深低温停循环脑损伤中GluK2受体小泛素化修饰蛋白2/3化的作用机制研究

Mechanisms of small ubiquitin-like modifier-2/3-ylation of GluK2 receptor in brain injury after deep hypothermic circulatory arrest

目的探究Glu K2受体小泛素化修饰蛋白(SUMO) 2/3化在猪模型深低温停循环(DHCA脑损伤中的作用机制。方法将18只中华小型猪随机分为体外循环组(CPB组)、DHCA组和选择性脑灌注(SACP)组,每组6只。建立猪DHCA和SACP模型,术后2 h处死猪留取海马区脑组织标本。蛋白质印迹法检测海马区脑组织中SUMO2/3化水平、Glu K2、p-MLK3、MLK3、p-JNK3、JNK3表达;免疫共沉淀检测Glu K2 SUMO2/3化的程度以及Glu K2和MLK3蛋白之间的相互作用。TUNEL法检测海马区脑组织凋亡情况;免疫组织化学染色检测Bax、Bcl-2和Caspase-3表达。结果 DHCA组海马区脑组织中SUMO2/3和Glu K2表达[(1. 438±0. 011)、(1. 965±0. 107)]较SACP组[(1. 220±0. 039)、(1. 372±0. 196),均P <0. 05]和CPB组[(0. 443±0. 025)、(0. 986±0. 181),均P <0. 01]明显增加。DHCA组海马区脑组织中Glu K2-SUMO2/3和Glu K2-MLK3表达较其他2组明显增加(均P <0. 01)。DHCA组p-MLK3/MLK3和p-JNK3/JNK3表达较其他2组明显增加(均P <0. 05)。TUNEL检测发现DHCA组海马区神经元凋亡最明显,凋亡率为64. 8%,较CPB组(15. 8%)和SACP组(47. 0%)明显增加(均P <0. 001)。免疫组织化学结果显示DHCA组海马区脑组织中Bax和Caspase-3表达较CPB组和SACP组明显增加,而Bcl-2表达较其他2组明显减少。结论 DHCA脑损伤时可能通过激活Glu K2受体发生SUMO2/3化,增加Glu K2受体和下游MLK3蛋白的相互作用,激活MLK3发生磷酸化,进一步激活下游的JNK3启动凋亡信号。

Objective To explore the role of small ubiquitin-like modifier-2/3( SUMO2/3)-ylation of Glu K2 receptor in brain injury after deep hypothermic circulatory arrest( DHCA) in piglet model. Methods Eighteen piglets were randomly divided into cardiopulmonary bypass( CPB) group,DHCA group and selective antegrade cerebral perfusion( SACP) group,with 6 piglets in each group. Two hours after making DHCA and SACP models,the animals were executed and hippocampal brain tissues were removed. Western blotting was used to test SUMO2/3-ylation,expressions of Glu K2,p-MLK3,MLK3,p-JNK3 and JNK3. Co-immunoprecipitation was used to detect the SUMO2/3-ylation of Glu K2 and the interaction between Glu K2 and MLK3. TUNEL was used to observe cell apoptosis in hippocampus and immunohistochemistry was used to detect expressions of Bax,Bcl-2 and Caspase-3.Results In hippocampus,DHCA was sufficient to significantly increase SUMO2/3-ylation and activation of Glu K2[( 1. 438 ± 0. 011),( 1. 965 ± 0. 107) ]compared with SACP group[( 1. 220 ± 0. 039),( 1. 372 ± 0. 196),P <0. 05] and CPB group(( 0. 443 ± 0. 025),( 0. 986 ± 0. 181),P < 0. 01)Expressions of Glu K2-SUMO2/3 and Glu K2-MLK3 were significantly higher in DHCA group than those in other two groups( P < 0. 01),which could activate phosphorylations of MLK3 and JNK3( P < 0. 05). TUNEL assay demonstrated more neuronal apoptosis( 64. 8%) of hippocampus in DHCA group than in CPB group( 15. 8%) and SACP group( 47. 0%)( P < 0. 001).Immunohistochemistry indicated high levels of Bax and Caspase-3 and low level of Bcl-2 in hippocampus in DHCA group than in other two groups. Conclusion SUMO2/3-ylation of Glu K2 in hippocampus may promote its binding with MLK3 to activate the phosphorylation of MLK3 and MLK3-JNK3 pathway,which may be responsible for brain injury after DHCA.