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. 2016 Jul;11(7):1090-8.
doi: 10.4103/1673-5374.187041.

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

Xiao-Lu Jin  1 , Peng-Fei Li  2 , Chun-Bing Zhang  3 , Jin-Ping Wu  4 , Xi-Lian Feng  1 , Ying Zhang  1 , Mei-Hong Shen  1
Affiliations

Electroacupuncture alleviates cerebral ischemia and reperfusion injury via modulation of the ERK1/2 signaling pathway

Xiao-Lu Jin et al. Neural Regen Res. 2016 Jul.

Abstract

Electroacupuncture (EA) has anti-oxidative and anti-inflammatory actions, but whether the neuroprotective effect of EA against cerebral ischemia-reperfusion (I/R) injury involves modulation of the extracellular regulated kinase 1/2 (ERK1/2) signaling pathway is unclear. Middle cerebral artery occlusion (MCAO) was performed in Sprague-Dawley rats for 2 hours followed by reperfusion for 24 hours. A 30-minute period of EA stimulation was applied to both Baihui (DU20) and Dazhui (DU14) acupoints in each rat (10 mm EA penetration depth, continuous wave with a frequency of 3 Hz, and a current intensity of 1-3 mA) when reperfusion was initiated. EA significantly reduced infarct volume, alleviated neuronal injury, and improved neurological function in rats with MCAO. Furthermore, high mRNA expression of Bax and low mRNA expression of Bcl-2 induced by MCAO was prevented by EA. EA substantially restored total glutathione reductase (GR), glutathione (GSH) and glutathione peroxidase (GSH-Px) levels. Additionally, Nrf2 and glutamylcysteine synthetase (GCS) expression levels were markedly increased by EA. Interestingly, the neuroprotective effects of EA were attenuated when ERK1/2 activity was blocked by PD98059 (a specific MEK inhibitor). Collectively, our findings indicate that activation of the ERK1/2 signaling pathway contributes to the neuroprotective effects of EA. Our study provides a better understanding of the regulatory mechanisms underlying the therapeutic effectiveness of EA.

Keywords: B cell lymphoma 2; electroacupuncture; glutamylcysteine synthetase; glutathione peroxidase; glutathione reductase; ischemia and reperfusion injury; middle cerebral artery occlusion; mitogen-activated protein kinase; nerve regeneration; nuclear factor erythroid 2-related factor 2; oxidative stress.

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Conflict of interest statement

Conflicts of Interest: None declared.

Figures

Figure 1
Figure 1
Baihui (DU20) and Dazhui (DU14) acupoints on the rat.
Figure 2
Figure 2
EA attenuates neurological deficits in rats with MCAO. (A) Neurological function scores 24 hours after reperfusion (n = 10 per group). A higher score indicates better neurological function. Representative 2,3,5-triphenyltetrazolium chloride (TTC) staining (B) and volume (%) (C) of the cerebral infarct in the rat brain (n = 5 per group). The infarcted tissue remained unstained (white), whereas normal tissue stained red. Results are expressed as the mean ± SEM. Paired t-test was used for comparing cerebral infarct volume, while one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test was used for comparing neurological deficit scores. **P < 0.01, vs. sham (sham-operation) group; ##P < 0.01, ###P < 0.001, vs. MCAO group; ††P < 0.01, vs. EA (MCAO + EA) group. MCAO: Middle cerebral artery occlusion; EA: electroacupuncture; EA plus PD98059: MCAO + EA + PD98059.
Figure 3
Figure 3
Evaluation of histopathological changes. HE staining was performed on sections from the ischemic cortex 24 hours after reperfusion in the sham (sham-operation), MCAO and EA (MCAO + EA) groups (n = 5 per group). There were no obvious pathological changes in rats in the sham group. In comparison, pyramidal cells in the MCAO group were disordered and sparsely distributed. In the EA group, edema was alleviated, and the loss of neurons and structural abnormalities were reduced in the cortex. Ultrastructural changes were observed by transmission electron microscope (× 5,800). Images show the ultrastructural changes in pyramidal cells, astrocytes and the blood-brain barrier (BBB) 24 hours after reperfusion in the different groups (n = 3 per group). The pyramidal cells in the sham group were characterized by a round nucleus, and a clear nucleolus and cytoplasm. In contrast, pyramidal cells in the MCAO group displayed a shrunken nucleus, swollen or ganelles and chromatin condensation. In the EA group, edema was reduced, and the organelles, cytoplasm and vasculature showed fewer pathological changes. MCAO: Middle cerebral artery occlusion; EA: electroacupuncture; HE: hematoxylin-eosin.
Figure 4
Figure 4
Effects of EA on mRNA expression levels of Bax, Bcl-2, GCS and Nrf2 in the ischemic cortex after MCAO. Bax (A), Bcl-2 (B), GCSh (D), GCSl (E) and Nrf2 (F) mRNA expression levels, as assessed by real-time PCR. The Bax/Bcl-2 ratio is shown in C. Data (n = 7 per group in A–C, n = 5 per group in D–F) are expressed as the mean ± SEM. Statistical significance was determined using analysis of variance followed by Tukey’s multiple comparison test for multiple comparisons. *P < 0.05, **P < 0.01, vs. sham (sham-operation) group; #P < 0.05, vs. MCAO group; †P < 0.05, vs. EA (MCAO + EA) group. MCAO: Middle cerebral artery occlusion; EA: electroacupuncture; EA plus PD98059: MCAO + EA + PD98059; GCSh: gamma-glutamylcysteine synthetase heavy subunit; GCSl: gamma-glutamylcysteine synthetase light subunit; Nrf2: nuclear factor erythroid 2-related factor 2.
Figure 5
Figure 5
Effects of EA on serum GR, GSH and GSH-Px levels. EA significantly increased the GSH (A), GSH-Px (B) and GR (C) levels, compared with the MCAO group (n = 10 animals per group). Data are expressed as the mean ± SEM. Statistical significance was determined using analysis of variance followed by Tukey’s multiple comparison test for multiple comparisons. *P < 0.05, vs. sham (sham-operation) group; #P < 0.05, vs. MCAO group. MCAO: Middle cerebral artery occlusion; EA: electroacupuncture; GR: glutathione reductase; GSH: glutathione; GSH-Px: glutathione peroxidase; EA plus PD98059: MCAO + EA + PD98059.
Figure 6
Figure 6
Effects of EA on GCS and Nrf2 immunoreactivities in the ischemic cortex. (A) Immunohistochemical staining for GCS and Nrf2 in the cortex in the different groups (× 400) (n = 5 per group). (B, C) Mean optical densities of GCS and Nrf2-immunoreactive cells. Data are expressed as the mean ± SEM. Paired t-test was used to compare two groups, while analysis of variance followed by Tukey’s multiple comparisons test was used for multiple comparisons. **P < 0.01, vs. sham (sham-operation) group; ##P < 0.01, vs. MCAO group; ††P < 0.01, vs. EA (MCAO + EA) group. EA: Electroacupuncture; GCS: glutamylcysteine synthetase; Nrf2: nuclear factor erythroid 2-related factor 2; MCAO: middle cerebral artery occlusion; EA plus PD98059: MCAO + EA + PD98059.
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