| Objective: This experimental study establishes diabetic nephropathy(DN) rats model by high sugar, high fat feed and small dose of STZ, Xiezhuojiedu Huoxuetongluo traditional Chinese medicine and irbesartan separated and combined applied to the intervention therapy, through observing the biochemical indexes of these model rats and renal Podocin m RNA expression to investigate the relationship between Podocin and DN, prove the intervention effect and possible mechanism of Xiezhuojiedu Huoxuetongluo traditional Chinese medicine on DN rats, so as to provide theoretical basis for clinical practice.Methods: Choose 85 Sprague-Dauley rats, all are clean healthy male, ordinary adaptive feed for a week and observe no matter urine protein or urine sugar negative, then selects 15 rats randomly which given ordinary feed for normal group(NG), the rest of the 70 rats given high sugar, high fat feed and the intraperitoneal injection of STZ 35mg·kg-1 by 12 weeks later, but corresponding volume of intraperitoneal injection of citrate buffer for the normal group rats. Measure fasting glucose caudal vein blood after 72 hours, which is greater than or equal to 16.7mmol·L-1 can be seen as diabetes building success. In the process of building model, two rats were died and one was rejected because of its blood sugar is not standard.Building these DN rats randomly assigned by weight: Model group(MG) with 17 rats, traditional Chinese medicine group(ZYG) and the western medicine group(XYG) also with 17, western medicine and Chinese medicine group(JYG) with 16, given drug intervention: the treatment group is given 4 ml/d corresponding drug lavage solution, but the normal group and model group are given corresponding physiological saline lavage, once per day, 16 weeks. During the treatment days of 4 weeks, 8 weeks and 12 weeks, take the rats, caudal vein of blood sugar and urinary protein in 24 hours. At the end of 16 th week treatment, each group is fasted, but not banned water. Collect rats urine in the metabolism of the cage for 24 hours to get the 24 h urinary total protein, weigh the rats and anaesthetize them by using 10% chloral hydrate abdominal aorta in blood, detect serum total protein(TP), albumin(ALB), triglycerides(TG), total cholesterol(TC), serum creatinine(Scr), urea nitrogen(BUN), and other biochemical indicators of rats, pick the right kidney and weigh it, calculate the hypertrophy index, observe the pathological changes of renal tissue in rats by light microscope and electron microscope, adopt immunohistochemistry technique and Real- time PCR method to detect the expression of kidney podocytes podocin protein in each group.Results: 1 The comparison of experimental rats in generalRats in normal group are good mental state, responsive, plump muscle, luster hair, normal diet and urine volume. Building model rats have typical symptoms of “Sanduo Yishao”: drink more, eat more, urine more but thin body. Over time some rats appear complications such as depression, hemiplegia, cataract, fester tail and so on. The most significant symptoms in model group. In the process of drug intervention treatment, the symptoms of the treatment group rats are more at ease than that of the model group. 2 Comparation of body weight, right kidney weight, and hypertrophy index(right kidney weight/body weight) in each groupCompared with NG, the body weight of building model group is reduced(P<0.05); No difference between MG and each treatment group(P>0.05). Right kidney weight is no obvious difference in each group(P>0.05). Compared with the NG, the hypertrophy index in MG and treatment group is higher(P<0.05); No difference in the MG and treatment group(P > 0.05). 3 Comparation of Hb A1 c in each groupCompared with NG, the Hb A1 c in treatment group and MG is increased(P<0.05); Compared with JYG and ZYG, the Hb A1 c in XYG and MG is higher(P<0.05); JYG and ZYG is no significant difference(P>0.05); XYG and MG has no significant difference(P>0.05). 4 Comparation of 24-hour urinary total protein(24h UTP)in each groupThe 24 h UTP in treatment group and MG is higher than NG(P<0.05); Compared with MG, the 24 h UTP in treatment group is decreased(P<0.05); Compared with ZYG and XYG, the 24 h UTP in JYG is decreased(P<0.05); There is no distinct difference between ZYG and XYG(P>0.05). 5 Comparation of serum TP, ALB, TC, TG in each groupCompared with NG, TP, ALB of MG and each treatment group is lower(P<0.05), while the TG, TC significantly increased(P<0.05); Compared with MG, TP,ALB of each treatment group is increased significantly(P<0.05), but TG, TC significantly decreased(P<0.05); Compared with JYG, lower TP and ALB for ZYG and XYG(P<0.05), while higher TG, TC(P<0.05); The comparison of TP, ALB, TG, TC in ZYG and XYG has no significant difference(P>0.05). 6 Comparation of Scr, BUN in each groupScr, BUN in each group has no significant difference(P> 0.05). 7 Kidney tissue pathological morphology observations 7.1 Light microscopy observation showsRats in NG have normal kidney tissue structure, normal glomerular size and the structure is clear and complete, no glomerular hypertrophy was observed, glomerular capillary basement membrane is no thicken and mesangial cell and mesangial matrix is no obvious changes. Model group rats’ glomerular volume increase visiblely, glomerular capillary basement membrane thickening obviously, glomerular cell proliferated and mesangial area widened significantly; Compared with NG, different degree of glomerular pathological changes can be seen in each treatment group, but less than MG. 7.2 Electron microscope observation showsNG rats have normal kidney tissue structure, uniform glomerularbasement membrane and no thickening, mesangial area did not see thepathological change, foot processes neatly and without fusion; Model grouprats, glomerular lesions clear, can observe glomerular capillary basementmembrane thickening visiblely, podocyte slit diaphragm broadening andwidely fusion, mesangial area broadening; Each treatment group has differentdegrees of pathological changes, JYG performance is less obvious.7.3 Immunohistochemical staining method to detect the expression of renaltissue PodocinPodocin can be expressed in NG and each treatment group, NG toexpress the strongest, MG is not obvious. Compared with NG, Podocinexpression decreased significantly in MG and treatment group(P<0.05);Compared with MG, Podocin can be expressed stronger in each treatmentgroup(P<0.05); Comparison between each treatment group, the strongestPodocin expression is in JYG(P<0.05); Both ZYG and XYG have theexpression, but no significant difference(P>0.05). 7.4 Use Real-time PCR method to detect renal tissue Podocin m RNA expressionCompared with NG, Podocin m RNA in renal tissue of MG and each treatment group expression is decreased(P<0.05); Compared with MG, Podocin m RNA in renal tissue of each treatment group expression is raised(P<0.05); Compared with JYG, Podocin m RNA in renal tissue of ZYG and XYG expression is decreased(P<0.05);ZYG and XYG, there is no obvious statistical significance(P>0.05).Conclusions:1 Xiezhuojiedu Huoxuetongluo traditional Chinese medicine can improve the general situation of DN rats, reduce the24-hour urinary total protein, regulate glucose metabolism and lipid metabolism, protect renal for work well.2 Xiezhuojiedu Huoxuetongluo traditional Chinese medicine can inhibit the thickening of basement membrane, mesangial matrix proliferation, foot process fusion, improve the pathological damage of diabetic nephropathy rats’ kidney tissue and delay worsening of it.3 Xiezhuojiedu Huoxuetongluo traditional Chinese medicine can raise Podocin m RNA expression of DN rats’ kidney, thereby reducing the leakage of proteinuria, protecting kidney.4 Both Xiezhuojiedu Huoxuetongluo traditional Chinese medicine and irbesartan have therapeutic effect on DN rats, but the combined treatment effect is better. |
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