝糖元和心肌糖原含量的影响.咸宁医学院学报,1998;12(4):223-226
26.蔡缨,沈忠松.茯苓对2型糖尿病患者的降糖作用.解放军医学杂志,2001;26(5):371—372
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