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

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.

Keywords: NF-κB/NLRP3; apoptosis; electroacupuncture; inflammatory response; spasticity after stroke.

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.