摘要
目的观察炎症分子核因子(NF)-κB、骨桥蛋白(OPN)及自噬相关分子微管相关蛋白1轻链3(LC3)、p62在糖尿病肾病(DN)大鼠肾小管间质表达的改变及外源性NF-κB抑制剂吡咯烷二硫代甲酸铵(PDTC)的干预作用。方法24只雄性Sprague—Dawley大鼠,其中12只给予60mg/kg链脲佐菌素(STZ)一次性腹腔注射制作糖尿病模型,成模后分为DN组、DN+PDTC组(PDTC腹腔注射,100mg·kg^-1·d^-1)各6只;另12只分为对照组及PDTC组,每组各6只。12周后检测血清肌酐(Scr)及24h尿蛋白,处死大鼠,观察肾病理改变,免疫组化检测NF—κBp65、OPN表达水平,电镜观察自噬改变,Western印迹检测肾皮质NF.KBp65、p62、OPN、LC3-Ⅱ/LC3-Ⅰ及细胞核磷酸化NF-κBp65(p-NF-κBp65)表达改变。结果各组大鼠Scr差异无统计学意义(P〉0.05)。DN组、DN+PDTC组尿蛋白水平明显高于其他两组(均P〈0.01),但DN+PDTC组明显低于DN组(P〈0.05)。DN组肾小管间质p65、OPN表达水平均明显高于对照组及PDTC组(均P〈0.05),肾皮质NF—κBp65、p62、OPN及核p—p65蛋白表达水平均高于对照组及PDTC组,LC3—Ⅱ/LC3-Ⅰ比值下降(均P〈0.05),而DN+PDTC组较DN组有明显改善。电镜下,DN组肾小管上皮细胞自噬泡明显减少、线粒体损伤增加,细胞核凋亡有明显增加,DN+PDTC组较DN组明显改善。相关性分析显示,肾皮质LC3—Ⅱ/LC3-Ⅰ与核p-p65、OPN表达呈负相关(r=-0.45,P=0.02;r=-0.50,P=0.01),p62表达与核p-p65、OPN表达呈正相关(r=0.33,P=0.01;r=0.41,P=0.01)。结论DN大鼠肾小管细胞炎症激活与自噬功能缺陷密切相关,PDTC可能通过阻断NF—κB活性而抑制炎症,增强小管细胞自噬能力,减轻小管间质损害。
Objective To investigate the effects of ammonium pyrrolidine dithiocarbamate (PDTC) on tubulointerstitial inflammatory molecules and autophagy in diabetic nephropathy ( DN ) rats. Methods Twenty-four male Sprague-Dawley rats were assigned to DN group ( n = 6) and DN + PDTC group ( n = 6, PDTC, ip, 100 mg · kg^-1 · d^-1 ), all received streptozotocin (STZ) 60 mg/kg intraperitoneally, and the other 12 rats were randomly divided into control group (n =6) and PDTC group (n =6). At the end of 12 weeks, after serum creatine (Scr) and 24-hour urinary protein were determined, rats were sacrificed to determined the renal pathological damages and the changes of nuclear factor (NF)-κB p65, p62, osteopontin (OPN), microtubule associated protein 1 light chain 3 (LC3)- Ⅱ/LC3-Ⅰ , nuclear p-NF-κB p65 by immunohistological stainning and Western blot, and ultrastructural changes of autophagic process was observed by electron microscopy (EM). Results Ser was similar among the four groups ( P 〉 0. 05 ). The levels of urinary protein in DN group and DN + PDTC group were significantly higher than the other twogroups (all P 〈 0. 01 ) , but the level of urinary protein in DN + PDTC group was lower than that of DN group (P 〈 0.05). DN + PDTC group had less tubulointerstitial damage compared with DN group ( P 〈 0.05 ). Among the four groups, expressions of p62, p65, OPN of tubulointerstitial area in DN group were significantly higher than that of the other groups ( all P 〈 0. 05 ), and Western blot showed that DN + PDTC group had less expressions of NF-κB p65, nuclear p-p65, OPN and more expresssion of LC3-Ⅱ/LC3-Ⅰ compared with DN group ( all P 〈 0. 05 ), which were consistent with the decreased autophagic vacuoles and increased mitochondria dysfunction revealed by EM. Correlation analysis showed that renal LC3-Ⅱ/LC3-Ⅰ was negatively correlated the expressions of nuclear p-p65 and OPN ( r = - 0. 45, P = 0. 02 ; r = - 0. 50, P = 0.01 ), and p62 was positively correlated the expressions of nuclear p-p65 and OPN ( r = 0. 33 ,P = 0. 01 ; r =0. 41 ,P =0. 01 ). Conclusion Tubular NF-κB activation is closely related to autophagy dysfunction in DN rats, and PDTC may enhance autophagy activity in tubule cells by blocking NF-κB activity.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2016年第44期3590-3595,共6页
National Medical Journal of China
基金
国家自然科学基金(31270791,30800529)
天津市卫生局重点攻关课题(11KG132)
天津市应用基础与前沿技术研究计划(14JCYBJC27900)