| Objective: Diabetic nephropathy(DN) is one of the microvascular complications of Diabetes mellitus(DM), that is, DN shows diabetic glomerulosclerosis in the late period, and it is the main cause of disability and death of DM. The mechanism of DN is complicated, and there are no special effective treatments for this disease at present. It is of great significance to find an effective therapy. The study was established by intraperitoneal injection of STZ to set up diabetic rat models, and then be applied with supplementing qi and nourishing yin resolving masses and dredging meridians (SNRD). By means of observing the differences in 24 hour urinary protein quantitative, renal function(Scr and BUN), glycosylated hemoglobin, renal pathology, and TNF-αlevels of different groups to make sure of the treatment of SNRD, to probe the possible mechanism, to provide an experimental and theoretical rationale for its application in clinic.Methods: We selected 85 healthy male Sprague-Dauley rats weighted 230g±20g for our study. After being fed for one week normally, the rats were carried out left-nephrectomy with their Urine sugar and urine protein negative. 2 weeks later, except the random 10 rats for normal group, the remains got intraperitoneal injection of STZ 40mg/kg and the rats in the normal group of the same volume of 0.1mol/L citric acid buffer solution as STZ into abdomen. After 72 hours, we measured the tail vein blood randomly for 3 times. If the blood glucose was≥16.7 mmol/L, the models were successful. Model rats were divided into 5 equal groups randomly(15 in each group): model group, supplementing qi and nourishing yin resolving masses and dredging meridians low dose group(SNRDL), middle dose group(SNRDM), high dose group(SNRDH), Irbesartan group. 1 week later, each group was given medicine once every day. SNRD low dose group, middle dose group, high dose group were intragastric gavaged herb 5.29g/kg/d, 10.58g/kg/d, 21.16g/kg/d separately, Irbesartan group was intragastric gavaged irbesartan 15mg/kg, normal and model groups were intragastric gavaged same quantitive saline for 12 weeks. The Rats were fed with normal fodder and drinking freely with room temperature 18-20℃and relative humidity 40%-70% and 12-hour alternating light and dark. During the experiment, tail vein blood glucose was tested sometimes. If blood glucose was higher than 26mmol/l or just the same to 26 mmol/L, we injected proper amount of long-acting insulin(0.5-4u) subcutaneously in order to avoid ketosis and reduce deaths. At the treatment of the twelve week, all rats drunk freely but were fed with no food. We collected 24 hour urine to measure 24 hour urinary protein quantative. And then the rats were weighted and anesthetized using 3% pentobarbital sodium to get the blood sample from arteria femoralis, to be taken out the right kidney for weighting and calculating hypertrophy indexes, to get the kidney tissue samples. Pathologic changes of the kidney were observed by light microscope and electron microscope. RNA was extracted from renal cortex of each group to detect the level of TNF-αmRNA using reverse transcription polymerase chain reaction (RT-PCR).Results:1 The body weight, kidney weight and hypertrophy index(kidney weight/body weight)in each groupThe weights of the model group and every treatment group were lighter than the normal group(P<0.05). The weights of treatment groups and the model group were of no differences(P>0.05). The weights of treatment groups had no differences(P>0.05). There were no obvious differences among the weights of right kidneys in each group(P>0.05). The hypertrophy indices of model group and treatment group were higher than the normal group(P<0.05). There were no significant differences between the model group and treatment groups, as well among treatment groups(P>0.05). 2 The 24 hour urinary protein quantity in each groupCompared with the normal group, this index of model group and treatment groups increased significantly(P<0.05). Compared with model group, the indices of SNRDL, SNRDM, SNRDH groups and Irbesartan group decreased significantly(P<0.05). There were no differences among each treatment groups(P>0.05).3 The HbA1C in each groupThe indices of the model group, Irbesartan group and SNRDL group, SNRDM group, SNRDH group were much higher than that of the normal group (P<0.05). There were no differences among Model group and treatment groups (P>0.05). There were no differences among each treatment groups (P>0.05).4 The renal function in each groupThe levels of Scr and BUN in the model group and each treatment group was significantly higher than that in the normal group (P<0.05). The indices of treatment groups were obviously lower than that of the model group (P<0.05). There were no differences among treatment groups (P>0.05).5 The pathomorphology changes of kidney5.1 Light microscrope observationWe can see that in the normal group: the structure of glomerulus was completed. There were no obvious changes in renal glomerulus, glomerular capillary basement membrane, mesangium and matrix. In the model group, we can see that: the glomerular hypertrophy, glomerular capillary basement membrane widened, and mesangial matrix accumulated. There were different extent pathomorphology changes among treatment groups, but the changes were less and slighter than the model group, i.e. glomerular hypertrophy slightly, less quantity mesangial cells and matrix accumulated slightly. There was no significant difference among each treatment group.5.2 transmission electron microscopy observationIn the normal group, the glomerular capillary basement memberance structural was integrity, capillary endothelial cells and podocyte were seen normal. In the model group, we can see that glomerular capillary basement memberance thickening, mesangial matrix increasing, accompanied with extensive podocyte fused. Each treatment group showed different extent pathomorphology changes, but the degree was slighter than the model group, i.e. glomerular capillary basement memberance thicken slightly, mesangial matrix accumulated slightly, accompanied with partial podocyte fusion. There was no significant difference among each treatment group.6 Levels of TNF-αmRNA in renal cortexThe indices of the model group and each treatment group increased significantly compared with the normal group(P<0.05). That of each treatment group decreased significantly compared with the model group(P<0.05). There were no significant differences among each treatment groups(P>0.05).Conclusions:1 SNRD recipe can delay the pathomorphology damages of the kidney, decrease 24 hour urinary protein quantity, decrease the levels of Scr and BUN, and protect renal function of DN rats.2 SNRD recipe can protect DN rats by means of reducing the level of TNF-αmRNA in renal tissues. It may be one of the mechanisms of its prevention and treatment in DN. |
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