苯并[a]芘对神经胶质细胞中胰岛素降解酶与脑啡肽酶表达的影响

Effects of benzo(a) pyrene on expressions of insulin-degrading enzyme and neprilysin in neuroglia cells

目的:探讨苯并[a]芘[benzo(a)pyrene,Ba P]对神经胶质细胞中具有β-淀粉体(β-amyloid,Aβ)清除作用的胰岛素降解酶(insulin-degrading enzyme,IDE)及脑啡肽酶(neprilysin,NEP)表达的影响。方法:制备新生SD大鼠原代神经胶质细胞混合培养体系,接受不同浓度的Ba P、Aβ1-42寡聚体、Aβ1-42纤维体单独或联合处理24 h后,采用细胞增殖-毒性检测试剂盒测定细胞存活率。随机分为对照组、Ba P(2.00μmol/L)组、Aβ1-42(20.00 mg/L)寡聚体组、Ba P+Aβ1-42寡聚体组、Aβ1-42(20.00 mg/L)纤维体组、Ba P+Aβ1-42纤维体组,其中Ba P均为预处理12 h后再与不同聚集状态的Aβ1-42共同作用。进一步采用实时荧光定量PCR及蛋白质免疫印迹技术检测体系中IDE、NEP的mRNA和蛋白表达水平。结果:20.00μmol/L及以下浓度的Ba P处理,20.00、40.00 mg/L的Aβ1-42寡聚体或Aβ1-42纤维体单独处理,Ba P与Aβ1-42寡聚体或Ba P与Aβ1-42纤维体联合处理对神经胶质细胞的存活率均无明显影响。与对照组相比,Ba P单独处理组中IDE、NEP的mRNA和蛋白水平均未明显改变;而Aβ1-42寡聚体单独作用可明显上调IDE的mRNA及蛋白水平(P<0.05),同时Ba P预处理可显著抑制Aβ1-42寡聚体作用引起的IDE表达水平上调(P<0.05);另一方面,Aβ1-42纤维体在单独处理或有Ba P预处理情况下,IDE、NEP的mRNA和蛋白水平均未发生明显改变。结论:在对细胞存活率无明显影响的前提下,Ba P预处理显著抑制了Aβ寡聚体作用后的IDE表达水平上调,提示苯并[a]芘可能干扰Aβ寡聚体降解途径导致Aβ增多聚集,进而可能促进认知功能降低及阿尔兹海默病的形成。

Objective： To investigate effects of benzo（ a） pyrene（ Ba P） on expressions of insulindegrading enzyme（ IDE） and neprilysin（ NEP） which have the ability to degrade β-amyloid（ Aβ） in neuroglia cells. Methods： Primary mix-neuroglia cells were cultured from newborn SD rats. After exposure to Ba P,Aβ1-42 oligomer or Aβ1-42 fiber individually or jointly for 24 h,the cell survival rate was measured by cell counting kit-8（ CCK-8）. Afterwards,the primary mix-neuroglia cells were divided randomly into six groups： Control group,Ba P group（ 2. 00 μmol/L）,Aβ1-42 oligomer group（ 20. 00 mg/L）,Ba P plus Aβ1-42 oligomer group,Aβ1-42 fiber group（ 20. 00 mg/L） and Ba P plus Aβ1-42 fiber group,of which Ba P was pretreated for 12 h followed by cotreatment with different aggregated Aβ1-42. The expressions of IDE and NEP were measured by quantitative real-time polymerase chain reaction（ qRT-PCR） for mRNA level and Western blotting for protein level. Results： The cell survival rate showed no significant differences after treatment with Ba P（ ≤ 20. 00 μmol/L）,Aβ1-42 oligomer（ 20. 00,40. 00 mg/L）,Aβ1-42 fiber（ 20. 00,40. 00 mg/L） or cotreatment with Ba P and Aβ1-42 oligomer or Ba P and Aβ1-42 fiber. Compared with the control group,expressions of IDE and NEP in Ba P-treated alone group had no obvious change; however,exposure to Aβ1-42 oligomer alone significantly increased the mRNA and protein level of IDE（ P〈0. 05）,and the Ba P pretreatment could significantly inhibit the up-regulated expressions of IDE by Aβ1-42 oligomer（ P〈0. 05）; on the other hand,exposure either to Aβ1-42 fiber alone or under the Ba P pretreatment did not change the mRNA and protein level of IDE and NEP obviously. Conclusion： On the premise of no significant change of cell survival rate,Ba P pretreatment inhibited the up-regulated expressions of IDE in primary mixed neuroglia cells under cotreatment with Aβ oligomer,indicating that Ba P may disturb degradation of Aβ oligomer and cause deposition of β-amyloid and further induce cognitive decline and acceleration of Alzheimer.