BRD4 inhibition attenuates inflammatory response in microglia and facilitates recovery after spinal cord injury in rats

Abstract

The pathophysiology of spinal cord injury (SCI) involves primary injury and secondary injury. For the irreversibility of primary injury, therapies of SCI mainly focus on secondary injury, whereas inflammation is considered to be a major target for secondary injury; however the regulation of inflammation in SCI is unclear and targeted therapies are still lacking. In this study, we found that the expression of BRD4 was correlated with pro-inflammatory cytokines after SCI in rats; in vitro study in microglia showed that BRD4 inhibition either by lentivirus or JQ1 may both suppress the MAPK and NF-κB signalling pathways, which are the two major signalling pathways involved in inflammatory response in microglia. BRD4 inhibition by JQ1 not only blocked microglial M1 polarization, but also repressed the level of pro-inflammatory cytokines in microglia in vitro and in vivo. Furthermore, BRD4 inhibition by JQ1 can improve functional recovery and structural disorder as well as reduce neuron loss in SCI rats. Overall, this study illustrates that microglial BRD4 level is increased after SCI and BRD4 inhibition is able to suppress M1 polarization and pro-inflammatory cytokine production in microglia which ultimately promotes functional recovery after SCI.

Keywords: BRD4; JQ1; inflammation; microglia; spinal cord injury.

Figure 1

The level of pro‐inflammatory cytokines was correlated with the expression of Bromodomain‐containing protein 4 (BRD4). (A, B, C) The levels of TNF‐α, IL‐1β and IL‐6 were detected using ELISA in damaged spinal cord at different time points after spinal cord injury (SCI). (D, E) Representative images of western blots and quantification data of BRD4 expression at different time points after traumatic SCI. (F, G) Representative western blots and quantification analysis of BRD4 expression in HAPI microglia cells treated with TNF‐α (50 ng/mL), IL‐1β (50 ng/mL) and IL‐6 (25 ng/mL) for 24 h. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 2

Bromodomain‐containing protein 4 knockdown and JQ1 block activation of MAPK signalling in microglia. (A, B) The BRD4 was silenced by lentivirus in HAPI microglia cells. (C‐F) Effects of BRD4 knockdown on phosphorylation of MAPKs (p38, JNK and ERK) in HAPI microglia cells. HAPI cells were administrated with LPS (100 ng/mL) for 1 h after pre‐treatment of JQ1 for 2 h. Then cells were collected and the cell lysate was subjected to western blots. (G‐J) Representative western blots and quantitative data for p‐p38, p‐38, p‐JNK, JNK, p‐ERK and ERK in HAPI microglia cells in the presence or absence of JQ1 or LPS. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 3

Bromodomain‐containing protein 4 knockdown and JQ1 alleviate the activation of NF‐κB signalling pathway in LPS‐treated microglia. (A, B) The effects of BRD4 knockdown on phosphorylation of p65 and the level of IκBα in LPS‐treated microglia. (C, D) Representative western blots and quantitative data for p‐p65 and IκBα in HAPI microglia cells with or without pre‐treatment of JQ1 for 2 h. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 4

Bromodomain‐containing protein 4 inhibition by JQ1 attenuates the microglial M1 polarization. A, Morphological results of microglia treated with JQ1 or LPS. Scale bar: 100 μmol/L. LPS‐treated microglia displayed retracted branches, whereas pre‐treatment of JQ1 extended microglial branches under LPS‐induced inflammatory condition. HAPI microglia cells were treated with LPS (1 μg/mL) for 24 h. (B‐D) Immunofluorescence staining and quantitative data for IBA‐1 in microglia from different groups. Scale bar: 100 μmol/L. (E‐G) Representative western blots and quantitative data for INOS and COX‐2 expression in microglia from different groups. HAPI microglia cells were treated with or without LPS for 6 h. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 5

Bromodomain‐containing protein 4 inhibition by JQ1 suppresses the expression of pro‐inflammatory cytokines in microglia. Before exposure to LPS (1 μg/mL) for 6 h, HAPI microglia cells were treated with JQ1 (200 nmol/L) for 2 h. (A, B, C) Real‐time PCR assay of Tnfa, Il1b and Il6 mRNA in HAPI microglia cells from each group as treated above. (D, E, F) ELISA measurements of TNF‐α, IL‐1β and IL‐6 from HAPI microglia cells in different groups. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 6

Bromodomain‐containing protein 4 inhibition by JQ1 suppresses inflammatory response after SCI. (A) Double immunofluorescence staining for CD68 (green) and IBA‐1 (red) positive microglia of sections from the tissue at 24 h after SCI. White arrows mark positive cells. Scale bar: 50 μmol/L. (B‐D) Quantification analysis of the levels of TNF‐α, IL‐1β and IL‐6 in spinal cord after 6 h after SCI. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 7

Bromodomain‐containing protein 4 inhibition by JQ1 improves functional recovery and attenuates structural disorder with less neuron loss in spinal cord after traumatic SCI. (A) The Basso, Beattie and Bresnahan (BBB) scores. (B, C) Quantification of BBB scores at 14 and 21 d. D, The footprint analysis of rats from Sham group, SCI group and SCI+JQ1 group. E, Representative images from haematoxylin and eosin and Nissl staining at 14 d after surgery. Scale bar: 1000 μmol/L. All experiments were performed as mean ± SD of three times in duplicates. *P < 0.05, **P < 0.01

Figure 8

The schematic diagram depicting the molecular mechanism underlying the role of Bromodomain‐containing protein 4 in microglial inflammatory response after traumatic spinal cord injury (SCI)