中国组织工程研究

中国组织工程研究 ›› 2020, Vol. 24 ›› Issue (在线): 1-.

• •    下一篇

雷公藤甲素干预脊髓损伤模型Thy-YFP转基因小鼠调节自噬及抑制细胞凋亡

朱  宁，杨新明，阮建伟   

  1. 河北北方学院附属第一医院，河北省张家口市  075000
  • 收稿日期:2019-12-10 修回日期:2019-12-14 接受日期:2020-03-03 出版日期:2020-12-08 发布日期:2020-05-08
  • 通讯作者: 杨新明，博士，教授，河北北方学院附属第一医院，河北省张家口市 075000
  • 作者简介:朱宁，女，1993年生，浙江省余姚市市，汉族，河北北方学院在读硕士，医师，主要从事脊髓损伤研究。
  • 基金资助:
    河北省卫生厅2011 年医学科学研究重点项目计划(20110176)；2013 年度河北北方学院创新人才培育基金项目(CXRC1322)

Triptolide improves spinal cord injury recovery via upregulation of autophagy and inhibition of apoptosis in Thy-yfp transgenic mice

Zhu Ning, Yang Xinming, Ruan Jianwei   

  1. First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, Hebei Province, China
  • Received:2019-12-10 Revised:2019-12-14 Accepted:2020-03-03 Online:2020-12-08 Published:2020-05-08
  • Contact: Yang Xinming, MD, Professor, First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, Hebei Province, China
  • About author:Zhu Ning, Master candidate, Physician, First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, Hebei Province, China
  • Supported by:
    2011 Medical Science Research Key Project of Hebei Provincial Department of Health, No. 20110176; 2013 Innovative Talent Cultivation Foundation of Hebei North University, No. CXRC1322

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摘要：

文题释义：

细胞自噬：自噬是一种细胞的自我保护机制,通过自噬体-溶酶体途径降解和再循环受损细胞器、有毒物质、错误折叠蛋白质等，在维持细胞稳态方面具有重要作用。

转基因小鼠：1974年，Rudolf Jaenisch通过将SV40 病毒的DNA注射到小鼠的囊胚中，创造了第一只携带外源基因的小鼠。后来又有研究人员把Murine leukemia病毒注射到小鼠胚胎得到了能通过生殖系统稳定遗传的小鼠，并且外源基因能在后代中稳定表达。这些能稳定遗传且表达外源基因的小鼠即现在一般意义上所说的转基因小鼠。

背景：研究表明，在脑卒中大鼠模型中，雷公藤甲素治疗减少了缺血性病变区域面积、水含量和神经细胞死亡。此外，雷公藤甲素能通过抑制星形胶质细胞增生、小胶质细胞活化和抑制炎症反应促进脊髓损伤修复。

目的：研究雷公藤甲素对Thy-YFP转基因小鼠脊髓损伤后细胞自噬和细胞凋亡的影响，探讨雷公藤甲素对脊髓损伤的保护作用及机制。

方法：将60只Thy-YFP转基因小鼠随机分为4组，分别是假手术组、二甲基亚砜组、甲基强的松龙组和雷公藤甲素组。假手术组只进行椎板切除术，不损伤脊髓；其他3组建立脊髓损伤模型。雷公藤甲素组、二甲基亚砜及假手术组小鼠术后立即分别腹腔注射雷公藤甲素 [0.2 mg/(kg·d)]或等量5%二甲基亚砜-生理盐水溶液，连续给药7 d；甲基强的松龙组小鼠于术后30 min、6 h、24 h腹腔注射甲基强的松龙溶液(30 mg/kg)。BMS评分检测小鼠运动功能恢复情况，苏木精-染色及Nissl染色法检测各组脊髓组织恢复情况，免疫印迹及免疫荧光染色检测各脊髓组织中自噬相关蛋白Beclin-1、LC3B、p62及凋亡相关蛋白Bcl-2、Bax、caspase-3的蛋白水平。动物伦理获得河北北方学院批准(W20200002)。

结果与结论：①雷公藤甲素及甲基强的松龙组脊髓损伤后运动功能改善，神经细胞死亡减少；②雷公藤甲素处理后自噬相关蛋白LC3B上调，p62降低，细胞凋亡相关蛋白caspase-3和Bax降低，抗凋亡蛋白Bcl-2升高；③结果表明，雷公藤甲素能促进脊髓损伤后运动功能恢复，其机制与促进自噬和抑制凋亡有关。结果提示雷公藤甲素可能对脊髓损伤具有潜在的神经保护作用。

关键词:

雷公藤甲素, 自噬, 凋亡, 脊髓损伤

Abstract:

BACKGROUND: Studies have shown that treatment with triptolide reduces ischemic lesion area, water content, and nerve cell death in a rat model of stroke. In addition, triptolide can promote the repair of spinal cord injury by inhibiting astrocyte hyperplasia, microglia activation and inflammatory response.

OBJECTIVE: To investigate the effect of triptolide on the autophagy and apoptosis in the spinal cord of Thy-YFP transgenic mice after spinal cord injury.

METHODS: Sixty Thy-YFP transgenic mice were randomly divided into four groups: sham, dimethyl sulfoxide (DMSO), triptolide and methylprednisolone groups. Following the establishment of the spinal cord injury models, mice in the triptolide, DMSO and sham group were immediately intraperitoneally injected with triptolide (0.2 mg/kg·d) or 5% DMSO-normal saline solution (0.2 mg/kg·d) for 7 days, whereas mice in the methylprednisolone group were intraperitoneally injected with methylprednisolone (30 mg/kg) at 30 minutes, 6 hours and 24 hours after surgery. BMS score was used to detect the recovery of motor function; hematoxylin-eosin staining and Nissl staining were used to explore tissue recovery after spinal cord injury; western blot and immunofluorescence staining were employed to assess the levels of autophagy-associated proteins, Beclin-1, LC3B, p62, and apoptosis-associated proteins, Bcl-2, Bax and caspase-3. An ethic approval for animal experiments was obtained from Hebei North University with an approval No. W20200002.

RESULTS AND CONCLUSION: Treatments with triptolide or methylprednisolone after spinal cord injury significantly improved motor function and reduced neuronal cell death. In addition, autophagy-associated protein LC3B was upregulated and p62 downregulated following triptolide treatment, apoptosis-associated proteins caspase-3 and Bax were reduced, while anti- apoptosis protein Bcl-2 was increased after triptolide treatment. To conclude, triptolide improves the motor function after spinal cord injury, which is related to upregulation of autophagy and inhibition of apoptosis. Triptolide may be a potential neuroprotective agent for spinal cord injury.

Key words: triptolide, autophagy, apoptosis, spinal cord injury

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