Protection And Mechanism Research Of Earthworm And Lumbrokinase On Diabetic Kidney Disease

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 changesPAS, 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 studiesDiabetes 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 BlotCollagen 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.