Knockdown of A20 attenuates microglial susceptibility to OGD/R-induced ferroptosis and upregulates inflammatory responses

Abstract

The A20 protein is considered to have a potent anti-inflammatory effect, but its mechanism of action in the regulation of ferroptosis and inflammation after stroke is still unknown. In this study, the A20-knockdown BV2 cell line (sh-A20 BV2) was constructed at first, and the oxygen-glucose deprivation/re-oxygenation (OGD/R) cell model was constructed. Both the BV2 and sh-A20 BV2 cells were treated with the ferroptosis inducer erastin for 48 h, the ferroptosis-related indicators were detected by western blot. The mechanism of ferroptosis was explored by western blot and immunofluorescence. Under OGD/R pressure, the oxidative stress level of sh-A20 BV2 cells was inhibited, but the secretion of the inflammatory factors TNF-α, IL-1β, and IL-6 was significantly upregulated. And sh-A20 BV2 cells had higher expression levels of GPX4 and NLRP3 proteins under OGD/R induction. Western blot further confirmed that sh-A20 BV2 cells inhibited OGD/R-induced ferroptosis. Under the effect of erastin of the ferroptosis inducer (0–1000 nM), sh-A20 BV2 cells had higher cell viability than wild-type BV2 cells and significantly inhibited the accumulation of ROS and the level of oxidative stress damage. It was confirmed that A20 could promote the activation of the IκBα/NFκB/iNOS pathway. It was confirmed by an iNOS inhibitor that iNOS inhibition could reverse the resistance effect of BV2 cells to OGD/R-induced ferroptosis after A20 knockdown. In conclusion, this study demonstrated that inhibition of A20 mediates a stronger inflammatory response while enhancing microglial resistance by knocking down A20 in BV2 cells.

Keywords:

• A20
• oxygen-glucose deprivation/re-oxygenation
• ferroptosis
• inflammatory responses
• iNOS

Author contributions

X. L and X. J designed the study. X. L, X. W, and X. Y drafted the original manuscript, collected the data and reviewed the literature. C. W, K. W and X. H interpreted the data and critically reviewed the manuscript. W. Z carried out the final check for all the data and the final approval of the version to be published. All authors read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Additional information

Funding

This work was supported by the Science and Technology Project of Suzhou Municipal Commission of Health and Family Planning (grant No.LCZX201924) and the Youth Science and Technology Project of Suzhou City (grant No.kjxw2018063).