摘要
糖尿病肾病(diabetic nephropathy, DN)是糖尿病慢性微血管并发症之一,是一类以进行性纤维化为病理特征的肾脏疾病,其发病机制复杂。近期研究发现,结缔组织生长因子(connective tissue growth factor, CTGF)在糖尿病肾病中可能是骨形态发生-7(bone morphor genetic p roteins-7, BMP-7)信号通路的抑制剂,两者在肾脏纤维化过程中均发挥关键作用。传统中药通心络已广泛用于心脑血管病的防治,随着对通心络新用途的不断开发,其临床应用也在不断扩大。本实验研究通心络对DM大鼠肾脏中BMP-7及CTGF的影响,探讨其对DN大鼠肾脏的保护作用及其可能机制。
目的研究通心络对糖尿病大鼠肾脏组织保护作用及结缔组织生长因子(CTGF)及骨形态蛋白7(BMP-7)表达的影响,并探讨其对DN大鼠肾脏的保护机制。
方法雄性Wistar大鼠70只,随机选出15只作为正常对照组(C组,n=15)。剩余55只采用链脲佐菌素(STZ)诱导建立糖尿病大鼠模型；禁食12小时,一次性尾静脉注射STZ 55mg／kg,72h后测定血糖,以空腹血糖水平≥16.7mmol／l(300mg／l)为糖尿病模型成功的标准。造模成功后大鼠随机分为糖尿病组(DM组),通心络治疗组(TXL组),于24周末处死大鼠检测各组大鼠24h尿微量蛋白、采血测空腹血糖(FPG)、糖化血红蛋白(HbAlc)、糖基化终末产物(AGEs)和肝肾功能等血清学指标,肾重／体质量(KW／BW),光镜PAS染色,观察肾脏病理改变及细胞外基质(ECM)含量的变化,采用逆转录-聚合酶链式反应(reversetranscriptase coupled to polymerase chain reaction, RT-PCR)和Western blotting分别用于大鼠肾脏组织CTGF和BMP-7mRNA和蛋白质表达水平。
结果1.一般指标及血清AGEs等的变化：C组大鼠表现活泼,体质量增长迅速；DM组大鼠出现多饮多食多尿及消瘦表现,较C组体质量明显下降(P<0.01)；TXL组DM组上述症状减轻,体质量明显升高(P<0.05)。DM组与C组比较,FPG、HbA1C、血清AGEs均明显升高(P<0.05)；TXL组较DM组有下降趋势,但差异无统计学意义(P>0.05)。2.各组大鼠肾功能的变化：DM组较C组大鼠BUN、Scr、Kw／Bw升高、24h尿微量蛋白升高,表明DM组大鼠存在肾脏肥大及早期肾病表现。TXL组与DM组比较,24 h尿微量蛋白含量减少,Kw／Bw亦降低,差别有统计学意义(P<0.05),其他肾功能指标的改变无统计学意义(P>0.05)。3.各组大鼠肾脏组织病理学观察镜下可见,C组大鼠肾小球及肾小管、间质未见明显病理改变。DM组大鼠大部分肾小球基底膜增厚,毛细血管腔受压变窄,肾小管上皮细胞空泡样变明显。TXL组大鼠肾小球球囊与正常组比较无明显增大,毛细血管丛内可见清晰的血管腔,部分毛细血管丛基底膜稍增厚,系膜区稍增宽,部分肾小管上皮细胞胞浆疏松,轻度水肿,未见空泡样改变。4.各组大鼠肾脏组织中ECM含量的变化DM组较C组大鼠肾脏组织ECM明显升高(P<0.01)。TXL组与DM组比较,ECM含量减少(P<0.05)。5.肾组织CTGF、BMP-7mRNA的变化RT-PCR结果示,DM组较C组CTGF mRNA上调、BMP-7mRNA下调明显(P<0.01)；TXL组较DM组CTGFmRNA下降,BMP-7mRNA上调,差异有统计学意义(P<0.05)。6.Western blot肾组织CTGF、BMP-7蛋白和内参β-actin蛋白检测条带光密度分析显示：DM组肾组织CTGF表达升高、BMP-7表达降低(P<0.01)；给药24周后可见,TXL组较DM组CTGF表达下降,BMP-7表达升高(P<0.05)。
结论1、通心络可以减轻糖尿病大鼠微量蛋白尿、改善肾脏组织病理及减少肾组织ECM含量,对DN大鼠早期具肾脏保护作用。2、通心络抑制糖尿病大鼠肾脏组织CTGF表达,增加BMP-7表达,可能为通心络对糖尿病肾病大鼠肾脏的保护机制。
Diabetic nephropathy (DN) is one of the chronic microvascular complications of diabetes and a kind for fibrosis to pathological features of renal disease, its pathogenesis is complexity, many mechanisms are still under explored. Recent studies found that connective tissue growth factor (CTGF) in diabetic nephropathy may be the bone morphogenetic-7 (BMP-7) signaling pathway inhibitor, The both play a key role in the kidney fibrosis. Tongxinluo,the components of traditional herb has been widely used in cardiovascular and Cerebrovascular disease prevention and treatment. With Tongxinluo continuous development of new uses, its clinical application are also expanding. This study on Tongxinluo is to explore the the protective effect of Diabetic mellitus (DM) rats and its possible mechanism through the changes of the expression of CTGF and BMP-7 in DN rats'kidney.
Objective To study the effects of Tongxinluo on the expression of connective tissue growth factor CTGF) and bone morphogenetic protein-7 (BMP-7) in Diabetic rats,and to explore the protective effects and mechanism on the kidney of Diabetic nephropathy rats.
Methods Randomly selected 15 as the normal control group (C group, n= 15) from 70 healthy male Wistar rats.The other 55 rats was established the diabetic (DM) rats model by one-dose introperitoneal injection of streptozotocin(STZ).After 72h,the determination of blood glucose to fasting glucose levels≥16.7mmol/1 (300mg/1) that were diabetes model of success. After animal models were successfully established,they were randomly divided into diabetic groups (DM group), Tongxinluo (500mg-kg-1-d-1) treated groups(TXL group). The ratio of kidney weight to body weight,24h urine microprotein, plasma fasting blood glucose (FPG), glycosylated hemoglobin (HbAlc), advanced glycation end products (AGEs) and kidney function were determined. The renal pathological changes were examined with light microscope PAS staining. The mRNA and protein expression of CTGF and BMP-7 in kidney were detected by reverse transcription-polymerase chain reaction(RT-PCR) and Western bloting.
Results 1. Weight and blood indices:Compared with C group,DM group weighed significantly less(P<0.01), After treated with Tongxinluo capsules for 24 weeks, rats in TXL group also weighed significantly less than those in C group (P <0.01),but weighed more than those in DM group with statistic significance(P<0.01). As to FPG、HbA1c and AGEs, DM group and C group, FBG, HbA1C, serum AGEs were significantly higher (P<0.05); TXL group was a downward trend than DM group, but the difference was not statistically significant.2. The changes of renal function in rats:DM group than in C group rats with decreased of plasma BUN、Scr and Kw/Bw higher,24h urine protein increased; TXL group than DM group,24h urine protein content decreased, Kw/Bw is also reduced (P<0.05).3. Light microscopic findings of renal morphological changes in rats (PAS×400):C group rats glomerular and tubular interstitial were no significant pathological changes.. In DM group, Most of the glomerular basement membrane were thickened, capillary was narrowed, tubular epithelial cells were vacuolated changes significantly. TXL group compared with DM group, some of glomerular basement membrane is thickened and widened mesangial areas slightly, some of renal tubular epithelial cells were no vacuolar changes.4.The changes of extracellular matrix (ECM) in the rars'kidney: DM group than C group, ECM were significantly higher (P<0.01); TXL group was reduced than DM group, (P<0.05).5.The expression of CTGF and BMP-7mRNA in kidney:RT-PCR results showed, The expression of CTGF mRNA in DM group than C group were significantly increased (P<0.01), TXL group than DM group decreased (P<0.05);The expression of BMP-7mRNA in DM group than C group were significantly decreased (P<0.01), TXL group than DM group increased (P <0.05).6.The expression of CTGF and BMP-7 protein in kidney:Western blotting results were that the expression of CTGF in DM group than C group, were significantly higher (P<0.01), TXL group was lower than the DM group (P<0.05); The expression of BMP-7 protein, DM group than C group, were significantly decreased (P<0.01), TXL group was increased than DM group (P<0.05).
Conclusion 1.Tongxinluo can reduced microalbuminuria in diabetic rats and improved the renal pathology changes, and decreased kidney deposition of ECM in diabetic nephropathy rats.2.Its protective effects and mechanism may be related to downregulating expression of CTGF and upregulating expression of BMP-7.
引文
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33.林成仁,王敏,庄百溪等.通心络胶囊对动脉粥样硬化闭塞症家兔ET、NO、SOD、MDA水平变化的影响.中成药.2008,30(8)：1114-1118
34.钱方方,陈军建,于焱等.通心络对糖尿病患者血小板活性的影响.江苏大学学报：医学版.2003，13(3)：、211-212
35.杨跃进,张健,吴永健等.通心络、卡维地洛、缬沙坦对兔急性心肌梗死晚期再灌注微血管内皮功能及完整性保护作用的对比.中国病理生理杂志.2006,22(7)：1366-1369
36.刘南海,黄晓峰,彭文杰等.通心络胶囊对短暂性脑缺血发作患者血浆溶血磷脂酸含量变化的影响.临床荟萃.2008,23(23)：1676-1678
37.梁景玉,吴以岭,贾振华等.通心络对气虚和气滞型血管损伤CoX-2和iNoS相互作用的影响.中成药.2009,31(6)：829-833
38.梁俊清,孙士然,吴以岭等.疲劳应激致血管内皮功能障碍与肾素—血管紧张素醛固酮系统变化的关系及通络方药的干预.中国病理生理杂志.2009,25(6)：1064-1069
39.曹刚,赵韶华.通心络胶囊对麻醉犬脑血管的影响..河北中医.2000,22(4)：315-317
40.赵岳中,许雁山.通心络对缺血性脑卒中患者血流变的影响.南华大学学报：医学版.2007,35(5)：768-769,771
41.马琦琳,孙明,杨天害等.通心络对血管紧张素Ⅱ诱导的血管内皮细胞活力及组织因子的影响.中南大学学报.2007,32(3)：485-489
42.关启刚,曾定尹,孙喜琢等.通心络抑制白细胞介素113介导的小型猪冠状动脉早期炎症反应及内膜增殖.中国动脉硬化杂志.2007,15(8)： 575-579
43.曹卫华,周力,王宇彬,等通心络对糖尿病肾病尿白蛋白排泄率的影响[J].潍坊医学院学报,2005,27(5)：176-177
44.梁小卫,田福利,梁春等.通心络体外促进人外周血内皮祖细胞的增殖、迁移、黏附的研究.国际心血管病杂志.2009,36(3)： 184-190
45.蒋海山,李求实,陆兵勋等.通心络对大鼠胚胎神经干细胞增殖和分化的影响.南方医科大学学报.2008,28(5)：679-683
46.赵玉霞,温登福,孙尚文.不同类型心绞痛患者血清中高敏C反应蛋白的含量及药物干预.南京中医药大学学报,2005,21(4)：226-227
47.赵玉霞,温登福,孙尚文.通心络胶囊对不稳定性心绞痛患者PAG及黏附分子水平的影响.上海中医药杂志,2004,38(10)：14-15
48.贾英,孙晓,葛华.通心络胶囊对不稳定型心绞痛血管内皮功能的影响.中国医科大学学报.2008,37(2)：285-285
49.秦廷兵.倍他乐克、通心络联合治疗冠心病心绞痛68例临床分析.中外医学研究.2009,7(11)：69-70
50.索龙,孙长华.通心络和鲁南欣康单用或合用治疗心绞痛的比较.中国医药指南：学术版.2009,7(12)：305-306
51.董丽华,.陈才.通心络胶囊联合硝苯地平治疗不稳定型心绞痛120例疗效观察.中国医药导报.2008,5(23)：76-76,79
52.于海青.通心络联合低分子肝素治疗不稳定型心绞痛.中西医结合心脑血管病杂志.2008,6(4)：469-470
53.孙运爱,丁国锋,张丽娜等.通心络与单硝酸异山梨酯合用治疗老年冠心病心绞痛疗效观察.临床医药实践.2003,12(9)：673-674
54.刘义桥.通心络胶囊治疗冠心病心绞痛临床观察.辽宁中医药大学学报.2008,10(11)：98-99
55.尤士杰,杨跃进,陈可冀等.通心络对急性心肌梗死患者再灌注后心肌和微血管的保护性研究.中华心血管病杂志.2005,33(5)：433-437
56.张伟.通心络胶囊对冠脉内支架置入术后患者凝血纤溶系统和血管紧张素Ⅱ的影响.河北医学.2006,12(1)：37-39
57.马利平,汪凤兰,王美玲.通心络对冠心病心力衰竭患者心功能和运动耐量的影响.中国临床康复.2003,7(21)：2975-2975
58.廖奕华,邓云梅等.通心络胶囊治疗慢性心力衰竭的疗效观察.中西医结合心脑血管病杂志.2003,1(1)：29-30
59.张晓峰,肖翠君,攸淑芳等.通心络胶囊对冠心病心力衰竭患者甲状腺激素水平影响的临床研究.疑难病杂志.2005,4(3)：129-131
60.徐贵成，齐晓琳等.通心络胶囊治疗风湿性心瓣膜病心力衰竭临床观察.浙江中西医结合杂志.2002,12(1)：5-7
61.左芳,赵玉霞.通心络胶囊对动脉粥样硬化斑块影响的临床研究.中西医结合心脑血管病杂志.2005,3(3)期：200-202
62.吴秋枫,李蕾.通心络对老年动脉粥样硬化治疗的临床观察.实用老年医学.2008,22(1)：24-26
63.黄晓松,杨期东,龙光喻等.通心络胶囊对颈动脉粥样硬化不稳定斑块影响的临床研究.疑难病杂志.2007,6(5)：267-269
64.苏晓燕,吕维红,张弈等.通心络胶囊对原发性肥厚型心肌病左室功能影响的观察.中国中西医结合急救杂志.2007,14(4)：231-232
66.傅志慧,张艳丽.通心络胶囊治疗椎-基底动脉系统短暂性脑缺血发作临床观察.中国中医急症.2009,18(2)：217-218
67.刘南海,黄晓峰,彭文杰.通心络胶囊对短暂性脑缺血发作患者血浆溶血磷脂酸含量变化的影响.临床荟萃.2008,23(23)：1676-1678
68.任金生,杨秋霞.通心络胶囊治疗急性脑梗死的疗效观察.中国实用神经疾病杂志.2008,11(4)：62-63
69.王薇.通心络胶囊对脑梗死康复期治疗作用评价.疑难病杂志.2006,5(5)：331-333
70.柯明远,吴秋英,杜志乾.通心络胶囊治疗急性脑出血的随机双盲对照研究.福建中医药.2005,36(3)：22-23
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72.周朝阳.通心络治疗肾病综合征高脂血症.临床肾脏病杂志2008,8(12)：570-571
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74.赵毅,张显林.通心络胶囊对糖尿病肾病患者血浆内皮素的影响.中国中西医结合杂志..2005,25(2)：131-133
75.陈阳,胡蕴刚,朱福.通心络胶囊联合依那普利治疗2型糖尿病肾病临察床观.疑难病杂志.2003,2(5)：277-279
76.古青.通心络胶囊联合开博通对早期糖尿病肾病尿微量白蛋白的影响.疑难病杂志.2008,7(10)：606-607
77.郭宏.中药通心络胶囊佐治糖尿病肾病的临床疗效观察.中国药物与临床.2009,9(B07)：82-83
78.焦秀坤,杨立波.]通心络联合甲钴胺治疗糖尿病周围神经病变的临床研究.中药药理与临床..2007,23(1)：71-73
79.魏耀周,韩璐荣,马大为.通心络胶囊治疗糖尿病周围神经病变32例临床观察.疑难病杂志.2004,3(6)：355-356
80.刘静芹,宗燕军,吕洪等.通心络胶囊对糖尿病自主神经病变心率变异的影响.中国现代医学杂志.2007,17(5)：601-604
81.莫云秋,伍松姣,王强.通心络胶囊佐治高血压肾损害患者的疗效观察.疑难病杂志.2009,8(11)：670-671
82.徐忠,王立真,候云.通心络胶囊对原发性高血压病病人内皮功能的影响.中西医结合心脑血管病杂志.2007,5(8)：677-679
83.徐丽林,管昌益,王朝阳等.通心络胶囊对高血压合并颈动脉内膜增厚患者C反应蛋白的影响.福建中医药.2009,40(5)：15-15,17
84.王以炳,夏蕾,刘莉敏.通心络对肺心病急性加重期患者血浆内皮素一氧化氮降钙素基因相关肽的影响.中国误诊学杂志..2007,7(18)：4195-4196
85.吴晓晖,马铮.通心络胶囊治疗慢性呼吸衰竭急性发作期30例观察.现代中西医结合杂志.2004,13(11)：1470-1471
86.杨大为,闫瑞萍,陈景丽.通心络治疗肺心病疗效观察.临床肺科杂志.2006,11(4)：444-444
87.赵龙现,潘华,张弛等.通心络胶囊治疗血管性头痛66例.陕西中医.2004,25(10)：872-873
88.童玖兴.通心络治疗偏头痛的TCD检测及疗效观察.中华现代内科学杂志 .2005,2(6)：513-515
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90.付宏娟,王东伟,徐燕梅.通心络治疗偏头痛56例临床观察.黑龙江医学.2008,32(7)：531-531
91.李庆,张国珺.通心络胶囊治疗喘息型支气管炎急性加重期伴呼吸衰竭的临床观察.中国全科医学.2005,8(6)：496-497
92.林志翔,戴禄寿,戴益琛等.通心络胶囊治疗类风湿关节炎的临床观察.临床军医杂志.2008,36(5)：732-734
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