转化生长因子β激活激酶1抑制剂对糖尿病小鼠肾脏的保护作用及机制

Renoprotective effect of transforming growth factor beta activator kinase 1 inhibitor in diabetic db/db mice and its mechanism

目的探讨转化生长因子β激活激酶1（TGF-βactivated kinase-1，TAK1）抑制剂（5Z-7-oxozeaenol，OZ）对糖尿病db／db小鼠肾脏的保护作用及机制。方法健康雄性db／db小鼠随机分为db／db组（n=12）和db／db＋OZ组（n=12），另取WT小鼠（n=12）作为对照组。OZ2mg／kg隔日腹腔注射。第8、12周末测定血糖、体质量、肾质量及24h尿白蛋白排泄率；光镜、电镜观察。肾组织病理改变；免疫组化检测NF—kBp65、单核细胞趋化蛋白1（MCP-1）与TNF-α表达；Western印迹检测磷酸化（P）-TAK1、转化生长因子B活化激酶结合蛋白1（TAB1）、p-p38MAPK及白细胞介素1p（IL-1p）蛋白表达；实时定量PCR检测细胞间细胞黏附分子1（ICAM-1）与MCP-1mRNA表达。结果8～12周时db／db小鼠血糖、体质量、肾质量及24h尿白蛋白排泄率显著高于对照组（P〈0．01），而db／db＋OZ组上述指标低于db／db组（P〈0．05）。8～12周db／db小鼠病理形态学表现为肾小球体积增大、细胞外基质增多，TAKl抑制剂可显著改善肾组织病理改变。免疫组化显示8～12周时db／db小鼠肾组织NF—KBp65、MCP-1和TNF-α表达显著高于对照组（P〈0．05），而db／db＋OZ组上述指标显著低于db／db组（P〈0．05）。Western印迹结果显示8—12周时db／db组小鼠p-TAKl、TAB1、p-p38MAPK和IL-1β蛋白表达显著高于对照组（P〈0．05），而db／db＋OZ组上述指标低于db／db组（P〈0．05）。实时定量PCR结果显示，db／db小鼠ICAM-1、MCP-1mRNA表达显著高于对照组（P〈0．01），而db／db＋OZ组上述指标显著低于db／db组（P〈0．05）。结论TAK1抑制剂可能通过抑制MAPK及NF—KB信号通路来抑制炎性反应，从而减轻糖尿病肾脏损伤。

Objective To investigate the renoprotective effect of transforming growth factor beta activator kinase 1 （TAK1） inhibitor 5Z- 7- oxozeaenol （OZ） in diabetic db/db mice and the mechanism. Methods Twenty-four male db/db mice were randomly divided into two groups： db/db mice （db/db, n=12） and db/db mice with 5Z-7-oxozeaenol treatment （db/db＋OZ, n=12）. Another group of wild type mice （n=12） was held as the control group. OZ 2 mg/kg was administrated by intraperitoneal injection every other day. At week 8 and 12 after 5Z- 7- oxozeaenol treatment, blood glucose （BG）, body weight （BW）, kidney weight （KW） and urinary albumin excretion rate （UAER） were evaluated. Kidney pathological lesions were detected by light and electron microscopy. NF-KB p65, monocyte chemotactic protein- 1 （MCP- 1） and tumor necrosis factor-ot （TNF- α） were detected by immunohistochemistry. Western blotting was used to detect p-TAK1, TAB1, p-p38MAPK and IL-1β expression, while ICAM- 1 and MCP-1 mRNA levels were evaluated by real- time PCR. Results Compared with control group, the levels of BG, BW, KW and UAER were higher （P 〈 0.01） in db/db mice group, while BW, KW and UAER levels were significantly decreased in db/db ＋ OZ group compared with that in db/db mice group （P 〈 0.05）. In week 8 and 12 db/db mice, glomernlar volume and extracellular matrix were increased, while pathological lesions in kidney tissue were positively improved by TAK1 inhibitor. Immunohistochemistry showed that NF-KB p65, MCP- 1 and TNF-ot expression levels were apparently increased in db/db mice group compared with that in control group （P 〈 0.05） and were significantly inhibited by TAK1 inhibitor （P 〈 0.05）. Western blotting showed that p-TAK1, TABI, p-p38MAPK and IL-1β expression levels were higher in db/db mice group than that in control group （P 〈 0.05） and lower in db/db＋ OZ group than that in db/db mice group （P 〈 0.05）. Moreover, real-time PCR showed that the expressions of ICAM-1 and MCP-1 mRNA were higher in db/db mice group than that in control group and lower in db/db＋OZ group than that in db/db mice group （P 〈 0.05）. Conclusions TAK1 Inhibitor can down-regulate MAPK and NF-KB pathway to restrain the reaction of inflammation and alleviate kidney injury in diabetic db/db mice.