肝脏缺血预处理过程中蛋白激酶C活性的变化

Changes of protein kinase C activity during hepatocyte ischemic precondition

目的:观察在肝脏缺血预处理过程中蛋白激酶C的活性改变及细胞内信号转导机制。方法:实验于2004-03/2005-03在南方医科大学珠江医院中心实验室完成。实验分组:36只大鼠随机分成6组,每组6只。①假手术组。②缺血再灌注组。③缺血预处理组。④缺血再灌注+豆蔻酸佛波酰乙脂组。⑤缺血预处理+白屈菜季铵碱组。⑥缺血预处理+PD98059组。实验干预:①假手术组仅分离肝十二指肠韧带,不阻断肝门,不进行其他干预处理。②缺血再灌注组在第一肝门用小血管夹阻断尾状叶及左肝叶血流40min松开血管夹肝脏再灌注3h,再灌注开始后关腹。③缺血预处理组先行3个循环的缺血预处理,阻断第一肝门10min,开放再灌注10min为1个循环,随后操作同缺血再灌注组。④缺血再灌注+豆蔻酸佛波酰乙脂组:按4μg/kg共0.5mL蛋白激酶C激动剂豆蔻酸佛波酰乙脂溶液,经静脉通道缓慢推注,推注10min,推注后等待10min,余处理同缺血再灌注组。⑤缺血预处理+白屈菜季铵碱组:按5mg/kg共0.5mL蛋白激酶C抑制剂白屈菜季铵碱溶液,同样用10min经静脉通道缓慢推注,推注后等待10min,其余处理同缺血预处理组。⑥缺血预处理+PD98059组:按5mg/kg共0.5mL丝裂原激活的蛋白激酶抑制剂PD98058溶液,同上速度缓慢静脉推注,推注后等待10min,余处理同缺血预处理组。实验评估:①在ECHNICONRA-1000全自动生化分析仪上采用速率法检测血清谷草转氨酶和血清谷丙转氨酶活性。②按蛋白激酶C活性测定试剂盒操作步骤检测肝组织蛋白激酶C活力。③Westernblot免疫印迹法检测肝组织磷酸P44/42MAPKs活性。结果:36只大鼠均进入结果分析,中途无脱落和死亡。①血清谷草转氨酶和血清谷丙转氨酶活性:缺血预处理组低于缺血再灌注组(P<0.01)。缺血预处理+白屈菜季铵碱组、缺血预处理+PD98059组显著高于缺血预处理组(P<0.01)。缺血再灌注+豆蔻酸佛波酰乙脂组显著低于缺血再灌注组(P<0.01)。②蛋白激酶C活力:缺血预处理组高于缺血再灌注组[(1877.2±81.0),(713.1±37.0)pkat/g,P<0.01];缺血再灌注+豆蔻酸佛波酰乙脂组亦显著高于缺血再灌注组[(2758.4±454.3),(713.1±37.0)pkat/g,P<0.01];缺血预处理+白屈菜季铵碱组明显低于缺血预处理组[(567.9±46.2),(1877.2±81.0)pkat/g,P<0.01]。③磷酸P44/42MAPKs活性:与假手术组比较,其在缺血再灌注组的表达量轻度升高;缺血预处理组较缺血再灌注组升高显著;缺血预处理+白屈菜季铵碱组较缺血预处理组明显降低,缺血预处理+PD98059组表达量下降更为显著;缺血再灌注+豆蔻酸佛波酰乙脂组表达量较缺血再灌注组明显升高。结论:缺血预处理保护效应中,蛋白激酶C对P44/42MAPKs信号通路的激活是细胞保护效应的一个重要环节。

AIM： To investigate the activity of protein kinase C （PKC） in liver ischemic preconditioning （IP） and the mechanism of its signal transduction. METHODS： The experiment was carried out from March 2004 to March 2005 at the Central Laboratory of Zhujiang Hospital, Southern Medical University. Experimental group： 36 rats were divided into six groups, six rats in each group, namely sham operation group, ischemia/reperfusion （fiR） group, IP group, I/R ＋ phorbol myristic acid ethyl ester group, IP ＋ chelerythdne group, and IP ＋ PD98059 group. Experiment intervention： （1）Sham operation group： Only hepatoduodenal ligament was separated, porta hepatis was not blocked up, without carrying out other intervention treatments. （2）I/R group： The blood of caudate lobe and left lobes of liver were obstructed for 40 minutes with small vessel clip at the first porta hepatis, then small vessel clip was taken away to recover liver reperfusion for 3 hours at the same time of closing rat abdomen. （3）IP group： The first porta hepatis was obstructed for 10 minutes followed by 10-minute opening, as 1 piece of circulation, which was repeated for 3 times, sequent treatment was identical with I/R group. （4）I/R ＋ phorbol myristic acid ethyl ester group： 0.5 mL phorbol myristic acid ethyl ester （4 μg/kg） was bloused slowly viavein channel for 10 minutes, followed by closing rat abdomen 10 minutes later.（5）lP ＋ chelerythrine group： 0.5 mL chelerythrine （5 mg/kg） was bloused slowly via vein channel later, （6）IP ＋ PD98059 group： 0.5 mL PD98059 （5 mg/kg） for 10 minutes, followed by closing rat abdomen 10 minutes was bloused slowly v/a vein channel for 10 minutes, other managements was identical with above. Experiment evaluation： （1）~The level of aspartate aminotransferase （AST） and alanine aminotransferase （ALT） in the serum of rats were detected by ECHNICONRA-1000 automatic biochemistry analyzer with velocity method.（2）PKC activity in the liver tissue was detected according to the introduction of PKC activity determination kit.（3）Phosphonic acid P44/42 MAPKs activity was detected by Western blot. RESULTS： AU 36 rats entered the consequence analysis. （1）The activity of AST and ALT was lower in IP group than in I/R group （P〈 0.01）, significantly higher in IP ＋ chelerythrine group and IP ＋ PD98059 group than in IP group （P 〈 0.01）, and significantly lower in I/R ＋ phorbol mydstic acid ethyl ester group than in I/R groupp （P 〈 0.01）.（2）PKC activity was higher in IP group than in I/R group [（1 877.2±81.0）, （713.1±37.0） pkat/g, P 〈 0.01], significantly higher in I/R ＋ phorbol myristic acid ethyl ester group than in I/R group [（2 758.4±454.3）, （713.1±37.0） pkat/g, ,D 〈 0.01], and significantly lower in IP ＋ chelerythdne group than in IP group [（567.9±_46.2）, （1 877.2±81.0） pkat/g, P 〈 0.01].（3）Phosphonic acid P44/42 MAPKs activity： Compared with sham operation group, it had a mild increasing in I/R group; Compared with I/R group, it increased significantly in IP group; in IP ＋ chelerythdne group and IP ＋ PD98059 group, it was significantly lower than that in IP group; Compared with I/R group, it increased obviously in I/R ＋ phorbol rnyristic acid ethyl ester group. CONCLUSION： PKC plays a pivotal role in the activation of P44/42 MAPKs signal pathway that participates in the preservation of liver ceils in IP protection effect.