| Objective: In this experiment, through established diabetes mellitus (DM)animal model, application of Huoxue–Huayu-Tongluo recipes interventiontreatment of animal model, and to observe the experimental animal24hoururinary protein quantity, renal function (SCr),glycosylated hemoglobin, renalpathological changes, immune staining in renal tissue angiotensin type II1receptor (AT1R) expression, PCR method was used to detect the expression ofmRNA in renal tissue of AT1R, Western-blot for the detection of renal tissueAT1R protein, to study the therapeutic effect of Huoxue Huayu Tongluorecipes on diabetic nephropathy and its possible mechanism, provide newtheoretical and experimental basis for the clinical application.Methods:There were80male Sprague-Dauley rats with the weight of90g～120g, ordinary forage adaptability feeding one week,15rats randomlyselected as normal control group, the remaining65rats were made into model,the model rats just eating for12hours then intraperitoneal injection of0.1Mcitric acid buffer (PH4.5) preparation the streptozotocin (STZ)55mg/kg, thenormal control group were injected with citrate buffer volume, After72hoursmeasured the tail vein blood glucose, three consecutive random blood glucosevalue≥16.7mmol/L as the DM model standard.3rats’ blood glucose valuedidn’t get the standard which is not selection. Then the DM models wererandomly divided into5groups again:14rats for model, group12rats foreach of Huoxue recipes, Huayu Tongluo recipes, Huoxue Huayu Tongluorecipes, Irbesartan recipes. After one week of DM models made successfully,the rats of the normal group, and model group were given2ml of drinkingwater daily, The does was converted according to body type coefficient of ratto hunman. danshen1.2g/kg/d, Chuanxiong0.96g/kg/d, dilong0.8g/kg/d, quanxie0.48g/kg/d, Iirbesartan0.024g/kg/d. Once daily administeredcontinuously for16weeks, collected24hours urine, for24hours urinaryprotein quantitative determination. At the end of16W,all rats were measuredthe blood glucose and glycosylated hemoglobin (HbA1c), and body weight,then took10%chloral hydrate anesthesia the animals, abdominal aortic blood,pick the right renal stripping film after weighing, the renal cortex samples,observed micro structural changes and the expression of AT1R observationrenal tissue.Results:1The general condition of the ratsThe control diet group rats were normal, normal urine volume, hair colorgloss, sensitive reaction, muscle fullness. Other groups appeared much drink,polyphagia, polyuria,gradually after injection of STZ, at the and of thisexperiment, compared with the blank control group, model group ratsunresponsive, apathetic, smaller, and the model group is most obvious. Duringadministration model rats in each group were died:6rats died in the modelgroup,1rat died in Huoxue Huayu Tongluo recipe treatment group,2rats diedin Irbesartan group, the cause of death may be due to high blood sugarultimate lead to multiple organ failure.2The body weight,the right kidney hypertrophy index (right kidney weight/body weight) comparisonCompared with the normal control group, the body weight of modelgroup and treatment groups significant lighter (P<0.05), the model group andthe treatment groups had no significant difference (P>0.05); the right kidneyweight of all groups were no significant difference (P>0.05); compared withthe normal group, hypertrophy index of the model group and other treatmentgroups was significantly higher (P<0.05), there was no significant differencebetween the model group and the treatment groups(P>0.05).3The24hour urinary protein quantity in each group(24h UTP)Compared with the normal group, model group and the treatment groupof24hour urinary protein quantitative increased significantly (P<0.05); compared with the model group, each group of24hours urine proteinincreased significantly (P<0.05); Huoxue recipes was obviouslyhigher(P<0.05).There was no statistical difference between Irbesartan recipes,Huayu Tongluo recipes and Huoxue Huayu Tongluo recipes(P>0.05).4Rats of each group of glycosylated hemoglobin (HbA1c) comparisonCompared with the normal control group, glycosylated hemoglobin ofmodel group and the treatment groups increased significantly (P<0.05), thedifference between model group and the treatment groups was not significant(P>0.05).5The serum creatinine of each groupNo significant difference in serum creatinine levels of rats in each group(P>0.05).6The pathomorphology change of kidney6.1Light microscope observationNormal group rats’ kidney glomerular structure clear and intact, noobvious hypertrophy, no basement membrane thickening, no mesangialproliferation; kidney glomeruli of rats in model group obviously hypertrophy,basement membrane thickening, visible mesangial proliferation, mesangialregion widen; compared with the model group, the therapy group glomerularrats were mild hypertrophy, glomerular basement membrane thickening wereseen were mild, mild mesangial proliferation, Huoxue recipes was obviouslyhigher than Irbesartan recipes, Huayu Tongluo recipes and Huoxue HuayuTongluo recipes.6.2Electron microscope observationThe normal group rats glomerular basement membrane structure integrity,podocyte foot process distribution is uniform, no fusion; glomerular basementmembrane; model group rats were significantly heterogeneous thickened, partof a domal uplift, podocyte foot process fusion; compared with the modelgroup, the rats in the treatment group also has the similar pathological changes,but to a lesser degree, Huoxue recipes was obviously higher than Irbesartanrecipes, Huayu Tongluo recipes and Huoxue Huayu Tongluo recipes. 7Immunohistochemical result of the expression of AT1R in kidneyExpression of AT1R in kidney of rat was mainly located in the renaltubules, proximal tubules, distal renal tubular, glomerular, renal vasculararound a small amount of expression. Compared with the normal group, modelgroup increased significantly (P<0.05), the treatment group also wassignificantly higher than normal group (P<0.05); compared with the modelgroup, the expression of each treatment group decreased significantly(P<0.05), Huoxue recipes was obviously higher than Irbesartan recipes,Huayu Tongluo recipes and Huoxue Huayu Tongluo recipes.(P<0.05).8Results of the expression of AT1R protein in renal tissue by Western-blotmethodCompared with the normal group, model group and treatment groupprotein expression were increased (P<0.05); compared with the model group,the treatment group were decreased the expression levels of protein (P<0.05),Huoxue recipes was obviously higher than Irbesartan recipes, Huayu Tongluorecipes and Huoxue Huayu Tongluo recipes.(P<0.05).9Results of the expression of AT1R mRNA in renal tissue by RT-PCRmethod methodsCompared with the normal group, model group and treatment group,AT1R mRNA expression was significantly increased (P<0.05); compared withthe model group, the expression of AT1R mRNA in the treatment group weredecreased (P<0.05), Huoxue recipes was obviously higher than Irbesartanrecipes, Huayu Tongluo recipes and Huoxue Huayu Tongluo recipes (P<0.05).Conclusions:1Huoxue-Huayu-Tongluo recipes has protective effect on kidney ofdiabetic nephropathy rats.2Huoxue-Huayu-Tongluo recipes can inhibit the expression of AT1,indicating that the treatment effect of Huoxue-Huayu-Tongluo recipes on DNmay be related with the approach for blocking RAS. |
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