雷公藤甲素与甲泼尼龙调节细胞自噬和凋亡促进脊髓损伤修复的比较研究

doi:10.3877/cma.j.issn.2095-9141.2022.03.002

中华神经创伤外科电子杂志 ›› 2022, Vol. 08 ›› Issue (03) : 132 -140. doi: 10.3877/cma.j.issn.2095-9141.2022.03.002

基础研究

雷公藤甲素与甲泼尼龙调节细胞自噬和凋亡促进脊髓损伤修复的比较研究

姚尧¹, 杨新明¹^,^†(

), 杜雅坤², 朱宁³, 阴彦林¹, 贾永利¹, 张瑛¹, 张培楠¹, 田野¹, 陈丽星¹

^1. 075000　张家口，河北北方学院附属第一医院骨科
^2. 050031　张家口，河北省儿童医院神经电生理科
^3. 075000　张家口，河北北方学院研究生学院

收稿日期:2022-05-11 出版日期:2022-06-15
通信作者: 杨新明
基金资助:
河北省省级科技计划-科技冬奥专项(20477707D); 河北省医学科学研究重点课题计划(20110176); 2022年张家口市科技计划财政资助项目(2022)

Comparison of triptolide and methylprednisolone in promoting the repair of spinal cord injury by regulating autophagy and apoptosis

Yao Yao¹, Xinming Yang¹^,^†(), Yakun Du², Ning Zhu³, Yanlin Yin¹, Yongli Jia¹, Ying Zhang¹, Peinan Zhang¹, Ye Tian¹, Lixing Chen¹

^1. Department of Orthopaedics, the First Affiliated Hospital of Hebei North University, Zhangjiakou 075000, China
^2. Department of Neurology, the Children's Hospital of Hebei Province, Shijiazhuang 050031, China
^3. Graduate School of Hebei North University, Zhangjiakou 075000, China

Received:2022-05-11 Published:2022-06-15
Corresponding author: Xinming Yang

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引用本文：

姚尧, 杨新明, 杜雅坤, 朱宁, 阴彦林, 贾永利, 张瑛, 张培楠, 田野, 陈丽星. 雷公藤甲素与甲泼尼龙调节细胞自噬和凋亡促进脊髓损伤修复的比较研究[J]. 中华神经创伤外科电子杂志, 2022, 08(03): 132-140.

Yao Yao, Xinming Yang, Yakun Du, Ning Zhu, Yanlin Yin, Yongli Jia, Ying Zhang, Peinan Zhang, Ye Tian, Lixing Chen. Comparison of triptolide and methylprednisolone in promoting the repair of spinal cord injury by regulating autophagy and apoptosis[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2022, 08(03): 132-140.

目的

探讨雷公藤甲素（TP）与甲泼尼龙（MP）调节细胞自噬和凋亡促进脊髓损伤（SCI）修复的作用机制，为临床替代MP治疗SCI提供理论依据和新的可选择性药物。

方法

选取60只雌性Thy-YFP转基因小鼠建立SCI模型，按照随机数字表法分为假手术组（Sham组）、DMSO溶液处理组（DMSO组）、MP实验组和TP实验组，每组15只。TP实验组、DMSO组及Sham组小鼠分别于术后立即腹腔注射TP（0.002 mg/10 g）、等量5%DMSO、0.9%NaCl溶液，连续给药7 d；MP实验组小鼠于术后30 min、6 h、24 h腹腔注射MP溶液（0.3 mg/10 g）。采用BMC评价运动功能，HE染色及Nissl染色法检测脊髓组织学变化，采用免疫印迹及免疫荧光染色检测自噬相关蛋白（Beclin-1、LC3B、p62）及凋亡相关蛋白（Bcl-2、Bax、caspase-3）的水平。

结果

TP实验组干预后SCI小鼠BMS运动功能评分随着时间推移逐渐升高，神经元数量增多，自噬相关蛋白Beclin-1、LC3B上调且p62降低，细胞凋亡相关蛋白caspase-3、Bax降低且抗凋亡蛋白Bcl-2升高；无论是运动功能评价、组织学变化，还是SCI后细胞自噬增强、细胞凋亡减少，TP实验组均优于DMSO组（P<0.05），但与MP实验组比较差异均无统计学意义（P>0.05）。

结论

TP在急性SCI中通过上调自噬、抑制凋亡可以促进损伤后的脊髓运动功能恢复，与MP相比对SCI具有相同的潜在保护作用。

关键词: 脊髓损伤, 雷公藤甲素, 甲泼尼龙, 修复, 细胞凋亡

Objective

To compare the mechanism of triptolide (TP) and methylprednisolone (MP) in regulating autophagy and apoptosis, and promoting the repair of spinal cord injury (SCI), so as to provide a theoretical basis and new selective drugs for new alternative drugs for clinical replacement of MP for SCI.

Methods

After establishing the SCI model, 60 THY-YFP transgenic mice were randomly divided into sham-operated group (Sham group), DMSO solution treatment group (DMSO group), MP experimental group and TP experimental group. Mice in TP experimental group, DMSO and Sham group were intraperitoneally injected with TP (0.002 mg/10 g), the same amount of 5% DMSO and 0.9% NaCl solution immediately after surgery, and had been continuously administrated for 7 d. Mice in MP experimental group were intraperitoneally injected with MP solution (0.3 mg/10 g) at 30 min, 6 h and 24 h after surgery. BMC was used to evaluate motor function, HE staining and Nissl staining were used to detect the histological changes of the spinal cord, Western blot and immunofluorescence staining were used to detect the levels of autophagy-related proteins (Beclin-1, LC3B, and p62) and apoptosis-related proteins (Bcl-2, Bax, and caspase-3).

Results

After TP intervention, the BMS motor function score of SCI mice gradually increased over time, the number of neurons increased, the autophagy-related protein, Beclin-1 and LC3B increased and p62 decreased, the apoptosis-related protein, caspase-3, Bax decreased and the anti-apoptotic protein Bcl-2 increased. Regardless of motor function evaluation, histological changes, enhanced autophagy and reduced apoptosis after SCI, TP experimental group was superior to DMSO group, but no significant difference was found between TP experimental group and MP experimental group (P>0.05).

Conclusion

TP can promote the recovery of spinal cord motor function after injury by up-regulating autophagy and inhibiting apoptosis in acute neck SCI, and has the same potential protective effect on SCI compared with MP.

Key words: Spinal cord injury, Triptolide, Methylprednisolone, Repair, Cell apoptosis

图1 小鼠脊髓损伤模型制备A：切除椎板，显露脊髓；B：脊髓右侧半切损伤

表1 4组小鼠造模后不同时间点的运动功能评估

组别	只数	第1天	第3天	第7天	第14天	第30天	第60天	χ²值	P值
Sham组	6	9	9	9	9	9	9
DMSO组	6	0	0^a	0(0,1.00)^a	2(1.00,2.00)^a	3(2.75,3.00)^a	4(3.00,4.00)^a	29.241	<0.001
MP实验组	6	0	0(0,1.00)^a	2(1.00,2.00)^a	3(3.00,4.00)^a	4.5(4.00,5.00)^a	7(6.00,7.25)^a	29.806	<0.001
TP实验组	6	0	0(0,1.00)^a	1.5(1.00,2.00)^a	3(2.75,3.00)^a	4(3.75,4.25)^a	6(5.75,7.00)^a	29.806	<0.001
χ²值			17.480	19.154	20.887	20.281	20.638
P值			0.001	<0.001	<0.001	<0.001	<0.001

表1 4组小鼠造模后不同时间点的运动功能评估

组别	只数	第1天	第3天	第7天	第14天	第30天	第60天	χ²值	P值
Sham组	6	9	9	9	9	9	9
DMSO组	6	0	0^a	0(0,1.00)^a	2(1.00,2.00)^a	3(2.75,3.00)^a	4(3.00,4.00)^a	29.241	<0.001
MP实验组	6	0	0(0,1.00)^a	2(1.00,2.00)^a	3(3.00,4.00)^a	4.5(4.00,5.00)^a	7(6.00,7.25)^a	29.806	<0.001
TP实验组	6	0	0(0,1.00)^a	1.5(1.00,2.00)^a	3(2.75,3.00)^a	4(3.75,4.25)^a	6(5.75,7.00)^a	29.806	<0.001
χ²值			17.480	19.154	20.887	20.281	20.638
P值			0.001	<0.001	<0.001	<0.001	<0.001

图2 HE染色及Nissl染色观察SCI组织学变化（×20）A：HE染色观察脊髓组织学结构；B：Nissl染色测定脊髓前角运动神经元数目；C：前角区运动神经元的数量；与Sham组比较，^aP<0.05；与DMSO组比较，^bP<0.05；DMSO：甲基亚砜；MP：甲泼尼龙；TP：雷公藤甲素；SCI：脊髓损伤

表2 4组大鼠的组织学表达结果比较

组别	只数	组织学表达
Sham组	6	40.67±3.33
DMSO组	6	16.83±1.47^a
MP实验组	6	27.33±2.73^ab
TP实验组	6	29.50±2.88^ab
F值		79.027
P值		<0.001

表2 4组大鼠的组织学表达结果比较

组别	只数	组织学表达
Sham组	6	40.67±3.33
DMSO组	6	16.83±1.47^a
MP实验组	6	27.33±2.73^ab
TP实验组	6	29.50±2.88^ab
F值		79.027
P值		<0.001

图3 免疫荧光检测脊髓前角区LC3B表达A：免疫荧光检测LC3B表达，红色代表脊髓前角运动神经元（第二排图是方框图放大的图片，×4）；B：具有代表性的TP实验组小鼠免疫荧光图；C：免疫荧光检测LC3B半定量分析结果；与Sham组比较，^aP<0.05；与DMSO组比较，^bP<0.05；DMSO：甲基亚砜；MP：甲泼尼龙；TP：雷公藤甲素

图4 免疫印迹检测LC3B、p62、Beclin-1表达情况A：免疫印迹检测自噬相关蛋白LC3B、p62、Beclin-1表达情况；B：LC3B、p62、Beclin-1的半定量分析；与Sham组比较，^aP<0.05；与DMSO组比较，^bP<0.05；DMSO：甲基亚砜；MP：甲泼尼龙；TP：雷公藤甲素

图5 免疫荧光及免疫印迹检测Bcl-2、caspase-3、Bax表达A：免疫荧光检测caspase-3表达（第二排图是方框图放大的图片，×4）；B：免疫荧光检测caspase-3半定量分析；C：免疫印迹检测凋亡相关蛋白（Bcl-2、caspase-3、Bax）表达情况；D：Bax、caspase-3、Bcl-2半定量分析；与Sham组比较，^aP<0.05；与DMSO组比较，^bP<0.05；DMSO：甲基亚砜；MP：甲泼尼龙；TP：雷公藤甲素

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Comparison of triptolide and methylprednisolone in promoting the repair of spinal cord injury by regulating autophagy and apoptosis

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Comparison of triptolide and methylprednisolone in promoting the repair of spinal cord injury by regulating autophagy and apoptosis