环氧合酶-2介导的低密度脂蛋白受体表达失调在糖尿病肾病早期足细胞损伤中的作用研究
发布时间：2019-05-23 19:21
【摘要】：目的观察环氧合酶-2(cyclooxygenase-2,COX-2)介导的低密度脂蛋白受体(low density lipoprotein receptor,LDLr)表达失调在糖尿病肾病(diabetic nephropathy,DN)早期足细胞损伤中的作用。方法选取8周龄雄性Sprague-Dawley(SD)大鼠30只,按照简单随机抽样原则分为对照组(Control)、糖尿病组(diabetic mellitus,DM)、糖尿病+阿司匹林组(DM+Aspirin),每组均为10只。采用链尿佐菌素(streptozotocin,STZ)诱导糖尿病大鼠模型,造模成功后Control组大鼠和DM组大鼠给予羧甲基纤维素钠灌胃,DM+Aspirin组大鼠给予Aspirin灌胃,直至第12周。于第12周收集24小时尿液、检测尿 ACR(microalbumin to creatinine ratio,ACR),第 12 周末处死大鼠,收集血标本,检测血脂谱,留取肾组织。通过油红O染色及胞内游离胆固醇定量测定法观察肾小球脂质沉积情况。通过透射电镜观察足细胞超微结构的改变,通过免疫组化染色及Western Blot观察足细胞特异性标志蛋白WT-1及nephrin在肾脏的表达情况。通过免疫组化染色及Western Blot观察COX-2、LDLr、胆固醇调节元件结合蛋白-2(sterol regulatory element-binding protein-2,SREBP-2)及其裂解激活蛋白(SREBP cleavage-activating protein,SCAP)在肾脏的表达情况,并分析COX-2与LDLr表达的相关性。通过Western Blot观察肾脏促炎因子表达情况,并通过免疫荧光共染色观察COX-2与WT-1在肾脏的共表达情况。结果与Control组大鼠相比,DM组大鼠血脂谱及尿ACR显著升高(P0.01);PAS染色(Periodic Acid-Schiff staining,PAS)可见肾小球肥大、系膜基质增多;免疫组化及Western Blot示肾脏WT-1、nephrin蛋白表达下降(P0.01),透射电镜观察到足细胞足突融合消失,肾小球基底膜增厚,提示DM组大鼠足细胞损伤加重;油红O染色可见肾小球内有显著的脂质沉积,免疫组化染色示肾小球COX-2及LDLr、SCAP、SREBP-2蛋白的表达增加(P0.01);相关性分析显示,COX-2蛋白表达与LDLr蛋白表达呈显著正相关(r=0.85,P0.01)。Western Blot示DM组大鼠促炎因子表达明显增加;激光共聚焦荧光显微镜观察进一步证实,大鼠肾小球COX-2与足细胞特异性标志物WT-1存在共表达。而在DM+Aspirin组大鼠,上述病理改变明显减轻(P0.05)。结论COX-2表达上调加重了 DN早期肾小球足细胞损伤,其机制可能与高糖条件下促炎因子表达增加,从而破坏LDLr负反馈调节通路,上调足细胞LDLr表达,增加足细胞内胆固醇摄入,致使足细胞脂质过度沉积有关;而抑制COX-2表达可以减轻足细胞损伤。
[Abstract]:Objective to observe the role of cyclooxygenase-2 (cyclooxygenase-2,COX-2)-mediated low density lipoprotein receptor (low density lipoprotein receptor,LDLr) expression disorder in early podocyte injury in diabetic nephropathy (diabetic nephropathy,DN). Methods according to the principle of simple random sampling, 30 8-week-old male Sprague-Dawley (SD) rats were randomly divided into control group (Control), diabetic group (diabetic mellitus,DM) and diabetic aspirin group (DM Aspirin), with 10 rats in each group. The diabetic rat model was induced by streptozotocin (streptozotocin,STZ). After successful establishment of the model, the rats in Control group and DM group were given sodium Carboxymethyl cellulose (, DM Aspirin) intragastrically with Aspirin until the 12th week. 24 hours urine was collected at the 12th week and urine ACR (microalbumin to creatinine ratio,ACR was detected. At the end of the 12th week, the rats were killed, blood samples were collected, blood lipid spectrum was detected, and renal tissue was taken. Renal lipid deposition was observed by oil red O staining and intracellular free cholesterol quantitative assay. The ultrastructure of podocytes was observed by transmission electron microscope, and the expression of podocyte specific marker proteins WT-1 and nephrin in kidney was observed by histochemical staining and Western Blot. The expression of COX-2,LDLr, cholesterol regulatory element binding protein-2 (sterol regulatory element-binding protein-2,SREBP-2 and its lytic activating protein (SREBP cleavage-activating protein,SCAP) in kidney was observed by Immunohistochemical staining and Western Blot. The correlation between COX-2 and LDLr expression was analyzed. The expression of pro-inflammatory factors in kidney was observed by Western Blot, and the co-expression of COX-2 and WT-1 in kidney was observed by immunofluorescence co-staining. Results compared with Control group, the blood lipid spectrum and urine ACR in DM group were significantly higher than those in DM group (P 0.01); PAS staining (Periodic Acid-Schiff staining,PAS), and the Mesangial matrix was increased. The expression of WT-1,nephrin protein in kidney decreased by immunohistochemistry and Western Blot (P 0.01). The fusion of podocytes disappeared and the basement membrane thickened by transmission electron microscope, suggesting that the injury of podocytes in DM group was aggravated. Oil red O staining showed significant lipid deposition in glomeruli, and immunohistochemical staining showed an increase in the expression of COX-2 and LDLr,SCAP,SREBP-2 protein in glomeruli (P01). Correlation analysis showed that the expression of COX-2 protein was positively correlated with the expression of LDLr protein (r 鈮，
本文编号：2484157
[Abstract]:Objective to observe the role of cyclooxygenase-2 (cyclooxygenase-2,COX-2)-mediated low density lipoprotein receptor (low density lipoprotein receptor,LDLr) expression disorder in early podocyte injury in diabetic nephropathy (diabetic nephropathy,DN). Methods according to the principle of simple random sampling, 30 8-week-old male Sprague-Dawley (SD) rats were randomly divided into control group (Control), diabetic group (diabetic mellitus,DM) and diabetic aspirin group (DM Aspirin), with 10 rats in each group. The diabetic rat model was induced by streptozotocin (streptozotocin,STZ). After successful establishment of the model, the rats in Control group and DM group were given sodium Carboxymethyl cellulose (, DM Aspirin) intragastrically with Aspirin until the 12th week. 24 hours urine was collected at the 12th week and urine ACR (microalbumin to creatinine ratio,ACR was detected. At the end of the 12th week, the rats were killed, blood samples were collected, blood lipid spectrum was detected, and renal tissue was taken. Renal lipid deposition was observed by oil red O staining and intracellular free cholesterol quantitative assay. The ultrastructure of podocytes was observed by transmission electron microscope, and the expression of podocyte specific marker proteins WT-1 and nephrin in kidney was observed by histochemical staining and Western Blot. The expression of COX-2,LDLr, cholesterol regulatory element binding protein-2 (sterol regulatory element-binding protein-2,SREBP-2 and its lytic activating protein (SREBP cleavage-activating protein,SCAP) in kidney was observed by Immunohistochemical staining and Western Blot. The correlation between COX-2 and LDLr expression was analyzed. The expression of pro-inflammatory factors in kidney was observed by Western Blot, and the co-expression of COX-2 and WT-1 in kidney was observed by immunofluorescence co-staining. Results compared with Control group, the blood lipid spectrum and urine ACR in DM group were significantly higher than those in DM group (P 0.01); PAS staining (Periodic Acid-Schiff staining,PAS), and the Mesangial matrix was increased. The expression of WT-1,nephrin protein in kidney decreased by immunohistochemistry and Western Blot (P 0.01). The fusion of podocytes disappeared and the basement membrane thickened by transmission electron microscope, suggesting that the injury of podocytes in DM group was aggravated. Oil red O staining showed significant lipid deposition in glomeruli, and immunohistochemical staining showed an increase in the expression of COX-2 and LDLr,SCAP,SREBP-2 protein in glomeruli (P01). Correlation analysis showed that the expression of COX-2 protein was positively correlated with the expression of LDLr protein (r 鈮，
本文编号：2484157
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