HMGB1 increases myelin debris phagocytosis and ICAM-1 expression in rat central nervous system endothelial cells in vitro via the TLR4/NF-κB pathway

doi:10.21203/rs.3.rs-1706741/v1

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HMGB1 increases myelin debris phagocytosis and ICAM-1 expression in rat central nervous system endothelial cells in vitro via the TLR4/NF-κB pathway

https://doi.org/10.21203/rs.3.rs-1706741/v1

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Objective

Myelin debris mediates the expression of intercellular adhesion molecule-1 (ICAM-1) in endothelial cells of the central nervous system (CNS) after disease and injury and plays a critical role in promoting the recruitment of peripheral immune cells. As an inflammatory necrosis factor, high mobility group box-1 (HMGB1) regulates the inflammatory response after CNS injury. Therefore, we aimed to determine the effect and mechanism of HMGB1 on the phagocytosis of myelin debris and the expression of ICAM-1 mediated by myelin debris in endothelial cells.

Methods

Rat brain and spinal cord microvascular endothelial cells were cultured in vitro. The expression of myelin basic protein (MBP), cathepsin D (CatD), and ICAM-1, as well as macrophage adhesion to endothelial cells, were studied in myelin debris-mediated endothelial cells with or without recombinant HMGB1 (rHMGB1) exposure, at different times. The role of the toll-like receptor 4 (TLR4) / nuclear factor-kappa B (NF-κB) signaling pathway in the regulation of MBP, CatD, and ICAM-1 expression by HMGB1 in myelin debris-mediated endothelial cells was also studied.

Results

The expression of MBP, CatD, and ICAM-1 in myelin debris-mediated endothelial cells was significantly increased from 12 to 72 h following rHMGB1 exposure, with a peak increase observed at the 24 h time point. The expression of MBP, CatD, ICAM-1, and macrophagocyte adhesion in myelin debris-mediated endothelial cells without rHMGB1 exposure was significantly reduced compared with those in the 24 h rHMGB1 + myelin debris group. Moreover, the expression of MBP, CatD, and ICAM-1, and macrophagocyte adhesion were significantly increased in endothelial cells first incubated with rHMGB1 for 24 h and then treated with myelin debris than in the myelin debris group. The HMGB1 effects on MBP, CatD, and ICAM-1 expression in myelin debris-mediated endothelial cells in vitro were mediated, at least in part, by activation of the TLR4/NF-κB signaling pathway.

Conclusion

HMGB1 increased MBP, CatD and ICAM-1 expression, as well as myelin debris phagocytosis and macrophage adhesion in myelin debris-mediated endothelial cells in vitro. These effects occur through the HMGB1/TLR4/NF-κB signaling pathway.

Spinal cord injury

high mobility group box-1

TLR4/NF-κB signaling pathway

myelin debris phagocytosis

macrophage adhesion

myelin basic protein

cathepsin D

intercellular adhesion molecule-1 expression

Spinal cord injury (SCI) is a serious central nervous system (CNS) injury that leads to microvascular destruction, myelin loss of spinal cord tissue, and inflammation (Ohnmar et al. 2009;Katoh et al. 2019༛Yousefifard et al. 2021). After SCI, blood vessels rupture, followed by the invasion of immune cells, resulting in an increased release of inflammatory cytokines, all of which require the involvement of endothelial cells(O'Shea et al. 2017༛Turtle et al. 2019). Endothelial cells are an important component of the blood spinal cord barrier (BSCB)(Zhou et al. 2019༛Reglero-Real et al. 2021), and play a crucial role in attracting the migration of immune cells(Sturtzel 2017). These events are mediated by a set of molecular and cellular interactions between endothelial and immune cells, including intercellular adhesion molecule-1 (ICAM-1)(Ley et al. 2007༛Rao et al. 2007༛Cerutti & Ridley 2017༛Harjunpää et al. 2019). ICAM-1 regulates the adhesion of immune cells to the vascular wall during their transendothelial migration (Harjunpää et al. 2019༛Ohta et al. 2020) .

Myelin is a protein lipid sheath that surrounds axons (Stadelmann et al. 2019). After SCI, oligodendrocyte death and myelin injury lead to chronic axonal injury (Wilkins et al. 2003). In addition, myelin debris actively participates in the inflammatory response at the site of SCI (Cunha et al. 2020;Grajchen et al. 2020༛Kopper et al. 2021). Myelin debris can be phagocytosed by endothelial cells, which promotes the expression of ICAM-1 in endothelial cells in SCI (Zhou et al. 2019). Myelin debris also increases the leakage of microvessels; therefore, it also has a certain influence on the integrity of BSCB(Zhou et al. 2019༛Yao et al. 2022)。

High mobility group box-1 (HMGB1) is a highly conserved non-histone DNA-binding, and multifaceted functional protein both inside and outside of the cell (Yang et al. 2020). It causes inflammation when cells die or it is released into the extracellular space through active secretion (Yang et al. 2020). After SCI, HMGB1 can aggravate the inflammatory response at the injury site, resulting in cell swelling, death, and apoptosis (Fan et al. 2020;Xia et al. 2020༛Yang et al. 2020). Toll-like receptor 4 (TLR4), a major HMGB1 receptor, is involved in many pathophysiological processes through the activation of nuclear factor-kappa B (NF-κB)(Sun et al. 2019༛Niu & Pan 2021). Various inflammatory factors can promote ICAM-1 expression through TLR4 receptors on endothelial cells (Lee et al. 2015༛Leng et al. 2018). Moreover, TLR4 receptors and their downstream pathways also play important roles in regulating cell phagocytosis(Li et al. 2021).

In this experiment, brain and spinal cord microvascular endothelial cells from rats were cultured. We studied the expression of myelin basic protein (MBP), cathepsin D (CatD), the cytophagocytic marker protein, ICAM-1, and macrophage adhesion to endothelial cells in myelin debris-mediated endothelial cells with or without recombinant HMGB1 exposure. The role of the TLR4/NF-κB signaling pathway in the regulation of MBP, CatD, and ICAM-1 expression by HMGB1 in myelin debris-mediated endothelial cells was also studied.

Experimental protocol

The expression of MBP, CatD, and ICAM-1 in endothelial cells mediated by myelin debris was observed at 12, 24, 48, and 72 h after exposure to recombinant HMGB1 (rHMGB1).

The expression of MBP, CatD, and ICAM-1, as well as macrophage adhesion to endothelial cells, was observed in myelin debris-mediated endothelial cells with or without rHMGB1 exposure. The groups consisted of the following: normal group, 24 h rHMGB1 (20 ng/mL) + myelin debris (1 mg/mL) group, myelin debris group, and pretreatment with rHMGB1 + myelin debris group. In the 24 h rHMGB1+ myelin debris group, endothelial cells were simultaneously incubated with both rHMGB1 and myelin debris for 24 h. In the myelin debris group, the endothelial cells were incubated with myelin debris for 24 h. In the pretreatment with rHMGB1 + myelin debris group, endothelial cells were first incubated with rHMGB1 for 24 h and then treated with myelin debris. The expression of MBP, CatD, and ICAM-1 in endothelial cells was evaluated by western blotting and immunofluorescence. Macrophagocyte adhesion to endothelial cells was observed using a fluorescence microscope.

Finally, the role of TLR4/NF-κB signaling in the regulation of MBP, CatD, and ICAM-1 expression by HMGB1 in myelin debris-mediated endothelial cells was studied. The groups were as follows: normal group, 24 h rHMGB1+ myelin debris group, 24 h rHMGB1+ myelin debris + CLI095 (TLR4 inhibitor, 5 μM) group, and 24 h rHMGB1+ myelin debris + BAY 11-7082 (NF-κB inhibitor, 5 μM) group. Endothelial cells were simultaneously incubated with rHMGB1, myelin debris, and an inhibitor for 24 h. The expression of TLR4, NF-κB, MBP, CatD, and ICAM-1 in endothelial cells was evaluated using western blotting, and the expression of MBP, CatD, and ICAM-1 was evaluated using immunofluorescence.

Special chemicals and antibodies

Special chemicals include CLI-095 (Invivo Gen, Cat# tlrl-cli95, USA), recombinant HMGB1 (ProSpec, Cat# pro-581-b, Israel), BAY 11-7082 (Sigma-Aldrich, Cat# B5556, USA), Dulbecco’s modified Eagle medium Nutnent Mixture F-12 (Gibco, Life Technologies, Cat# C11330500BT, USA), fetal bovine serum (Gibco, Life Technologies, Cat# 16000-044, USA), collagenase type II (Sigma-Aldrich, Cat# C6885,USA), bovine serum albumin (Biosharp, Cat# BS114,China), BCA protein Assay Kit (Beyotime, Cat# P0012, China), enhanced chemiluminescence (Affinity, Cat#KF003, USA), CellTracker™ fluorescent probes (invitrogen，Cat# C7025，USA), and Complete culture medium for rat bone marrow derived macrophages (Procell，Cat# CM-R141，China). RIPA Lysis Buffer (Biosharp, Cat# BL504A, China).

Primary antibodies: rabbit polyclonal anti-ICAM-1 antibody (Sigma, Cat# SAB5700809, USA); rabbit polyclonal anti-Myelin Basic Protein antibody (anti-MBP, Abcam, Cat# ab216590, USA); rabbit polyclonal anti-Cathepsin D antibody (anti-CatD, Abcam, Cat#ab217310, USA); rabbit polyclonal anti-TLR4 antibody (Abcam, Cat#ab217310, USA ); rabbit monoclonal anti-NF-κB antibody (Cell Signaling Technology, Cat# 4764, USA); rabbit monoclonal anti-GAPDH antibody (Bioworld, Cat# AP0063, China); mouse monoclonal anti-Histone H3 antibody (Beyotime, Cat# AF0009, China); mouse polyclonal anti-CD31 antibody (Santa Cruz Biotechnology, Cat# cs-376764, USA).CD68(Proteintech, Cat# 25747-1-AP, China)

Secondary antibodies ：horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Affinity, Cat# s0001, USA), horseradish peroxidase-conjugated goat anti-mouse secondary antibody (Affinity, Cat# s0002, USA), FITC-conjugated goat anti-mouse secondary antibody (Affinity, Cat# s0007, USA), and FITC-conjugated goat anti- rabbit secondary antibody (Affinity, Cat# s0008, USA).

Rat brain and spinal cord endothelial cells culture

After anesthesia and disinfection, rat brain and spinal cord tissue of 10-day-old neonatal rats (Shanxi Medical University, Taiyuan, China) were isolated, and the meningeal and visible large blood vessels were removed. The gray matter and spinal cord were shredded and centrifuged.(Liu et al. 2013). The precipitate was resuspended in 20% bovine serum albumin (BSA) and then centrifuged at 600 × g for 10 min. The lowest layer of microvascular particles was transferred to a new centrifuge tube, and the remaining precipitate was remixed and centrifuged in the same manner two to three times. The obtained precipitate was digested with 0.1% type II collagenase at 37 °C for 35 min. The digested microvascular particles were centrifuged at 150 × g for 5 min to obtain a microvascular precipitate. Microvascular segments were cultured at 37 °C, 5% CO₂, and 95% air in a culture flask containing 15% fetal bovine serum (FBS) and inoculated with 0.2% rat tail gel. The medium was changed every 2–3 days. After fusion of the cultured cells, purity was determined using a CD31 antibody. Cultured third-generation microvascular endothelial cells were used in this experiment.

Preparation of myelin debris

As previously described (Norton & Poduslo 1973), myelin was isolated from the brains of 3-month-old rats by sucrose density gradient centrifugation (0.32 M and 0.85 M) at 25000 × g. The myelin debris content was determined using the BCA method. The concentration of endotoxin in myelin debris was below the detection limit of the Limulus Amebocyte Lysate assay. Myelin debris was used at a concentration of 1 mg /mL in all experiments.

Macrophage culture, staining and co-culture

Bone marrow fluid from the femurs and tibias of 14–20-day-old rats was collected and filtered through a 200-mesh cell screen. The precipitates were centrifuged, and macrophages derived from rat bone marrow were obtained using a lymphocyte separation solution. Rat bone marrow-derived macrophages were used to induce cell precipitation in complete culture medium. The cells were inoculated in petri dishes and cultured at 37 °C in a 5% CO₂ constant temperature incubator, and the solution was changed every 3 days. CD68 was used for immunofluorescence identification after 9 days of culture. After digestion and centrifugation, the macrophages were incubated at 37 °C for 30 min with 0.5–25 µM fluorescence probe and centrifuged for precipitation. The pre-inoculated endothelial cells in the 24-well plate were washed, and 1 × 10⁵ /500 µL labeled macrophages were added to each well and incubated at 37 °C for 30 min. After fixation with 4% paraformaldehyde, the fluorescence intensity of adherent cells was observed under a fluorescence microscope and statistically analyzed.

Western Blot

The cells were washed with phosphate buffered saline (PBS) and dissolved in RIPA lysis buffer containing phosphatase and protease inhibitors. The lysate protein concentration was then determined using a BCA Protein Assay Kit, and protein samples were subjected to sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on the gel were transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon, USA), which were blocked with 5% milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 2 h at RT. The membranes were incubated with the indicated primary antibodies at 4 °C overnight, as well as anti-ICAM-1(1:500, Sigma), anti-myelin basic protein (1: 1000, Abcam), anti-cathepsin D (1: 1000, Abcam), anti-TLR4 (1: 1000, Abcam), anti-GAPDH (1: 10000, Bioworld), anti-NF-κB (1: 1000, CST), and anti-histone H3 (1: 1000, Beyotime). After being washed with TBST, the membranes were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (1：3000) or HRP-conjugated goat anti-mouse secondary antibodies (1：3000) for 2 h at RT. The immunoreactive bands were detected using enhanced chemiluminescence (ECL) and analyzed using Image J. All values were normalized to GAPDH, as indicated in the figure legends。

Immunofluorescence

Cells were seeded in 24-well plates at 1 × 10⁵ cells/well and fixed in 4% paraformaldehyde for 15 min at 4 °C. Cells were washed with PBS three times, permeabilized with 0.1% Triton X-100 at RT, blocked with 5% goat serum for 20 min at 37 °C, and incubated with primary antibody at 4 °C overnight. The following antibodies were used: anti-ICAM-1(1:100, Sigma), anti-myelin basic protein (1: 100, Abcam), anti-cathepsin D (1: 100, Abcam), and anti-CD31(1:100, Santa) and cells were incubated with secondary antibody for 1.5 h at 37 °C. The cells were incubated with DAPI for 5 min. Immunostaining was observed under a fluorescence microscope and analyzed using ImageJ.

Statistical Analysis

All data are presented as the mean ± standard deviation (SD). Data analysis was performed using one-way ANOVA followed by Tukey's post hoc test using SPSS (version 24.0; IBM, https://www.ibm.com). Statistical significance was set at p < 0.05.

Identification of rat brain and spinal cord microvascular endothelial cells

The cultured brain and spinal cord microvascular endothelial cells of the rats were identified by immunofluorescence staining with CD31, an endothelial cell marker. The results showed that CD31-labeled endothelial cells accounted for more than 95% of the total cells. (Fig. 1).

Expression of MBP, CatD, and ICAM-1 in myelin debris-mediated endothelial cells following rHMGB1 exposure

To evaluate the effect of rHMGB1 on myelin debris-mediated MBP, CatD, and ICAM-1 expression in endothelial cells, we performed western blotting and immunofluorescence test. Western blot analysis demonstrated MBP expression in myelin debris-mediated endothelial cells from 12 to 72 h following rHMGB1 exposure, with a peak increase observed at 24 h (p < 0.05, Fig. 2 a). The expression of CatD and ICAM-1 was significantly increased in myelin debris-mediated endothelial cells from 12 to 72 h following rHMGB1 exposure than in the normal group, with a peak increase observed at 24 h (p < 0.05, Fig. 2 b, c). Immunofluorescence also showed that MBP, CatD, and ICAM-1 expression was significantly increased in myelin debris-mediated endothelial cells from 12 to 72 h following rHMGB1 exposure, with a peak increase observed at 24 h (p < 0.05, Fig. 2 d-f).

Effects of rHMGB1 on the expression of MBP, CatD, and ICAM-1 in myelin debris-mediated endothelial cells, as well as macrophagocyte adhesion to endothelial cells

We examined the effect of rHMGB1 on MBP, CatD, and ICAM-1 expression in myelin debris-mediated endothelial cells. Western blot analysis demonstrated that MBP, CatD, and ICAM-1 expression in myelin debris-mediated endothelial cells was significantly increased in the 24 h rHMGB1 + myelin debris group (p < 0.05), and without rHMGB1, these increases were significantly attenuated in the myelin debris group when compared with those in the 24 h rHMGB1+ myelin debris group (p < 0.05, Fig. 3 a-c). Moreover, the expression of MBP, CatD, and ICAM-1 was significantly increased in the pretreatment with rHMGB1 + myelin debris group than in the myelin debris group (p < 0.05, Fig. 3 a-c). Immunofluorescence results for MBP, CatD, and ICAM-1 are shown in Fig. 3 d-f. The mean optical density values of MBP, CatD, and ICAM-1 were significantly increased in both the 24 h rHMGB1 + myelin debris group and the pretreatment with rHMGB1 + myelin debris group compared to those in the myelin debris group (p < 0.05).

To determine macrophagocyte adhesion to endothelial cells, the fluorescence microscopy results are shown in Fig. 3 g. The number of macrophagocytes adhering to endothelial cells increased in both the 24 h rHMGB1 + myelin debris group and the pretreatment with rHMGB1 + myelin debris group compared with that in the myelin debris group. (P<0.05).

Role of TLR4/NF-κB signaling pathway in the regulation of MBP, CatD, and ICAM-1 expression by HMGB1 in myelin debris-mediated endothelial cells

The role of TLR4/NF-κB signaling pathway in the regulation of MBP, CatD, and ICAM-1 expression by HMGB1 in myelin debris-mediated endothelial cells 24 h following rHMGB1 exposure was investigated using western blotting and immunofluorescence test. Western blot analysis demonstrated that CLI-095 reduced TLR4 expression, while both CLI-095 and BAY 11-7082 reduced NF-κB expression (p < 0.05, Fig. 4 a, b). MBP, CatD, and ICAM-1 expression decreased after inhibition of TLR4 and NF-κB when compared with that in the 24 h rHMGB1+ myelin debris group (p < 0.05, Fig. 4 c-e). Immunofluorescence test results also showed that the mean optical density values of MBP, CatD, and ICAM-1 significantly decreased after inhibition of TLR4 and NF-κB compared with those in the 24 h rHMGB1+ myelin debris group.

Our study demonstrated that HMGB1 increased MBP, CatD, and ICAM-1 expression in myelin debris-mediated endothelial cells and promoted phagocytosis of myelin debris and peripheral macrophage adhesion in endothelial cells. In addition, HMGB1 is closely related to the activation of the TLR4/NF-κB pathway in myelin debris-mediated endothelial phagocytosis and ICAM-1 expression.

SCI includes primary and secondary injury, in which the latter induces the damaged tissue to release inflammatory factors and promotes the invasion of peripheral immune cells into the spinal cord tissue, thereby aggravating SCI(Anjum et al. 2020;Bloom et al. 2020༛Liu et al. 2021). Following SCI, various immune cells migrate to the surface of endothelial cells and eventually cross the vascular barrier into the damaged tissue (Guo et al. 2016༛Wettschureck et al. 2019༛Wimmer et al. 2019༛Zivkovic et al. 2021). Macrophages participate in the repair of the damaged tissues (Gensel & Zhang 2015). In addition, BSCB permeability increases, resulting in more peripheral immune cell exudation after SCI (Kumar et al. 2018).

Endothelial cells in the BSCB play an important role in the exudation of peripheral immune cells (Couty et al. 2007;Bartanusz et al. 2011). Under the action of various inflammatory factors following SCI, endothelial cells express ICAM-1 to bind peripheral immune cells (such as macrophages) on the surface of endothelial cells, and immune cells finally exude into the tissue after crawling and migrating (Wettschureck et al. 2019). Lee et al. and Wang et al. reported that the increased expression of ICAM-1 in endothelial cells can promote the recruitment of more peripheral macrophages in damaged tissues (Lee et al. 2015༛Wang et al. 2015). Similar results were found in our study; when the expression of ICAM-1 in endothelial cells increased, the adhesion of macrophages was promoted, especially after exposure to HMGB1.

After CNS injury, the demyelination of neurons produces a large amount of myelin debris, leading to a significant inhibition of axonal regeneration (Mar et al. 2016;Uyeda & Muramatsu 2020). Microvascular endothelial cells can phagocytose myelin debris, which promotes the expression of ICAM-1 and VCAM-1 and the recruitment of macrophages after nerve injury (Zhou et al. 2019༛Yao et al. 2022). In our study, the expression of MBP, CatD, and ICAM-1 in endothelial cells was significantly increased via myelin debris. These results indicate that endothelial cells can phagocytose myelin debris, which leads to an increase in ICAM-1 expression after intracellular digestion and promotes the recruitment of peripheral macrophages.

HMGB1 is a highly conserved nuclear protein present in all cell types. It acts as a critical damage-associated molecular pattern molecule (DAMP) after release from cells, and TLR4 acts as a major receptor for HMGB1, which is associated with inflammatory responses(Andersson & Tracey 2011;Yang et al. 2020). After CNS injury, extracellular HMGB1 mainly originates from microglia, astrocytes, neurons, and necrotic cells (Nishibori et al. 2019༛Nishibori et al. 2020). Following SCI, the increase in HMGB1 can promote the release of a variety of inflammatory factors as well as apoptosis of cells, especially neurons, and enhance BSCB permeability(Kang et al. 2014༛Nishibori et al. 2020༛Yang et al. 2020). Previously, we found that HMGB1 inhibition reduced early spinal cord edema and decreased astrocyte activation and aquaporin-4 expression in rats after SCI(Sun et al. 2017༛Sun et al. 2019). In the current study, we found that HMGB1 could promote the expression of MBP, CatD, and ICAM-1 in endothelial cells mediated by myelin debris. When the endothelial cells were pretreated with HMGB1, the expression of MBP, CatD, and ICAM-1 was significantly increased. The results showed that HMGB1 promoted phagocytosis of myelin debris and adhesion of peripheral macrophages to endothelial cells. In addition, endothelial cells serve as a complementary pathway for the clearance of myelin debris, and HMGB1 plays an important role in accelerating the clearance of myelin debris by endothelial cells and alleviating the inhibitory effect of myelin on axon regeneration.

TLR4 is a key player in the innate immune response as it recognizes DAMP and this interaction in turn activates pro-inflammatory NF-κB (Shao et al. 2019;Xu et al. 2020). Previous studies have shown that TLR4 and its downstream pathway promote the expression of ICAM-1 and VCAM-1 in endothelial cells, participate in the migration of neutrophils and monocytes, and affect the tight connections between endothelial cells(McDonald et al. 2013༛Pirvulescu et al. 2014༛Lv et al. 2019༛Huang et al. 2020༛Zhang et al. 2021). However, the effects of TLR4 and its downstream pathways on endothelial phagocytosis have not been discussed in recent studies. In this study, we found that the expression of MBP, CatD, and ICAM-1 in myelin debris-mediated endothelial cells decreased upon the inhibition of TLR4 and NF-κB. We believe that the TLR4/NF-κB signaling pathway activated by HMGB1 is involved in the phagocytosis of myelin debris and the adhesion of peripheral macrophages and may influence the adhesion function of endothelial cells by enhancing the phagocytosis of endothelial cells..

The present study has some limitations. First, we only studied the effect of HMGB1 on myelin debris-mediated endothelial phagocytosis and cell adhesion in vitro. Further studies on the effect of HMGB1 on myelin debris-mediated endothelial phagocytosis and cell adhesion in vivo after SCI are needed. Second, in this study, we studied the effect of HMGB1 on myelin debris in endothelial cells between 12 and 72 h. The effect of HMGB1 on myelin debris in endothelial cells should be studied at other time points as well. Finally, this study only investigated the effect of HMGB1 on the phagocytosis and adhesion of myelin debris-mediated endothelial cells via the TLR4/NF-κB pathway. Understanding the effect of HMGB1 on the phagocytosis and adhesion of myelin debris-mediated endothelial cells through other pathways and their mechanisms warrant further study

HMGB1 can increase the expression of MBP, CatD, and ICAM-1 in endothelial cells mediated by myelin debris and enhance the adhesion of endothelial cells to macrophages, partly or entirely through the TLR4/NF-κB pathway. HMGB1 is crucial in clearing myelin debris from endothelial cells after SCI and could therefore be a new target for studying functional recovery after SCI.

HMGB1, high mobility group box-1; ICAM-1, intercellular adhesion molecule-1;MBP ,myelin basic protein ;CatD ,Cathepsin D;CNS, Central nervous system;SCI, Spinal cord injury; TLR4,toll-like receptor 4; NF-κB,nuclear factor-kappa B; BSCB,blood spinal cord barrier; VCAM-1, vascular cell adhesion molecule-1；; rHMGB1: recombinant HMGB1

Acknowledgements

This study was supported by grant from the National Natural Science Foundation of China (No.: 81870976), and Shanxi Provincial Health Commission Key Projects to Tackle Key Problems (No.: 2020XM27).

Conflict of interest

The authors declare no conflict of interest.

Author contributions

Z-xQ and LS conceived, designed, and coordinated the study. Z-xQ performed the experiments and analyzed the data. L-pW and Z-qW helped with cells culture.J-mL, CD and LD wrote, revised, and checked the data analysis. All the authors revised and approved the final version of the manuscript.

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HMGB1 increases myelin debris phagocytosis and ICAM-1 expression in rat central nervous system endothelial cells in vitro via the TLR4/NF-κB pathway

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