肝细胞缺血预处理期间信号转导中蛋白激酶C依赖性激活的P44/42丝裂原活化蛋白激酶和热休克蛋白70
Protein kinase C-dependent activation of P44/42 mitogen-activated protein kinase and heat shock protein 70 in signal transduction during hepatocyte ischemic preconditioning.
作者信息
Gao Yi, Shan Yu-Qiang, Pan Ming-Xin, Wang Yu, Tang Li-Jun, Li Hao, Zhang Zhi
机构信息
Department of Hepatobiliary Surgery, Zhujiang Hospital, 253 Gongye Road, Guangzhou 510282, Guangdong Province, China.
出版信息
World J Gastroenterol. 2004 Apr 1;10(7):1019-27. doi: 10.3748/wjg.v10.i7.1019.
AIM
To investigate the significance of protein kinase C(PKC), P44/42 mitogen-activated protein kinase (MAPKs) and heat shock protein (HSP)70 signal transduction during hepatocyte ischemic preconditioning.
METHODS
In this study we used an in vitro ischemic preconditioning (IP) model for hepatocytes and an in vivo model for rat liver to investigate the significance of protein kinase C (PKC), P44/42 mitogen-activated protein kinase (P44/42 MAPKs) and heat shock protein 70 (HSP70) signal transduction in IP. Through a normal liver cell hypoxic preconditioning (HP) model in which cultured normal liver cells were subjected to 3 cycles of 5 min of incubation under hypoxic conditions followed by 5 min of reoxygenation and subsequently exposed to hypoxia and reoxygenation for 6 h and 9 h respectively. PKC inhibitor, activator and MEK inhibitor were utilized to analyze the phosphorylation of PKC, the expression of P44/42 MAPKs and HSP70. Viability and cellular ultrastructure were also observed. By using rat liver as an in vivo model of liver preconditioning (3 cycles of 10-min occlusion and 10-min reperfusion), in vivo phosphorylation of PKC and P44/42MAPKs, HSP70 expression were further analyzed. AST/ALT concentration, cellular structure and ultrastructure were also observed. All the data were statistically analyzed.
RESULTS
Similar results were obtained in both in vivo and in vitro IP models. Compared with the control without IP (or HP), the phosphorylation of PKC and P44/42 MAPKs and the expression of HSP70 were obviously increased in IP (or HP) treated model in which cytoprotection could be found. The effects of preconditioning were mimicked by stimulating PKC with 4beta phorobol-12-myristate13-acetate (PMA). Conversely, inhibiting PKC with chelerythrine abolished the protection given by preconditioning. PD98059, inhibitor of MEK (the upstream kinase of P44/42MAPKs), also reverted the cytoprotection exerted by preconditioning.
CONCLUSION
The results demonstrate that preconditioning induces a rapid activation of P44/42MAPKs and PKC activation plays a pivotal role in the activation of P44/42 MAPKs pathway that participates in the preservation of liver cells. HSP expression is regulated by signals in PKC dependent P44/42 MAPKs pathway.
目的
探讨蛋白激酶C（PKC）、P44/42丝裂原活化蛋白激酶（MAPKs）和热休克蛋白（HSP）70信号转导在肝细胞缺血预处理中的意义。
方法
本研究采用肝细胞体外缺血预处理（IP）模型和大鼠肝脏体内模型，以研究蛋白激酶C（PKC）、P44/42丝裂原活化蛋白激酶（P44/42 MAPKs）和热休克蛋白70（HSP70）信号转导在IP中的意义。通过正常肝细胞缺氧预处理（HP）模型，将培养的正常肝细胞在缺氧条件下孵育3个循环，每个循环5分钟，随后复氧5分钟，然后分别再暴露于缺氧和复氧6小时和9小时。使用PKC抑制剂、激活剂和MEK抑制剂分析PKC的磷酸化、P44/42 MAPKs和HSP70的表达。还观察了细胞活力和细胞超微结构。以大鼠肝脏作为肝脏预处理的体内模型（10分钟阻断和10分钟再灌注3个循环），进一步分析PKC和P44/42MAPKs的体内磷酸化、HSP70表达。还观察了谷草转氨酶/谷丙转氨酶浓度、细胞结构和超微结构。所有数据均进行统计学分析。
结果
体内和体外IP模型均获得相似结果。与未进行IP（或HP）的对照组相比，在可发现细胞保护作用的IP（或HP）处理模型中，PKC和P44/42 MAPKs的磷酸化以及HSP70的表达明显增加。用4β佛波醇-12-肉豆蔻酸13-乙酸酯（PMA）刺激PKC可模拟预处理的效果。相反，用白屈菜红碱抑制PKC可消除预处理给予的保护作用。MEK（P44/42MAPKs的上游激酶）抑制剂PD98059也可逆转预处理所发挥的细胞保护作用。
结论
结果表明，预处理可诱导P44/42MAPKs的快速激活，PKC激活在参与肝细胞保护的P44/42 MAPKs途径激活中起关键作用。HSP表达受PKC依赖性P44/42 MAPKs途径中的信号调节。
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