电针预处理通过抑制NF-kB/NLRP3信号通路介导炎症和凋亡改善大鼠脑卒中后痉挛

doi:10.12122/j.issn.1673-4254.2024.11.06

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (11): 2102-2109.doi: 10.12122/j.issn.1673-4254.2024.11.06

• • 上一篇

电针预处理通过抑制NF-kB/NLRP3信号通路介导炎症和凋亡改善大鼠脑卒中后痉挛

孙秀颀¹(), 蔡静¹, 张安邦¹, 庞博¹, 陈春艳², 查琪琪², 全菲², 叶涛²()

^1.贵州中医药大学第一附属医院，神经内科，贵州贵阳 550001
^2.贵州中医药大学第一附属医院，康复科，贵州贵阳 550001

收稿日期:2024-07-25 出版日期:2024-11-20 发布日期:2024-11-29
通讯作者: 叶涛 E-mail:1229078575@qq.com;yetao008@gzy.edu.cn
作者简介:孙秀颀，博士，主治医师，E-mail: 1229078575@qq.com
基金资助:
国家自然科学基金(82060896);贵州省科技计划基础研究（科学技术基金）项目(黔科合基础-ZK[2021]一般505）;贵州省教育厅青年科技人才成长项目(黔教合KY字[2021]210);贵州省中医药管理局中医药、民族医药科学技术研究课题(QZYY-2021-015);贵州省卫生健康委科学技术基金项目(gzwkj2021-072)

Electroacupuncture pretreatment alleviates post-stroke spasticity in rats by inhibiting NF‑κB/NLRP3 signaling pathway-mediated inflammation and neuronal apoptosis

Xiuqi SUN¹(), Jing CAI¹, Anbang ZHANG¹, Bo PANG¹, Chunyan CHENG², Qiqi CHA², Fei QUAN², Tao YE²()

^1.Department of Neurology, First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China
^2.Department of Rehabilitation Medicine, First Affiliated Hospital of Guizhou University of Traditional Chinese Medicine, Guiyang 550001, China

Received:2024-07-25 Online:2024-11-20 Published:2024-11-29
Contact: Tao YE E-mail:1229078575@qq.com;yetao008@gzy.edu.cn
Supported by:
National Natural Science Foundation of China(82060896)

摘要/Abstract

摘要：

目的探讨电针预处理诱导NF-κB/NLRP3信号通路及炎症和凋亡在脑卒中后挛治疗中的潜在机制。方法 SPF级雄性SD大鼠18只随机分为：假手术组（sham），模型组（I/R），电针治疗组（I/R+EP），每组6只。假手术组仅暴露不做阻塞，模型组进行中动脉闭塞模型，电针组使用电针治疗曲鬓穴和百会穴，持续治疗3 d。采用HE、Nissl、TUNEL染色检测大鼠脑组织病理变化情况。ELISA法检测各组IL-4、IL-6、TNF-α、TMAO的水平变化情况。采用qRT-PCR、Western blotting和免疫组化检测NF-κB p65、NLRP3、caspase-3、caspase-9 mRNA和蛋白水平。结果与假手术组相比，模型组的神经功能评分增加（P<0.05），肌张力增增加（P<0.05），新事物识别时间减少（P<0.05），海马细胞排列不均、松散和轮廓不清晰（P<0.05），细胞凋亡数量显着增加（P<0.05），促炎因子IL-6和TNF-α，NF-B p65、NLRP3、caspase-3和caspase-9水平上升（P<0.05）。与模型组相比，电针治疗组神经功能评分下降，肌张力评分下降，新事物识别时间增加（P>0.05），海马细胞排列紧密规则，染色均匀，轮廓清晰（P<0.05），活神经元数量增加（P<0.05），细胞凋亡明显减少（P<0.05），促炎因子IL-6和TNF-α水平下降，抑炎因子IL-4水平上升（P<0.05）。此外，电针治疗抑制了脑组织中NLRP3、caspase-3、caspase-9和NF-B p65的表达（P<0.05）。结论电针预处理有助于缓解大鼠脑卒中后痉挛，这是因为它阻断了NF-B/NLRP3通路，抑制炎症反应和细胞凋亡。

关键词: 脑卒中后痉挛, 电针预处理, NF-κB/NLRP3信号通路, 炎症反应, 细胞凋亡

Abstract:

Objective To explore the mechanism of electroacupuncture pretreatment (EP) for relieving post-stroke spasticity in rats. Methods Eighteen rats were randomized equally into sham-operated group, middle cerebral artery occlusion (MCAO) group, and MCAO+EP group. In MCAO+EP group, the rats received electroacupuncture at the acupoints Qubin and Baihui for 3 consecutive days prior to MCAO. Neurological deficits and cognitive function of the rats were evaluated, and pathologies in the hippocampus were examined using HE, Nissl, and TUNEL staining. The expressions of IL-4, IL-6, TNF‑α, and TMAO in the brain tissues were detected with ELISA, and the mRNA and protein expression levels of NF-κB p65, NLRP3, caspase-3, and caspase-9 were determined with qRT-PCR, Western blotting, and immunohistochemistry. Results The rats receiving MCAO had significantly increased neurological deficit scores and showed increased muscle tension, number of apoptotic neurons, and expressions of IL-6, TNF-α, NF-κB p65, NLRP3, caspase-3 and caspase-9 in the hippocampus and significantly reduced length of time for new object recognition. Microscopically, the cells in the hippocampus of the MCAO rats showed uneven and loosened arrangement and unclear cell boundaries. In contrast, the rats in I/R+EP group showed significantly lowered neurological deficit scores and dystonia rating scores, reduced cell apoptosis, lowered hippocampal expressions of IL-6, TNF-α, caspase-3, caspase-9, and NF‑κB p65, increased time for new object recognition, tightly arranged and uniformly stained hippocampal cells with clear boundaries, with also an increased number of active neurons and enhanced expression of IL-4 in the hippocampus. Conclusion EP alleviates post-stroke spasticity in rats by inhibiting inflammatory responses and hippocampal neuronal apoptosis mediated by the NF-κB/NLRP3 signaling pathway.

Key words: spasticity after stroke, electroacupuncture, NF-κB/NLRP3, inflammatory response, apoptosis

孙秀颀, 蔡静, 张安邦, 庞博, 陈春艳, 查琪琪, 全菲, 叶涛. 电针预处理通过抑制NF-kB/NLRP3信号通路介导炎症和凋亡改善大鼠脑卒中后痉挛[J]. 南方医科大学学报, 2024, 44(11): 2102-2109.

Xiuqi SUN, Jing CAI, Anbang ZHANG, Bo PANG, Chunyan CHENG, Qiqi CHA, Fei QUAN, Tao YE. Electroacupuncture pretreatment alleviates post-stroke spasticity in rats by inhibiting NF‑κB/NLRP3 signaling pathway-mediated inflammation and neuronal apoptosis[J]. Journal of Southern Medical University, 2024, 44(11): 2102-2109.

图/表 9

表1 引物序列

Tab.1 Primers used for RT-qPCR in this study

Primer name	Primer sequence (5'-3')	Product length (bp)	Annealing temperature (℃)
NF-κB p65 F	GCAAAAGGACCTACGAGACC	20	57.48
NF-κB p65 R	CGGGAAGGCACAGCAATA	18	57.48
NLRP3 F	CTCAACAGACGCTACACCCA	20	58.64
NLRP3 R	CCACATCTTAGTCCTGCCAAT	21	58.64
Caspase-3 F	AACGAACGGACCTGTGGAC	19	59.45
Caspase-3 R	GGAATAGTAACCGGGTGCG	19	59.45
Caspase-9 F	CAGGGAAGATCGAGAGACATGC	22	59.46
Caspase-9 R	CAAAGCCGTGACCATTTTCTTAG	23	59.46
β-actin F	GCCATGTACGTAGCCATCCA	20	59.50
β-actin R	GAACCGCTCATTGCCGATAG	20	59.50

图 1 24 h I/R后 (A)神经功能评分、(B)肌张力障碍评分和(C)新物体识别时间

Fig.1 Neurological function scores, dystonia rating scores and time for new object recognition 24 h after cerebral ischemia-reperfusion (I/R). *P<0.05 vs sham group, #P<0.05 vs I/R group.

图2 海马HE染色

Fig.2 HE staining of the hippocampus in each group (Original magnification: ×400). A: Sham group. B: I/R group. C: I/R+EP group.

图3 HE染色大鼠脑组织

Fig.3 HE staining of the infarct area in each group (×400). A: Sham group. B: I/R group. C: I/R+EP group.

图4 大鼠神经元Nissl染色

Fig.4 Nissl staining of the hippocampal neurons in each group (×400). A: Sham group. B: I/R group. C: I/R+EP group. D: Comparison of the number of positively stained cells. *P<0.05 vs sham group; #P<0.05 vs I/R group.

图5 大鼠脑TUNEL染色

Fig.5 TUNEL staining of the infarct area in each group (×400). A: Sham group. B: I/R group. C: I/R+EP group. D: Comparison of the number of apoptotic cells. *P<0.05 vs sham group; #P<0.05 vs I/R group.

图6 ELISA法测定大鼠IL-4、IL-6、TNF-α水平

Fig.6 Expression levels of IL-4 (A), IL-6 (B) and TNF-α (C) in the brain tissues of the rats measured by ELISA. *P<0.05 vs sham group; #P<0.05 vs I/R group.

图7 免疫组化检测大鼠脑组织NF-κB p65、NLRP3、caspase-3、caspase-9

Fig.7 Immunohistochemistry for detecting NF-κB p65, NLRP3, caspase-3 and caspase-9 in rat brain tissues (×400).

图8 大鼠脑组织中NF‑κB p65、NLRP3、caspase-3和caspase-9的mRNA和蛋白水平

Fig.8 　The mRNA (A) and protein (B) expression ofNF-κB p65, NLRP3, caspase-3 and caspase-9 in ratbrain tissues detected by RT-qPCR and Westernblotting and their relative protein expressionslevels (C). *P<0.05 vs sham group; #P<0.05 vs I/Rgroup.

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电针预处理通过抑制NF-kB/NLRP3信号通路介导炎症和凋亡改善大鼠脑卒中后痉挛

Electroacupuncture pretreatment alleviates post-stroke spasticity in rats by inhibiting NF‑κB/NLRP3 signaling pathway-mediated inflammation and neuronal apoptosis

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