The Protective Effect And Mechanism Of Ze-Huang Apozeme On Rats With Experimental Diabetes Renal Injury

Diabetes mellitus (DM) is a kind of lifetime and general disease that severely threatens human healthy, with its incidence increasing year by year. WHO estimates that by 2025 the DM patients in the world will reach 299 million. DM is rising to the fifth major cause for death. The newly increased DM patients are mainly from developing countries like China and African states, of which 90% is 2 Type DM. Diabetic nephropathy (DN) is one of the most common chronic capillary complications as well as the main cause for terminal stage renal failure. It is becoming an imperative clinical topic to effectively prevent and treat it for the medical field. Based on the traditional Chinese science pathoginesis of deficiency of both vital energy and Yin, and blood stasis and composition of supplementing Qi and nourishing Yin, Ze-Huang apozeme( ZHA) is pure traditional Chinese compound preparation summarized from years'clinical experience. Clinical observation confirmed that it had curative effect in promoting symptoms such as oedema, haemorheology, lower fasting plasma glucose( FPG), urea nitrogen, serum creatinine(Scr), 24h urea protein excretion, and etc. in early DN patients. In order to further explore the clinical effect mechanism of ZHA, we investigated the protection of ZHA to DN by adopting immunohistochemical method, RT-PCR and pathological method, thus providing pharmacological evidence and experiment basis for the clinical application, development and generalization of this preparation. Our main observations were reported as follows.ExprimentⅠThe renal injury effect of ZHA to experimental diabetes renal injury rats and its regulation to extracellular matrix expression.Objective: To investigate the effect of Ze-Huang-Jian-Ji (ZHA) on extracellular matrix (ECM) expression in rats with diabetes mellitus (DM) induced by streptozotocin (STZ).Methods:60 male SD rats were randomized into normal control group as the control group (10) and diabetes model groups as experiment group (50). Conventional forage was feed to the control group, and the high glucose and grease forage to the experiment group. 4 weeks later, the animals showed insulin resistance. Then low dosage (30mg/kg) Streptozotocin (STZ)was administered with vena caudalis intravenous injection to destroy the abdominal salivary so that insulin exist with comp paracrisis, and finally, the 2 type DM model was established. The successful criterion were fasting plasma glucose>7.8mmol/L and positive urine glucose. 30 rats were random selected from the model and divided into diabetes and renal injury group ( model group), Glipizide treatment group( control group) and ZHA treatment group( observation group), with 10 rats in each group. The immunohistological method was employed to determine distribution and expression of MMP-2, TIMP-1, TGF-β1, COL-Ⅳ, LN and FN in nephridial tissue of rats and detected indexes in rats of each group, including FPG, FINS, ISI, Scr, BUN, 24h urine protein excretion, TG, TC, FFA and etc. Results: General observation of experiment animals: the diabetic rats were characterized by polydipsia, polyphagia, diuresis, gaining weights at various degrees at early stage, 4 weeks later, weights lowered, emaciated, scratch, tarnish and even abdominal distension was observed in late stage.Rats in model groups had higher blood glucose, Scr, BUN, TG, TC, FFA and 24h urine protein excretion compared with control group (P<0.05); blood glucose, Scr, BUN, TG, TC, FFA, GHb and 24h urine protein excretion significantly decreased in the control group and the observation group (P<0.05), of which the observation group decreased much significant (P<0.05).In control group, the glomeruli structure, regular arrayed renal cells, complete basal laminal, no inflammatory cell infiltration and fibroplasias can be clearly detected, while in the model group, obviously larger glomeruli, wider mesangial region, intercapillary cells proliferation, groundmass increase, thickening vessel wall, great amount of akaryocyte siltation, diffuse vacuole-like denaturation and swell of renal cells and protein cast diffuse in nephric tubule were detected. In the control group, epithelium vacuole degen foliated, the akaryocyte siltation was observed in glomeruli. Nephric tubule showed epitheliums desquamate, and protein cast. The observation groups was found focus shape denaturation only under renal capsule, rare blood siltation in glomeruli, few epitheliums desquamated and scarce protein cast in tubule.MMP-2 showed obvious expression in normal nephridial tissue, with buffy strong positive staining in glomeruli, nephric tubule and mesenchymal; while MMP-2 in the model group only demonstrated weakly positive expression.TIMP-1 mainly expressed in masangial cells, endotheliocyte and epithelium in ren. The TIMP-1 was higher expressed in model group than in normal group (P<0.05).Small quantity of buffy staining of COL-Ⅳcan be seen in normal nephridial tissue. TGF-β1 and COL-Ⅳwas high expressed in model group, with a lot of buffy staining in glomeruli and renal interstitium. COL-Ⅳshowed strong positive. LN and FN distributed foliated in mesangial region and linearly on Bowman's bag basal lamina; the model groups was higher expressed than the normal group (P<0.05).MMP-2 in the control group and observation group significantly rose (P<0.05), with TIMP-1, TGF-β1, COL-Ⅳ, LN and FN significantly decreased, among which FN was most significant (P<0.01)ExprimentⅡThe regulation of ZHA to AQP2 protein in experiment diabetes renal injury rats nephridial tissue and its gene levelObjective: To observe the regulation of Ze-Huang-Jian-Ji (ZHA) to AQP2 in diabetes renal injury rats nephridial tissue and its gene level.Methods:The animal model construction was the same as previous stated, reference to the previous literature [43].AQP2 immunohistochemistry detection: distribution in kidney of the rats in each group and protein expression. RT-PCR was used to determine AQP2 mRNA expression of rats, and quantitate withβ-actin.Results: AQP2 expressed in ren collecting tube, while not expressed in glomeruli or renal interstitium. The buffy granula of AQP2 protein in model group rats ren distributed along collecting tube epithelium had a darker color and thickening than the normal group, with wider distribution area. The buffy AQP2 in control group and observation group was significantly lightened than that of the model group, with decreasing area (P<0.05).RT-PCR product eletrophoresis observations demonstrated that AQP2 mRNA expression in model group rats ren was significantly higher than that of the normal group, with 0.81±0.059 and 0.51±0.004, respectively (P<0.01). The observation group (0.70±0.027) was significantly lower than the model group (P<0.05).Conclusion:1. ZHA can reduce the blood glucose, the blood fats, reduces 24-hour Urine proteins excretion rate, reduces UN,serum creatinine and so on , alleviated these symptoms polydipsia,polyphagia, urorrhagia and so on , increase the animals' weight, improved the animals' roughly condition.2. ZHA reduces the expression of COL-Ⅳ, LN and FN in the DN rats' nephridial tissue, delayed the development of glomcrulus'sclerosis, has obvious protective function to the kidney pellet,which is one of effective mechanisms to prevent and cure the DN . ZHA reduces ECM mainly relate to adjust MMP-2,TIMP-1,TGF-β1 protein'expression and activeness.3. The AQP2 protein and the AQP2 mRNA expression in The DM rats' nephridial tissue raising up suggest that nephridial tissue dropsy and the cells engorge correlate to the Changes of kidney AQP2 expression.ZHA can reduce the expressions of AQP2 protein and the AQP2 mRNA expression in the DN rats' nephridial tissue,which probably is the mer-mechanism to mitigate nephridial tissue dropsy.4. In the nephridial tissue, AQP are multi-hypotype and multi-location,these characters probably is the Chinese medicine "the kidney host water" modern medicine understanding foundation; ZHA can reduce red cell stasis in the glomerul-micrangium,adjust AQP2 expression in nephridial tissue,which probably is one of effect modern medicine essences of the praeparatum "Hua-Yu-Li-Shui" .5. ZHA reduces AQP2 probably related to Urine protein excretion, whether has other mechanisms await further investigation.