针刺对中型创伤性颅脑损伤后BDNF/TrkB信号通路的影响

doi:10.3877/cma.j.issn.2095-9141.2023.04.002

中华神经创伤外科电子杂志 ›› 2023, Vol. 09 ›› Issue (04) : 199 -205. doi: 10.3877/cma.j.issn.2095-9141.2023.04.002

基础研究

针刺对中型创伤性颅脑损伤后BDNF/TrkB信号通路的影响

王景景, 符锋, 李建伟, 任党利, 陈翀, 刘慧, 孙洪涛, 涂悦^†(

)

300162　天津，中国人民武装警察部队特色医学中心神经创伤及修复研究所
100024　北京，北京朝阳中西医结合急诊抢救医院脑病医学部

收稿日期:2023-03-06 出版日期:2023-08-15
通信作者: 涂悦

Effect of acupuncture on BDNF/TrkB signaling pathway after moderate traumatic brain injury

Jingjing Wang, Feng Fu, Jianwei Li, Dangli Ren, Chong Chen, Hui Liu, Hongtao Sun, Yue Tu^†()

Institute of Neurotrauma and Repair, Characteristic Medical Center of Chinese People's Armed Police Forces, Tianjin 300162, China
Department of Brain Disease Medicine, Beijing Chaoyang Emergency Rescue Hospital of Integrated Traditional Chinese and Western Medicine, Beijing 100024, China

Received:2023-03-06 Published:2023-08-15
Corresponding author: Yue Tu
Supported by:
National Natural Science Foundation of China(332070791); Science and Technology Project of Tianjin(21JCQNJC01140); The Innovation Team of Characteristic Medical Center of People's Armed Police Forces(KYCXTD0201, KYCXTD0207)

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引用本文：

王景景, 符锋, 李建伟, 任党利, 陈翀, 刘慧, 孙洪涛, 涂悦. 针刺对中型创伤性颅脑损伤后BDNF/TrkB信号通路的影响[J]. 中华神经创伤外科电子杂志, 2023, 09(04): 199-205.

Jingjing Wang, Feng Fu, Jianwei Li, Dangli Ren, Chong Chen, Hui Liu, Hongtao Sun, Yue Tu. Effect of acupuncture on BDNF/TrkB signaling pathway after moderate traumatic brain injury[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2023, 09(04): 199-205.

目的

探讨针刺对中型创伤性颅脑损伤（TBI）后脑源性神经营养因子（BDNF）表达和激活酪氨酸激酶受体B（TrkB）活性的影响。

方法

42只成年SD雄性大鼠按照随机数字表法分为TBI组（21只）和TBI+针刺组（21只）。2组大鼠均采用液压冲击法制备中型TBI模型，TBI组仅造模，TBI+针刺组在建立TBI模型后给予针刺治疗。造模后21 d获取2组大鼠脑组织匀浆液和切片；采用ELISA法检测BDNF的表达水平；采用免疫荧光染色在中型TBI后6、12 h检测TrkB蛋白的表达水平；采用Western blot在中型TBI后48、96和168 h检测TrkB蛋白表达水平，在12、48 h检测蛋白激酶B（Akt）和细胞外调节蛋白激酶1/2（ERK1/2）的表达情况。

结果

2组大鼠的BDNF蛋白表达水平在中型TBI后12 h时达到高峰，48 h逐渐下降。TBI+针刺组的BDNF蛋白水平在TBI后6、48、96、168 h均高于TBI组，差异具有统计学意义（P<0.05）；在12 h的BDNF蛋白水平比较差异无统计学意义（P>0.05）。TBI+针刺组的p-TrkB蛋白水平在6、12、48、96、168 h均高于TBI组，p-Akt、p-Erk1/2蛋白在12、48 h均高于TBI组，差异均具有统计学意义（P<0.05）。

结论

针刺疗法可上调中型TBI后BDNF蛋白的表达水平，还可通过TrkB蛋白磷酸化进而活化TrkB蛋白，最终激活其下游的信号通路。

关键词: 创伤性颅脑损伤, 针刺疗法, 脑源性神经营养因子, 激活酪氨酸激酶受体B

Objective

To investigate the effect of acupuncture on brain derived neurotrophic factor (BDNF) expression and receptor tyrosine kinase B (TrkB) activity after moderate traumatic brain injury (TBI).

Methods

Forty-two adult SD male rats were randomly divided into TBI group (21 rats) and TBI+acupuncture group (21 rats) according to random number table method. The moderate TBI model was established by fluid percussion instrument in both groups. The TBI group was only modeled, and the TBI+acupuncture group was given acupuncture treatment after the TBI model was established. Tissue homogenates and sections were harvested 21 d after moderate TBI. The level of BDNF was examined by ELISA. Immunofluorescence staining was used to detect the expression level of TrkB protein at 6 and 12 h after moderate TBI, Western blot was used to detect the expression levels of TrkB protein at 48, 96, and 168 h after moderate TBI, and the expression of protein kinase B (Akt) and extracellular regulated protein kinase 1/2 (ERK1/2) at 12 and 48 h.

Results

The expression level of BDNF protein in two groups of rats reached its peak at 12 h after moderate TBI and gradually decreased at 48 h. The BDNF protein levels in the TBI+acupuncture group were higher than those in the TBI group at 6, 48, 96, 168 h after TBI, with statistical significance (P<0.05), there was no statistically significant difference in BDNF protein levels at 12 h (P>0.05). The levels of p-TrkB protein in the TBI+acupuncture group were higher than those in the TBI group at 6, 12, 48, 96, 168 h, while p-Akt and p-Erk1/2 protein levels were higher than those in the TBI group at 12, 48 h, with statistical significance (P<0.05).

Conclusion

Acupuncture therapy can upregulate the expression level of BDNF protein after moderate TBI, and activate TrkB protein by phosphorylation, ultimately activating its downstream signaling pathway.

Key words: Traumatic brain injury, Acupuncture therapy, Brain-derived neurotrophic factor, Tyrosine kinase receptor B

表1 2组大鼠在中型TBI后各时间点BDNF蛋白表达量比较（pg/mg，±s）

Tab.1 Comparison of BDNF protein expression levels between two groups of rats at different time points after moderate TBI (pg/mg, Mean±SD)

组别	只数	6 h（n=3）	12 h（n=3）	48 h（n=3）	96 h（n=3）	168 h（n=3）	F值	P值
TBI组	15	26.36±3.97	46.24±6.35	14.89±2.31	14.44±1.65	13.96±1.46	43.770	0.001
TBI+针刺组	15	37.12±4.68	54.01±6.42	41.21±5.63	38.45±4.65	37.01±4.23	5.726	0.011
t值		3.039	1.491	7.474	8.429	8.920
P值		0.038	0.210	<0.001	<0.001	<0.001

表1 2组大鼠在中型TBI后各时间点BDNF蛋白表达量比较（pg/mg，±s）

Tab.1 Comparison of BDNF protein expression levels between two groups of rats at different time points after moderate TBI (pg/mg, Mean±SD)

组别	只数	6 h（n=3）	12 h（n=3）	48 h（n=3）	96 h（n=3）	168 h（n=3）	F值	P值
TBI组	15	26.36±3.97	46.24±6.35	14.89±2.31	14.44±1.65	13.96±1.46	43.770	0.001
TBI+针刺组	15	37.12±4.68	54.01±6.42	41.21±5.63	38.45±4.65	37.01±4.23	5.726	0.011
t值		3.039	1.491	7.474	8.429	8.920
P值		0.038	0.210	<0.001	<0.001	<0.001

图1 中型TBI急性期大脑皮质p-TrkB蛋白免疫荧光染色结果（400×）

Fig.1 Immunofluorescence staining results of p-TrkB protein in the acute phase of cerebral cortex after moderate TBI (400×)

表2 2组大鼠TBI急性期大脑皮质p-TrkB蛋白荧光强度的比较（±s）

Tab.2 Comparison of p-TrkB protein fluorescence intensity in the cerebral cortex between two groups of rats during acute phase (Mean±SD)

组别	只数	6 h（n=3）	12 h（n=3）	t值	P值
TBI组	6	126.50±11.15	190.59±13.23	6.417	0.003
TBI+针刺组	6	176.68±18.25	225.99±16.33	3.488	0.025
t值		4.063	2.918
P值		0.015	0.043

表2 2组大鼠TBI急性期大脑皮质p-TrkB蛋白荧光强度的比较（±s）

Tab.2 Comparison of p-TrkB protein fluorescence intensity in the cerebral cortex between two groups of rats during acute phase (Mean±SD)

组别	只数	6 h（n=3）	12 h（n=3）	t值	P值
TBI组	6	126.50±11.15	190.59±13.23	6.417	0.003
TBI+针刺组	6	176.68±18.25	225.99±16.33	3.488	0.025
t值		4.063	2.918
P值		0.015	0.043

图2 2组大鼠中型TBI后48、96和168 h p-TrkB、TrkB的Western blot结果

Fig.2 Western blot results of p-TrkB and TrkB at 48, 96, and 168 h after moderate TBI in two groups of rats

表3 2组大鼠中型TBI后48、96和168 h p-TrkB蛋白表达量的比较（±s）

Tab.3 Comparison of p-TrkB protein expression levels between two groups of rats at 48, 96, and 168 h after moderate TBI (Mean±SD)

组别	只数	48 h（n=3）	96 h（n=3）	168 h（n=3）	F值	P值
TBI	9	0.412±0.050	0.354±0.049	0.432±0.050	2.037	0.211
TBI+针刺组	9	1.421±0.201	1.435±0.192	1.201±0.190	1.368	0.324
t值		8.454	9.452	6.762
P值		0.001	0.001	0.002

表3 2组大鼠中型TBI后48、96和168 h p-TrkB蛋白表达量的比较（±s）

Tab.3 Comparison of p-TrkB protein expression levels between two groups of rats at 48, 96, and 168 h after moderate TBI (Mean±SD)

组别	只数	48 h（n=3）	96 h（n=3）	168 h（n=3）	F值	P值
TBI	9	0.412±0.050	0.354±0.049	0.432±0.050	2.037	0.211
TBI+针刺组	9	1.421±0.201	1.435±0.192	1.201±0.190	1.368	0.324
t值		8.454	9.452	6.762
P值		0.001	0.001	0.002

图3 2组大鼠中型TBI后12、48 h p-Akt、Akt的Western blot结果

Fig.3 Western blot results of p-Akt and Akt at 12 and 48 h after moderate TBI in two groups of rats

表4 2组大鼠中型TBI后12、48 h p-Akt蛋白表达量比较（±s）

Tab.4 Comparison of p-Akt protein expression levels between two groups of rats at 12 and 48 h after moderate TBI (Mean±SD)

组别	只数	12 h（n=3）	48 h（n=3）	t值	P值
TBI组	6	0.60±0.08	0.21±0.03	8.490	0.001
TBI+针刺组	6	1.42±0.19	1.25±0.17	1.146	0.316
t值		7.040	10.610
P值		0.002	<0.001

表4 2组大鼠中型TBI后12、48 h p-Akt蛋白表达量比较（±s）

Tab.4 Comparison of p-Akt protein expression levels between two groups of rats at 12 and 48 h after moderate TBI (Mean±SD)

组别	只数	12 h（n=3）	48 h（n=3）	t值	P值
TBI组	6	0.60±0.08	0.21±0.03	8.490	0.001
TBI+针刺组	6	1.42±0.19	1.25±0.17	1.146	0.316
t值		7.040	10.610
P值		0.002	<0.001

图4 2组大鼠中型TBI后12、48 h p-Erk1/2、Erk1/2的Western blot结果

Fig.4 Western blot results of p-Erk1/2 and Erk1/2 at 12 and 48 h after moderate TBI in two groups of rats

表5 2组大鼠中型TBI后12、48 h p-Erk1/2蛋白表达量比较（±s）

Tab.5 Comparison of p-Erk1/2 protein expression levels between two groups of rats at 12 and 48 h after moderate TBI (Mean±SD)

组别	只数	12 h（n=3）	48 h（n=3）	t值	P值
TBI组	6	0.45±0.06	0.20±0.03	6.753	0.003
TBI+针刺组	6	1.15±0.20	0.95±0.10	1.603	0.184
t值		5.819	12.090
P值		0.004	<0.001

表5 2组大鼠中型TBI后12、48 h p-Erk1/2蛋白表达量比较（±s）

Tab.5 Comparison of p-Erk1/2 protein expression levels between two groups of rats at 12 and 48 h after moderate TBI (Mean±SD)

组别	只数	12 h（n=3）	48 h（n=3）	t值	P值
TBI组	6	0.45±0.06	0.20±0.03	6.753	0.003
TBI+针刺组	6	1.15±0.20	0.95±0.10	1.603	0.184
t值		5.819	12.090
P值		0.004	<0.001

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Effect of acupuncture on BDNF/TrkB signaling pathway after moderate traumatic brain injury

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Effect of acupuncture on BDNF/TrkB signaling pathway after moderate traumatic brain injury