对氧磷酶2对氧化低密度脂蛋白诱导的乳鼠心肌细胞损伤的影响

目的 探讨对氧磷酶2(paraoxonase 2,PON2)在氧化低密度脂蛋白(oxidized low density lipoprotein,ox-LDL)诱导的乳鼠心肌细胞(neonatal rat ventricular cells,NRVCs)损伤中的作用及机制.方法 分离SD大鼠(出生1～3d,雌雄不限,共90只)心肌细胞.细胞分为:①对照组(未进行任何干预)、②0.1 μmol/Lox-LDL组(ox-LDL干预24 h)、③10 μmol/Lsalubrinal+ox-LDL组(salubrinal预处理1h后加入ox-LDL干预24 h)、④20 μg/mLPON2+ox-LDL组(PON2预处理1h后加入ox-LDL干预24 h)、⑤2.5μmol/LMn(Ⅲ)TBAP+ ox-LDL组(Mn(Ⅲ)TBAP预处理1h后加入ox-LDL干预24 h].通过MTT法和Caspase-3/7活性检测试剂盒分别检测细胞活性和细胞凋亡变化(n=6);Western blot检测心肌细胞内质网应激和氧化应激蛋白表达情况(n=4).结果 与对照组相比,ox-LDL明显降低心肌细胞活性(0.417 ±0.047) vs(O.883 ±0.064),P ＜0.01]显著增加心肌细胞凋亡(11 505 ±817)vs(6417 ±439),P ＜0.01].与ox-LDL组相比,PON2、salubrinal和Mn(Ⅲ)TBAP干预后,细胞活性显著升高(0.567 ±0.066) 、0.649 ±0.082) 、0.539 ±0.049) vs0.417 ±0.047),P ＜0.01],细胞凋亡显著下降(7 776 ±488) 、(7 545 ±547)、(7 960±480)vs (11 505 ±817),P ＜0.01).与对照组相比,ox-LDL显著增加PON2蛋白表达(0.86 ±0.223) vs (0.561 ±0.13),P ＜0.05]、内质网应激蛋白eIF-2αphox、caspase-12和氧化应激蛋白Nox2/gp91 phox、p47 phox表达(1.309 ±0.274) vs (0.780 ±0.186) 、(1.294±0.273) vs (0.606 ±0.064) 、(1.342 ±0.274) vs(0.788 ±0.137) 、(1.178 ±0.194) vs (0.629 ±0.125),P＜0.01].与ox-LDL组相比,PON2干预后,PON2蛋白表达明显升高(1.190 ±0.151) vs (0.860±0.222),P＜0.05];eIF-2αphox 、caspase-12、Nox2/gp91 phox和p47 phox蛋白表达明显降低(0.924 ±0.170) vs(1.309 ±0.274) 、(0.895 ±0.202) vs (1.294 ±0.273) 、(0.957 ±0.190) vs (1.342 ±0.274) 、(0.799±0.232) vs(1.178 ±0.194),P ＜0.05].与PON2作用类似,salubrinal干预后,eIF-2αphox和caspase-12蛋白表达明显降低(1.010±0.096) vs(1.309±0.274)、(0.788±0.042)vs(1.294±0.273),P＜0.01],Mn(Ⅲ)TBAP干预后,Nox2/gp91 phox和p47 phox蛋白表达明显降低(0.898 ±0.190) vs(1.342±0.274) 、(0.769±0.158) vs(1.178 ±0.194),P ＜0.01],二者均显著升高PON2蛋白表达(1.248±0.259) 、(1.176 ±0.248) vs(0.860 ±0.222),P ＜0.05].结论 PON2可能通过代偿性升高抑制氧化应激和内质网应激而减轻ox-LDL诱导的心肌细胞损伤.

Protective effect of paraoxonase 2 against oxidized low-density lipoprotein-induced neonatal rat myocardial cell injury in vitro

Objective To investigate the effects of paraoxonase 2 (PON2) in ameliorating neonatal rat ventricular cell (NRVC) injury induced by oxidized low density lipoprotein (ox-LDL) and explore the underlying mechanism.Methods NRVCs isolated from 90 neonatal (1 ～ 3 days old) Sprague-Dawley rats were exposed to 0.1 μmol/L ox-LDL with or without pretreatment for 1 h with 10 μmol/L salubrinal,20 μmol/L PON2,or 2.5 μmol/L Mn (Ⅲ) TBAP.The cell viability and apoptosis were detected with MTT assay and caspase-3/7 activity assay kit,and Western blotting was used to detect the expression of proteins related with endoplasmic reticulum stress (ERS) and oxidative stress (OS).Results Exposure to ox-LDL resulted in significantly reduced cell viability (0.417 ± 0.047 vs O.883 ± 0.064,P ＜ 0.01) and increased number of apoptotic cells (11 505 ± 817 vs 6 417 ± 439,P ＜ 0.01) in neonatal rat cardiac cells.Compared with cells exposed to ox-LDL alone,the cells pretreated with PON2,salubrinal or Mn (Ⅲ) TBAP exhibited significantly increased viability (0.567 ± 0.066,0.649 ± 0.082,and 0.539 ± 0.049,respectively;P ＜0.01) and decreased number of apoptotic cells (7 776 ± 488,7 545 ± 547,and 7 960 ± 480,respectively;P ＜0.01).Compared with the control cells,exposure to ox-LDL markedly increased the expression of PON2 in the cardiac cells (P ＜ 0.05),ERS-related proteins eIF-2αphox (P ＜ 0.01) and caspsae-12 (P ＜ 0.01),and OS-related proteins Nox2/gp91phox (P ＜ 0.01) and p47phox (P ＜ 0.01);pretreatment of the cells with PON2 significantly decreased the protein expressions of eIF-2αphox,caspase-12,Nox2/gp91phox,and p47phox and enhanced PON2 expression (P ＜ 0.05);pretreatment with salubrinal significantly decreased the protein expressions of eIF-2αphox and caspsae-12 (P ＜ 0.01),Mn (Ⅲ) TBAP significantly decreased the protein expressions of Nox2/gp91 phox and p47phox (P ＜ 0.01),and they both significantly increased the expression of PON2 (P ＜ 0.05).Conclusion PON2 may provide cardioprotection against NRVC injury induced by ox-LDL by the compensatory elevation of PON2 to inhibit oxidative stress and endoplasmic reticulum stress.