| Objective: In this experiment, we first established diabetes mellitus (DM) animal model, then development for diabetic nephropathy (DN), and carried on the intervention treatment using the safflower yellow injection to the model. Through observed experimental animal's 24 hour urinary protein quantitative, glycohemoglobin, blood lipid, renal function, pathomorphology change of kidney and the expression of AngiotensinⅡType 1 Receptor(AT1R) in renal tissue, we researched safflower yellow's therapeutic action to DN and the possible mechanism, and provided the experiment foundation and the theory basis for its clinical application.Methods:There were 46 male Sprague-Dauley rats with the weight of 200±10g selected to this experiment. The rats were fed in ordinary ways for one week. After 7 days, their urine glucose and urine protein were detected, the result was negative. According to the weight, the animals were randomly divided into four groups: 10 in normal group, 12 in the model group, 12 in benazepril group and 12 in safflower yellow group. Besides the normal group, the rats of other groups were by streptozotocin(STZ) once intraperitoneal injection, 60 mg/kg, the normal group were given the corresponding quantity's physiological saline, after 72 hours, measured their blood glucose, took"≥16.7mmol/L"as the DM model standard. According to the blood glucose level, the rats of three DM model were randomly divided into three groups again, the category and the animal number were invariable. After one week of DM models made successfully, the drugs started be given to the rats according to adult dosage 20 times, daily one time. The rats of safflower yellow group were given the safflower yellow by intraperitoneal injection, 26.7mg/kg, the benazepril group were administrated with benazepril by gavage, 10mg/kg, the normal group and the model group were given corresponding quantity's physiological saline by intraperitoneal injection. Each group was given the medicine for 23 weeks. After 24 week of DM models made successfully, all rats were put into metabolism cage to obtain 24 hour urine to detect 24 hour urinary protein quantitative. All animals were killed. Glycohemoglobin(HbA1C), total cholesterol(TC), triglyceride (TG), serum creatinine (Scr), urea nitrogen (BUN) were assayed with the automaticbiochemistry analyzer. Weighed the weight of kidney and calculated hypertrophy index. Pathomorphology change of kidney were observed with light microscope and electron microscope. The expression of AngiotensinⅡType 1 Receptor in renal tissue was observed with the immunohistochemical staining and was analyzed with the pathology image analysis system. Results:1 The general condition of the ratsThe rats of normal group were eusitia, whose eye is bright, had an agility reaction, gloss color pattern, thick subcutaneous fat, and its dejecta was granular and the amount of urine was normal. After STZ injection, the rats of other three groups all presented obviously polydipsia, polyphagia, polyuria, and presented slowly the energetic dispirited, the slow reaction, little moves hugs the group. In last week of the experiment, compares with the normal group, the rats of DN model were obviously thin and small, the muscle are few, the above symptom were most remarkable by the model group. There were rat death in treatment period among three DN model groups, the model group was three, the benazepril group was one and the safflower yellow group was two. The estimated cause of death was the internal organs failure caused by the metabolic disorder for the hyperglycemia.2 The body weight, right kidney weight and hypertrophy index (kidney weight/body weight) of each groupCompared with normal group, the body weight of the rats in model group, benazepril group and safflower yellow group was obviously lighter (P<0.01), there was no significant difference among three DN model groups (P>0.05). The right kidney weight of the rats in all four groups had no significant difference (P>0.05). The hypertrophy index of the rats in model group, benazepril group and safflower yellow group was obviously higher than normal group (P<0.01), but there was no significant difference among three DN model groups (P>0.05).3 The 24 hour urinary protein quantitative of each groupThe 24 hour urinary protein quantitative of the rats in model group, benazepril group and safflower yellow group was significantly higher than normal group (P<0.01), compared with model group, the 24 hour urinary protein quantitative of benazepril group and safflower yellow group was obviously lower (P<0.01). There was no statistical difference between benazepril group and safflower yellow group (P>0.05).4 The HbA1C of each groupCompared with normal group, the HbA1C of the rats in model group, benazepril group and safflower yellow group was significantly higher (P<0.01), but there was no statistical difference among model group, benazepril group and safflower yellow group (P>0.05).5 The TC and TG of each groupThe TC of the rats in model group, benazepril group and safflower yellow group was significantly higher than normal group (P<0.01), compared with model group, the TC of safflower yellow group was obviously lower (P<0.01), but there was no significant difference between benazepril group and model group (P>0.05). The TG of the rats in normal group, benazepril group and safflower yellow group was significantly lower than model group (P<0.01), there was no statistical difference among normal group, benazepril group and safflower yellow group (P>0.05).6 The Scr and BUN of each groupCompared with model group, the Scr of the rats in normal group, benazepril group and safflower yellow group was significantly lower (P<0.01), and benazepril group was obviously higher than normal group (P<0.05), there was no significant difference between benazepril group and safflower yellow group (P>0.05). The BUN of the rats in normal group, benazepril group and safflower yellow group was significantly lower than model group (P<0.01), compared with normal group, the BUN of benazepril group and safflower yellow group was significantly higher (P<0.01), and there was no statistical difference between benazepril group and safflower yellow group (P>0.05).7 The pathomorphology change of kidney7.1 Light microscope observationThe normal group showed that: the structure of renal glomerulus was complete, glomerular capillary basement membrane, mesangial and matrix did not show abnormal change. The model group showed that: glomerular hypertrophy, thickening of glomerular capillary basement membrane, mesangial cell proliferation, mesangial matrix increased, mesangial area broadening. The benazepril group and safflower yellow group also showed different degree of pathological changes, but the pathological degree was obviously lighter than the model group. 7.2 Observations under transmission electron microscopyThe normal group showed that: the structure of glomerular capillary basement membrane was clear and complete, microcirculatary endothelial cell, foot processes did not show abnormal change. The model group: the most of glomerular capillary basement membrane showed significant homogeneous thicking, partial basement membrane showed elevation in the shape of hills; microcirculatary endothelial cell significantly confluence and the most of window structure disappeared; the fusion of the foot processes was extensive. The benazepril group and safflower yellow group also showed the similar pathological changes, but the pathological degree was lighter than the model group.8 Immunohistochemical result of the expression of AT1R in renal tissueAT1R was mainly expressed in proximal renal tubular and weakly expressed in distal renal tubular, also expressed around of renal vascular, but nearly not expressed in glomerulus. AT1R could express in all four groups. The result of semi-quantitative analysis showed that: compared with the normal group, the model group AT1R had stronger expression (P<0.01), the benazepril group and safflower yellow group also was stronger than the normal group (P<0.01);compared with the model group, the expression of the two treatment groups was lower (P<0.01), and the expression was not statistical difference between the two treatment groups (P>0.05). Conclusions:1 The safflower yellow injection can reduce DN rat's proteinuria; can cut down TC, TG and improve the lipid metabolism; can decrease Scr and BUN to protect renal function.2 The safflower yellow injection can effectively inhibit DN rat's thickening of glomerular capillary basement membrane, mesangial matrix increased, fusion of foot processes of podocytes to delay the pathologic development of kindey.3 The safflower yellow injection can diminution the expression of AT1R in DN rat's renal tissue, which suggests that the treatment effect of safflower yellow on DN is possibly related with the approach for blocking RAS.
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