KDM4A, involved in the inflammatory and oxidative stress caused by traumatic brain injury-hemorrhagic shock, partly through the regulation of the microglia M1 polarization

Abstract

Background: Microglial polarization and the subsequent neuroinflammatory response and oxidative stress are contributing factors for traumatic brain injury (TBI) plus hemorrhagic shock (HS) induced brain injury. In the present work, we have explored whether Lysine (K)-specific demethylase 4 A (KDM4A) modulates microglia M1 polarization in the TBI and HS mice.

Results: Male C57BL/6J mice were used to investigate the microglia polarization in the TBI + HS model in vivo. Lipopolysaccharide (LPS)-induced BV2 cells were used to examine the mechanism of KDM4A in regulating microglia polarization in vitro. We found that TBI + HS resulted in neuronal loss and microglia M1 polarization in vivo, reflected by the increased level of Iba1, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, malondialdehyde (MDA) and the decreased level of reduced glutathione (GSH). Additionally, KDM4A was upregulated in response to TBI + HS and microglia were among the cell types showing the increased level of KDM4A. Similar to the results in vivo, KDM4A also highly expressed in LPS-induced BV2 cells. LPS-induced BV2 cells exhibited enhanced microglia M1 polarization, and enhanced level of pro-inflammatory cytokines, oxidative stress and reactive oxygen species (ROS), while this enhancement was abolished by the suppression of KDM4A.

Conclusion: Accordingly, our findings indicated that KDM4A was upregulated in response to TBI + HS and microglia were among the cell types showing the increased level of KDM4A. The important role of KDM4A in TBI + HS-induced inflammatory response and oxidative stress was at least partially realized through regulating microglia M1 polarization.

Keywords: HS; KDM4A; M1 polarization; Microglia; TBI.

Fig. 1

TBI + HS accelerated microglia M1 polarization and aggravated the microglia-mediated inflammatory response and oxidative stress. (A). The lesions in the brain sections of the sham and TBI + HS mice was measured by Nissl-staining. (B). Enlargement Nissl staining images of neuronal loss in the ischemic penumbra area (the red box area in A) of sham and TBI + HS mice. (C). The images of double immunofluorescent staining for NeuN (green) and TUNEL (red) in sham and TBI + HS mice. (D-G). The content of TNF-α, IL-1β, MDA and GSH in sham and TBI + HS mice were measured by detection kits (n = 6). Unpaired t-test was used to analyze data. (H). The images of double immunofluorescent staining for CD16/32 (green) and Iba1 (red) in sham and TBI + HS mice. Blue fluorescence represented DAPI. Data were collected from three independent experiments and expressed as mean ± SD.

Fig. 2

KDM4A was upregulated in the activated microglia of TBI + HS mice. (A-B). The mRNA and protein level of KDM4A in the mice cerebral cortex tissues were accessed by qRT-PCR and western blot (n = 6). Unpaired t-test was used to analyze data. (C). The images of double immunofluorescent staining for KDM4A (green) and Iba1 (red) in sham and TBI + HS mice. Blue fluorescence represented DAPI. Data were collected from three independent experiments and expressed as mean ± SD. Full-length blots are presented in Supplementary Figure S1

Fig. 3

KDM4A was upregulated in LPS-induced BV2 cells. (A-D). The content of TNF-α, IL-1β, MDA and GSH in LPS-induced BV2 cells was measured by detection kits (n = 3). (E-F). The mRNA and protein level of KDM4A in LPS-induced BV2 cells was accessed by qRT-PCR and western blot (n = 3). One-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test were used to analyze data. Data were collected from three independent experiments and expressed as mean ± SD. Full-length blots are presented in Supplementary Figure S2

Fig. 4

KDM4A involved in the inflammation and oxidative stress in LPS-induced BV2 cells. (A). The mRNA and protein expression levels of KDM4A in KDM4A down-regulated BV2 cells (n = 3). (B). The mRNA and protein expression levels of KDM4A in KDM4A down-regulated LPS-induced BV2 cells (n = 3). (C-F). The content of TNF-α, IL-1β, MDA and GSH in KDM4A inhibited LPS-induced BV2 cells were measured by detection kits (n = 3). ANOVA and Tukey’s multiple comparisons test were used to analyze data. (G). ROS level in KDM4A down-regulated LPS-induced BV2 cells. Data were collected from three independent experiments and expressed as mean ± SD. Full-length blots are presented in Supplementary Figure S3

Fig. 5

Suppressed KDM4A abolished microglia M1 polarization in LPS-induced BV2 cells. (A-B). The mRNA and protein expression levels of iNOS and COX-2 in KDM4A down-regulated BV2 cells (n = 3). ANOVA and Tukey’s multiple comparisons test were used to analyze data. (C). Images of double immunofluorescent staining for KDM4A (red) and CD16/32 (green) in KDM4A inhibited LPS-induced BV2 cells. Blue fluorescence represented DAPI. Data were collected from three independent experiments and expressed as mean ± SD. Full-length blots are presented in Supplementary Figure S4

Fig. 6

The simple scheme indicated that TBI + HS resulted in enhanced microglia M1 polarization. KDM4A was upregulated in response to TBI + HS and microglia were among the cell types showing the increased level of KDM4A. Inhibition of KDM4A alleviated the inflammation and oxidative stress, which was related with microglia activation. Accordingly, our findings indicated that the important role of KDM4A in TBI + HS induced inflammatory response and oxidative stress was at least partially realized through microglia M1 polarization