Department of Surgery, University of Michigan, Ann Arbor, MI.
Department of Anesthesia, Critical Care and Pain Medicine, Massachusetts General Hospital/Harvard Medical School, Charlestown, MA.
Surgery. 2014 Aug;156(2):221-8. doi: 10.1016/j.surg.2014.03.038. Epub 2014 Jun 18.
Therapeutic hypothermia and histone deacetylase inhibitors, such as valproic acid (VPA), independently have been shown to have neuroprotective properties in models of cerebral ischemic and traumatic brain injury. However, the depth of hypothermia and the dose of VPA needed to achieve the desired result are logistically challenging. It remains unknown whether these two promising strategies can be combined to yield synergistic results. We designed an experiment to answer this question by subjecting hippocampal-derived HT22 cells to severe hypoxia in vitro.
Mouse hippocampal HT22 cells were exposed to 200 μM cobalt chloride (CoCl(2)), which created hypoxic conditions in vitro. Cells were incubated for 6 or 30 hours under the following conditions: (1) Dulbecco's Modified Eagle Medium; (2) 200 μM CoCl(2); (3) 200 μM CoCl(2) plus 1 mmol/L VPA; (4) 200 μM CoCl(2) plus 32°C hypothermia; and (5) 200 μM CoCl(2) plus both 1 mmol/L VPA and 32°C hypothermia. Cellular viability was evaluated by (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and lactate dehydrogenase release assays at 30 hours after treatment. Levels of acetylated histone H3, hypoxia-inducible factor-1α, phospho-GSK-3β, β-catenin, and high-mobility group box-1 were measured by Western blotting.
High levels of acetylated histone H3 were detected in the VPA-treated cells. The release of lactate dehydrogenase was greatly suppressed after the combined hypothermia + VPA treatment (0.269 ± 0.003) versus VPA (0.836 ± 0.026) or hypothermia (0.451 ± 0.005) treatments alone (n = 3, P = .0001). (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay showed that the number of viable cells was increased by 17.6 % when VPA and hypothermia were used in combination (n = 5, P = .0001). Hypoxia-inducible factor-1α and phospho-GSK-3β expression were synergistically affected by the combination treatment, whereas high-mobility group box-1 was increased by VPA treatment, and inhibited by the hypothermia.
This is the first study to demonstrate that the neuroprotective effects of VPA and hypothermia are synergistic. This novel approach can be used to develop more effective therapies for the prevention of neuronal death.
在脑缺血和创伤性脑损伤的模型中，已证明治疗性低温和组蛋白去乙酰化酶抑制剂（如丙戊酸（VPA））具有神经保护作用。然而，需要达到所需效果的低温深度和 VPA 剂量在后勤上具有挑战性。目前尚不清楚这两种有前途的策略是否可以结合使用以产生协同作用。我们设计了一项实验，通过将海马源性 HT22 细胞在体外进行严重缺氧来回答这个问题。
将小鼠海马 HT22 细胞暴露于 200μM 氯化钴（CoCl 2 ）中，在体外产生缺氧条件。在以下条件下孵育细胞 6 或 30 小时：（1）杜尔贝科改良伊格尔培养基；（2）200μM CoCl 2 ；（3）200μM CoCl 2 加 1mmol/L VPA；（4）200μM CoCl 2 加 32°C 低温；和（5）200μM CoCl 2 加 1mmol/L VPA 和 32°C 低温。在治疗后 30 小时通过（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）和乳酸脱氢酶释放测定评估细胞活力。通过 Western 印迹测定乙酰化组蛋白 H3、缺氧诱导因子-1α、磷酸化 GSK-3β、β-连环蛋白和高迁移率族蛋白盒 1 的水平。
在 VPA 处理的细胞中检测到高水平的乙酰化组蛋白 H3。与 VPA（0.836±0.026）或低温（0.451±0.005）单独处理相比，联合低温+VPA 处理后乳酸脱氢酶的释放大大抑制（0.269±0.003）（n=3，P=0.0001）。（3-（4,5-二甲基噻唑-2-基）-2,5-二苯基四唑溴化物）测定表明，当 VPA 和低温联合使用时，存活细胞数增加了 17.6%（n=5，P=0.0001）。缺氧诱导因子-1α和磷酸化 GSK-3β的表达受联合处理的协同影响，而高迁移率族蛋白盒 1 受 VPA 处理的影响，受低温的抑制。
这是第一项表明 VPA 和低温的神经保护作用具有协同作用的研究。这种新方法可用于开发更有效的预防神经元死亡的治疗方法。
