| Objective: Diabetic nephropathy(DN) is kind of renal damage, which belongs to metabolic diseases. It is a serious chronic complication of diabetes. In this study, the DM rat model was established by high-fat diet combined with low-dose streptozotocin intraperitoneal injection. Application of Huayu Recipe or Tongluo Recipe or irbesartan intervented the model. By observing the general conditions of rats, the pathological changes of kidney, the blood regular test, the blood coagulation, the biochemical index of the rats, the quantitative measurement of 24 hours urinary protein, and the expression level of fibronectin(FN) and laminin(LN), to study the intervention of Huayu or Tongluo traditional Chinese medicine, and to explore its possible mechanism.Methods: Fifty-five healthy male Sprague-Dauley rats, weight of 100±20g, fed adaptability for one week. Determination of urinary protein, urine and urine red blood cells were all negative. Randomly selected 10 rats as normal control group(C), fed by normal diet, the remaining rats were fed by high diet. After four weeks, the rats which fed by high diet were fasted for 12 hours or more, 35mg/kg STZ was given by intraperitoneal injection, measured rats’ tail vein random blood glucose ≥16.7mmol / L three times, after 72 hours for successful DM model. Successful model rats were randomly divided into four groups, model group(M), Huayu recipe treatment group(H), Tuolong recipe treatment group(T), Irbesartan group(I). Each treatment group give corresponding drug lavage, once daily for 16 weeks. At the end of the 16 th week, collected rats′ 24-hour urine to test the 24 h urine protein detection. Rats were fasted 12 hours, measured rats′ body weiht, after anesthesiaed rats, blood was collected from abdominal aortic to test the blood regular test, the blood coagulation and the blood biochemical index of the rats. Remained renal tissue specimens for light microscopy, electron microscopy, immunohistochemistry and RT-PCR.Results: 1 The comparison of the general state of the rats in each groupNormal control group of rats generally in good condition, rats of model group and treatment group appeared typically symptoms of eating more, drinking more, urinating more and becoming thinner. The most significant in the model group, and the rats′ symptoms of treatment group eased during treatment. One rat of normal control group escaped during the experiment, model group three died, Huayu recipe treatment group one died, irbesartan group one died. 2 The comparison of rats′ body weight and the kidney hypertrophy index(right kidney weight /body weight)Compared with the normal control group, the body weight of model group was significantly lighter(P<0.05), hypertrophy index of the model group was significantly higher(P<0.05); compared among model group and treatment groups, difference of the body weight and the kidney hypertrophy index was not statistically significant(P>0.05). 3 The comparison of rats′24 hours urinary protein quantity(24h UTP)Compared with the normal control group, 24 h UTP of model group was singificantly increased(P<0.05); compared with the model group, 24 h UTP of treatment groups were reduced(P<0.05); compared among treatment groups, it was not statistically significant(P>0.05). 4 The comparison of blood test results 4.1 The comparison of PLT in blood resultsCompared with the normal control group, the platelet count of model group increased significantly(P<0.05); Compared with the model group, the platelet count of treatment groups were significantly reduced(P<0.05); No significant difference among the treatment groups(P>0.05). 4.2 The comparison of TG in blood biochemical testCompared with the normal control group, TG levels of model group was significantly increased(P<0.05); compared with the model group, TG levels of treatment groups were significantly reduced(P<0.05); No significant difference among the treatment groups(P>0.05). 4.3 The comparison of LDL in blood biochemical testCompared with the normal control group, LDL levels of model group was significantly increased(P<0.05); No significant difference among the model group and the treatment groups(P>0.05). 4.4 The comparison of TP in blood biochemical testCompared with the normal control group, TP level of model group, the Huayu recipe treatment group, and the Tongluo recipe treatment group was reduced(P<0.05); Compared with the model group, TP level of the Huayu recipe treatment group, the Tongluo recipe treatment group and the irbesartan treatment group was increased(P<0.05); TP level between the irbesartan group and the Tongluo recipe treatment group was not statistically significant(P>0.05); TP level between the irbesartan group and the normal control group was not statistically significant(P>0.05). 4.5 The comparison of Scr in blood biochemical testCompared with the normal control group, Scr level of model group was significantly increased(P<0.05); Compared with the model group, Scr level of treatment groups were reduced, but no significant difference among the treatment groups(P>0.05). 4.6 The comparison of PT in hemagglutination testCompared with the normal control group, PT of model group was reduced(P<0.05); compared with the model group, PT of treatment groups were increased(P<0.05); No significant difference among the treatment groups(P>0.05). 5 Morphology of renal pathology 5.1 Light microscopyThe rats of normal control group had normal and clear kidney tissue structure; The rats of model group showed kidney structure disorder, renal capule were crack-like changes, the basement membrane thickening, mesangial matrix accumulation, even balloon adhesions; compared with the model group, each treatment groups′ pathological changes were significantly reduced; compared among groups, the Tongluo recipe treatment group ′s pathological change is slightly lighter than any other treatment groups. 5.2 Electron microscopyThe rats of normal control group had normal and clear kidney tissue structure; The rats of model group′s glomerular basement membrane thickening, mesangial matrix increased, podocyte foot processes were fusion; compared with the model group, each treatment group has a similar pathology, but to a lesser extent; Compared among treatment groups, the Tongluo recipe treatment group′s pathology performance is slightly lighter than any other treatment groups. 6 The expression of FN and LN in kidney by Immunohistochemical method 6.1 FN results show: In normal control group, FN was little expressed in glomerular mesangial and basement membrane. Compared with the control group, the expression of FN was significantly increased in model group(P<0.05); compared with the model group, the expression of FN was significantly decreased in each treatment group(P<0.05); Compared among treatment groups, FN was lower expression in Tongluo recipe treatment group and Irbesartan treatment group, which is significant difference in Huayu recipe treatment(P<0.05). 6.2 LN results show: In normal control group, LN was little expressed in tubular and interstitial. Compared with the control group, the expression of LN was significantly increased in model group(P<0.05); compared with the model group, the expression of LN was significantly decreased in each treatment group(P<0.05); Compared among treatment groups, LN was lower expression in Tongluo recipe treatment group and Irbesartan treatment group, which is significant difference in Huayu recipe treatment(P<0.05). 7 RT-PCR detection of renal tissue FN m RNA、LN m RNA expression 7.1 FN results show: Compared with the normal control group, model group FN m RNA expression was significantly increased(P<0.05); Compared with the model group, FN m RNA expression in treatment groups were significantly reduced(P<0.05); Compared among treatment groups, FN m RNA was lower expression in Tongluo recipe treatment group and Irbesartan treatment group, which is significant difference in Huayu recipe treatment(P<0.05). 7.2 LN results show: Compared with the normal control group, model group LN m RNA expression was significantly increased(P<0.05); Compared with the model group, LN m RNA expression in treatment groups were significantly reduced(P<0.05); Compared among treatment groups, LN m RNA was lower expression in Tongluo recipe treatment group and Irbesartan treatment group, which is significant difference in Huayu recipe treatment(P<0.05).Conclusions:1 Huayu medicine and Tongluo medicine can improve the general status of diabetic nephropathy rats, decrease the urine protein, maintain the plasma protein content, show a protective effect in the DN rats kidney.2 Huayu medicine and Tongluo medicine can decrease the number of platelet, reduce its activity, preventing diabetic nephropathy reduce to the complication of thrombosis and embolism.3 Huayu medicine and Tongluo medicine can regulate lipid metabolism in diabetic by lowering plasma triglyceride levels.4 Huayu medicine and Tongluo medicine can reduce FN and LN expression in the kidney of diabetic nephropathy rats, relieve pathology of diabetic nephropathy, suggesting its therapeutic effect on DN may be related to delay renal fibrosis.5 According to morphology of renal pathology and the expression of FN and LN in kidney, Tongluo medicine is superior to Huayu medicine in preventing kidney. |
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