Inhibition of HMGB1 reduces rat spinal cord astrocytic swelling and AQP4 expression after oxygen-glucose deprivation and reoxygenation via TLR4 and NF-κB signaling in an IL-6-dependent manner

Abstract

Background: Spinal cord astrocyte swelling is an important component to spinal cord edema and is associated with poor functional recovery as well as therapeutic resistance after spinal cord injury (SCI). High mobility group box-1 (HMGB1) is a mediator of inflammatory responses in the central nervous system and plays a critical role after SCI. Given this, we sought to identify both the role and underlying mechanisms of HMGB1 in cellular swelling and aquaporin 4 (AQP4) expression in cultured rat spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R).

Methods: The post-natal day 1-2 Sprague-Dawley rat spinal cord astrocytes were cultured in vitro, and the OGD/R model was induced. We first investigated the effects of OGD/R on spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. We then studied the effects of HMGB1 inhibition on cellular swelling, HMGB1 and AQP4 expression, and HMGB1 release. The roles of both toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway and interleukin-6 (IL-6) in reducing cellular swelling resulting from HMGB1 inhibition in spinal cord astrocytes after OGD/R were studied. Intergroup data were compared using one-way analysis of variance (ANOVA) followed by Dunnett's test.

Results: The OGD/R increased spinal cord astrocytic swelling and HMGB1 and AQP4 expression, as well as HMGB1 release. Inhibition of HMGB1 using either HMGB1 shRNA or ethyl pyruvate resulted in reduced cellular volume, mitochondrial and endoplasmic reticulum swelling, and lysosome number and decreased upregulation of both HMGB1 and AQP4 in spinal cord astrocytes, as well as HMGB1 release. The HMGB1 effects on spinal cord astrocytic swelling and AQP4 upregulation after OGD/R were mediated-at least in part-via activation of TLR4, myeloid differentiation primary response gene 88 (MyD88), and NF-κB. These activation effects can be repressed by TLR4 inhibition using CLI-095 or C34, or by NF-κB inhibition using BAY 11-7082. Furthermore, either OGD/R or HMGB1 inhibition resulted in changes in IL-6 release. IL-6 was also shown to mediate AQP4 expression in spinal cord astrocytes.

Conclusions: HMGB1 upregulates AQP4 expression and promotes cell swelling in cultured spinal cord astrocytes after OGD/R, which is mediated through HMGB1/TLR4/MyD88/NF-κB signaling and in an IL-6-dependent manner.

Keywords: Aquaporin-4; Astrocyte swelling; High mobility group box-1; Interleukin-6; Nuclear factor-kappa B; Spinal cord injury; Toll-like receptor 4.

Fig. 1

Spinal cord astrocyte identification and high mobility group box-1 (HMGB1) knockdown. a Spinal cord astrocytes were identified using immunofluorescence. The percentage of cells stained with the astrocytic marker S100β, which were identified as astrocytes, was more than 95% of the total cells (three replicates). b HMGB1 knockdown efficiency in the plasma membrane and cytoplasm of spinal cord astrocytes was evaluated using Western blot for HMGB1 protein levels. Results were obtained after 72 h of specific HMGB1 shRNA treatment. HMGB1 protein levels were decreased to approximately 30% of normal levels with shRNA multiplicity of infection 60 as compared to normal astrocytes. *P < 0.05 vs. normal group (three replicates)

Fig. 2

Effects of oxygen-glucose deprivation/reoxygenation (OGD/R) on cellular swelling, high mobility group box-1 (HMGB1), and aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes as well as levels of HMGB1 and interleukin-6 (IL-6) released into the surrounding medium. a Astrocyte volume measurement was performed using a Live Cell Imaging System. Cellular volume was calculated by the average value of four measured diameters of the largest compiled Z-slice image. Cellular volumes of spinal cord astrocytes were significantly increased at 2, 6, 12, 24, and 48 h during reoxygenation after OGD when compared with normal astrocytes. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). b Membrane and cytoplasmic HMGB1 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). c Membrane and cytoplasmic AQP4 expression was significantly increased in spinal cord astrocytes at different time points after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). d HMGB1 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates). e IL-6 levels in the surrounding medium of spinal cord astrocytes were significantly increased at 6, 12, and 24 h during reoxygenation after OGD/R. #P < 0.05 vs. normal group; *P < 0.05 vs. OGD6h/R24h group (three replicates)

Fig. 3

Effects of inhibiting high mobility group box-1 (HMGB1) on cellular swelling in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R). a Astrocyte volume analysis was performed using a Live Cell Imaging System, and cellular volume was calculated by the average value of four measured diameters. Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly blocked increases in cellular volume of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation when compared with astrocytes of the OGD/R group. *P < 0.05 vs. OGD/R group (three replicates). b—d Effects of inhibiting HMGB1 on spinal cord astrocytic morphology and ultrastructure were evaluated using transmission electron microscopy at 6, 12, and 24 h during reoxygenation after OGD. After OGD/R, spinal cord astrocytes showed swelling at 6, 12, and 24 h during reoxygenation. The mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and an increase in the number of lysosomes (L) were concurrent with this observation. However, astrocytic swelling, mitochondrial (M) swelling, endoplasmic reticulum (ER) swelling and fragmentation, and the increase in lysosome (L) number after OGD/R were reduced by HMGB1 inhibition using either HMGB1 shRNA or EP (× 50,000, bar equal to 1 μm, three replicates)

Fig. 4

Effects of inhibiting high mobility group box-1 (HMGB1) on HMGB1, aquaporin-4 (AQP4), and toll-like receptor-4 (TLR4) expression in cultured spinal cord astrocytes after oxygen-glucose deprivation/reoxygenation (OGD/R) as well as levels of HMGB1 and interleukin-6 (IL-6) release into the surrounding medium. a Inhibiting HMGB1 using either HMGB1 shRNA or ethyl pyruvate (EP) significantly suppressed the increased levels of HMGB1 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. *P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 significantly suppressed the increased levels of AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. *P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 significantly suppressed increased levels of TLR4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 6, 12, and 24 h during reoxygenation after OGD. *P < 0.05 vs. OGD/R group (three replicates). d HMGB1, AQP4, and TLR4 immunofluorescence on spinal cord astrocytes at 24 h into the reoxygenation process after OGD showed significantly increased membrane and cytoplasmic levels of HMGB1, AQP4, and TLR4 in the OGD/R group when compared with those in the normal group. These were markedly suppressed in both the OGD/R + HMGB1 shRNA and OGD/R + EP groups (× 200, bar equal to 100 μm). *P < 0.05 vs. OGD/R group (three replicates). e, f Inhibiting HMGB1 mitigated increases in levels of HMGB1 and IL-6 in the surrounding medium when compared with levels in the OGD/R group at 6, 12, and 24 h during reoxygenation after OGD. *P < 0.05 vs. OGD/R group (three replicates)

Fig. 5

Effects of either inhibiting high mobility group box-1 (HMGB1) or toll-like receptor-4 (TLR4) on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, TLR4, myeloid differentiation primary response gene 88 (MyD88), aquaporin-4 (AQP4) upregulation, and nuclear factor-kappa B (NF-κB) activation as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a Inhibiting HMGB1 (using either HMGB1 shRNA or ethyl pyruvate (EP)) or TLR4 (using CLI-095 or C34) significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those in the OGD/R group. *P < 0.05 vs. OGD/R group (three replicates). b Inhibiting HMGB1 or TLR4 significantly suppressed the increased levels of TLR4, MyD88, and AQP4 in both the plasma membrane and cytoplasm of spinal cord astrocytes at 24 h during the reoxygenation process after OGD. *P < 0.05 vs. OGD/R group (three replicates). c Inhibiting HMGB1 or TLR4 significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation process after OGD. *P < 0.05 vs. OGD/R group (three replicates). d Immunofluorescence results showed that either inhibiting HMGB1 or TLR4 decreased membrane and cytoplasmic TLR4 and AQP4 upregulation and attenuated the increases of nuclear NF-κB when compared with the OGD/R group at 24 h during reoxygenation (× 200, bar equal to 100 μm). *P < 0.05 vs. OGD/R group (three replicates). e Inhibiting HMGB1 or TLR4 reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. *P < 0.05 vs. OGD/R group (three replicates)

Fig. 6

Effects of nuclear factor-kappa B (NF-κB) inhibition on oxygen-glucose deprivation/reoxygenation (OGD/R)-induced astrocytic swelling, NF-κB activation, and aquaporin-4 (AQP4) upregulation, as well as levels of interleukin-6 (IL-6) released into the surrounding medium. a NF-κB inhibition (using BAY 11-7082) significantly suppressed the increased nuclear levels of NF-κB and the upregulation of cytoplasmic p-IκBα in spinal cord astrocytes after 24 h of the reoxygenation phase after OGD. *P < 0.05 vs. OGD/R group (three replicates). b NF-κB and AQP4 immunofluorescence in spinal cord astrocytes after 24 h of the reoxygenation process after OGD showed significantly increased nuclear levels of NF-κB and membrane and cytoplasmic levels of AQP4 in the OGD/R group. Levels were markedly attenuated in the OGD/R + HMGB1 shRNA, OGD/R + BAY 11-7082, and OGD/R + EP groups (× 200, bar equal to 100 μm). *P < 0.05 vs. OGD/R group (three replicates). c NF-κB inhibition significantly reduced the increase in cellular volume of spinal cord astrocytes at 24 h during the reoxygenation process after OGD when compared with those of the OGD/R group. *P < 0.05 vs. OGD/R group (three replicates). d NF-κB inhibition significantly suppressed increased AQP4 levels in both the plasma membrane and cytoplasm of spinal cord astrocytes after 24 h of the reoxygenation process after OGD. *P < 0.05 vs. OGD/R group (three replicates). e NF-κB inhibition reduced increased levels of IL-6 in the surrounding medium when compared with those of the OGD/R group after 24 h of the reoxygenation process after OGD. *P < 0.05 vs. OGD/R group (three replicates)

Fig. 7

Effects of recombinant HMGB1 (rHMGB1) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. Incubation of cultured spinal cord astrocytes with rHMGB1 (0, 0.1, 1, 10, and 20 ng/ml) for 24 h did not induce dose-dependent increases in the membrane and cytoplasmic AQP4 expression (three replicates)

Fig. 8

Effects of interleukin-6 (IL-6) on aquaporin-4 (AQP4) expression in cultured spinal cord astrocytes. a Spinal cord astrocytes were exposed to exogenous IL-6 at 0, 0.1, 1, or 10 ng/ml. After 24 h exposure, the membrane and cytoplasmic AQP4 expression in spinal cord astrocytes were markedly increased in the IL-6 0.1 ng/ml group, IL-6 1 ng/ml group, and IL-6 10 ng/ml group. *P < 0.05 vs. 0 ng/ml group (three replicates). b IL-6 levels increased in the surrounding medium of the OGD/R group after 24 h of the reoxygenation process after OGD. In comparison, this increase was significantly reduced in the OGD/R + HMGB1 shRNA group. *P < 0.05 vs. OGD/R group (three replicates). c The effects of astrocyte conditioned medium (ACM) on AQP4 expression in cultured spinal cord astrocytes. Twenty-four hours exposure of spinal cord astrocytes to the ACM obtained from the OGD/R group significantly increased the membrane and cytoplasmic AQP4 expression when compared with astrocytes incubated with the ACM obtained from the OGD/R + HMGB1 shRNA group. *P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates). d Western blot analysis showed that the neutralizing anti-rat-IL-6 antibody could significantly reverse the upregulation effect of exogenous IL-6 or OGD/R ACM containing increased IL-6 on AQP4 expression in cultured spinal cord astrocytes. #P < 0.05 vs. astrocytes + IL-6 group; *P < 0.05 vs. astrocytes + OGD6h/R24h ACM group (three replicates)