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

doi:10.12122/j.issn.1673-4254.2024.11.06

Abstract

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

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.

Figures/Tables 9

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

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.

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.

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

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.

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.

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.

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

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.

References 30

1	Wang QJ, Zhang X, Suo YJ, et al. Normobaric hyperoxia therapy in acute ischemic stroke: a literature review[J]. Heliyon, 2024, 10(1): e23744.
2	Tang YS, Xu AP, Shao SJ, et al. Electroacupuncture ameliorates cognitive impairment by inhibiting the JNK signaling pathway in a mouse model of Alzheimer's disease[J]. Front Aging Neurosci, 2020, 12: 23.
3	母晓艺, 宋艳华, 张蒂, 等. 针刺内关穴预处理改善心肌缺血再灌注损伤机制的研究进展[J]. 针灸临床杂志, 2018, 34(10): 75-9.
4	宁文华, 李礼, 郭扬, 等. 电针预处理脑保护作用机制研究进展[J]. 中国康复理论与实践, 2019, 25(11): 1315-9.
5	叶涛, 朱路文, 唐强, 等. 电针预处理对脑缺血再灌注大鼠缺血半暗区细胞凋亡及凋亡相关蛋白表达的影响[J]. 中国康复理论与实践, 2018, 24(1): 54-9.
6	叶涛, 朱路文, 唐强, 等. 电针预处理对大鼠脑缺血再灌注损伤后脑梗死体积及血清TNF-α、IL-10含量的影响[J]. 中国针灸, 2017, 37(10): 1093-8.
7	叶涛, 朱路文, 唐强, 等. 电针预处理对脑缺血再灌注损伤大鼠神经功能和缺血半暗区Tolls样受体4、核转录因子κB蛋白的影响[J]. 中国康复理论与实践, 2017, 23(7): 745-9.
8	何琦. 针刺经筋结点法治疗卒中后上肢痉挛性瘫痪的临床疗效观察[D]. 天津: 天津中医药大学, 2022: 3-4.
9	Xie XL, Wang F, Li XJ. Inhibition of TRIM14 protects cerebral ischemia/reperfusion injury through regulating NF‑κB/NLRP3 pathway-mediated inflammation and apoptosis[J]. J Recept Signal Transduct Res, 2022, 42(2): 197-205.
10	Ye T, Zhang N, Zhang AB, et al. Electroacupuncture pretreatment alleviates rats cerebral ischemia-reperfusion injury by inhibiting ferroptosis[J]. Heliyon, 2024, 10(9): e30418.
11	Longa EZ, Weinstein PR, Carlson S, et al. Reversible middle cerebral artery occlusion without craniectomy in rats[J]. Stroke, 1989, 20(1): 84-91.
12	Wang JX, Mu JD, Ma LX, et al. Waggle needling wields preferable neuroprotective and anti-spastic effects on post-stroke spasticity rats by attenuating γ-aminobutyric acid transaminase and enhancing γ-aminobutyric acid[J]. Neuroreport, 2020, 31(10): 708-16.
13	武凤, 刘俊岑, 徐红丹, 等. 补骨脂提取物对APP/PS1小鼠学习记忆能力及海马中雌激素受体表达的影响[J]. 中药材, 2022, 45(7): 1699-704.
14	张秦墉, 马良宵, 王俊翔, 等. 运动头针法抗卒中后痉挛效应的电生理机制研究[J]. 针刺研究, 2023, 48(10): 986-92.
15	王艺莹, 李瑞青, 李婧雯, 等. 督脉电针对脑缺血再灌注后肢体痉挛大鼠大脑皮层氯离子稳态的影响[J]. 针刺研究, 2023, 48(9): 860-9.
16	Ma Z, Zhang ZL, Bai FH, et al. Electroacupuncture pretreatment alleviates cerebral ischemic injury through α7 nicotinic acetylcholine receptor-mediated phenotypic conversion of microglia[J]. Front Cell Neurosci, 2019, 13: 537.
17	张建云, 梅紧紧, 张帅迪, 等. 基于谷胱甘肽抗氧化系统探讨督脉电针对脑卒中后肢体痉挛大鼠氧化应激的影响[J]. 北京中医药大学学报, 2024, 47(1): 124-32.
18	刘丹妮, 周君, 黄夏荣, 等. 电针 “水沟” “百会” 穴对脑缺血再灌注损伤大鼠海马神经元自噬的影响[J]. 针刺研究, 2022, 47(6): 491-6.
19	Qi YC, Xiao XJ, Duan RS, et al. Effect of acupuncture on inflammatory cytokines expression of spastic cerebral palsy rats[J]. Asian Pac J Trop Med, 2014, 7(6): 492-5.
20	张小超, 陈蓬, 孔琼荣, 等. 紫草婴儿软膏对湿疹豚鼠皮损组织中CD4、CD8、IL-4及TNF-α表达的影响[J]. 时珍国医国药, 2020, 31(4): 789-92.
21	Yuan ZY, Li M. Arecoline promotes fibroblast activation and M2-macrophage polarization by up-regulating the expression of IL-4[J]. Arch Oral Biol, 2024, 167: 106052.
22	van Panhuys N, Tang SC, Prout M, et al. In vivo studies fail to reveal a role for IL-4 or STAT6 signaling in Th2 lymphocyte differentiation[J]. Proc Natl Acad Sci U S A, 2008, 105(34): 12423-8.
23	Wei B, Billman ZP, Nozaki K, et al. NLRP3, NLRP6, and NLRP12 are inflammasomes with distinct expression patterns[J]. Front Immunol, 2024, 15: 1418290.
24	游小芳, 林万庆, 李萍萍, 等. 电针对脑缺血再灌注大鼠NLRP3/Caspase-1/GSDMD轴及神经功能的影响[J]. 中国针灸, 2023, 43(6): 661-8.
25	Jiang T, Wu MY, Zhang ZQ, et al. Electroacupuncture attenuated cerebral ischemic injury and neuroinflammation through α7nAChR-mediated inhibition of NLRP3 inflammasome in stroke rats[J]. Mol Med, 2019, 25(1): 22.
26	Sun XQ, Zhang AB, Pang B, et al. Electroacupuncture pretreatment alleviates spasticity after stroke in rats by inducing the NF‑κB/NLRP3 signaling pathway and the gut-brain axis[J]. Brain Res, 2024, 1822: 148643.
27	Li D, Zhang KX, Xu CC, et al. Cypermethrin induces apoptosis, autophagy and inflammation via ERS-ROS-NF‑κB axis in hepatocytes of carp (Cyprinus carpio)[J]. Pestic Biochem Physiol, 2023, 196: 105625.
28	耿翊宁. 温针灸辅以中药汤剂对老年脑卒中后肢体痉挛患者神经递质及细胞凋亡相关指标的影响[J]. 中国老年学杂志, 2020, 40(16): 3383-7.
29	Fu CF, Ji WB, Cui QW, et al. GSDME-mediated pyroptosis promotes anti-tumor immunity of neoadjuvant chemotherapy in breast cancer[J]. Cancer Immunol Immunother, 2024, 73(9): 177.
30	张春华, 胡凌云, 解云, 等. 刺芒柄花素通过阻断海马组织NF-κB通路抑制炎症因子释放改善慢性轻度不可预见性应激老龄大鼠认知行为[J]. 细胞与分子免疫学杂志, 2023, 39(7): 610-6.

[1]	Hanjun ZUO, Zhaoda DUAN, Zhao WANG, Tao GUO, Jinsha SHI, Haolong SHI, Juanjuan LI. Gastrodin improves microglia-mediated inflammatory response after hypoxic-ischemic brain damage in neonatal rats via PI3K/AKT pathway [J]. Journal of Southern Medical University, 2024, 44(9): 1712-1719.
[2]	Shuo LIU, Jing LI, Xingwang WU. Swertiamarin ameliorates 2,4,6-trinitrobenzenesulfonic acid-induced colitis in mice by inhibiting intestinal epithelial cell apoptosis [J]. Journal of Southern Medical University, 2024, 44(8): 1545-1552.
[3]	Xiaofan CONG, Teng CHEN, Shuo LI, Yuanyuan WANG, Longyun ZHOU, Xiaolong LI, Pei ZHANG, Xiaojin SUN, Surong ZHAO. Dihydroartemisinin enhances sensitivity of nasopharyngeal carcinoma HNE1/DDP cells to cisplatin-induced apoptosis by promoting ROS production [J]. Journal of Southern Medical University, 2024, 44(8): 1553-1560.
[4]	Linyu XIAO, Ting DUAN, Yongsheng XIA, Yue CHEN, Yang SUN, Yibo XU, Lei XU, Xingzhou YAN, Jianguo HU. Linarin inhibits microglia activation-mediated neuroinflammation and neuronal apoptosis in mouse spinal cord injury by inhibiting the TLR4/NF-κB pathway [J]. Journal of Southern Medical University, 2024, 44(8): 1589-1598.
[5]	Mengdong ZHENG, Yan LIU, Jiaojiao LIU, Qiaozhen KANG, Ting WANG. Effect of deletion of protein 4.1R on proliferation, apoptosis and glycolysis of hepatocyte HL-7702 cells [J]. Journal of Southern Medical University, 2024, 44(7): 1355-1360.
[6]	Yuanguo WANG, Peng ZHANG. Ferroptosis suppressor genes are highly expressed in esophageal cancer to inhibit tumor cell ferroptosis [J]. Journal of Southern Medical University, 2024, 44(7): 1389-1396.
[7]	Zhijun REN, Jianxin DIAO, Yiting WANG. Xionggui Decoction alleviates heart failure in mice with myocardial infarction by inhibiting oxidative stress-induced cardiomyocyte apoptosis [J]. Journal of Southern Medical University, 2024, 44(7): 1416-1424.
[8]	Guiling CHEN, Xiaofeng LIAO, Pengtao SUN, Huan CEN, Shengchun SHU, Bijing LI, Jinhua LI. Solasonine promotes apoptosis of non-small cell lung cancer cells by regulating the Bcl-2/Bax/caspase-3 pathway [J]. Journal of Southern Medical University, 2024, 44(6): 1109-1116.
[9]	Lingjun LU, Xiaodi YANG, Huaping ZHANG, Yuan LIANG, Xiulan SHI, Xin ZHOU. Recombinant Schistosoma japonicum cystatin alleviates acute liver injury in mice by inhibiting endoplasmic reticulum stress, inflammation and hepatocyte apoptosis [J]. Journal of Southern Medical University, 2024, 44(6): 1126-1134.
[10]	Guoxin LIANG, Hongyue TANG, Chang GUO, Mingming ZHANG. MiR-224-5p overexpression inhibits oxidative stress by regulating the PI3K/Akt/FoxO1 axis to attenuate hypoxia/reoxygenation-induced cardiomyocyte injury [J]. Journal of Southern Medical University, 2024, 44(6): 1173-1181.
[11]	HE Cheng, CHEN Wei, ZHANG Nianzhi, LUAN Jun, WANG Sanfeng, ZHANG You. Shenqi Chongcao Formula ameliorates inflammatory response in rats with pulmonary fibrosis by activating the ASS1/src/STAT3 signaling pathway [J]. Journal of Southern Medical University, 2024, 44(4): 644-651.
[12]	BAO Hansheng, WANG Sutong, LÜ Mujie, WANG Yongcheng, JIANG Ping, LI Xiao. Activation of α7 nAChR improves white fat homeostasis and promotes beige adipogenesis and thermogenesis in obese mice [J]. Journal of Southern Medical University, 2024, 44(3): 499-506.
[13]	ZHANG Xiaoyan, WANG Xie, WANG Jie, SHAO Nan, CAI Biao, XIE Daojun. Huangpu Tongqiao Capsule improves cognitive impairment in rats with Wilson disease by inhibiting endoplasmic reticulum stress-mediated apoptosis pathway [J]. Journal of Southern Medical University, 2024, 44(3): 447-454.
[14]	CHEN Guodong, LUO Suxin. Colchicine alleviates myocardial ischemia-reperfusion injury in mice by activating AMPK [J]. Journal of Southern Medical University, 2024, 44(2): 226-235.
[15]	Xuerou LIU, Yumei YANG, Wei LIU, Zhen ZHANG, Xingqi ZHOU, Wenyu XIE, Lin SHEN, Mengxiao ZHANG, Xian LI, Jialan ZANG, Shanshan LI. Euphorbia helioscopia inhibits proliferation, invasion, and migration and promotes apoptosis of non-small cell lung cancer cells [J]. Journal of Southern Medical University, 2024, 44(10): 1918-1925.