Downregulation of High mobility group box 2 relieves spinal cord injury by inhibiting microglia-mediated neuroinflammation

Abstract

Spinal cord injury (SCI), characterized by sensory disturbance and motor deficits, is associated with excessive inflammatory cytokine production of microglial cells. Previous studies have demonstrated High mobility group box 2 (HMGB2) as a microglial pro-inflammatory factor in stroke. This present study aims to evaluate the function of HMGB2 in a SCI rat model induced by striking the spinal cord at T9 to T12 using a rod. Our results showed that the levels of HMGB2 were significantly increased in the spinal cord tissues of SCI rats. Besides, HMGB2 downregulation was achieved by receiving an injection of lentivirus encoding HMGB2 shRNA in the spinal cord. Knockdown of HMGB2 suppressed SCI-induced microglial activation and neuroinflammation, as well as alleviated neuronal loss. In addition, we confirmed that HMGB2 silencing lessened lipopolysaccharide (LPS)-induced neuroinflammation in BV-2 cells. Furthermore, our findings demonstrated that HMGB2 knockdown suppressed the canonical nuclear factor of kB (NF-κB) signaling pathway both in vivo and in vitro. Collectively, this study manifested strong anti-inflammatory roles of HMGB2 knockdown on microglia-mediated neuroinflammation and suggested that HMGB2 might serve as a potential target for SCI therapy.

Keywords: High mobility group box 2; microglia; neuroinflammation; nuclear factor of κB; spinal cord injury.

Fig. 1.

High mobility group box 2 (HMGB2) was upregulated in the spinal cord tissues of rats. (A) The mRNA level of Hmgb2 was detected using real-time PCR. (B) Western blot was used to test the protein level of HMGB2. Data are presented as the mean ± SD (n=3) and were analyzed using two-way analyses of variance (ANOVA) followed by Bonferroni’s post hoc test, *P<0.05, **P<0.01. (C) Co-labeling of Iba-1 with HMGB2 in the spinal cord tissues of spinal cord injury (SCI) rats.

Fig. 2.

High mobility group box 2 (HMGB2) knockdown alleviated neuronal loss 3 days after spinal cord injury (SCI). (A) Real-time PCR was performed to determine the mRNA level of Hmgb2. (B) The protein level of HMGB2 was accessed using Western blot. Data are expressed as means ± SD (n=6) and were compared among groups using one-way analyses of variance (ANOVA) followed by Tukey’s post hoc test, **P<0.01. (C) The Basso Beattie Bresnahan (BBB) score of rats with SCI. Two-way ANOVA followed by Tukey’s post hoc test. Mean ± SD (n=6) **P<0.01. (D) LFB, H&E, and Nissl staining of the spinal cord tissues of rats with SCI. Black arrows showed neurons and red arrows showed demyelination vacuolations. (E) Specificity of NeuN was detected in the spinal cord tissues.

Fig. 3.

Reduction of High mobility group box 2 (HMGB2) restrained neuroinflammation. Immunofluorescence images of Iba-1 in the spinal cord tissues of spinal cord injury (SCI) rats. (B) The protein levels of p-NF-κB p65, NF-κB p65, p-IκBα, IκBα, and (C) nuclear NF-κB p65 were analyzed by Western blot analysis. Concentrations of (D) TNF-α, (E) IL-1β, and (F) IL-6 were evaluated using ELISA. All results are presented as the mean ± SD (n=6) and assessed by one-way analyses of variance (ANOVA) followed by Tukey’s post hoc test, *P<0.05, **P<0.01.

Fig. 4.

High mobility group box 2 (HMGB2) silencing decreased lipopolysaccharide (LPS)-induced inflammation in BV-2 cells. (A) Immunofluorescence staining of Iba-1 in BV-2 cells. (B) The levels of TNF-α, IL-1β, and IL-6 in supernatants collected from BV-2 cells were evaluated using ELISA. (C) Real-time PCR was used to test the mRNA levels of TNF-α, IL-1β, and IL-6. Western blot analysis was used to monitor the protein expression of (D) HMGB2 and nuclear NF-κB p65, (E) p-NF-κB p65, NF-κB p65, p-IκBα, and IκBα. (F) Immunofluorescence staining of NF-κB p65 in BV-2 cells. All number values are reported as the mean ± SD (n=3) and all data were analyzed using one-way analyses of variance (ANOVA) followed by Tukey’s post hoc test, **P<0.01.

Fig. 5.

The function of High mobility group box 2 (HMGB2) in spinal cord injury (SCI). Knockdown of HMGB2 attenuated SCI by suppressing microglia activation and neuroinflammation via inhibiting the NF-κB signaling pathway.