Circular RNA DLGAP4 Inhibits Ischemic Stroke-Induced Microglia M1 Polarization and Proinflammatory Cytokine Production, Possibly through the NF-κB Pathway

Circular RNA DLGAP4 Inhibits Ischemic Stroke-Induced Microglia M1 Polarization and Proinflammatory Cytokine Production, Possibly through the NF-κB Pathway

Ji Ding,¹ Yun Zhang¹ and Min Xu¹

¹Department of Neurology, Tong Ren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China

Circular RNA DLGAP4 (circ_DLGAP4) participates in the progression of ischemic stroke (IS), but whether it could regulate microglia activation to affect IS injury is unclear. This study aimed to explore the effect of circ_DLGAP4 on IS-induced microglia polarization and inflammatory cytokines, and the underlying mechanism. BV-2 cells (microglia) were transfected with circ_DLGAP4 overexpression (oeCirc), short hairpin RNA plasmid (shCirc), or corresponding negative control plasmids (oeNC and shNC). oeCirc or oeNC transfected cells were also treated with phorbol 12-myristate 13-acetate (PMA). Subsequently, BV-2 cells were treated with oxygen-glucose deprivation and reperfusion (OGD/R) to mimic IS. Circ_DLGAP4 was reduced in OGD/R-stimulated microglia versus normal microglia. Circ_DLGAP4 overexpression decreased cluster of differentiation (CD)68 and CD86, but increased CD206 and arginase-1 in OGD/R-stimulated microglia, suggesting that circ_DLGAP4 overexpression might inhibit M1 but facilitate M2 polarization of microglia. Besides, circ_DLGAP4 overexpression reduced tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, but elevated IL-10 in OGD/R-stimulated microglia, indicating that circ_DLGAP4 overexpression reduced proinflammatory cytokines but facilitated anti-inflammatory cytokines. Circ_DLGAP4 overexpression decreased p-nuclear factor kappa-B (NF-κB) and p-NF-κB inhibitor (IκB)-α in OGD/R-stimulated microglia, suggesting its inhibition of the NF-κB pathway. Notably, circ_DLGAP4 downregulation reversed the above phenomenon. PMA facilitated M1 polarization and proinflammatory cytokines but inhibited M2 polarization and anti-inflammatory cytokines in OGD/R-stimulated microglia. Interestingly, PMA attenuated the effect of circ_DLGAP4 overexpression on the above-mentioned processes in OGD/R-stimulated microglia. In conclusion, circ_DLGAP4 may attenuate IS injury by inhibiting microglia M1 polarization and proinflammatory cytokine production, which may be attributed to the inactivation of the NF-κB pathway.

Key words —— circular RNA DLGAP4; inflammatory cytokine; ischemic stroke; microglia polarization; NF-κB pathway

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Tohoku J. Exp. Med., 2024 August, 263(4), 277-285.

*These two authors contributed equally to this work.

Correspondence: Min Xu, Department of Neurology, Tong Ren Hospital, Shanghai Jiao Tong University School of Medicine, No.1111, Xianxia Road, Shanghai 200050, China.

e-mail: xm19760925@126.com