Yin Cheng, Huang Guang-Fu, Sun Xiao-Chuan, Guo Zongduo, Zhang John H
Departments of Anesthesiology and Physiology, Loma Linda University School of Medicine, Loma Linda, CA, USA; Department of Neurosurgery, Affiliated Hospital of the University of Electronic Science and Technology of China, Sichuan Provincial People's Hospital, Chengdu, China.
Department of Neurosurgery, Affiliated Hospital of the University of Electronic Science and Technology of China, Sichuan Provincial People's Hospital, Chengdu, China.
Neurobiol Dis. 2017 Jul;103:133-143. doi: 10.1016/j.nbd.2017.04.006. Epub 2017 Apr 8.
Dual leucine zipper kinase (DLK/MA3K12) has been reported involved in apoptosis and neuronal degeneration during neural development and traumatic brain injury. This study was designed to investigate the role of DLK with its adaptor protein JNK interacting protein-3 (JIP3), and its downstream MA2K7/JNK signaling pathway in early brain injury (EBI) after subarachnoid hemorrhage (SAH) in a rat model.
Controlled in vivo laboratory study.
Animal research laboratory.
Two hundred and twenty-three adult male Sprague-Dawley rats weighing 280-320g.
SAH was induced by endovascular perforation in rats. The SAH grade, neurological score, and brain water content were measured at 24 and 72h after SAH. Immunofluorescence staining was used to detect the cells that expressed DLK. The terminal deoxynucleotid transferase-deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) was used to detect the neuronal apoptosis. In mechanism research, the expression of DLK, JIP3, phosphorylated-JNK (p-JNK)/JNK, and cleaved caspase-3 (CC-3) were analyzed by western blot at 24h after SAH. The DLK small interfering RNA (siRNA), JIP3 siRNA, MA2K7 siRNA and recombinant DLK protein which injected intracerebroventricularly were given as the interventions.
The DLK expression was increased in the left cortex neurons and peaked at 24h after SAH. DLK siRNA attenuated brain edema, reduced neuronal apoptosis, and improved the neurobehavioral functions after SAH, but the recombinant DLK protein deteriorated neurobehavioral functions and brain edema. DLK siRNA decreased and recombinant DLK protein increased the expression of MA2K7/p-JNK/CC-3 at 24h after SAH. The JIP3 siRNA reduced the level of JIP3/MA2K7/p-JNK/CC-3, combined DLK siRNA and JIP3 siRNA further decreased the expression of DLK/MA2K7/p-JNK/CC-3, and MA2K7 siRNA lowered the amount of MA2K7/p-JNK/CC-3 at 24h after SAH.
As a negative role, DLK was involved in EBI after SAH, possibly mediated by its adaptor protein JIP3 and MA2K7/JNK signaling pathways. To reduce the level of DLK may be a new target as intervention for SAH.
双亮氨酸拉链激酶（DLK/MA3K12）已被报道参与神经发育和创伤性脑损伤过程中的细胞凋亡及神经元变性。本研究旨在探讨DLK及其衔接蛋白JNK相互作用蛋白3（JIP3），以及其下游的MA2K7/JNK信号通路在大鼠蛛网膜下腔出血（SAH）后早期脑损伤（EBI）中的作用。
体内对照实验室研究。
动物研究实验室。
223只体重280 - 320g的成年雄性Sprague-Dawley大鼠。
通过血管内穿刺诱导大鼠SAH。在SAH后24小时和72小时测量SAH分级、神经功能评分及脑含水量。采用免疫荧光染色检测表达DLK的细胞。用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）检测神经元凋亡。在机制研究中，于SAH后24小时通过蛋白质免疫印迹法分析DLK、JIP3、磷酸化JNK（p-JNK）/JNK及裂解的半胱天冬酶-3（CC-3）的表达。将脑室内注射的DLK小干扰RNA（siRNA）、JIP3 siRNA、MA2K7 siRNA及重组DLK蛋白作为干预手段。
SAH后左侧皮质神经元中DLK表达增加，并在24小时达到峰值。DLK siRNA减轻了SAH后的脑水肿，减少了神经元凋亡，改善了神经行为功能，但重组DLK蛋白使神经行为功能和脑水肿恶化。SAH后24小时，DLK siRNA降低而重组DLK蛋白增加了MA2K7/p-JNK/CC-3的表达。JIP3 siRNA降低了JIP3/MA2K7/p-JNK/CC-3的水平，联合使用DLK siRNA和JIP3 siRNA进一步降低了DLK/MA2K7/p-JNK/CC-3的表达，且MA2K7 siRNA降低了SAH后24小时MA2K7/p-JNK/CC-3的量。
作为一种负面作用，DLK参与了SAH后的EBI，可能是通过其衔接蛋白JIP3和MA2K7/JNK信号通路介导的。降低DLK水平可能成为SAH干预的新靶点。
