从冷诱导RNA结合蛋白表达探讨参附方对颅脑损伤大鼠亚低温治疗的神经保护机制

Mild hypothermia therapy has became one of the most important and common treatment for Trauma Brain Injury patients during acuted period. This menthod demonstrated effective functions, however, it had some disadvantages including the shortage of neuro-protection of TBI patients. In order to keep the advantage of mild hypothermia, our team initially distinguished the symptoms of TBI patients in mild hypothermia using TCM, finding those patients in the single case as the result of medical control, and then We used the shenfu decoction cure TBI patients in mild hypothermia, showed the significant effection of the neuro-protection. The study intent to demonstrate the mechanism for this decoction to mild hypothermia by the way of the expression of the key protein, CIRP, expressed differently in diverses area in rats' brain of TBI in mild hypothermia ,such as higher expression in hypothalamus than hippocampus and cortex during the early study.At same time, the early study showed that the neuro-protection of CIRP to mild hypothermia may rely on the adjustion for ERK pathway. Above those studies and theories, we draw a hypothesis that the mechanism for this decoction to hypothermia by promoted the expression of CIRP in hippocampus and cortex to directly stimulate the ERK pathway to achieve the function of neuron-protection, and verified by the RNAi experiment. Through this study we want to explicitly demonstrate the key pathway of the regulation for the shenfu decoction to the special symptom of TBI patients in mild hypothermia and proclaim its mechanism in order to prefer the theoretical sustention to using TCM make up the shortage of mild hypothermia treatment.

亚低温已成为TBI患者急性期最重要、最普遍治疗措施之一，虽其有效性得到认可，但也存在不足之处，主要包括对患者某些重要神经功能保护欠佳。前期研究针对TBI患者亚低温状态进行了中医辨证分型，发现因医源性调控，此类患者处于寒淤闭证的特殊单一证候，在此基础上辨证应用参附方可弥补亚低温缺陷，使患者神经功能得到显著改善。CIRP是温度依赖调控蛋白，在亚低温神经保护中发挥关键作用，在亚低温期不同部位脑组织存在表达差异，此点与亚低温神经保护作用不足之处相对应，同时ERK通路是CIRP发挥亚低温保护作用的调控通路，本次研究推测该方作用机制可能为：通过促进皮层、海马等不同部位脑组织内CIRP表达进而直接激活ERK信号途径发挥神经保护作用，并应用基因静默体内实验进行验证。通过本次研究明确参附方对TBI患者亚低温期这一特殊中医证型的关键调控作用点并揭示其机制，为中药辨证应用弥补亚低温治疗重要缺陷提供基础理论支撑。

项目的背景：经前期临床观察及研究显示，应用参附方对亚低温治疗TBI患者具有显著而确切的神经保护作用，但其分子生物学及药物动力学作用机制不详，本次研究拟通过对TBI亚低温期脑组织不同部位差异表达、亚低温作用关键蛋白CIRP及可能发挥作用的ERK调控通路为观察目标，在药物作用前后各部位脑组织进行检验对比研究，明确参附方对TBI患者亚低温期的关键调控作用点。研究内容：将大鼠随机分为非转染对照组、AD5-GFP转染组及AD5-GFP-CIRP-SiRNA转染组，再将3组分为模型亚组、中药治疗低、高剂量亚组，取大鼠脑皮质、海马区、下丘脑部位脑组织，用TUNEL法测定脑细胞凋亡指数， RT-PCR测定CIRP mRNA表达， Western Blot法测定Ras、Shc、PKC、ERK、 MEK蛋白表达量。结果显示：非转染对照组、AD5-GFP转染组中低、高剂量药物治疗亚组脑皮质、海马区、下丘脑AI均较模型组降低，CIRP mRNA表达均较模型组升高；AD5-GFP-CIRP-SiRNA转染组中模型组和低、高剂量药物治疗亚组脑皮质、海马区、下丘脑AI及CIRP mRNA表达差异均无统计学意义，但AI均明显高于非转染对照组和AD5-GFP转染组相应组，CIRP mRNA明显低于非转染对照组和AD5-GFP转染组相应组。各组各部位Ras、Shc、PKC、MEK 蛋白表达水平比较差异均无统计学意义（均P＞0.05），非转染对照组、AD5-GFP转染组低、高剂量药物治疗亚组脑皮质、海马区、下丘脑p-ERK/ERK均较模型组降低，且以高剂量亚组降低更显著〔脑皮质：非转染对照组为7.2±1.0：15.3±1.8，AD5-GFP转染组为8.1±0.7：16.2±1.5；海马区：非转染对照组为6.6±0.8：14.7±2.0，AD5-GFP转染组为6.8±1.0：14.9±1.3；下丘脑：非转染对照组为9.4±1.1：12.7±1.7，AD5-GFP转染组为10.6±1.3：9.4±1.1，均P＜0.05〕；AD5-GFP-CIRP-SiRNA转染组各亚组各部位p-ERK/ERK比较差异均无统计学意义（均P＞0.05）。科学意义：参附方可能通过促进CIRP过表达，降低ERK表达，抑制继发的转录因子磷酸化的启动信号转导，从而减少神经细胞凋亡，发挥其辅助亚低温治疗的抗凋亡作用。