摘要
目的：从冷诱导RNA结合蛋白（CIRP）表达推测参附方辅助亚低温治疗颅脑损伤的作用机制。方法将SD大鼠按随机数字表法分为非转染对照组、腺病毒介导免疫荧光逆转录病毒组（AD5-GFP转染组）及腺病毒介导携CIRP静默表达基因免疫荧光逆转录病毒组（AD5-GFP-CIRP-SiRNA转染组），每组30只；再将3组分为颅脑损伤和亚低温治疗模型组、中药低、高剂量组，每组10只。中药组于亚低温治疗48 h后经尾静脉注射参附注射液1 mL/kg（低剂量组）和5 mL/kg（高剂量组），模型组注射1 mL/kg生理盐水，均每日1次，连用2 d。两个转染组先采取病毒载体大鼠鞘内给药复制病毒转染模型〔分别一次性鞘内注射0.1 mL空白AD5-GFP和0.1 mL（1×1010 pfu/mL）AD5-GFP-CIRP-SiRNA，非转染对照组给予0.1 mL生理盐水〕。取大鼠脑皮质、海马区、下丘脑部位脑组织，用原位末端缺刻标记试验（TUNEL）测定脑细胞凋亡情况，反转录-聚合酶链反应（RT-PCR）测定CIRP mRNA表达，蛋白质免疫印迹试验（Western Blot）测定大鼠肉瘤蛋白（Ras）、 Ras活化相关因子（Raf）、细胞外信号调节激酶（ERK）、磷酸化ERK（p-ERK）、丝裂原活化蛋白激酶 ERK（MEK）、磷酸化MEK（p-MEK）的蛋白表达量。结果非转染对照组、AD5-GFP转染组中药低、高剂量组脑皮质、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转染组中模型组和中药低、高剂量组脑皮质、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Raf/Ras、p-MEK/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转染组中药低、高剂量组脑皮质、海马区、下丘脑p-ERK/ERK均较模型组降低，且以中药高剂量组降低更显著〔脑皮质：非转染对照组为7.2±1.0比15.3±1.8，AD5-GFP转染组为8.1±0.7比16.2±1.5；海马区：非转染对照组为6.6±0.8比14.7±2.0，AD5-GFP转染组为6.8±1.0比14.9±1.3；下丘脑：非转染对照组为9.4±1.1比12.7±1.7，AD5-GFP转染组为10.6±1.3比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）。结论参附注射液可能通过促进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。
ObjectiveFrom the changes of expression of cold-inducible RNA-binding protein （CIRP） in rats with traumatic brain injury under mild hypothermia treatment with Shenfu decoction as a subsidiary, to speculate the mechanism of protective effect of the decoction on the injury.Methods Ninety Sprague-Dawley （SD） rats were divided into three groups by random number table： non-transfection control group, adenovirus mediated immune flourescent reverse transcription virus group （blank AD5-GFP transfection group） and adenovirus mediated immune flourescent reverse transcription virus carrying CIRP silent expression gene group （AD5-GFP-CIRP-SiRNA transfection group）, 30 rats in each groups. Then, each group was subdivided into three subgroups： model group, traditional Chinese medicine （TCM） low and high dose groups, 10 rats in each subgroup. After the mild hypothermia treatment for 48 hours, in the TCM low dose group and high dose group, a dose of TCM 1 mL/kg and 5 mL/kg was injected via a tail vein into the rat respectively, while in the model group, 1 mL/kg normal saline was injected into the same vein, once a day for consecutive 2 days in all the groups. Before modeling in the blank AD5-GFP transfection group and AD5-GFP-CIRP-SiRNA transfection group, virus transfection models were reproduced at first by one-time intrathecal injection of 0.1 mL AD5-GFP and 0.1 mL （1×1010 pfu/mL） AD5-GFP-CIRP-SiRNA virus vector respectively, and in model group, 0.1 mL normal saline was given. The rat cortex, hippocampus and hypothalamus part were collected, the brain cell apoptosis was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling （TUNEL）, the CIRP mRNA expression in the cortex, hippocampus and hypothalamus part was measured by reverse transcription-polymerase chain reaction （RT-PCR）, the protein expressions of rat sarcoma protein Raf, Ras, extracellular signal-regulated kinase （ERK）, phosphorylation ERK （p-ERK）, mitogen activated protein kinase （MEK）, p-MEK were determined by Western Blot.Results The brain tissue cell apoptosis indexes （AI） in the cortex, hippocampus and hypothalamus part in TCM low, high dose group of non-transfection control and blank AD5-GFP transfection group were lower than those in model group, and the expressions of CIRP mRNA were higher than those in model group, there were no significant differences in AI and CIRP mRNA in the cortex, hippocampus and hypothalamus between model, TCM low and high dose groups of AD5-GFP-CIRP-SiRNA transfection group, but AI was significantly higher and CIRP mRNA was significantly lower than that in corresponding subgroups of AD5-GFP transfection control group and blank AD5-GFP transfection group. Western Blot detection showed that： Raf/Ras, p-MEK/MEK protein expressions revealed no statistical significant differences in different parts of each group （allP 〉 0.05）, the p-ERK/ERK protein expression in the cortex, hippocampus, and hypothalamus part was significantly lower in TCM low and high dose group than that in the model group of non-transfection control group and blank AD5-GFP transfection group, the degree of descent in the TCM high dose group being more significant （the cortex： non-transfection control group was 7.2±1.0 vs. 15.3±1.8, AD5-GFP transfection group was 8.1±0.7 vs. 16.2±1.5; hippocampus part： non-transfection control group was 6.6±0.8 vs. 14.7±2.0, AD5-GFP transfection group was 6.8±1.0 vs. 14.9±1.3; hypothalamus part： non-transfection control group was 9.4±1.1 vs. 12.7±1.7, AD5-GFP transfection group was 10.6±1.3 vs. 9.4±1.1, allP 0.05）.Conclusions The Shenfu decoction used in rats with brain trauma under treatment of mild hypothermia is possibly by promoting CIRP over-expression, lowering ERK expression and inhibiting the initiation of signal transduction of the secondary transcription factor phosphorylation, thereby the neural cell apoptosis is decreased and play a subsidiary role of anti-apoptosis of mild hypothermia.
出处
《中国中西医结合急救杂志》
CAS
北大核心
2016年第5期479-483,共5页
Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care
基金
国家自然科学基金资助项目（81303091）
关键词
颅脑损伤
亚低温
冷诱导RNA结合蛋白
细胞外信号调节激酶
参附方
Trauma brain injury
Mild hypothermia
Cold-inducible RNA-binding protein
Extracellular regulated protein kinases
Shenfu decoction
