| Diabetes mellitus (DM) is a lifetime systemic disease as a serious threat to human health, and the incidence rate is increasing progressively. According to WHO reports, there were 125 million diabetic patients worldwide in 1995, the number of diabetic patients will reach 299 million in 2025. DM has become the world's fifth leading causes of death. Diabetic nephropathy (DN) is one of the most serious and common chronic complications of DM, and also a major cause of death in DM patients. Therefore, the effective prevention and treatment of DN, a serious and refractory microvascular complication of DM, has become an urgent clinical issue to be resolved. Zehuang Granule is a Chinese medicine preparation with the main pharmacodynamic effects to dissipate blood stasis and alleviate water retention, which was developed based on the theory that blood stasis is the basic pathology of DN and combined with clinical experience. Clinical observation confirmed that Zehuang Granule had good therapeutic effects in the improvement of edema, hemorheology and other symptoms, and reduction of the BUN, Scr, Upro in patients with early-stage DN. To further study the mechanism of Zehuang Granule, we explored the protective effects and mechanism of Zehuang Granule on DN by means of immunohistochemistry, Real-time PCR, Western-Blot, ELASA, pathological examination and other research methods, so as to provide pharmacological and experimental evidence for the clinical application, development and promotion of this preparation. The main results were reported as follows:Experiment 1 General pharmacodynamic observation of Zehuang Granule in DM ratsObjective: To investigate the mechanism of the protective effects of Zehuang Granule on renal injury in STZ-induced DM rats and the regulatory role on general pharmacodynamic and hemorheological index.Methods: A total of 50 SPF-class male Sprague-Dawlay (SD) rats aged 4 weeks, weighing 180-200g, were randomly divided into normal group (n=10) and DM model group (n=40). Control group were fed with normal diet; diabetic model group were fed with high fat and high glucose diet, after fed with high fat and high glucose diet for 4 weeks, rats received a bolus intraperitoneal injection of STZ (45mg/kg body weight) at fasting, blood glucose was monitored after 72h. Rats with fasting blood glucose≥16.7 mmol/L for continuous three times, urine sugar positive and urinary albumin qualitative measurement positive were regarded as successful diabetes model. 35 DM rats were randomly divided into diabetic group (DM group, n =12), Zehuang granule treatment group (Z group, n =12) and glipizide group (G group, n =11). Renal pathological changes were examined with light microscope and electron microscopy, and BG, HbAlc, Scr, Ucr, BUN, UAER, TG, TC and blood rheology index were measured.Results: DM rats manifested significant polydipsia, polyphagia, polyuria, body weight increased at early stage followed by weight loss and the deterioration of general condition, fur dullness, obvious stinging hair and vulnerability to infection. BG, Hba1c, BUN, UAER, TG, TC, left kidney weight / body weight, whole blood viscosity, plasma viscosity of diabetic rats were significantly higher than normal group; while Ccr was significantly lower than normal rats (all P <0.05). BG, Hba1c, BUN, UAER, TG, TC, left kidney weight / body weight, whole blood viscosity, plasma viscosity of Z group and G group were significantly decreased than DM group (P <0.05), the decrease of Z group was more obvious than G group (P <0.05). Ccr was significantly higher in Z group and G rats than DM group, but the increase in Z group was more significant than G group.HE staining found clear structure of glomerular, tubular epithelial cells lined up in order with integrated basement membrane and no interstitial inflammatory cells infiltration or fibrous tissue hyperplasia in N group rats; in DM rats, we found glomerular hypertrophy, widened mesangial region, mesangial cell proliferation, matrix increase, the deposition of a large number of red blood cells within glomerular, swelling and exfoliation of renal tubular epithelial cells, vacuolar degeneration and obvious sugar matrix deposition; in Z group rats, the deposition of red blood cells within glomerular was rare, there was no tubular epithelial cell exfoliation, protein casts were rarely seen in kidney tubules. In G group rats, the deposition of red blood cells within glomerular was observed, epithelial cells showed foliated vacuolar degeneration, tubular epithelial cells exfoliation was occasionally identified.Transmission electron microscope observation : in N group rats, the glomerular structure was normal, foot processes were well-distributed, slit membrane structure was clear; DM group rats showed basement membrane thickening, obviously enlarged mesangial region, diffusely widened mesangial cells and matrix, swelling of foot processes, fusion of some segments, reduction and disappearance of local slit membrane; the pathological changes in Z group and G group was alleviated compared with DM group, showed that the widened mesangial areas and foot process fusion was alleviated and slit membrane was mostly restored.Experiment 2 Regulatory effects of Zehuang Granule on renal AQP1, AQP2 and urine AQP2 expressionObjective: To observe the regulatory effects of Zehuang Granule on renal AQP1, AQP2 and urine AQP2 expressionMethods: The animal model was established as previously described. Immunohistochemistry, Western-Blot, Real-time PCR were used to detect the distribution and expression of AQP1, AQP2 in kidney, ELASA was used to detect the concentration of AQP2 in rat urine.Results: AQP1 was expressed on the apical membrane surface of cells in renal cortex proximal tubule and descending thin section of medullary loop, no expression of AQP1 was detected in glomeruli. AQP1 protein and mRNA expression levels in kidney of four groups of rats had no significant change (all P> 0.05).AQP2 protein was expressed in kidney collecting ducts, distal tubules, but not expressed in glomeruli. Kidney AQP2 protein, mRNA expression and urinary AQP2 levels were significantly lower in Z group rats compared with DM rats (P <0.05); kidney AQP2 protein content was positively correlated with the concentration of urine AQP2 (r = 0.703, p <0.01). Experiment 3 Regulatory effects of Zuhuang Granule on the expression of podocytes associated protein Nephrin, Podocin in kidney of diabetic ratsObjective: To observe the regulatory effects of Zuhuang Granule on the expression of podocytes associated protein Nephrin, Podocin in kidney of diabetic ratsMethods: The animal model was established as previously described. Immunohistochemistry, Western-Blot, Real-time PCR were used to detect the distribution and expression of nephrin and podocin in kidney.Results: Nephrin and podocin were expressed in the glomerular slit membrane, with uniform linear distribution along the glomerular capillary loop, no expression detected in renal tubular and renal cysts. Nephrin and podocin protein, mRNA expression were significantly increased in Z group compared with DM group (P <0.05).Conclusion:1. Zehuang Granule can reduce BG, Hba1c, BUN, UAER, TG, TC, left kidney weight/body weight, whole blood viscosity, and plasma viscosity index, effectively alleviate symptoms including polydipsia, polyphagia and polyuria, steadily increase body weight and improve the general conditions of diabetic rats.2. Zehuang Granule decreased renal AQP2 protein and mRNA expression in DM rats, which may be a part of the mechanism of the pharmacological effects to alleviate water retention; Zehuang Granule also significantly decreased urinary volume and urinary AQP2 levels in DM rats, and rat kidney AQP2 protein level was positively correlated with AQP2 concentration in urine. We established a method for urinary AQP2 detection, which provides a new clinical detection and evaluation index to reveal the traditional Chinese medicine nature of DM,"a water retention disease"and for the study of"blood stasis dissipation and water retention alleviation treatment".3. Zehuang Granule had no significant effects on the expression of AQP1 in renal tissue of diabetic rats, whether AQP1 is involved in water metabolism of DM needs to be further explored.4. The protective effects of Zehuang Granule on kidney may be through increasing the expression of nephrin and podocin, improve cell ultrastructure of podocytes, and this may also be part of its mechanism to"dissipate blood stasis". |
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