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. 2019 Aug 1;9(41):23916-23924.
doi: 10.1039/c9ra03614h. eCollection 2019 Jul 29.

Retracted Article: Elevation of USP4 antagonizes oxygen glucose deprivation/reoxygenation-evoked microglia activation and neuroinflammation-mediated neurotoxicity via the TRAF6-NF-κB signaling

Zhaoxia Wang  1 , Xinming Li  2 , Zhixing Shao  1 , ZhengFang  1 , Yueping Zhai  2
Affiliations

Retracted Article: Elevation of USP4 antagonizes oxygen glucose deprivation/reoxygenation-evoked microglia activation and neuroinflammation-mediated neurotoxicity via the TRAF6-NF-κB signaling

Zhaoxia Wang et al. RSC Adv. .

Retraction in

Abstract

An ischemic stroke is a devastating neurological disease with the typical occurrence of brain ischemia/reperfusion (I/R) injury, and it has high mortality and disability globally. Microglia activation after a stroke results in the release of pro-inflammatory cytokines that can further aggravate brain damage. A recent study confirmed the potential role of ubiquitin-specific peptidase 4 (USP4) in the injury process. Nevertheless, the role and mechanism of USP4 during an ischemic stroke remain elusive. In this research, we simulated an I/R injury by oxygen glucose deprivation/reoxygenation (OGD/R) in vitro and confirmed the obvious down-regulation of USP4 in microglia under OGD/R conditions. Moreover, USP4 elevation antagonized the OGD/R-induced microglia proliferation and activation by suppressing the NO levels and the expression of the microglial marker IBA-1. Additionally, the overexpression of USP4 suppressed the release of microglia activation-induced pro-inflammatory cytokines, including IL-1β, IL-6, and TNF-α. Intriguingly, incubation with the conditioned medium from the microglia under OGD/R conditions induced neurotoxicity by inhibiting cell viability and increasing the LDH release, apoptosis, and caspase-3 activity, which were reversed following USP4 overexpression. Mechanism analysis corroborated that USP4 up-regulation repressed the OGD/R-induced activation of TRAF6-NF-κB signaling. Notably, restoring the TRAF6 signaling ameliorated the suppressive effects of USP4 elevation on microglia activation, inflammation, and the subsequent neuron injury. These findings suggest that USP4 may alleviate ischemic stroke by restraining microglia-mediated neuro-inflammation and neurotoxicity via the TRAF6-NF-κB pathway, due to which it is a promising therapeutic agent against strokes.

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Conflict of interest statement

There are no conflicts of interest concerning this article.

Figures

Fig. 1
Fig. 1. The expression of USP4 decreased in microglia under OGD/R conditions. (A) Microglia and neuronal morphology under light microscopy after 5 days of culture. Scale bars, 40 μM. (B) For identification, cells were separately stained with IBA-1 and MAP-2; then, they were observed by a fluorescence microscope. Scale bars, 10 μM. (C) The isolated microglial cells were incubated with an oxygen glucose deprivation condition for 3 h to simulate an ischemic insult. Then, the cells were exposed to normoxic conditions (95% air, 5% CO2) to mimic reperfusion. The mRNA levels of USP4 were determined by qRT-PCR. (D) The protein expression of USP4 was analyzed by western blotting. *P < 0.05 versus control groups.
Fig. 2
Fig. 2. Overexpression of USP4 suppressed microglia proliferation and activation under OGD/R conditions. (A) Microglia were transfected with the recombinant USP4 vector or empty vector and the transcriptional levels of USP4 mRNA were analyzed. (B) Western blotting was performed to determine the corresponding protein levels of USP4. (C) Cells transfected with USP4 plasmids were exposed to OGD/R and the cell proliferation was then evaluated using an MTT assay. (D and E) The effects on microglia marker IBA mRNA (D) and protein expression (E) were detected. (F) The contents of NO were measured using commercial kits. *P < 0.05 vs. control groups, #P < 0.05 vs. OGD/R groups.
Fig. 3
Fig. 3. USP4 elevation inhibited the OGD/R-induced inflammatory response in microglia. Cells were transfected with pcDNA-USP4 plasmids prior to OGD/R exposure. Then, the transcriptional levels of IL-1β (A), IL-6 (B) and TNF-α (C) were determined by qRT-PCR. (D–F) An ELISA assay was conducted to measure the concentrations of IL-1β (A), IL-6 (B) and TNF-α (C) in USP4-overexpressed microglia under OGD/R conditions. *P < 0.05, #P < 0.05.
Fig. 4
Fig. 4. Enhancement of USP4 alleviated neurotoxicity induced by incubation with a conditioned medium (CM) from OGD/R-activated microglia. (A) The conditioned medium (CM) was collected from OGD/R-activated microglia that were pre-transfected with pcDNA-USP4 vectors. Then, the primary neurons were incubated with CM from microglia. Cell viability was evaluated using an MTT assay. (B) LDH release was then tested using a colorimetric assay kit. (C) A flow cytometer was applied to analyze cell apoptosis. (D) The effects on caspase-3 activity were also assessed. *P < 0.05, #P < 0.05.
Fig. 5
Fig. 5. USP4 regulated microglia activation-induced inflammation and neuron injury by blocking the TRAF6-NF-κB signaling. (A) After transfection with the recombinant USP4 vectors, microglia were exposed to OGD/R. Then, the expression of TRAF6, p-p65 NF-κB was detected by western blotting. (B and C) The corresponding quantified analysis of TRAF6, p-p65 NF-κB was evaluated by the Image J software. (D and E) Effects of pcDNA-TRAF6 transfection on the protein levels of TRAF6 and p-p65NF-κB. (F) The mRNA levels of IBA-1 were measured by qRT-PCR. (G–I) The concentrations of IL-1β (G), IL-6 (H) and TNF-α (I) were detected by an ELISA assay. (J–L) The subsequent effects on cell viability (J), LDH release (K) and apoptosis (L) were also evaluated. *P < 0.05 vs. control groups, #P < 0.05 vs. OGD/R groups, and $P < 0.05 vs. OGD/R + USP4 groups.
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