吡咯烷二硫代甲酸铵对糖尿病肾病大鼠肾小管间质炎症分子表达及自噬的影响

Effects of ammonium pyrrolidine dithiocarbamate （PDTC） on osteopontin expression and autophagy in tubular cells in streptozotocin-induced diabetic nephropathy rat

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.