12／15-脂氧酶反义寡核苷酸对氧糖剥夺损伤皮质神经元中PPARγ核移位的影响

Effects of 12/15-1ipoxygenase antisense oligonucleofide on peroxisome proliferator-activated receptor γ translocation in primarily cultured cortical neurons after oxygen-glucose deprivation

目的观察12／15－脂氧酶反义寡核苷酸（asON-12／15-LOX）在体外培养皮质神经元中对氧糖剥夺损伤（OGD）诱导的过氧化物酶体增殖物激活受体（PPARγ）表达和核移位的影响。方法体外原代培养SD大鼠皮质神经元，随机分为正常对照组、氧糖剥夺损伤组、干预组，其中正常对照组5皿神经元、氧糖剥夺损伤组9皿神经元、干预组5皿神经元。制作体外培养皮质神经元氧糖剥夺3h再灌注24h损伤模型（OGD），干预组12／15－LOX反义寡核苷酸预处理48h后，给予OGD处理。分别采用Western印迹法和免疫荧光染色法检测12／15．LOX和PPARγ的改变。结果（1）Western印迹和免疫荧光检测显示，与正常对照组（0．25±0．14）相比，OGD组（1．73±0．78）12／15-LOX蛋白表达增加（t=-3．72，P=0．03）。Western印迹检测显示，与OGD组（1．32±0．20）相比，12／15-LOX反义寡核苷酸干预组（0．81±0．02）12／15-LOX蛋白表达降低（t=5．03，P=0．02）。差异均有统计学意义。（2）Western印迹和免疫荧光检测显示，与正常对照组（0．43±0．14）相比，OGD组（2．63±0．63）PPARγ总蛋白表达增加（t=-6．79，P=0．04）；与正常对照组（0．41±0．17）相比，OGD组（2．60±0．93）PPARγ核蛋白表达增加（t=-4．67，P=0．02）；与正常对照组（1．88±0．79）相比，OGD组（0．50±0．21）PPARγ浆蛋白表达降低（t=3．40，P=0．04）（即核移位增加）。差异均有统计学意义。（3）Western印迹和免疫荧光检测显示，与OGD组（0．23±0．08）相比，12／15-LOX反义寡聚核苷酸组（0．11±0．03）PPAR7总蛋白水平降低（t=2．83，P=0．04）。与OGD组（2．58±0．60）相比，12／15-LOX反义寡聚核苷酸组（0．44±0．10）PPARγ核蛋白表达水平降低（t=7．05，P=0．01）；与OGD组（0．18±0．11）相比，12／15-LOX反义寡聚核苷酸组（2．78±0．94）PPARγ浆蛋白表达水平增加（t=5．47，P＝0．01）（即核移位抑制）。差异均具有统计学意义。结论12／15-LOX反义寡核苷酸抑制OGD诱导的体外培养皮质神经元PPARγ表达和核移位。

Objective To explore the effects of 12/15-1ipoxygenase antisense oligonucleotide （asON-12/15-LOX） on OGD （oxygen-glucose deprivation ）-induced PPARγ （peroxisome proliferator- activated receptor 7） expression and nuclear translocation in primarily cultured cortical neurons. Methods After a 48-h pre-treatment of 12/15-LOX antisense oligonucleotide （ asON ） , primarily cultured cortical neurons underwent 3-hour OGD followed by a 24-hour reperfusion. Immunofloreseent staining and Western blot were used to evaluate the expressions of 12/15-LOX and PPARγ as well as the nuclear translocation of PPARγ. Results Compared with the control group, the expressions of 12/15-LOX and PPARγ whole protein were enhanced in OGD group （ t = - 3.72 and - 6. 79, P = 0. 03 and 0. 04）. And an increase of PPAR＇y in nucleus （ t = - 4. 67, P = 0. 02） could be noted with a simultaneous reduction in cytosol （ t = 3.40, P = 0. 04） after OGD, indicating an induction of nuclear translocation by OGD. Compared with OGD group, a pre-treatment of asON-12/15-LOX dramatically attenuated OGD-induced increase in 12/15-LOX whole protein expression （t = 5.03, P = 0. 02）. Compared with OGD group, a pre-treatment of asON-12/15- LOX greatly reduced OGD-induced increase in PPARγ total protein expression （ t = 2. 83, P = 0. 04 ） and nuclear translocation （ t = 7.05, P = 0. 01 for nuclear protein ; t = - 5.47, P = 0. 01 for cytosol protein）. It indicated a possible link between 12/15-LOX and PPARγ. Conclusion 12/15-LOX antisense oligonucleotide suppresses the expression and nuclear translocation of PPARγ in primarily cultured cortical neurons after OGD.