12-脂氧化酶对糖尿病肾病鼠肾小球p27^kap1表达的影响

Impact of 12-1ipoxygenase on p27^kap1 expression in experimental diabetic glomeruli

目的探讨12-脂氧化酶（12-LO）对糖尿病肾病肾小球内p27^kap1表达的影响。方法：在有或无p38MAPK（p38）抑制剂（SB203580，1μmol／L）的条件下，用12-LO作用产物12羟二十烷四烯酸[12（S）-HETE，10^-7mmol／L]刺激系膜细胞24h。雄性SD大鼠分为普通饮食对照组、普通饮食＋12-L0抑制剂（CDC，8mg／kg，3次／周，皮下注射）处理组、高脂饮食结合小剂量链脲菌素（STZ，35mg／kg，腹腔注射）诱导2型糖尿病组、高脂饮食结合小剂量STZ诱导2型糖尿病＋CDC处理组，连续皮下注射CDC1个月。野生型和12-LO基因敲除C57BL／6小鼠随机分成野生型对照组、12-L0基因敲除组、野生型STZ（200mg／kg，腹腔注射）诱导1型糖尿病组、12-LO基因敲除STZ诱导1型糖尿病组，饲养16周。实验结束后收集尿、血液、提取肾脏，用系列过筛方法分离肾小球。Western印迹和免疫组织化学方法检测p38活性和p27^kap1蛋白表达的变化。结果抑制p38活性可有效阻断12（S）-HETE诱导的系膜细胞内p27^kap1的表达（P〈0．01）。CDC可抑制2型糖尿病大鼠肾小球体积增大，降低尿白蛋白量（24h），阻止肾小球内p38活性和p27^kap1蛋白表达增加，与CDC未处理2型糖尿病大鼠比较差异均有统计学意义（均P〈0．01）。与野生型糖尿病小鼠比较，12-LO基因敲除糖尿病小鼠p38活性、p27^kap1蛋白表达及细胞外基质积聚显著减少，差异有统计学意义（均P〈0．01）。结论12-LO可通过p38通路上调糖尿病肾小球内p27^kap1的表达。

Objective To investigate the effect of 12-1ipoxygenase（12-LO） on the p27^kap1 expression in diabetic glomeruli. Methods Mesangia] cells were exposed to 12-LO product 12 （S）-HETE （10-7 mmol/L） with or without p38 MAPK （p38） inhibitor （SB203580, 1 μmol/L） for 24 hours. Rats fed with high fat diet received low dose streptozotoein （STZ, 35 mg/kg, IP injection） to develop type 2 diabetes and were divided into 2 groups： low dose STZ, low dose STZ＋12-LO inhibitor einnamyl-3,4-dihydroxy-ct-cynanocinnamate （CDC, 8 mg/kg） treatment. Rats fed with regular chow were divided into two groups： controls, CDC treatment. The rats received injection of CDC or vehicle subcutaneously in the hind leg. CDC or vehicle injection was performed three times weekly on alternate days. All the rats were sacrificed after 4 weeks. Wild type and 12-LO knockout C57BL/6 mice were divided into 4 groups： wild type control, 12-LO knockout, STZ- induced wild type type 1 diabetes and STZ-induced 12-LO knockout type 1 diabetes. All the mice were sacrificed after 16 weeks. Urine, blood, kidney cortical tissue and isolated glomeruli by sieving method were collected at the end of study respectively. Western blot and immunohistochemistry for target protein were performed respectively. Results Inhibition of p38 activation could significantly reduce p27^kap1 expression induced by 12 （S）-HETE in mesangial ceils （P〈0.01）. Increased glomerular volume, microalbuminuria, elevated glomeluli p38 activation, p27^kap1 expresssion in type 2 diabetic glomeruli was decreased after CDC treatment （P〈0.01）. Compared with wild type diabetic mice, glomerular p38 activation, p27^kap1 expresssion and extracellular matrix accumulation in the 12-LO knockout diabetic mice were significantly decreased （P 〈0.01, respectively ）. Conclusions 12-LO induces p27kipl expression via p38 pathway in diabetic glomeruli.