Objective

2

To observe the effects of electroacupuncture (EA) with different waveforms on nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome and pyroptosis in the cerebral cortex of rats with cerebral ischemia-reperfusion injury (CIRI)

and explore the potential mechanisms of EA in treating CIRI.

Methods

2

A total of 70 male SD rats were selected. Twelve rats were randomly assigned to a sham operation group

while the remaining rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) model by reforming Longa method. Forty-eight successfully modeled rats were randomly divided into a model group

an EA-continuous wave (EA-A) group

an EA-discontinuous wave (EA-B) group

and an EA-dense-disperse wave (EA-C) group

with 12 rats in each group. EA was performed at "Baihui" (GV20)

"Dazhui" (GV14)

and bilateral "Zusanli" (ST36) once daily for 20 min for 7 d. The EA-A group used a continuous wave (15 Hz)

the EA-B group used a discontinuous wave (15 Hz with 1.5 s intervals)

and the EA-C group used a dense-disperse wave (alternating 3 Hz/15 Hz). Neurological deficit scores were recorded before and after intervention. After intervention

TTC staining was used to assess cerebral infarct volume. HE staining was performed to observe the morphological changes in the ischemic cortical brain tissue. ELISA was used to measure interleukin-1β (IL-1β) and interleukin-18 (IL-18) levels in the ischemic cortex. Western blot was used to detect the protein expression of NLRP3

Caspase-1

gasdermin D (GSDMD)

and apoptosis-associated speck-like protein (ASC)

and qRT-PCR was used to detect the mRNA expression of NLRP3

Caspase-1

GSDMD and ASC.

Results

2

Compared with the sham operation group

the model group showed significantly higher neurological deficit score and infarct volume percentage (

P

<

0.01); histological analysis revealed loose tissue structure

widened cytoplasmic gaps

and vacuolar degeneration in the model group. The IL-1β and IL-18 levels

and both p

rotein and mRNA expression of NLRP3

Caspase-1

GSDMD

and ASC in the model group were significantly higher than those in the sham operation group (

P

<

0.01). Compared with the model group

all three EA groups showed reduced neurological deficit scores and infarct volume percentage (

P

<

0.01)

improved tissue damage

and decreased levels of IL-1β and IL-18 (

P

<

0.01). Protein and mRNA expression of NLRP3

Caspase-1

GSDMD

and ASC were also lower than those in the model group (

P

<

0.05

P

<

0.01). Compared with the EA-A group

the EA-B group had lower IL-1β level (

P

<

0.05)

and the EA-C group had significantly lower levels of IL-1β and IL-18 (

P

<

0.01

P

<

0.05)

as well as decreased GSDMD and ASC protein expression and reduced Caspase-1 and ASC mRNA expression (

P

<

0.05

P

<

0.01). Compared with the EA-B group

the EA-C group showed further decreases in ASC protein expression and Caspase-1

ASC mRNA expression (

P

<

0.05

P

<

0.01).

Conclusion

2

Different EA waveforms could all alleviate neurological deficits and reduce cerebral infarct volume in CIRI rats. The mechanism may be related to inhibition of NLRP3 inflammasome-mediated pyroptosis.