A20-Binding Inhibitor of NF- κ B 1 Ameliorates Neuroinflammation and Mediates Antineuroinflammatory Effect of Electroacupuncture in Cerebral Ischemia/Reperfusion Rats

Abstract

A20-binding inhibitor of NF-κB 1 (ABIN1) is an inhibitor of NF-κB and exerts anti-inflammatory effect. Electroacupuncture (EA) is considered as a neuroprotective strategy by inhibiting neuroinflammatory damage after cerebral ischemia. This study was performed to explore the role of ABIN1 and investigate whether the ABIN1 is involved in the mechanism of EA in cerebral ischemia/reperfusion (I/R) rats. Male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) and received EA after reperfusion once a day. Lentivirus-mediated ABIN1 gene knockdown was used to detect the role of ABIN1 in neuroinflammation after I/R. ABIN1 expression, proinflammatory cytokine levels, microglial activation, neurological function, infarct volumes, and NF-κB activation were assessed. ABIN1 expression was elevated in the peri-infarct cortex and was further upregulated by EA. ABIN1 knockdown increased the levels of proinflammatory cytokines and activation of microglia, worsened neurological deficits, and enlarged the infarct volume. Moreover, ABIN1 was blocked to partially reverse the neuroprotective effect of EA, and this treatment weakened the ability of EA to suppress NF-κB activity. Based on these findings, ABIN1 is a potential suppressor of neuroinflammation and ABIN1 mediates the antineuroinflammatory effect of EA in cerebral I/R rats.

Figure 1

Schematic diagram of GV20, LI 4, and LR 3 acupoints of rat.

Figure 2

ABIN1 expression in the peri-infarct area at different time points. (a) The core and peri-infarct areas of a MCAO/R rat. (b–d) RT-qPCR and western blot were used to, respectively, detect the levels of the ABIN1 mRNA and protein in the peri-infarct cortex at 6 h, 12 h, 24 h, 48 h, and 72 h after reperfusion (n = 5 rats per group). The expression of ABIN1 was normalized to GAPDH. (e) ABIN1 (red) and DAPI (blue) immunofluorescence staining present the distribution of ABIN1 at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (f) Column chart presenting the ABIN1⁺ cell counts in the four groups (^∗P < 0.05 and ^∗∗P < 0.01 compared to the sham group; #P < 0.05 and ^##P < 0.01 compared to the MCAO/R group).

Figure 3

ABIN1 is colocated with A20 and NeuN and Iba-1, respectively, in the peri-infarct cortex. (a) Double immunofluorescence staining for ABIN1 and A20 in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (b) Coimmunoprecipitation of ABIN1 and A20 in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). (c) Double immunofluorescence labeling of ABIN1 (red) and NeuN (green, neurons), Iba-1 (green, microglia), and GFAP (green, astrocytes), respectively (n = 3 rats per group). White arrows show that ABIN1 is colocalized with NeuN and Iba-1, respectively. Scale bar = 50 μm. (d) Comparisons of the percentage of ABIN1⁺ NeuN⁺ cells among ABIN1⁺ cells and ABIN1⁺ Iba-1⁺ cells among ABIN1⁺ cells in the peri-infarct cortex. ^∗∗P < 0.01 compared to ABIN1⁺ NeuN⁺/ABIN1⁺.

Figure 4

ABIN1 knockdown increases proinflammatory cytokine production and microglial activation. (a-b) Levels of the ABIN1 mRNA and protein were detected 24 h after reperfusion using RT-qPCR and western blot, respectively, to confirm the efficiency of ABIN1 gene knockdown (n = 5 rats per group). (c) ELISA was used to detect the concentrations of TNF-α, IL-1β and MCP-1 in the peri-infarct cortex at 24 h after reperfusion (n = 5 rats per group). (d) Microglial morphology was observed using immunofluorescence staining for Iba-1 (green) in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (e and f) Quantification of microglia process endpoints and cell and process lengths/cell (^∗∗P < 0.01 compared to the sham group; ^##P < 0.01 compared to the MCAO/R group).

Figure 5

ABIN1 knockdown exacerbates the neurological deficits and enlarges the infarct volume. (a and b) The mNSS and MST were, respectively, analyzed at 72 h before and 24, 48, and 72 h after reperfusion (n = 5 rats per group). (c) Brain tissue sections were stained with TTC at 72 h after reperfusion (n = 5 rats per group). (d) The infarct volume is presented as a percentage of the intact hemisphere (^∗∗P < 0.01 compared to the sham group; ^#P < 0.05 compared to the MCAO/R group).

Figure 6

ABIN1 knockdown impairs the antineuroinflammatory effect of EA. (a) The concentrations of TNF-α, IL-1β, and MCP-1 in the peri-infarct cortex were detected using ELISAs at 24 h after reperfusion (n = 5 rats per group). (b) Microglial morphology was observed by conducting immunofluorescence staining for Iba-1 (green) in the peri-infarct cortex at 24 h after reperfusion (n = 3 rats per group). Scale bar = 50 μm. (c and d) Quantification of microglial endpoints/cell and process length/cell (^∗∗P < 0.01 compared to the MCAO/R group; ^#P < 0.05 and ^##P < 0.01 compared to the MCAO/R + EA group).

Figure 7

ABIN1 knockdown inhibits the neuroprotective effect of EA. (a-b) The mNSS and MST were recorded at 72 h before and 24, 48, and 72 h after reperfusion (n = 5 rats per group). (c) Brain tissue sections were stained with TTC at 72 h after reperfusion (n = 5 rats per group). (d) The infarct volume is presented as a percentage of the intact hemisphere (^∗P < 0.05 and ^∗∗P < 0.01 compared to the MCAO/R group; ^#P < 0.05 compared to the MCAO/R + EA group).

Figure 8

EA prevents NF-κB activation by upregulating ABIN1 expression. (a-c) The levels of ABIN1, p-IκBα, IκBα, nuclear NF-κB p65, and cytoplasmic NF-κB p65 proteins in the peri-infarct cortex at 24 h after reperfusion were detected using western blot. GAPDH served as the internal reference for total and cytoplasmic proteins, and histone H3 served as the internal reference for nuclear proteins (n = 5 rats per group). (d) The nuclear translocation of NF-κB p65 was observed with immunofluorescence staining in the peri-infarct area at 24 h after reperfusion (n = 3 rats per group). Scale bar = 20 μm. Column chart presenting the NF-κB p65⁺ cell counts in the four groups (^∗P < 0.05 and ^∗∗P < 0.01 compared to the MCAO/R group; ^#P < 0.05 and ^##P < 0.01 compared to the MCAO/R + EA group).