摘要
目的：
本课题拟通过链脲佐菌素(streptozotocin, STZ)诱导建立糖尿病SD大鼠模型,并采用灌胃给予地龙煎剂、蚓激酶溶液、ACEI溶液的方法,观察治疗后糖尿病大鼠肾脏组织中基质金属蛋白酶MMP-2、MMP-9、IV型胶原蛋白表达水平的变化,探讨地龙及其粗提取物蚓激酶对糖尿病大鼠肾脏的保护作用及相关作用机制；通过观察糖尿病肾病3-4期患者使用地龙针剂前后,中医瘀血症状及体征、尿微量白蛋白、血糖、胰岛素、C肽、糖化血红蛋白及相关生化指标的变化情况,评估糖尿病肾病的病情变化情况,明确地龙针剂的疗效,为人类糖尿病肾病的早期防治提供理论依据。
方法：
1、实验研究
清洁级健康雄性SD大鼠,体重在200-230g之间,禁食16h后将STZ(临用前以0. lmol/L, pH4.2柠檬酸缓冲液溶解新鲜配置)按剂量60mg/kg体重一次性腹腔注射,于开始注射STZ后72h后取大鼠尾静脉血测血糖,凡血糖≥16.7mmol/L确定为糖尿病大鼠模型制作成功。
将成模大鼠随机分为糖尿病组、1g／kg地龙治疗组,3g／kg地龙治疗组,蚓激酶治疗组,ACEI+地龙治疗组,ACEI治疗组,每组12只,另外选取12只正常大鼠做正常组。其中正常组和糖尿病模型组予生理盐水每日灌胃,1g／kg地龙治疗组给予地龙汤剂1g-kg-1 d-1灌胃；3g／kg地龙治疗组地龙汤剂3g-kg-1-d-1灌胃；蚓激酶治疗组给予蚓激酶60万U·kg-1·d-1灌胃,ACEI+地龙治疗组予培哚普利4mg及地龙3g-kg-1d-1灌胃,ACEI治疗组给予培哚普利4mg·g-1·d-1灌胃。共用药干预12周。蚓激酶溶解于反渗水中。灌胃的剂量根据大鼠体重每周调整。所有大鼠自由饮食和饮水。动物饲养室恒温21℃,昼夜各12小时。
于灌胃0w、2w、4w、8w、12周末测血压、收集血、尿及肾组织标本,检测血肌酐、血尿素氮、谷丙转氨酶、谷草转氨酶、甘油三酯、胆固醇、24h尿蛋白定量及肾重／体重比值：石蜡切片行HE、PAS、PASM、Masson染色和免疫组化染色,肾组织蛋白用Western-bloting法检测,观察肾组织Ⅳ-型胶原蛋白、MMP-2、MMP-9表达情况。
结果用均数±标准差(x±s)表示,两组以上均数比较采用单因素方差分析,其后进行的两者之间比较应用最小极差分析。通过SPSS-13.0统计软件处理,以P<0.05为有显著性差异的标准。
2、临床研究
将我院2009年6月至2010年5月临床糖尿病肾病3-4期住院患者,根据纳入标准和排除标准,将入选患者随机分为2组,地龙治疗组18例、对照组25例。按照课题设计要求地龙治疗组在对照组一般治疗基础上,给予静滴地龙针剂6ml+250ml葡萄糖溶液中静滴治疗,疗程2w。观察治疗前后患者血糖、糖化血红蛋白、尿微量白蛋白、血浆蛋白、肝、肾功能、血脂、血常规等指标的变化情况。评估治疗前后的效果。
结果：
1、实验研究
(1)12 w末,糖尿病大鼠较正常对照组大鼠肾／体比值,24h尿量、饮水量、饮食量,24h尿微量白蛋白均有显著升高(P<0.01)。
糖尿病大鼠各时间点血压均较正常组大鼠降低(P<0.05), ACEI组大鼠较模型组血压降低有统计学意义(P<0.05),其中蚓激酶组、ACEI+地龙组均较模型组血压有降低趋势,但无统计学意义。单纯模型组大鼠血压与病程总体上呈递减趋势。
12周末,糖尿病大鼠ALT.AST.BUN.TG.TC均较正常组大鼠升高(P<0.05),模型组与各治疗组之间差异不明显。正常、模型及治疗组之间Cr差异不明显。单纯模型组大鼠ALT.AST.Cr.BUN.TG.TC均较Ow点大鼠增高(P<0.05)。
12w末,模型组大鼠蛋白尿明显高于正常组(P<0.05),各治疗组均有降低尿蛋白的作用,且与模型组相比均有统计学意义(P<0.05)。其中以ACEI组、ACEI+地龙组疗效最显著,其次为蚓激酶治疗组及3g／kg地龙组。据各时间点观察,各组大鼠于8w前尿蛋白差异不明显,模型组大鼠尿蛋白于8w是呈现增高趋势,12w时明显增高,有统计学意义(P<0.05)。
(2)病理组织学改变
PAS及PASM染色结果显示,与正常组大鼠相比,糖尿病组大鼠肾小球系膜基质增多,系膜扩张。蚓激酶组、1g／kg地龙组、3g／kg地龙组系膜基质增多较糖尿病组明显减轻。肾小球硬化半定量评分结果表明,与糖尿病组比较,蚓激酶治疗组,1g／kg地龙组、3g／kg地龙组能明显降低肾小球硬化指数(p<0.05)。与正常组大鼠相比,糖尿病组大鼠肾小管萎缩,扩张,空泡变性,管型形成均明显增加。肾小管损伤半定量评分结果表明,蚓激酶治疗组、1g／kg地龙组、3g／kg地龙组、ACEI+地龙组均能明显降低糖尿病大鼠肾小管损伤指数(p<0.05)。
MASSON染色结果显示,与正常组大鼠相比,糖尿病组大鼠肾小球系膜区、肾小管间质胶原沉积明显增加。MASSON染色半定量结果表明,与糖尿病组比较,蚓激酶治疗组、1g／kg地龙组、3g／kg地龙组、ACEI+地龙组能明显降低肾小管间质胶原沉积(p<0.05)。
(3)免疫组织化学
Ⅳ型胶原蛋白的表达正常组大鼠肾小球及系膜区Ⅳ型胶原表达较少,糖尿病组大鼠肾小球系膜区Ⅳ型胶原表达明显增多,蚓激酶治疗组、1g／kg地龙组、3g／kg地龙组、ACEI+地龙组Ⅳ型胶原表达较糖尿病组减少,差异有显著性(P<0.05)。
金属基质蛋白酶-2(MMP-2)的表达正常组大鼠肾小球、近端小管、远端小管、集合管MMP-2表达较多,糖尿病组大鼠肾小球、近端小管、远端小管、集合管表达MMP-2明显较少,蚓激酶治疗组、lg／kg地龙组、3g／kg地龙组、ACEI+地龙组MMP-2表达较糖尿病组增多,差异有显著性(P<0.05)。
金属基质蛋白酶-9(MMP-9)的表达正常组大鼠肾小球、近端小管、远端小管、集合管MMP-9表达较多,糖尿病组大鼠肾小球、近端小管、远端小管、集合管MMP-9表达明显较少,蚓激酶治疗组、1g／kg地龙组、3g／kg地龙组、ACEI+地龙组MMP-2表达较糖尿病组增多,差异有显著性(P<0.05)。
(4) Western Blot
Ⅳ型胶原蛋白表达与正常组比较,糖尿病大鼠肾组织Ⅳ型胶原蛋白表达水平明显升高,3g／kg地龙组及蚓激酶组Ⅳ型胶原蛋白表达水平较糖尿病组明显减少,差异有显著性(P<0.05)。
MMP-9蛋白表达与正常组比较,糖尿病大鼠肾组织MMP-9蛋白表达水平明显降低,3g／kg地龙组及蚓激酶组MMP-9蛋白表达水平较糖尿病组明显增加,差异有显著性(P<0.05)。
2、临床研究
两组治疗后瘀血证候评分、尿微量白蛋白、血脂异常均较治疗前明显改善,地龙治疗组较对照组疗效更为显著(P<0.05)。
结论：
1.糖尿病肾病时,金属基质蛋白酶MMP-2、MMP-9表达减少,细胞外基质Ⅳ型胶原蛋白表达增多,与肾脏细胞外基质积聚、肾脏肥大及肾功能受损相一致,提示MMP-2、MMP-9,Ⅳ型胶原蛋白在糖尿病肾病的发生发展中起作用。
2：地龙煎剂、蚓激酶可显著减少实验性糖尿病大鼠模型肾小球系膜区细胞外基质Ⅳ型胶原的蛋白的表达,纠正金属基质蛋白酶MMP-2、MMP-9表达的减少,促进糖尿病大鼠肾小球内细胞外基质的降解,减轻肾小球硬化和肾小管损伤,减少蛋白尿的生成。
3.地龙煎剂及其粗提取物蚓激酶对糖尿病肾脏保护作用的机制可能是：提供纤溶酶源激活物(e-PA),激活纤溶酶,促进金属基质蛋白酶MMP活化,降解细胞外基质,从而改善糖尿病过程中肾小球系膜基质的沉积,基底膜增厚,通过改善细胞外基质代谢从而延缓糖尿病肾病肾小球硬化、肾小管纤维化的进展。
4.地龙针剂临床观察,结果显示：地龙针剂可以通过改善糖尿病患者血瘀证候,改善血脂紊乱,降低尿微量白蛋白排泄率,从而达到治疗和预防糖尿病肾病的作用。
Objective:To investigate the possible protective effects and relevant mechanism of earthworm and lumbrokinase on diabetic kidney desease. Experimental diabetes was induced by a single intraperitoneal injection of 60 mg/kg body weight Streptozotocin after a 16h overnight fast. Diabetic rats were treated with earthworm decoction, lumbrokinase and perindopril intragastrically. Expression of extracellular matrix associated protein, collagenⅣ, Matrix metalloproteinases, MMP-2, MMP-9 were investigated by immunochemistry and wsternblot.43 patients of DN in 3-4 stage were observed to evaluate the protection of earthworm on diabetic kidney disease. Blood stasis symptom integral UAE、HbAlc、blood glucose、renal function lipids and blood regular test were all adopted before and after treatment.
Methods:
1. Experimental study:
Male Sprague-Dawley rats, weighting 200-220g, were induced by a single intraperitoneal injection of 60 mg/kg body weight Streptozotocin (STZ, Sigma Aldrich, St. Louis, MI, USA) in 0.01 mol/1 citrate buffer (pH 4.2) after a 16h overnight fast. The induction of the diabetic state was confirmed by the blood glucose level measurement on the third day after STZ administration. The rats with fasting blood glucose concentrations>16.7 mmol/L were classified as diabetic and used in the study.
The rats were randomly allocated into the following experimental groups:untreated diabetic rats (Diabeitc group, n=12); diabetic rats treated with earthworm decoction at a dose of lg/kg body weight (Diabetic +lg/kg earth worm decoction group, n=12); diabetic rats treated with earthworm decoction at a dose of 3g/kg body weight (Diabetic + 3g/kg earth worm decoction group, n=12);diabetic rats treated with lumbrokinase(Lum, Bokang Pharmaceutical Company, Zhuhai, China) at a dose of 600 000 U/Kg body weight (Diabetic+ lumbrokinse group, n=12); diabetic rats treated with ACEI and earthworm decoction at a dose of 3g/kg body weight earthworm decoction and 4mg/kg body weight perindoril (Diabetic + ACEI and earth worm decoction group, n=12); diabetic rats treated with the ACE inhibitor, perindopril (PER, Servier Laboratories, Neuilly, France) at a dose of 4 mg/kg body weight (Diabeitc +ACEI group, n=12). Medications were administered intragastrically. In addition, non-diabetic rats served as control (Non-diabetic group, n=12) and were studied concurrently. The animals were housed at constant room temperature (21°C) under a controlled 12h light to 12h dark cycle. The animals had free access to water and standard laboratory diet.
Body weight, kidney/body ration, the values of plasma creatinine (Cr), urea nitrogen (BUN), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TC), cholesterol (TG),systolic blood pressure (SBP),and 24h UAE were all measured at Ow,2w,4w,8w,12w. At 12 weeks after administration of lumbrokinse, earthworm decotion and PER, rats were anesthetized with pentobarbital sodium. The kidneys were removed immediately, weighed and rinsed in PBS buffer. Sections of the kidneys were stored in 10% buffered formaldehyde for subsequent histopathological examinations and immunohistochemical studies. The rest of the kidneys were stored at-80℃for the later analysis. Immunohistological and western blot examinations were performed with antibodies of CoIV, MMP-9 and MMP-2.
Data were expressed as mean±SEM. Group differences were analysed by one-way ANOVA with correction for multiple comparisions using the Least-Significant Difference post hoc test for multiple comparisons. A p value of less than 0.05 was considered statistically significant.
2. Clinical research:
Select 43 patients of DN in 3-4 stage. Divide them into treatment group (18 persons) and control group (25 persons) randomly. Both groups were given the same therapy such as controlling the blood pressure and glucose, and regulating lipids. Give earth worm injection to the treatment group everyday for 14 days. Observe the change of blood stasis symptom integral、
Results:
1. Experimental study:
(1)Compared to the non-diabetic rats, diabetic rats had polyuria, polydipsia, polyphagia, increased kidney weight, tardy weight gain and 24h UAE at 12 weeks (P< 0.01). No differences in body weight, kidney index, 24 h urine, water or food intake were observed among the treatment groups.
Diabetic rats had lower systolic blood pressure than non-diabetic rats in our study (P< 0.05). Compared to untreated diabetic rats, ACEI group had decreased blood pressure (P< 0.05).
The Cr had no differences between diabetic group and other treatment groups and little distinctions about BUN, ALT, AST, TC and TG among treatment groups also were observed.
At 12 weeks, diabetic rats had higher urinary albumin excretion than non-diabetic rats (P<0.05). Supplementation of lumbrokinase, earthworm decoction and ACEI could inhibit the rise in urinary albumin excretion (P< 0.05).The ACEI and ACEI+earthworm group had obvious results, lumbrokinase and 3g/kg earthworm group were the latter.
(2) histopathological changes
PAS, PASM and MASSON staining tests showed diabetic rats had more glycogen (collagen) deposits in glomerular mesangium and basement membrane. Thickening basement membrane of and mesangial expansion were obvious in diabetic rats. The severity of lesions was most pronounced in the diabetic group, whereas supplementation with lumbrokinase and earthworm decoction attenuated these changes. Diabetic rats had moderate glomerulosclerosis and tubulointerstitial fibrosis, as evidenced by mesangial expansion and deposition of extracellular matrix and the tubulointerstitial injury, respectively. Quantitative analyses of these changes showed that diabetes was associated with an increase in the glomerulosclerosis index (GSI) (P< 0.05) and tubulointerstitial fibrosis index (TIFI) (P< 0.05). Lumbrokinase supplementation attenuated these changes (P< 0.05).
(3) immunohistochemical studies
Diabetes was associated with an apparent increase in the intensity of immunostaining for collagen IV in these areas, and most intensely in the expanded mesangial areas. Lumbrokinase and earthworm decoction supplementation resulted in a decrease of immunolocalization for collagenⅣ(P<0.05).
MMP-2 and MMP-9 were immunolocalized to proximal and distal tubules, collecting ducts and the mesangial areas in the glomerulus. Diabetes was associated with an apparent overall reduction in the intensity of immunolocalization for MMP-2 and MMP-9. In diabetic rats receiving lumbrokinse and earthworm decoction supplementation, the pattern of immunostaining for both MMP-2 and MMP-9 was increased (P<0.05).
(4) Western Blot
Collagen typeⅣwas detected as a band of 200 kDa. The expression of collagenⅣwas increased in the diabetic kidneys compared with the non-diabetic, while lumbrokinase and earthworm decoction supplementation could partially attenuate this increase in collagenⅣprotein expression (P<0.05). MMP-9 was detected as a band of 98 kDa. There was a decrease of MMP-9 in diabetic kidneys compared to non-diabetic, while lumbrokinse and earthworm decoction supplementation could ameliorate these changes (P<0.05).
2. Clinical research:
Earth worm injection could obviously change the blood stasis symptom integral,24hUAE and lipids disorder of DN patients (P<0.05).
Conclusion:
1. Glomerular ECM accumulation is mediated by a plasmin/MMPs cascade in diabetic nephropathy. Decreased degradation of the glomerular extracellular matrix (ECM), collagenⅣand decreased total glomerular proteolytic activity and specific glomerular protease activity such as MMP-2, MMP-9 were thought to contribute to the development and progression of glomerulosclerosis and tubulointerstitial fibrosis in diabetic nephropathy.
2. Data from the present study firstly demonstrate that the renoprotection and associated mechanism of lumbrokinase and earthworm decoction. In the present study, we demonstrate that in the STZ-induced diabetic rats, supplementation with lumbrokinase and earthworm decoction for 12 weeks is renoprotective, by reducing urine albumin excretion (UAE) and collagenⅣdeposition as well as up-regulating expression of MMP-2 and MMP-9.
3. Lumbrokinase and earthworm decoction attenuated glomerulosclerosis and tubulointerstitial fibrosis associated with diabetic nephropathy, via reducing ECM deposition. These findings suggested that lumbrokinase and earthworm supplementation was beneficial in preventing the development ofdiabetic renal complications.
4. Earth worm injection could active the blood stasis, regulate the disturbance of lipids and reduce urine albumin excretion (UAE), so as to preventing the development of diabetic renal complications.
引文
[1]NORDT TK, SAWA H, FUJ II S, et al. Augmentation of arterial endothelial cell exp ression of the plasm inogen activator inhibitor type-1 (PAI-1) geneby proinsulin and insulin in vivo [J]. J Mol Cell Cardiol,1998,30:1535-1543
[2]BASTARD J P, PIERON IL, HA INQUE B. Relationship between plasma plasminegen activator inhibitor I and insulin resislance, diabetes [J]. Metab Res REV,2000,16:192-201
[3]ASO Y, MATSURMOTO S, FUJ IWARA Y, et al. Impaired fibrinolytic compensation for hypercoagulability in obese patients with type 2 diabetes: association with increased plasminogen activator inhibitor-1[J]. Metabolism,2002,51:471-476
[4]CARR M E. Diabetes mellitus:a hepercoagulable state [J]. Journal of Diabetes & Its Comp lations,2001,15 (1):44-54
[5]CEC ILE D, BERYL V C, FRANCOISE V K, et al. Clinical correlates of advanced retinopathy in 2 diabetic patients:implications for screening [J]. J Chin Ep idem id,1996,49:679-680
[6]CEC ILE D, BERYL V C, FRANCO ISE V K, et al. Clinical correlates of advanced retinopathy in 2 diabetic patients:implications for screening [J]. J Chin Epidem id,1996,49:679-680
[7]JOHANSSON L, JANSSON J H, BOMANT K, et al. Tissue Plasm inogen activator or plasm ingen activator inhibitor 1 and tPA/PAI-1 complex as risk factors for the development of a first stroke [J]. Stroke,2000,31:26-32
[8]WANG W, BOFFA M B, BJAZAR L, et al. A study of the mechanism of inhibition of fibrinolysis by activated thrombin activable fibrinlysis inhibitor [J]. B iol Chem,1998,273 (42):27176-27181
[9]HOR I Y, CABAZZA E C, YANO Y, et al. Insulin resistance is associated with increased circulating level of thrombin-activatabl fibrinolysis inhibitor in type 2 diabetic patients [J]. J Clin Endocrinol M etab.2002, 87 (2):660-665
[10]郭洪海.2型糖尿病肾病患者凝血及纤溶指标变化的临床意义[J].中国误诊学杂志,2007,(14)
[11]陈小敏, 杨香玖.2型糖尿病肾病的相关危险因素[J].福建医科大学学报, 2005, (02)
[12]Peter GT, Xiao RH, Micheal CB, et al. P-selectin directs Tlymphocyte-mediated injury in delayed-type hypersensitivity responses: studies in glomerulonephritis and cutaneous delayedtyped hypersensitivity [J]. Eur J I m m unol,1996,26 (2):4542460
[13]Omoto S, Nomura S, Shouzu Z, et al. Significance of platelet-derived microparticles and activated platelets in diabetic nephropathy [J]. Nephron, 1999,81 (3):2712277
[14]Thoenes W, Rumpelt JH (1977) The obsolescent renal glomerulus collapse, sclerosis, hyalinosis, fibrosis, Virchows Arch [A]377:1-15
[15]Striker LM-M, Killen PD, Chi E, Striker GE (1984) The composition of glomerulosclerosis. I. Studies in focal sclerosis, crescentic glomerulonephritis and membranoproliferative glomerulonephritis. Lab Invest 51:181-192
[16]Oomura A, Nakamura T, Arakawa M, Ooshima A, Isemura I (1989) Alterations in the extracellular matrix components in human glomerular disease. V~rchows Arch [A] 415:151-159
[17]Mignatti P. Extracellular matrix remodeling by metalloproteinases and plasminogen activators. Kidney Int Suppl,1995,49:S12214.
[18]Carmeliet P, Collen D. Development and disease in proteinase-deficient mice role of plasminogen matrix metalloproteinase and coagulation system. Thromb Res,1998,91:2552285.
[19]Kazes I, Delarue F, Hagege J, et al. Soluble latent membrane-type 1 matrix metalloprotease secreted by human mesangial cells is activated by urokinase. Kidney Int,1998,54:197621984.
[20]Cossins J, Dudgeon TJ, Catlin G, et al. Biochem Biophys Res Connun, 1996 Nov 12; 228(2):494～8
[21]Li L, Arers K, Eisen AZ, et al.Exp Cell Res,1997; 232:322～330
[22]Wilhelm SM, Collier IE, Marmer BL, et al. J Biol Chem,1989; 264:17213～ 221
[23]Parsons SL, Watson SA , et al. Br J Surg,1997; 84 (2):160～166
[24]Parsons SL, Watson SA , et al. Br J Surg,1997; 84 (2):160～166
[25]Mattei M G, Roeckl N , Olsen BR, et al. Genomics,1997 Feb 15; 40(1)
[26] Korzus E, Natase H, Rydell R,et al. J Biol Chem, 1997 10; 272(2):1188～96
[27]Vincenti MP, White LA, Schroen Dj, et al. Crit Rev Eukaryot Gene Expr,1996; 6(4):391-411
[28]Maisi P, Prikk K, Sepper R, et al. Soluble merebran2type lmatrix metallop roteinase (MT1-MMP) and gelatinase A(MMP-2) in induced sputum and bronchoalveolar lavage fluid of human bronchial asthma and ronchiectasis [J]. A PMIS,2002,110(11):771
[29]Yigzaw Y, Gielens C, Preaux G. Isolation and characterization of an alpha macroglobulin from the gastropod mollusc Helix pomatia with tetrameric structure and preserved activity after methylamine treatment. Biochim Biophys Acta,2001,1545:1042113.
[30]Beekman B, Drijfhout JW, Ronday HK, et al. Fluorogenic MMP activity assay for plasma including MMPs complexed to alpha 2-macroglobulin. Ann N YAcad Sci,1999,878:1502158.
[31]Keski-Oja J, Lohi J, Tuuttila A, Tryggvason K, VartioT (1992) Proteolytic processing of the 72,000-Da type IV collagenase by urokinase plasminogen activator. Exp Cell Res 202:471-476
[32]Baricos WH, Cortez SL, El-Dahr SS, Schnaper HW (1995) ECM degradation by cultured human mesangial cells is mediated by a plasminogen activator/plasmin/matrix metalloproteinase 2 cas-cade. Kidney Int
[33]Desrochers PE, Jeffrey JJ, Weiss SJ (1991) Interstitial collagenase (matrix metalloproteinase-1) expresses serpinase activity. J Clin Invest 87:2258-2265
[34]Marcotte PA, Kozan IM, Dorwin SA, Ryan JM (1992) The matrix metalloproteinase PUMP-1 catalyzes formation of low molecular weight (pro)urokinase in cultures of normal human kidney cells. J Biol Chem 267:13 803-13 806
[35]任淼,李聪爽.地龙退热121服液的制备及药理研究[J].医学理论与实践,1996,9(7)：334.
[36]黄志力,桂常青,刘锡玖,等.生石膏及地龙对小青龙汤解热的增强作用[J].中药药理与临床,1997,13(5)：12-15.
[37]陈斌艳,张雷,王竞,等.地龙粉针对大鼠小鼠与兔的解热镇痛作用[J].上海医科大学学报,1996,13(3)：115.
[38]Mihara H, et al. GB,21 165 65A.
[39]陈遗发,刘事顺.蚓激酶治疗老年性脑血栓形成55例临床分析[J].新医学, 1998,29(4)：196.
[40]梁健,韦新.地龙注射液治疗不稳定心绞痛疗效观察[J].广西医学,2001,23(6)：1374.
[41]潘建生,周阿旺,方云,等.复方地龙胶囊治疗性脑梗塞40例临床分析[J].中华新医学,2002,3(6)：530.
[42]张凤春,陈云峰,苏颜珍,等.地龙对巨噬细胞免疫活性的增强作用[J].中国药学杂志,1998,33(9)：532-535
[43]吴朝晖,张国平,张明,等.蚯蚓制剂(普恩复)对小鼠游泳时间和丙二醛影响的初步研究[J].中国微循环,1998,2(2)：82—84.
[44]鲍世铨,曾耀辉.蚯蚓在医药保健方面的综合利用研究[J].中国生化药物杂志,1994,15(3)：165.
[45]张风春,陈云峰,苏颜珍,等.地龙促进大白兔背部创伤伤12l愈合收缩的实验研究[J].中国中药杂志,1998,23(9)：560—561.
[46]张风春,陈云峰,苏颜珍,等.地龙对新西兰大白鼠背部创伤愈合作用的机制研究[J].中国药学杂志,1999,34(1)：93.
[47]张复夏,王西发,袁克平,等.蚯蚓体内杀精物质的实验研究[J]. 陕西中医,1996,17(5)：234.
[48]郭宝珠,张复夏,王惠云,等.蚯蚓提取物体外杀精及抗阴道毛滴虫作用的实验研究[J].中医药研究,1997,13(4)：39—41.
[49]张复夏,邵道通.地龙粉治疗男性不育症30例[J].陕西中医1996,17(10)：438.
[50]李淑兰,谢桂芹,曲竞南,等.地龙降压作用的研究[J].中医药信息,1995,3：11.
[51]程能能,马越呜.地龙中降太的类血小板活化因子物质[J].中国中药杂志,1993,18(12)：747.
[52]韩苇.地龙胶囊(912)透析组分对M Gc803胃癌细胞DNA合成的抑制作用[J].中国肿瘤临床,1991(3)：152—153.
[53]徐德门.用动态观察方法判断地龙胶囊(912)与放疗综合治疗食管癌的近期效果[J].中国肿瘤临床,1991(3)：149.
[54]曾子澜,张祖,碧玉,等.蚯蚓提取物对多种瘤细胞的作用[J].山西医学院学报,1995,26(2)：81.
[55]林建海,刘宝路.平喘中药对致敏性哮喘豚鼠气道的作用[J].上海医学,1996,19(11)：638.
[56]张理平,李如辉.地龙药理研究与临床应用的概况[J].福建中医药.1990,21(6)：52.
[57]Mihara H, Sumi H, Akazawa T, et al. Fibrinolytic enzyme extracted from the earthworm. Thromb Haemostas,1983,50: 258-263
[58]Mihara. H, et al, Thrombotic proteases from earthworm. Eur PatAppl EP 105,092 C1C12 N9647,11, Apr 1984, JP Aprl,82 (?) 173 669, 02 Oct.1982
[59]杨嘉树等.蚯蚓体内一种纤溶酶原激活剂(e2PA)对BAEE的降解.中国生物化学和分子生物学报,1998,Vol.14,No.4,412-416
[60]杨嘉树等.蚯蚓体内一种纤溶酶原激活剂(e2PA)对ATEE的降解.中国生物化学和分子生物学报,1998,Vol.14,No.4,417-421
[61]Nakajima N, Sugimoto M, Ishihara K, et al. Fut her characterization of eart hworm serine poteases:cleavage specificity against peptide subst rates and on autolysis[J]. B iosci B iotech B iochem,1999 63 (11):2031.
[62]Tang Y, et al. Crystral structure of earthworm fibrinolytic enzyme component a:revealing the structural determinants of its dual-fibrinolytic activity. J Mol Biol,2002, Aug 2; 321 (1) 57～68
[63]姜东胜,张国桢,王宝珍,等.赤子爱胜蚓纤溶酶的药理性质探讨.上海医科大学学报,1993,20(1)：16219.
[64]张国平,钱睿哲,杨诗春等.蚓激酶抑制血小板聚集的实验观察[J].中华医学杂志,1998,78(5)：394
[65]段平,胡淑丽,曾耀辉.蚯蚓溶栓酶对大鼠凝血及纤溶指标的影响[J].时珍国药研究,1997,8(1)：14.
[66]何红,车庆明,孙启时.地龙提取物的抗凝血作用[J].中草药,2007,(05))
[67]朱燕,何执中,孙可迪,王少飞.蚓激酶对实验性血栓的影响[J].中国药科大学学报,2000,(01)
[68]张丽萍,牟善初,余霞君.蚓激酶对实验性血栓的预防作用[J].中国循环杂志,1995,(11)
[69]徐宗佩,王益民,张吉正,宫涛,李百臣张伯礼.地龙提取物对健康小鼠红细胞变形性的影响[J].天津中医,2000,(03)
[70]吕中明,石根勇,胡启之,李贵山,薄丽津,任倩.蚯蚓提取物对微循环障碍形成的保护作用[J].中国生化药物杂志,2003,(02)
[71]Ji H Mechanisms of lumbrokinase in protection of cerebral ischemia. Eur J Pharmacol.2008V590N1-3:281-289
[72]吴勇飞.蚓激酶对实验动物兔脑循环的影响[A].浙江省中西医结合学会神经 内科专业委员会第五次学术研讨会中西医结合神经内科学习班资料汇编[C],2006
[73]孙宏丽, 焦军东, 潘振伟, 董德利, 杨宝峰.蚓激酶的心肌保护作用及机制[J].药学学报,2006,(03)
[74]王华光, 龙江, 王玲, 杨宝峰.蚓激酶对脑缺血的保护作用[A].中国病理生理学会动脉粥样硬化专业委员会五届一次会议论文集,2002,(总第)临床研究
[75]Mihara. H, et al. Noval thrombolytic therapy discovered from traditional oriental medicine using the earthworm. Southeast Asia J Trop Med Public Health,1992,23 Suppl.2:131～140
[76]Zhang ZX, et al. Effects of crude extract of earthworm on promoting blood circulation to removing stasis. ZhongGuo Zhong Xi Yi Jiehe Zazhi,1992, Dec.; 12 (12):741-743
[77]段平等.时珍国药研究,1997,Vol.8,No.1,14-16
[78]欧柏青,杨玉莲,陈智敏,等.蚓激酶对冠脉介入术(PCI)后血纤维蛋白原浓度(Fg)变化的影响[J].中国医学理论与实践,2004,14(7)：903-904.
[79]丁铭臣,庞式琪,谢淑萍,王拥军.蚓激酶胶囊治疗脑梗塞1500例临床总结.中华精神神经科杂志1995；28：5-7.
[80]张昆南,王祖福,高幼奇,赖曰文瑾,谢旭芳,屈新辉.博洛克治疗急性缺血性脑血管病血浆中t-PA,PAI,PL,FDP的变化.中华精神神经科杂志1995；28：8-9.)
[81]金莉蓉,徐桂芝,张国平等.蚓激酶(普恩复)治疗脑梗塞时抗凝和纤溶变化的临床研究[J].中国微循环,1998,2(3)：157
[82]马培志,邵凤民.蚯蚓提取物对原发性肾病综合征患者凝血系统的影响[J].中国中西医结合肾病杂志,2002,(09)
[83]邵凤民,王小阳,张小玲,邢清和,朱长连,程银桢,张明轩,蚯蚓提取物治疗肾实质性高血压的研究[J].中国中西医结合肾病杂志,2001,(02)
[84]梁舒,田明华1视网膜静脉阻塞血液流变学检测及蚓激酶治疗[J]1南通医学院学报,1999,19(2)：188
[85]杨丽娜.蚓激酶对糖尿病并发症患者血脂及血液流变学指标的影响[J].山东医药,2007,(17)
[86]程震锋,王文华,何建方.蚓激酶对糖尿病患者血浆血小板颗粒膜蛋白-140和血栓烷B_2的影响[J].新医学,1998,(05)
[1]Bai Y, Wang L, Li Y, et al.High ambient glucose levels modulates the production of MMP-9 and alpha5(IV) collagen by cultured podocytes[J].Cell Physiol Biochem,2006, 17(1-2):57-68.
[2]陆亚琴,刘顺英,陈洪,等.地龙2号抑制大鼠肝纤维化的研究[J].胃肠病学及肝病学杂志,2004,13(3)：225-227.
[3]陈洪,陆亚琴,刘顺英,等.地龙2号对大鼠肝纤维化TGF2 β1, MMP-13及TIMP-1 mRNA和蛋白表达的影响[J].世界华人消化杂志,2004,12(10)：2333-2337.
[4]Nakagawa T, Kosugi T, Haneda M.Abnormal angiogenesis in diabetic nephropathy[J].Diabetes,2009,58(7):1471-1478.
[5]McLennan SV, Martell SY, Yue DK.High glucose concentration inhibits the expression of membrane type metalloproteinase by mesangial cells:possible role in mesangium accumulation[J]. Diabetologia,2000,43 (5):642-648.
[6]Breyer MD, Bottinger E, Brosius FC 3rd, et al.Mouse models of diabetic nephropathy[J]. J Am Soc Nephrol,2005,16(1):27-45.
[7]Sun HL, Sun L, Li YY, et al.ACE-inhibitor suppresses the apoptosis induced by endoplasmic reticulum stress in renal tubular in experimental diabetic rats[J]. Exp Clin Endocrinol Diabetes,2009,117 (7):336-344.
[8]Magnatri P, Fisher SJ, Globus RK.Extracellular matrix remodeling by metalloproteinases and plasminogen activator [J].Kidney Int,1995,47 (Suppl):12-14.
[9]Baricos WH, Cortez SL, el-Dahr SS, et al.ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade [J].Kidney Int,1995,47 (4):1039-1047.
[10]Rondeau E, Sraer JD, Schleuning WD.The renal plasminogenactivating system, in Molecular Nephrology: Kidney Function in Health and Diseases[M]. New York:Marcel Dekker Inc,1995:699-715.