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. 2016 Oct 3;13(1):258.
doi: 10.1186/s12974-016-0731-3.

Upregulation of neuronal zinc finger protein A20 expression is required for electroacupuncture to attenuate the cerebral inflammatory injury mediated by the nuclear factor-kB signaling pathway in cerebral ischemia/reperfusion rats

Jian Zhan  1   2   3 , Wenyi Qin  4 , Ying Zhang  1   2 , Jing Jiang  1   2 , Hongmei Ma  1   2 , Qiongli Li  1   2 , Yong Luo  5   6
Affiliations

Upregulation of neuronal zinc finger protein A20 expression is required for electroacupuncture to attenuate the cerebral inflammatory injury mediated by the nuclear factor-kB signaling pathway in cerebral ischemia/reperfusion rats

Jian Zhan et al. J Neuroinflammation. .

Abstract

Background: Zinc finger protein A20 (tumor necrosis factor alpha-induced protein 3) functions as a potent negative feedback inhibitor of the nuclear factor-kB (NF-kB) signaling. It exerts these effects by interrupting the activation of IkB kinase beta (IKKβ), the most critical kinase in upstream of NF-kB, and thereby controlling inflammatory homeostasis. We reported previously that electroacupuncture (EA) could effectively suppress IKKβ activation. However, the mechanism underlying these effects was unclear. Therefore, the current study further explored the effects of EA on A20 expression in rat brain and investigated the possible mechanism of A20 in anti-neuroinflammation mediated by EA using transient middle cerebral artery occlusion (MCAO) rats.

Methods: Rats were treated with EA at the "Baihui (GV20)," "Hegu (L14)," and "Taichong (Liv3)" acupoints once a day starting 2 h after focal cerebral ischemia. The spatiotemporal expression of A20, neurobehavioral scores, infarction volumes, cytokine levels, glial cell activation, and the NF-kB signaling were assessed at the indicated time points. A20 gene interference (overexpression and silencing) was used to investigate the role of A20 in mediating the neuroprotective effects of EA and in regulating the interaction between neuronal and glial cells by suppressing neuronal NF-kB signaling during cerebral ischemia/reperfusion-induced neuroinflammation.

Results: EA treatment increased A20 expression with an earlier peak and longer lasting upregulation. The upregulated A20 protein was predominantly located in neurons in the cortical zone of the ischemia/reperfusion. Furthermore, neuronal A20 cell counts were positively correlated with neurobehavioral scores but negatively correlated with infarct volume, the accumulation of pro-inflammatory cytokines, and glial cell activation. Moreover, the effects of EA on improving the neurological outcome and suppressing neuroinflammation in the brain were reversed by A20 silencing. Finally, A20 silencing also suppressed the ability of EA to inhibit neuronal NF-kB signaling pathway.

Conclusions: Ischemia/reperfusion cortical neurons in MCAO rats are the main cell types that express A20, and there is a correlation between A20 expression and the suppression of neuroinflammation and the resulting neuroprotective effects. EA upregulated neuronal A20 expression, which played an essential role in the anti-inflammatory effects of EA by suppressing the neuronal NF-kB signaling pathway in the brains of MCAO rats.

Keywords: Cerebral ischemia; Electroacupuncture; NF-kB signaling pathway; Neuroinflammation; Zinc finger protein A20.

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Figures

Fig. 1
Fig. 1
Effects of EA on A20 expression in the rat brain after I/R. a A20 mRNA levels in the rat brain were measured at the indicated time points using RT-qPCR. The graph shows the relative mRNA levels after normalization to the housekeeping gene β-actin. Data are presented as means ± SEM; n = 5 per time point per group. ***P < 0.001 vs. the MCAO group; ### P < 0.001 vs. the sham group. b Western blots showing the expression of A20 in rat brains at the indicated time points. The housekeeping protein β-actin was used as a loading control. c Graph showing the relative A20 protein levels after normalized to β-actin. Data are presented as means ± SEM; n = 3 per time point per group. **P < 0.01, ***P < 0.001 vs. the MCAO group; # P < 0.05, ### P < 0.001 vs. the sham group. d Immunofluorescence staining of A20 expression in the ischemia/reperfusion cortex of MCAO and MCAO + EA group rats 24 h after reperfusion and in the cortex of the sham group rats. Scale bar = 50 μm. e The A20-positive cell counts in the ischemia/reperfusion cortex of MCAO and MCAO + EA rats and the cortex of rats in the sham group. The A20-positive cell counts were expressed as number/in.2. Data are presented as means ± SEM; n = 5 per group. ### P < 0.001 vs. the sham group; ***P < 0.001 vs. the MCAO group
Fig. 2
Fig. 2
The spatial distribution and cell-type location of A20 in the rat brain after I/R. Brain sections of the ischemia/reperfusion cortex of rats in the MCAO + EA group 24 h after reperfusion. Sections were stained with double immunofluorescence labeling (a–d), which showed the upregulated expression of A20 protein (red) in neurons (green, identified using NeuN). e–h Very few of A20-positive (red) astrocytes (green, identified using GFAP) were observed. i–l A20 (red) was barely presented in microglial cells (green, identified using iba-I). Scale bar = 50 μm. 1–3 were the magnified parts from the merged images of d, h, l which more clearly showed the location relationship between A20 and neurons, astrocytes, and microglia, respectively. Scale bar = 25 μm
Fig. 3
Fig. 3
Neuroprotective effects of A20 on focal cerebral ischemia reperfusion injury. The neurological score and brain infarction volume were evaluated 72 h after reperfusion in rats in the four groups (MCAO, MCAO + LV-A20, MCAO + LV-shA20, and MCAO + vehicle). a Neurological scores are presented as median (interquartile range), n = 10 per group. *P < 0.05, # P < 0.05 vs. the MCAO group. b Brain infarct volume presented as a percentage of the intact hemisphere. Data are presented as means ± SEM, n = 10 per group. ***P < 0.001, ### P < 0.001 vs. the MCAO group. c Images of cerebral infarction (white, infarct tissue; red, non-infarct tissue) were stained using 2,3,5-triphenyltetrazolium chloride in coronal sections of rat brains
Fig. 4
Fig. 4
A20 silencing inhibits the neuroprotective effects of EA on focal cerebral ischemia reperfusion injury. Neurological score and brain infarction volume were evaluated 72 h after reperfusion in rats in the four groups (MCAO, MCAO + EA, MCAO + EA + LV-shA20, MCAO + EA + vehicle). a Neurological scores are presented as median (interquartile range), n = 10 per group. *P < 0.05 vs. the MCAO group; ### P < 0.001 vs. the MCAO + EA group. b Brain infarction volume presented as a percentage of the intact hemisphere. Data are presented as means ± SEM, n = 10 per group. ***P < 0.001 vs. the MCAO group; ### P < 0.001 vs. the MCAO + EA group. c Images of cerebral infarction (white, infarct tissue; red, non-infarct tissue) were stained using 2,3,5-triphenyltetrazolium chloride in coronal sections of rat brains
Fig. 5
Fig. 5
A20 silencing prevents the anti-inflammatory effects of EA after focal cerebral ischemia reperfusion. Experiments were performed 24 h after reperfusion in the ischemia/reperfusion cortex, n = 5 per group. a TNF-α and b IL-1β content was measured using ELISA. $$$ P < 0.001 vs. the sham group; *P < 0.05, ***P < 0.001 vs. the MCAO group; ### P < 0.001 vs. the MCAO + EA group. The relative mRNA levels of c A1 and d GFAP were measured using RT-qPCR. $$$ P < 0.001 vs. the sham group; ***P < 0.001, *P < 0.05 vs. the MCAO group; ### P < 0.001 vs. the MCAO + EA group. The activated morphology of astrocytes and microglia was demonstrated using e GFAP and f Iba-1 immunofluorescence staining (green), respectively. Yellow arrows indicate reactive astrocytes with thick cell bodies, and red arrows indicate hypertrophied activated microglia, noted by their stout, dense appearance with shorter and thicker branched projections. Scale bar = 50 μm
Fig. 6
Fig. 6
The correlations between A20-positive neuronal cell count and the inflammatory response and neurological outcome. a The relationship between the number of A20-positive neurons and neurological scores (n = 20 pairs, r = 0.8140, P < 0.0001). b The relationship between the number of A20-positive neurons and the relative infarct volume (n = 20 pairs, r = −0.7591, P < 0.0001). c The relationship between the number of A20-positive neurons and TNF-α content (n = 20 pairs, r = −0.8504, P < 0.0001). d The relationship between the number of A20-positive neurons and IL-1β content (n = 20 pairs, r = −0.8362, P < 0.0001). e The relationship between the number of A20-positive neurons and the relative A1 mRNA level (n = 20 pairs, r = −0.8704, P < 0.0001). f The relationship between the number of A20-positive neurons and the relative GFAP mRNA level (n = 20 pairs, r = −0.8209, P < 0.0001)
Fig. 7
Fig. 7
EA suppressed NF-kB signaling by upregulating A20. a Western blots showing the expression of A20, p-IKKβ, IKKβ, p-IkBα, IkBα, nuclear p65, and cytoplasmic p65 in ischemia/reperfusion cortical extracts 24 h after reperfusion. β-actin was used as the loading controls for total and cytoplasm proteins, and lamin B was used as the nuclear loading control. b The phosphorylation ratio of IKKβ and IkBα is presented as the p-IKKβ/IKKβ and p-IkBα/IkBα ratios, respectively. The nuclear translocation ratio of NF-kB p65 was indicated as nucleus-p65/cytoplasm-p65. n = 5 per group. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the MCAO group; ### P < 0.001 vs. the MCAO + EA group. c At 24 h after reperfusion, NF-kB p65 immunohistochemistry (brown) was performed to show NF-kB p65 nuclear translocation in the ischemia/reperfusion cortex of rats from the four groups (sham, MCAO, MCAO + EA, MCAO + EA + LV-shA20, n = 5 per group). Yellow arrows indicate NF-kB p65 mainly in cytoplasm, and red arrows indicate NF-kB p65 both in cytoplasm and nucleus. Scale bar = 50 μm. d Immunofluorescence staining was performed to demonstrate NF-kB p65 expression (red) in neurons (green) of ischemia/reperfusion cortex of rats from the four groups (sham, MCAO, MCAO + EA, MCAO + EA + LV-shA20, n = 5 per group) at reperfusion 24 h. Yellow arrows indicate NF-kB p65 mainly in cytoplasm, and red arrows indicate NF-kB p65 both in cytoplasm and nucleus. Scale bar = 50 μm
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