阿托伐他汀对糖尿病大鼠肾脏eNOS表达的影响及其可能的肾脏保护作用

发布时间：2018-05-10 22:40

本文选题：糖尿病 + 肾病　；参考：《河北医科大学》2017年硕士论文

【摘要】：目的:糖尿病肾病(Diabetic Kidney Disease,DKD)是糖尿病(Diabetes Mellitus,DM)常见的微血管并发症之一,晚期可以进展至终末肾功能衰竭(end-stage renal failure,ESRF),导致患者死亡,成为严重危害人类生活质量并威胁人类生命的疾病。近年来研究发现他汀类药物除突出的降脂作用以外,其对糖尿病患者肾脏的非降脂的保护作用受到越来越多的关注。一氧化氮(nitric oxide,NO)作为人体内重要的血管舒张因子,可以扩张肾小球入球小动脉及出球小动脉,降低肾血管阻力,改善肾小球滤过率和肾脏血流灌注,以维持肾脏良好的血流动力学状态及血管张力。本实验通过观察阿托伐他汀对2型糖尿病大鼠肾组织中内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)表达水平、NO含量等的影响,探讨其是否会通过干预eNOS-NO通路减慢糖尿病肾病进展,从而为他汀类药物对临床糖尿病肾脏保护提供理论依据。方法:选取160-200g健康清洁级雌性SD大鼠54只,随机分为两组,一组为正常对照组(NC组),另一组为高糖高脂喂养同时联合小剂量链脲佐菌素(STZ)(30 mg/kg)一次性腹腔注射建立的2型糖尿病大鼠模型组。于实验第10w末称取大鼠体重(BW),取下腔静脉血标本检测空腹胰岛素(FINS)、空腹血糖(FBG)、总胆固醇(TC)和甘油三酯(TG),并根据FBG和FINS计算胰岛素抵抗指数(HOMA-IR)。之后将2型糖尿病大鼠随机分为糖尿病组(DM组)和阿托伐他汀干预组(A组)。每周监测血糖、BW。实验第21w末称取各组大鼠体重(BW),取大鼠下腔静脉血测定FINS、FBG、TC、TG、糖化血红蛋白(Hb A1c)、谷丙转氨酶(ALT)、谷草转氨酶(AST)、肌酐(SCr)、尿素(UREA),计算胰岛素抵抗指数(HOMA-IR);取出大鼠肾脏后,去除被膜、脂肪等肾脏以外组织后称取双肾重量(KW),计算肾脏指数(Kidney Index,KI=KW/BW);肾脏组织石蜡切片行HE染色、PAS染色观察大鼠肾脏病理改变;取部分肾皮质匀浆以硝酸还原法测定NO含量;应用real-time PCR检测肾皮质eNOS m RNA含量;免疫组织化学染色检测eNOS在肾皮质中的表达。应用SPSS 21.0统计软件进行数据分析,数据描述用均数±标准差(`x±s)表示。两组间样本均数比较采用独立样本t检验,多组间样本均数的多重比较采用LSD、SNK-q、Tamhane检验。P0.05认为差异具有统计学意义。结果:1 BW变化实验第10w末,糖尿病大鼠的BW明显高于NC组大鼠(P0.05)。第21w末,与NC组比较,DM组大鼠和A组大鼠的BW均明显下降(P0.01);DM组大鼠和A组大鼠相比,体重无显著差异(P0.05)。2血生化指标检测实验第10w末,糖尿病大鼠的FBG、TG、TC、FINS均明显高于同期NC组大鼠(P0.01)。第21w末,DM组和A组大鼠FINS、FBG、TC、TG、Hb A1c与同期NC组大鼠相比均明显升高(P0.01,P0.05),而DM组与A组大鼠上述指标差异无统计学意义(P0.05);DM组和A组大鼠UREA水平均高于同期NC组大鼠(P0.05),A组大鼠和DM组大鼠相比,UREA水平差异无统计学意义(P0.05);3组大鼠SCr、ALT、AST均无显著差异(P0.05)。3胰岛素敏感性测定实验第10w末,糖尿病大鼠的HOMA-IR显著高于同期NC组(P0.01),证实糖尿病大鼠存在胰岛素抵抗,为2型糖尿病大鼠。第21周末,DM组、A组大鼠HOMA-IR仍显著高于同期NC组大鼠(P0.01),DM组与A组相比较HOMA-IR无显著差异(P0.05)。4肾脏指数测定实验第21w末,DM组和A组大鼠的双肾重量(KW)和肾脏指数(Kidney Index,KI=KW(mg)/BW(g))均明显高于NC组大鼠(P0.01),A组大鼠上述指标较DM组大鼠均降低,差异有统计学意义(P0.01)。5肾脏病理改变NC组大鼠肾小球及肾小管形态正常。DM组大鼠肾小球肥大,系膜区扩张,基底膜不规则增厚,PAS阳性物质沉积增多。部分肾小管出现细胞空泡样变性,管腔变窄。A组大鼠肾脏病变较DM组稍有减轻,表现为肾小球轻度增大,基底膜轻微增厚,PAS阳性物质沉积有所减少,偶可见肾小管细胞空泡样变性及管腔变窄。6肾皮质NO含量检测DM组、A组大鼠肾皮质NO含量低于同期NC组大鼠(P0.01),DM组大鼠肾皮质NO含量低于A组大鼠,差异有统计学意义(P0.01)。7肾皮质eNOS免疫组化染色胞浆中呈棕黄色染色颗粒者为eNOS阳性表达。DM组和A组大鼠肾皮质eNOS表达均显著低于NC组大鼠(P0.01,P0.05),A组大鼠肾皮质eNOS表达高于DM组大鼠,差异有统计学意义(P0.05)。8肾皮质eNOS m RNA real-time PCR结果与NC组相比,DM组和A组大鼠肾皮质中eNOS m RNA含量均降低(P0.01);其中A组大鼠eNOS m RNA含量高于DM组大鼠(P0.01)。结论:1在高糖高脂饮食的基础上联合小剂量链脲佐菌素一次性腹腔注射能够成功建立2型糖尿病大鼠模型,随着病程的进展,其肾脏组织学改变基本具备人类2型糖尿病肾病的典型病理特征。2与正常对照组相比,糖尿病大鼠肾皮质eNOS表达下降,肾皮质中NO含量降低,与DM组相比,应用阿托伐他汀对FBG、Hb A1C、HOMA-IR等虽无显著影响,但其可以明显改善肾脏病理学变化,上调肾皮质中eNOS的表达,增加NO合成,提示阿托伐他汀对糖尿病大鼠肾脏可能具有非降脂的保护作用。
[Abstract]:Objective: Diabetic Kidney Disease (DKD) is one of the common microvascular complications of diabetes (Diabetes Mellitus, DM). In the late stage, it can advance to terminal renal failure (end-stage renal failure, ESRF), causing death and becoming a serious threat to human life and threat to human life. In recent years, research has been found. In addition to the protruding effect of lipid lowering, statins have attracted more and more attention to the non lipid lowering effect of kidney in diabetic patients. Nitric oxide (NO), as an important vascular diastolic factor in the human body, can dilate the glomerular arteriole and arteriole, reduce renal vascular resistance and improve glomerular filtration. The effect of atorvastatin on the expression of endothelial nitric oxide synthase (eNOS) and the content of NO in renal tissue of type 2 diabetic rats was investigated to investigate whether it could interfere with eNOS-NO through the observation of the effect of atorvastatin on the expression of endothelial nitric oxide synthase (oxide synthase, eNOS) in the renal tissue of type 2 diabetic rats. The progress of diabetic nephropathy was slowed down to provide a theoretical basis for statins to protect the kidney from clinical diabetes. Methods: 54 healthy and clean female SD rats of 160-200g were randomly divided into two groups, one group was the normal control group (group NC) and the other group was combined with low dose streptozotocin (STZ) (30 mg/kg) at the same time. The rat model group of type 2 diabetic rats was established by intraperitoneal injection. The rat body weight (BW) was measured at the end of the experiment at the end of the experiment. The fasting insulin (FINS), fasting blood glucose (FBG), total cholesterol (TC) and triglyceride (TG) were measured in the inferior vena cava blood samples, and the insulin resistance index (HOMA-IR) was calculated according to FBG and FINS. Then, type 2 diabetic rats were randomly divided into sugars. Urine disease group (group DM) and atorvastatin intervention group (group A) monitoring blood sugar weekly, BW. experiment 21W end 21W was called group rats weight (BW), the rat inferior vena cava blood was taken to determine FINS, FBG, TC, TG, glycosylated hemoglobin (Hb A1c), glutamic aminotransferase (ALT), cereal transaminase, creatinine, urea and insulin resistance index. After removing the kidneys of the rats, the kidney index (KW) was removed and the kidney index (Kidney Index, KI=KW/BW) was calculated after removal of the membrane, fat and other tissues outside the kidney. The renal tissue paraffin sections were stained with HE, and the renal pathological changes were observed by PAS staining. Some renal cortex homogenates were measured with nitric acid reduction method and NO content was measured. Real-time PCR was used to detect eNOS m R. ENOS of real-time PCR was used to detect eNOS m R. NA content; immunohistochemical staining was used to detect the expression of eNOS in the renal cortex. The data was analyzed with SPSS 21 statistical software, and the data description was expressed with mean mean standard deviation (`x + s). The average number of samples between the two groups was compared with the independent sample t test. The multiple comparison of the average numbers of the multiple groups adopted LSD, SNK-q, and Tamhane test.P0.05. The results were statistically significant. Results: the BW of diabetic rats was significantly higher than that in group NC (P0.05) at the end of the 1 BW change experiment. At the end of 21W, the BW of both DM and A rats decreased significantly (P0.01) compared with those in the NC group, and there was no significant difference in weight between the rats of the DM group and the rats. TG, TC and FINS were significantly higher than that of NC rats in the same period (P0.01). At the end of 21W, FINS, FBG, TC, TG, and A groups were significantly higher than those in the same period. There was no significant difference in UREA level in the group of rats (P0.05), and there was no significant difference between the 3 groups of rats SCr, ALT and AST (P0.05).3 insulin sensitivity test 10W, and the HOMA-IR in diabetic rats was significantly higher than that of the NC group (P0.01) in the same period. It was confirmed that the diabetic rats had insulin resistance, which was type 2 diabetic rats. Twenty-first weekend, DM group, and rats were the rats. A-IR was still significantly higher than that of NC group (P0.01), and there was no significant difference in HOMA-IR between group DM and A group (P0.05).4 kidney index test, and both kidney weight (KW) and kidney index in DM and A group were significantly higher than that of group rats. There were statistically significant (P0.01).5 renal pathological changes in group NC, glomeruli and renal tubules in normal.DM rats, glomerular hypertrophy, mesangial region dilation, irregular thickening of basement membrane, and increased PAS positive substance deposition. Some renal tubules appeared vacuolated degeneration of cells and renal lesions in group.A group were slightly lessened than those in the DM group, showing kidneys. Slight enlargement of small ball, slight thickening of basement membrane and decrease of PAS positive substance deposition, NO content in renal cortex of renal tubule cells and narrowing of.6 in group DM, NO content in renal cortex of A group was lower than that of NC group (P0.01) in A group, and the content of NO content in renal cortex of DM group was lower than that of A group, the difference was statistically significant (P0.01) kidney The expression of eNOS expression in.DM group and A group was significantly lower than that of NC group (P0.01, P0.05) in.DM group and A group. The expression of eNOS expression in renal cortex of A group was higher than that of DM group, and the difference was statistically significant (P0.05) compared with that of the group of NC. The content of eNOS m RNA in renal cortex of group and A rats decreased (P0.01), and eNOS m RNA content in group A rats was higher than that of group DM rats (P0.01). Conclusion: 1 a one-time injection of small dose streptozotocin combined with small dose of streptozotocin on the basis of high glucose and high fat diet can successfully establish a model of type 2 diabetic rats. With the progress of the course of disease, the kidney histology changes The typical pathological features of human type 2 diabetic nephropathy had a typical pathological feature.2, compared with the normal control group, the expression of eNOS in the renal cortex of diabetic rats decreased and the NO content in the renal cortex decreased. Compared with the DM group, the use of atorvastatin had no significant influence on FBG, Hb A1C, HOMA-IR and so on, but it could obviously improve the renal pathological changes and up the renal cortex. The expression of eNOS increased NO synthesis, suggesting that atorvastatin may protect the kidneys of diabetic rats from lipid lowering.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

【参考文献】