氯化锂抑制血管平滑肌细胞增殖和迁移以及抗神经细胞损伤的分子机制研究

【作者】 王竹瑶；

【导师】 刘畅；

【作者基本信息】 南京师范大学 ， 细胞生物学， 2014， 博士

【摘要】 作为化学元素周期表中最轻的元素之一,锂离子用于治疗精神疾病的历史已过百年。锂离子既能有效控制急性躁狂和急性抑郁,又能预防躁狂和抑郁反复发作,因此是治疗双相和单相抑郁性障碍的首选药物之一。血管平滑肌细胞(vascular smooth muscle cells,VSMCs)的病理性增殖和迁移是导致诸多心血管病变的重要诱因,同时也是发生支架内再狭窄的独立风险性因素。近年来的研究显示,锂离子对心血管系统同样具有保护作用。然而,目前有关这方面的报道还主要停留在现象的描述上,缺乏深入细致的机制探讨。转录共激活因子PGC-1α(peroxisome proliferator activated receptor y coactivator1α)是近年来发现的最为重要的能量代谢调节因子。近期我们注意到,PGC-1α在调控VSMCs功能方面同样有着活跃的表现,提高PGC-1α的表达对内膜增生的治疗具有重要的应用价值。我们通过实验发现,预孵氯化锂(lithium chloride, LiCl)可以剂量依赖性地提高PGC1α的蛋白表达量以及核定位。四甲基偶氮唑盐(MTT)法和乙炔脱氧尿嘧啶核苷(EdU)法证明LiCl可以抑制血清诱导的VSMCs增殖。相似地,划痕癒合(wound healing)实验和迁移小室(Transwell)实验证实LiCl可以抑制血清诱导的VSMCs迁移。LiC1同样可以阻滞血清诱导的氧自由基(reactive oxygen species, ROS)产生和细胞周期的病理性进程。在分子水平上,LiC能够减少细胞重新进入细胞周期、粘附、炎症和迁移所需的蛋白表达量。此外,在体内水平,大鼠接受连续14天的LiCl灌胃,减缓了球囊损伤模型后内膜增生的病理性变化。更为重要的是,通过PGC-1α siRNA干扰PGC-1α的表达,能够明显削弱LiCl在体内体外水平上对于VSMCs的保护效果。局部麻醉对于脊椎麻醉和病痛缓解有着至关重要的作用。但是,运用局部麻醉可能会引起神经毒性并导致手术后病人神经学上的并发症。我们探索了LiCl对于神经毒性的保护作用。在我们的研究中采用体外培养的小鼠神经母细胞瘤细胞株(mouse neuroblastoma neuro2a, N2a),探讨LiCl对其突触生长的影响及其机制。结果显示,预孵LiCl可以减少布比卡因引起的N2a细胞损伤,其作用逆转了布比卡因所导致的Akt (threonine-serine protein kinase B)以及ERKs (extracellular-signal regulated kinases)信号通路的下降并阻滞线粒体膜电位的降低。我们还观察到,Akt以及ERKs信号通路的抑制剂可以削弱LiCl对于布比卡因所致神经损伤的保护作用。综上所述,我们的研究表明,LiCl对于体外水平的VSMCs增殖和迁移,以及体内水平的内膜增生病理模型有着重要的保护意义。另外,LiCL可以同时激活N2a细胞内Akt以及ERKs信号通路。LiCl主要通过Akt以及ERKs信号通路保护N2a细胞神经突触免受布比卡因的细胞毒性。更多还原

【Abstract】 As one of the lightest solid elements, lithium has been used as a mood stabilizer for more than one century. Studies in recent years have shown that lithium also plays a protective role on the cardiovascular system.The proliferation and migration of vascular smooth muscle cells (VSMCs) is a major pathophysiological process leading to various cardiovascular disorders such as in-stent restenosis. However, previous studies stay in the descriptive stage and the direct molecular target responsible for the beneficial action of lithium in cardiovascular system remains unknown.Peroxisome proliferator-activated receptor coactivator-la (PGC-la) is a transcriptional coactivator intensively involved in the regulation of cellular energy metabolism. Recently, We have noted that PGC-la could negatively regulate the pathological activation of VSMCs and increase of PGC-la expression may have great potentials to treat restenosis.We found that pretreatment of LiCl increased PGC-laprotein expression and nuclear translocation in a dose-dependent manner. MTT and EdU incorporation assays indicated that LiCl inhibited serum-induced VSMC proliferation. Similarly, deceleration of VSMC migration was confirmed by wound healing and transwell assays. LiCl also suppressed serum-induced ROS generation and cell cycle progression. At the molecular level, LiCl reduced the protein which involved in the cell cycle re-entry, adhesion, inflammation and motility. In addition, in vivo administration of LiCl alleviated the pathophysiological changes in balloon injury-induced neointima hyperplasia. More importantly, knockdown of PGC-laby siRNA significantly attenuated the beneficial effects of LiCl on VSMCs both in vitro and in vivo.Local anesthetics (LAs) are necessary for the regional anesthesia, spinal anesthesia, and pain management. However, the application of LAs may cause neurotoxicity and result in postoperative neurological complications. In the second part, we evaluated the effects of LiCl on bupivacaine (a frequently used LAs) induced injury in mouse neuroblastoma neuro2a(N2a) cells. Pretreatment of N2a cells with LiCl significantly attenuated bupivacaine-induced cell injury. LiCl pretreatment completely reversed the suppression of Akt and ERKs signalings and significantly prevented the decline of transmembrane potentialin N2a cells after bupivacaine treatment. More importantly, pharmacological inhibition of Akt and ERKs diminished the protective effect of LiCl against bupivacaine-induced neuronal death.In conclusion, our research shows that LiCl has great potentials on the prevention and treatment of cardiovascular diseases related to VSMC abnormal proliferation and migration. In addition, LiCl increases the phosphorylation of ERKs and Akt. LiCl pretreatment provides a protective effect on bupivacaine induced neuronal cell injury. This action of LiCl is mediated through, at least in part, the activating of Akt-and ERKs-dependent mechanisms.更多还原