不同浓度CB2受体激动剂预处理对大鼠海马神经元凋亡的保护作用及机制

Neuroprotection of CB2 agonist preconditioning of different concentration on rat hippocampal neurons apoptosis and its mechanism

目的探讨CB2受体激动剂AM1241预处理对缺氧/复氧后大鼠海马神经元凋亡的影响。方法离体培养的大鼠海马神经元,随机分成6组:对照组(Con组),细胞未做特殊处理。CoCl_2组:300μM CoCl_2预处理4 h后,正常培养基继续培养24 h,然后用无血清培养基培养。AM1241 1μmol/L组、AM1241 3μmol/L组、AM1241 5μmol/L组、AM1241 10μmol/L组:培养孔中分别加入浓度为1、3、5、10μM AM1241处理2 h后,加入300μM的Co Cl2处理4 h,然后更换正常的培养基培养24 h,之后更换无血清的培养基培养。MTT法测定细胞增殖,RT-PCR技术检测bcl-2、caspase-3、iNOS mRNA的表达。结果与AM1241 1μmol/L组和AM12413μmol/L比较,AM1241 5μmol/L组和AM1241 10μmol/L组预处理明显增加海马神经元的增殖能力(P<0.05或P<0.01),上调bcl-2 mRNA的表达,下调促凋亡基因caspase-3和iNOS mRNA的表达(P<0.05或P<0.01)。结论 5μM和10μM AM1241预处理通过对凋亡相关基因的调控,对缺氧/复氧后的海马神经元有保护作用。

Objective To determine the effects of CB2 agonist AM1241 preconditioning on rat hippocampal neurons apoptosis induced by anoxia-reoxygenation. Methods Hippocampal neurons were cultured and randomly assigned to six groups ： control group ; CoCl2 group, in which cells were treated with 300 μM CoCl2 （ chemical anoxia analogue） for 4 h, cultured with normal medium for 24 h, and then stimulated with out serum media;AM1241 1 μmol/L, AM1241 3 μmo1/L,AM1241 5 p, mol/L and AM1241 10 μmol/L group：the neurons were pretreated with 1,3,5 ,and 10μM AM1241 ,then 300 μM CoC12 were added after 2 h,then the others were the same as CoC12 group. MTT method was used to detect the proliferation of neurons. Then RT-PCR method was used to determine the expression of bcl-2,caspase-3 ,iNOS mRNA. Results Compared with AM1241 1 μmol/L and AM1241 3μmol/L group,when pretreated with 5μM and 10μM AM1241, cell proliferation increased （ P 〈 0. 05 or P 〈 0.01 ）. The bcl-2 mRNA was upregulated, and caspase-3 and iNOS mRNA were down-regulated （ P 〈 0. 05 or P 〈 0. 01 ）. Conclusion Preconditioning with 5 μM and 10 μM AM1241 can protect hippocampal neurons against anoxia-reoxygenation injury by attenuating apoptosis.