【摘要】：目的觀察環(huán)氧合酶-2(cyclooxygenase-2,COX-2)介導(dǎo)的低密度脂蛋白受體(low density lipoprotein receptor,LDLr)表達失調(diào)在糖尿病腎病(diabetic nephropathy,DN)早期足細胞損傷中的作用。方法選取8周齡雄性Sprague-Dawley(SD)大鼠30只,按照簡單隨機抽樣原則分為對照組(Control)、糖尿病組(diabetic mellitus,DM)、糖尿病+阿司匹林組(DM+Aspirin),每組均為10只。采用鏈尿佐菌素(streptozotocin,STZ)誘導(dǎo)糖尿病大鼠模型,造模成功后Control組大鼠和DM組大鼠給予羧甲基纖維素鈉灌胃,DM+Aspirin組大鼠給予Aspirin灌胃,直至第12周。于第12周收集24小時尿液、檢測尿 ACR(microalbumin to creatinine ratio,ACR),第 12 周末處死大鼠,收集血標(biāo)本,檢測血脂譜,留取腎組織。通過油紅O染色及胞內(nèi)游離膽固醇定量測定法觀察腎小球脂質(zhì)沉積情況。通過透射電鏡觀察足細胞超微結(jié)構(gòu)的改變,通過免疫組化染色及Western Blot觀察足細胞特異性標(biāo)志蛋白WT-1及nephrin在腎臟的表達情況。通過免疫組化染色及Western Blot觀察COX-2、LDLr、膽固醇調(diào)節(jié)元件結(jié)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)在腎臟的表達情況,并分析COX-2與LDLr表達的相關(guān)性。通過Western Blot觀察腎臟促炎因子表達情況,并通過免疫熒光共染色觀察COX-2與WT-1在腎臟的共表達情況。結(jié)果與Control組大鼠相比,DM組大鼠血脂譜及尿ACR顯著升高(P0.01);PAS染色(Periodic Acid-Schiff staining,PAS)可見腎小球肥大、系膜基質(zhì)增多;免疫組化及Western Blot示腎臟WT-1、nephrin蛋白表達下降(P0.01),透射電鏡觀察到足細胞足突融合消失,腎小球基底膜增厚,提示DM組大鼠足細胞損傷加重;油紅O染色可見腎小球內(nèi)有顯著的脂質(zhì)沉積,免疫組化染色示腎小球COX-2及LDLr、SCAP、SREBP-2蛋白的表達增加(P0.01);相關(guān)性分析顯示,COX-2蛋白表達與LDLr蛋白表達呈顯著正相關(guān)(r=0.85,P0.01)。Western Blot示DM組大鼠促炎因子表達明顯增加;激光共聚焦熒光顯微鏡觀察進一步證實,大鼠腎小球COX-2與足細胞特異性標(biāo)志物WT-1存在共表達。而在DM+Aspirin組大鼠,上述病理改變明顯減輕(P0.05)。結(jié)論COX-2表達上調(diào)加重了 DN早期腎小球足細胞損傷,其機制可能與高糖條件下促炎因子表達增加,從而破壞LDLr負反饋調(diào)節(jié)通路,上調(diào)足細胞LDLr表達,增加足細胞內(nèi)膽固醇攝入,致使足細胞脂質(zhì)過度沉積有關(guān);而抑制COX-2表達可以減輕足細胞損傷。
[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，

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