深低温停循环猪脑组织中小泛素样修饰蛋白表达实验研究

Expression of small ubiquitin-like modifiers in brain tissue after deep hypothermic circulatory arrest in piglet models

目的观察深低温停循环后猪脑组织中小泛素样修饰蛋白(small ubiquitin-like modifier,SUMO)2/3水平的表达。方法 24只中华小型猪中,6只行体外循环1h(体外循环组);其余18只行深低温停循环1h,其中仅行深低温停循环6只(停循环组);深低温停循环过程中行顺行脑灌注,灌注流量25mL/(kg·min)为脑灌注25组6只,灌注流量50mL/(kg·min)为脑灌注50组6只。术后2h处死猪留取海马及皮质脑组织标本,采用Western blot法检测SUMO2/3水平、SUMO特异性结合酶(E2)Ubc9、SUMO特异性蛋白酶(sentrin-specific protease,SENP)、凋亡因子Bax和caspase-3、抗凋亡因子Bcl-2表达水平。结果停循环组海马区及皮质区SUMO2/3(2 505.80±140.85、2 516.20±169.17)、Ubc9(646.32±18.46、868.39±20.63)、Bax(645.91±8.89、784.31±13.03)、caspase-3(765.80±20.84、818.06±10.86)表达水平明显高于体外循环组[SUMO2/3(889.12±5.71、886.73±10.32)、Ubc9(218.37±6.74、291.58±4.05)、Bax(202.26±2.05、300.40±3.69)、caspase-3(129.45±14.20、292.15±5.41)]、脑灌注25组[SUMO2/3(1 587.50±135.10、1 656.74±120.00)、Ubc9(522.60±14.42、569.30±19.09)、Bax(506.05±1.78、650.23±24.40)、caspase-3(566.41±20.67、408.66±13.85)]和脑灌注50组[SUMO2/3(1 207.91±115.78、1 196.63±28.37)、Ubc9(350.48±14.70、454.71±17.74)、Bax(462.20±6.86、502.49±16.73)、caspase-3(449.45±20.67、357.75±18.87)](P<0.01);SENP(251.99±12.50、340.27±16.39)、Bcl-2(431.78±19.77、224.36±14.53)表达水平明显低于体外循环组[SENP(752.26±4.45、874.10±7.93)、Bcl-2(761.33±5.77、791.97±7.27)]、脑灌注25组[SENP(380.60±13.20、411.13±11.68)、Bcl-2(535.85±18.00、420.35±21.47)]和脑灌注50组[SENP(426.63±16.42、599.30±17.55)、Bcl-2(634.86±14.84、624.67±13.15)](P<0.01);脑灌注50组海马和皮质区脑组织中SUMO2/3、Ubc9、Bax和caspase-3表达水平明显低于脑灌注25组(P<0.01),高于体外循环组(P<0.01),SENP和Bcl-2表达水平高于脑灌注25组(P<0.01),低于体外循环组(P<0.01)。结论在猪的深低温停循环模型中,选择性顺行脑灌注能起到较好的脑保护作用,其中灌注流量50mL/(kg·min)较25mL/(kg·min)具有更好的保护效果;SUMO2/3水平增加是深低温停循环脑损伤时启动的内源性应激反应,通过增加SUMO化、减少去SUMO化共同实现。

Objective To observe the expression of small ubiquitin-like modifier（SUMO）2/3in brain tissue after deep hypothermic circulatory arrest（DHCA）in piglet models.Methods In 24 piglets,6received cardiopulmonary bypass（CPB）for one hour（CPB group）,the other 18 piglets received DHCA for one hour at 18 ℃,in which 6piglets only received DHCA（DHCA group）,6received selective antegrade cerebral perfusion（SACP）at 25mL/（kg·min）during DHCA（SACP25group）,and 6piglets received SACP at 50 mL/（kg·min）during DHCA（SACP50group）.Two hours after operation,all piglets were sacrificed to get hippocampus and cerebral cortex specimens.The levels of SUMO2/3,Ubc9,sentrin-specific protease（SENP）,Bax,Bcl-2and caspase-3were detected by Western blot technique.Results In the hippocampus and cerebral cortex,the expressions of SUMO2/3（2 505.80±140.85,2 516.20±169.17）,Ubc9（646.32±18.46,868.39±20.63）,Bax（645.91±8.89,784.31±13.03）and caspase-3（765.80±20.84,818.06±10.86）were significantly higher in CPB group than those in DHCA group（SUMO2/3：889.12±5.71,886.73±10.32;Ubc9：218.37±6.74,291.58±4.05;Bax：202.26±2.05,300.40±3.69;caspase-3：129.45±14.20,292.15±5.41）,SACP25group（SUMO2/3：1 587.50±135.10,1 656.74±120.00;Ubc9：522.60±14.42,569.30±19.09;Bax：506.05±1.78,650.23±24.40;caspase-3：566.41±20.67,408.66±13.85）and SACP50group（SUMO2/3：1 207.91±115.78,1 196.63±28.37;Ubc9：350.48±14.70,454.71±17.74;Bax：462.20±6.86,502.49±16.73;caspase-3：449.45±20.67,357.75±18.87）（p〈0.01）.The expressions of SENP and Bcl-2 were significantly lower in CPB group（251.99±12.50,340.27±16.39;431.78±19.77,224.36±14.53）than those in DHCA group（SENP：752.26±4.45,874.10±7.93;Bcl-2：761.33±5.77,791.97±7.27）,SACP25group（SENP：380.60±13.20,411.13±11.68;Bcl-2：535.85±18.00,420.35±21.47）and SACP50group（SENP：426.63±16.42,599.30±17.55;Bcl-2：634.86±14.84,624.67±13.15）（p〈0.01）.The expressions of SUMO2/3,Ubc9,Bax and caspase-3were significantly lower in SACP50 group than those in SACP25 group and higher than those in DHCA group（p〈0.01）,and the expressions of SENP and Bcl-2were higher in SACP50 group than those in SACP25 group and lower than those in DHCA group（p〈0.01）.Conclusion SACP plays a good cerebral protection role in DHCA piglet models,and SACP at 50mL/（kg·min）is superior to SACP at 25mL/（kg·min）.The activation of SUMO2/3conjugation is believed to be an endogenous stress response after DHCA by acceleration of the conjugating process and inhibition of the deconjugating process.