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Chinese Journal of Traumatology logoLink to Chinese Journal of Traumatology
. 2025 Mar 22;28(5):361–369. doi: 10.1016/j.cjtee.2024.10.007

Dimethyloxalylglycine improves functional recovery through inhibiting cell apoptosis and enhancing blood-spinal cord barrier repair after spinal cord injury

Wen Han a,⁎, Chao-chao Ding a, Jie Wei b, Dan-Dan Dai a, Nan Wang a, Jian-Min Ren a, Hai-Lin Chen a, Ling Xie c,⁎⁎
PMCID: PMC12489486  PMID: 40274522

Abstract

Purpose

The secondary damage of spinal cord injury (SCI) starts from the collapse of the blood spinal cord barrier (BSCB) to chronic and devastating neurological deficits. Thereby, the retention of the integrity and permeability of BSCB is well-recognized as one of the major therapies to promote functional recovery after SCI. Previous studies have demonstrated that activation of hypoxia inducible factor-1α (HIF-1α) provides anti-apoptosis and neuroprotection in SCI. Endogenous HIF-1α, rapidly degraded by prolylhydroxylase, is insufficient for promoting functional recovery. Dimethyloxalylglycine (DMOG), a highly selective inhibitor of prolylhydroxylase, has been reported to have a positive effect on axon regeneration. However, the roles and underlying mechanisms of DMOG in BSCB restoration remain unclear. Herein, we aim to investigate pathological changes of BSCB restoration in rats with SCI treated by DOMG and evaluate the therapeutic effects of DMOG.

Methods

The work was performed from 2022 to 2023. In this study, Allen's impact model and human umbilical vein endothelial cells were employed to explore the mechanism of DMOG. In the phenotypic validation experiment, the rats were randomly divided into 3 groups: sham group, SCI group, and SCI + DMOG group (10 rats for each). Histological analysis via Nissl staining, Basso-Beattie-Bresnahan scale, and footprint analysis was used to evaluate the functional recovery after SCI. Western blotting, TUNEL assay, and immunofluorescence staining were employed to exhibit levels of tight junction and adhesion junction of BSCB, HIF-1α, cell apoptosis, and endoplasmic reticulum (ER) stress. The one-way ANOVA test was used for statistical analysis. The difference was considered statistically significant at p < 0.05.

Results

In this study, we observed the expression of HIF-1α reduced in the SCI model. DMOG treatment remarkably augmented HIF-1α level, alleviated endothelial cells apoptosis and disruption of BSCB, and enhanced functional recovery post-SCI. Besides, the administration of DMOG offset the activation of ER stress induced by SCI, but this phenomenon was blocked by tunicamycin (an ER stress activator). Finally, we disclosed that DMOG maintained the integrity and permeability of BSCB by inhibiting ER stress, and inhibition of HIF-1α erased the protection from DMOG.

Conclusions

Our findings illustrate that the administration of DMOG alleviates the devastation of BSCB and HIF-1α-induced inhibition of ER stress.

Keywords: Spinal cord injury, Dimethyloxalylglycine, Hypoxia inducible factor-1α, Blood spinal cord barrier, Endoplasmic reticulum stress

1. Introduction

It has been reported that spinal cord injury (SCI) causes long-term disabilities for over 27 million patients worldwide.1 As one of the most serious traumatic diseases in spinal surgery, SCI often leads to severe functional impairment in and below the injured segment, resulting in a lasting and devastating mental and economic burden on the patient's daily life.2,3 Therefore, the clearness of its pathological mechanism and effective therapy has always been the hotspot in this field.

In adult mammals, secondary functional impairment of SCI begins with the disruption of the blood spinal cord barrier (BSCB), leading to loss of integrity and increased permeability.4,5 Accordingly, a lot of toxic substances, inflammatory factors, and factors that harm the central nervous system (CNS), enter into spinal cord tissue further exacerbating the damage.6 A series of pathological and physiological reactions occur immediately, such as bleeding, oxidative stress, endoplasmic reticulum (ER) stress, and excessive inflammatory response.7,8 Therefore, it is well accepted that the retention of the integrity and permeability of BSCB is well-recognized as one of the major therapies to promote functional recovery after SCI.

As a special structure in the spinal cord circulatory system, BSCB regulates the molecular exchange between the blood and parenchyma.6,9 Meanwhile, the barrier function of BSCB is a key and main aspect of spinal homeostasis.10 It reports that BSCB is made up of several types of cells and intercellular junction complexes. Cells contain endothelial cells (ECs), pericytes, basement membrane, and astrocyte pods. Notably, as the first and most important basic cellular structure, ECs are a dynamic and highly regulated interface in the blood and spinal cord nervous system. In contrast to peripheral endothelial cells, there is only the lowest level of pinocytosis and tight connection between cells as well as no fenestral structure in ECs of BSCB.6,11 Besides, the tight junctions including tight junction (TJ) and adhesion junction (AJ) between ECs, are critical to regulating the permeability of BSCB, mainly restricting the interaction between cell solutes, ions, and water. Thereby, research on the changes and repair mechanisms of BSCB after SCI and inhibition of apoptosis of ECs are an effective potential strategy to maintain the function of the spinal cord nervous system and promote the repair and reconstruction of SCI.

Hypoxia inducible factors (HIFs) compose HIF-α and HIF-β, with 3 isoforms (HIF-1, HIF-2, and HIF-3).12 Under normal conditions, HIFs are synthesized and hydrolyzed by prolyl hydroxylase and only a very low level of HIFs is kept to maintain cellular oxygen balance. When it is hypoxic, although activated, HIFs are quickly hydrolyzed by prolylhydroxylase (PHD) and cannot play a positive role.13 HIFs are reported to have great goodness in the transcriptional response to hypoxia and anti-apoptosis.14 Among them, HIF-1α is the most important isoform for the regulation genes related to the ability of cell adaptation to hypoxia. Moreover, the inhibition of PHD or activation of HIF-1α has been proven to be effective in many disorders.15,16 DMOG, a PHD inhibitor, has been reported to have potency and acts as a HIF-1α activator in various disease models. In murine periodontitis, DMOG was proven to inhibit alveolar bone resorption by regulating macrophage polarization.17 For chronic wound healing, DMOG@ZIF-8/Gelatin-PCL electrospinning dressing was reported to have effects on sequential anti-infection and proangiogenesis.18 DMOG also ameliorates endotoxin-induced acute lung injury through the HIF-1a/HO-1 signaling pathway.19 Gupta et al.20 illustrated that DMOG protected against skin inflammation. Nagamine et al.21 proved that PHD inhibitor, DMOG attenuates apoptosis and lung injury in mice. What is more, SCI leads to a microenvironment of ischemia and hypoxia. HIF-1α stabilization was confirmed to augment axonal regeneration and functional recovery by inhibiting autophagy after SCI.22 Therefore, the stabilization of HIF-1α in hypoxia has become a novelty in curing SCI. Apoptosis of ECs in BSCB plays a critical role in SCI. The activation of HIF-1α by inhibition of PHD is a potential therapeutic approach to ameliorate apoptosis and damage of BSCB after SCI.

ER is a crucial subcellular organelle to handle the synthesis, modification, and folding of proteins, which are vital in cellular function and survival activities.23 The unfolded protein response (UPR) induces the proteostasis of ER. When damage occurs, the cytoplasm is infiltrated by various factors, leading to the activation of ER stress.24 Once ER stress is activated, lots of UPR occurs, ultimately resulting in cell death. Many UPR-related proteins trigger the process of cell death in ER stress, such as the chaperone GRP78 (a cell sensor to apoptosis),25 the proapoptotic factor (C/EBP homologous protein) chop,26 and transcription factor 6 (ATF6).27 Therefore, the surveillance of changes in the expression of these proteins is the appropriate way to evaluate the activity of ER stress. Emerging evidence has proved that ER stress plays a key role in many diseases, and its alleviation favors the recovery, such as stroke, neurodegenation disorders, and diabetes as well as SCI.28,29 Previous studies on SCI have reported that ER stress is involved in SCI and contributes to the disintegration of BSCB,30 but whether it takes part in DMOG induced functional repair remains unclear.

Herein, we assumed that DMOG application may inhibit ER stress by upregulating the expression of HIF-1α to attenuate apoptosis and enhance BSCB restoration after SCI in vivo and in vitro. Our study also investigated the potential role of ER stress during DMOG treatment or loss of TJ and AJ proteins including ZO-1, P120, β-catenin, and occluding.

2. Methods

2.1. SCI model and drug administration

Adult female Sprague Dawley rats (n = 120, weighted 250 – 300 g, aged 6 – 8 weeks) were purchased from the Animal Center of the Chinese Academy of Science (Hangzhou, China). The SCI protocol for adult female Sprague Dawley rats under sterile conditions was applied Allen's smashing model, as described earlier.22 Rats were sedated with 2% pentobarbital sodium (40 mg/kg, intraperitoneal) with the skin along the midline of the back cleaned and incised as well as muscles expanded, and a laminectomy was conducted at T9-T10 level. After surgery, a moderate contusion on the thoracic spinal cord was operated by MACSIS/NYU impactor (10 g forces, 3 cm height). The rats were randomly and equally divided into 3 groups: sham group, SCI group, and SCI + DMOG group. Five rats were allocated for Western blot analysis, and another 5 rats were designated for histology and immunofluorescence assays. No damage was performed on rats in the sham group after laminectomy. Afterward, animals in SCI + DMOG group were orally treated with DMOG (10 mg/kg/d in vitro, 1 mM in vivo) daily for up to 28 days after surgery. The positive control group was administrated with YC-1 (a HIF-1α inhibitor, 30 mg/kg/d in vitro, 30 μM in vivo) or/and tunicamycin (TM, an ER stress activator, 10 μg/kg in vitro, 3 μM in vivo). The sham group received equal saline. Finally, the rats were euthanized on 1, 3, 5, 7, 14, and 28 days after injury.

2.2. Oxygen-glucose deprivation (OGD) model

The medium was replaced with glucose-free Dulbecco's Modified Eagle Medium, and the cells were exposed to an anaerobic environment containing 5% CO2 and 95% N2 for 2 h. Following this, the medium was replaced with a normal medium for 12 h. In this period, the cells were pretreated with DMOG (1 mM for 2 h before OGD), and the treatment was maintained throughout the reoxygenation phase.

2.3. Tissue collection and preparation

All rats sacrificed at specific time points following SCI were anesthetized with 2% pentobarbital sodium (40 mg/kg, intraperitoneal). Spinal cord segments (around 1 cm) from the injury site as the center were dissected. For pathological section staining such as Nissl staining and immunofluorescence staining, the tissue above was immediately fixed by 4% paraformaldehyde for 6 h and post-embedded in paraffin. Slices (5 μM thick, vertically or horizontally) were mounted on slides, and then dried naturally air and stored at room temperature for subsequent staining. For Western blot, segments were stored at −80 °C immediately.

2.4. Western blot

Proteins of animals or human umbilical vein endothelial cells (HUVECs) were first quantified with bicinchoninic acid reagents and 80 μg proteins were mixed into a target system. Then, after being separated on 10% (w/v) gels, the system was transferred onto PVDF membrane (Bio-Rad, Hercules, CA, USA), and next, after blocked with 5% (w/v) non-fat milk (Bio-Rad) in Tris-buffered saline mixed with 0.1% Tween-20 for 2 h at room temperature. Then those were incubated overnight at 4 °C with primary antibodies (P120 (1:2000, Abcam), Occludin (1:2000, Cell Signaling Technologies), ZO-1 (1:200 Sigma Aldrich), HIF-1α (1:200, Abcam), ATF6 (1:2000, Proteintech), GRP78 (1:2000, Proteintech), Chop (1:2000, Abcam), Bax (1:2000, Abcam), Bcl2 (1:2000, Abcam), and Cleaved caspase3 (1:2000, Abcam)). Finally, the membranes were treated with a second antibody coupled with horseradish peroxidase for 60 min. Chemi DocXRS + Imaging System (Bio-Rad) was employed to visualize all result signals and Image J was to analyze. All experiments were repeated 3 times to maintain accuracy.

2.5. Histology and immunofluorescence assay

All the slices (5 μM thick, vertically) were taken from preservation tissue at rostral 5 mm and caudal 5 mm of the spinal cord. The slices for histological analysis were treated by Nissl staining with the manufacturer's instructions. Light microscopy was used to obtain Brightfield images for Nissl staining. For immunofluorescence, after a series of operations such as dewaxing and hydration, slices were treated with primary antibodies against the following proteins overnight at 4 °C: β-catenin (1:500, Abcam), HIF-1α (1:400, Abcam), Chop (1:400, Abcam), and Cleaved caspase3 (1:400, Abcam). Washed with PBST 4 times for 3 min each, the sections were then incubated with AlexaFluor 488 or AlexaFluor 568 donkey antirabbit/rat secondary antibodies for 1 h at 37 °C. After repeating the washing procedure, the slices were incubated with dihydrochloride (4′,6-Diamidino-2-phenylindole dihydrochloride) solution for 7 min, followed by another wash, and then mounted with a coverslip for sealing. A confocal fluorescence microscope (Nikon, Japan) was used to capture all images, and Image J was analyzed.

2.6. TUNEL assay

TUNEL staining assay (Beyotime Biotechnology company) was a typical method to measure apoptotic levels. Sections were obtained and were performed at 1 dpi with the instructions. Nikon ECLIPSE Ti microscope (Nikon, Japan) was employed to capture the images. Three rats were measured in each group. TUNEL-positive cells were counted from 5 to 10 randomly selected slices to calculate the average number in each group.

2.7. Locomotion recovery assessment

Basso-Beattie-Bresnahan (BBB) locomotion and footprint analysis were 2 main and critical tools for locomotion recovery, which were conducted by independent 4 staff. Rats moved freely in an open experimental field for 5 min. Walking ability was assessed by the BBB scale (at optimal timepoint: 0, 1, 3, 5, 7, 14, 21, and 28 dpi) ranging from 0 (no limb movement or weight support) to 21 (normal walking). The footprint analysis was performed at 14 dpi, staining the animal's posterior limb in red and the fore limb in blue.

2.8. Statistical analysis

All data were collected and collated as mean ± standard error of the mean (SEM). Differences between groups in BBB scores were organized with a one-way analysis of variance (ANOVA) followed by Tukey's Multiple comparison test. Statistical analysis of other data was performed using one-way ANOVA. All analyses were conducted using GraphPad Prism 9 for Windows was hired to statistically analyze the data. There is a significant statistical difference when p < 0.05.

3. Results

3.1. DMOG favors functional repair after SCI in rats

To explore the effects of DMOG on SCI, rats were randomly dispensed to 3 groups: sham, SCI, or SCI + DMOG groups (DMOG treatment group) (n = 5), and the following tests were performed: BBB score and footprint analysis for locomotor recovery assignment, and Nissl staining for neuron survival observation. BBB scores ranged from 0 (representing complete functional loss of hind limbs after SCI) to 21 (indicating normal function). Footprint analysis recorded the coordination, stability, and balance of walking. As shown in Fig. 1A, the healthy function was observed in the sham group throughout the whole experiment, while an entire loss of motor function immediately following SCI was shown in other groups. No notable differences between the other 2 groups at the timepoint of 1, 3, or 5 days post-injury (dpi), while a similar significant functional improvement was demonstrated between SCI and SCI + DMOG groups at 7, 14, and 28 dpi. Those indicated a therapeutic effect of DMOG on SCI. Meanwhile, the finding above was consistent with the results of the footprint test at 14 dpi. Rats in the sham group exhibited coordinated, balanced, and stable walking with a clear vision of the front and rear toes. Compared to them, rats in the SCI group dragged their hind limbs, while those in the DMOG group emerged with a harmonious crawling and slightly staggering gait (Fig. 1B). These results suggested DMOG treatment favored locomotor functional restoration after SCI. To confirm the finding, Nissl staining was employed to assess the effect on neuron loss. As shown in Fig. 1C and D, the number of ventral motor neurons in the sham group reflected a normal level of apoptosis in the intact tissue. However, the number of ventral motor neurons met a remarkable surge at 7 dpi after SCI surgery and DMOG treatment significantly offset this trend. Therefore, DMOG can enhance neuronal survival and functional recovery after SCI.

Fig. 1.

Fig. 1

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DMOG favors functional repair after SCI in rats (n = 5). (A) The BBB score of rats at specific days 1, 3, 5, 7, 14, 28. (B) The footprint analysis at 14 dpi. (C & D) Nissl staining at 7 dpi. Scale bar = 50 μm. All data are presented as the mean ± SEM.

∗ vs. sham: ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001; #vs. SCI:#p < 0.05, ##p < 0.01.

DMOG: dimethyloxalylglycine; SCI: spinal cord injury; BBB: Basso-Beattie-Bresnahan; VMN: ventral motor neurons; SEM: standard error of the mean.

3.2. DMOG lessens the loss of connecting proteins in the BSCB

To identify the role of DMOG in blood-spinal cord barrier disruption, Western blot was used to evaluate the expression levels of TJ (Occludin and ZO-1) and AJ (P120 and β-catenin) proteins after DMOG treatment for SCI. The results demonstrated that compared with the sham group, the levels of those proteins in the spinal cord were strikingly reduced after SCI, which was remarkably blocked by DMOG treatment (Fig. 2A–E). The finding was then vindicated by immunofluorescence staining of β-catenin in vitro. An OGD model was to mimic SCI in vitro. As shown in Fig. 2F and G, the administration of DMOG displayed a protective effect against OGD-induced loss of β-catenin. It revealed that DMOG treatment impeded the loss of TJs and AJs, improving the integrity of BSCB.

Fig. 2.

Fig. 2

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DMOG lessens the loss of connecting proteins in the BSCB (n = 5). (A–E) Western blot analysis of AJ and TJ proteins (ZO-1, P120, Occludin and β-catenin). (F & G) Immunofluorescence staining of β-catenin) at 1 dpi. Scale bar = 100 μm. All data are presented as the mean ± SEM. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SCI: spinal cord injury; DMOG: dimethyloxalylglycine; BSCB: blood spinal cord barrier; AJ: adhesion junction; TJ: tight junction; SEM: standard error of the mean.

3.3. DMOG hinders cell apoptosis induced by SCI

Apoptosis runs throughout the entire process of SCI recovery. Endothelial cells are the main cytoskeleton of BSCB, and thus the retainment of the survival of endothelial cells is critical for BSCB integrity. To figure out whether DMOG promoted endothelial cell survival after SCI, TUNEL assay, Western blot, and immunofluorescence staining of proteins linked with apoptosis (Bax, Bcl2, and cleaved-caspase3) were performed at 7 dpi. Compared with the sham group, the expression of Bax and cleaved-caspase3, which reflected the activity of cell apoptosis, was notably augmented by SCI. However, this trend was significantly twisted by DMOG treatment. Meanwhile, the level of Bcl2 (the sign of inhibition of dormancy of apoptosis) increased after treatment with DMOG which notably decreased by SCI (Fig. 3A–C). DMOG was implied to attenuate cell death after SCI, which was further validated by TUNEL staining. As shown in Fig. 3D and E, the number of TUNEL-positive cells markedly surged by SCI compared with the sham group, while it was remarkably reduced in the SCI + DMOG group. Similarly, the result of immunofluorescence staining of cleaved-caspase3 (green) in vivo exhibited parallelism. Although the immunofluorescence of cleaved-caspase3 displayed a significant increase in the OGD condition, a sharp decrease was observed when treated with DMOG (Fig. 2F and G). Overall, DMOG can weaken SCI-induced apoptosis and enhance the integrity of BSCB.

Fig. 3.

Fig. 3

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DMOG hinders cell apoptosis induced by SCI (n = 5). (A–C) Western blot analysis of cell apoptosis proteins (Bax, Bcl2 and cleaved-caspase3). (D & E) TUNEL assay in rats at 1 dpi. Scale bar = 100 μm. (F & G) Immunofluorescence staining of cleaved-caspase3 on HUVECs in vivo. Scale bar = 100 μm. All data are presented as the mean ± SEM. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

GAPDH: glyceraldehyde-3-phosphate dehydrogenase; SCI: spinal cord injury; DMOG: dimethyloxalylglycine; SCI: spinal cord injury, SEM: standard error of the mean.

3.4. HIF-1α stabilization is the key to DMOG-induced BSCB recovery

PHD inhibitors have been reported to have a future in neuroprotection through HIF-dependent mechanisms. Thereby, the level of HIF-1α expression was assessed to explore its role in the anti-SCI effect of DMOG. As shown in Fig. 4A and B, the level of HIF-1α exhibited a time-dependent manner in vivo when incubated with DMOG in OGD condition. Then a well-known HIF-1α inhibitor-YC-1 was employed to further define the effect of DMOG on HIF-1α expression and BSCB. The results of the Western blot showed that under OGD conditions, the level of HIF-1α, ZO-1, and P120 expression in HUVECs decreased significantly in contrast with that in the control group. However, after co-incubation with DMOG for 4 h, the expression level of HIF-1α notably offset the decrease caused by OGD, as this reversal was markedly blocked by YC-1 (Fig. 4C–F). Therefore, DMOG could contribute to BSCB restoration via HIF-1α stabilization under the OGD condition.

Fig. 4.

Fig. 4

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Hif-1α stabilization is the key to DMOG-induced BSCB recovery (n = 5). (A & B) Western blot analysis of Hif-1α in time-dependent manner. (C–F) Western blot analysis of Hif-1α and BSCB-associated proteins (ZO-1 and P120). All data are presented as the mean ± SEM. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OGD: oxygen-glucose deprivation; Hif-1α: hypoxia inducible factor-1α; DMOG: dimethyloxalylglycine; BSCB: blood spinal cord barrier, SEM: standard error of the mean.

3.5. DMOG inhibits ER stress through stabilizing HIF-1α expression

Emerging evidence has proved that ER stress has a bearing on SCI and plays a pivotal role in maintaining the stability of BSCB and cellular homeostasis. To ensure the effect of DMOG on ER stress induced by SCI, proteins that related to ER stress (ATF6, GRP78, and chop) were first tested. Compared with the control group, the expression of ATF6 and GRP78 remarkably raised, prompting that OGD condition can activate ER stress. In contrast, DMOG administration downregulated the level of those 2 proteins (Fig. 5A–C). This finding was then confirmed by immunofluorescence of chop. As shown in Fig. 5D and E, the intensity of chop in the OGD group met a significant increase, whereas in the DMOG co-incubation group, the intensity of chop markedly reduced. These results demonstrated that DMOG blocked OGD-induced ER stress. TM as an ER stress activator along with YC-1 was used to explore the potential mechanism between DMOG and ER stress. Compared with the DMOG group, the level of ATF6 and GRP78 was significantly upregulated, suggesting that TM stimulation reversed DMOG-inhibited ER stress. However, this phenomenon was then eliminated by TM and DMOG co-incubation. Besides, YC-1 stimulation inhibited the level of ATF6 and GRP78, suggesting a close role in DMOG. However, TM treatment did not impact the expression of HIF-1α. In conclusion, DMOG inhibited ER stress by stabilizing HIF-1α.

Fig. 5.

Fig. 5

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DMOG inhibits ER stress through stabilizing Hif-1α expression in vivo (n = 5). (A–C) Western blot analysis of ATF6 and GRP78 reflecting the activity of ER stress. (C & D) Immunofluorescence staining of chop. Scale bar = 100 μm. (F–I) Western blot analysis of Hif-1α, ATF6 and GRP78. YC-1 TM as positive control. All data are presented as the mean ± SEM. ∗p < 0.5, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001

GAPDH: glyceraldehyde-3-phosphate dehydrogenase; OGD: oxygen-glucose deprivation; DAPI: dihydrochloride (4′,6-Diamidino-2-phenylindole dihydrochloride) solution; TM: tunicamycin; DMOG: dimethyloxalylglycine; ER: endoplasmic reticulum; Hif-1α: hypoxia inducible factor-1α, SEM: standard error of the mean.

4. Discussion

SCI contains a series of primary damage events (which are considered irreversible) and secondary damage events (which lead to devastating results in functional dysfunction).8 The disruption of the BSCB initiates a cascade of pathological events, permitting the infiltration of toxic substances, inflammatory factors, and reactive oxygen species into the spinal cord, which subsequently triggers multiple neural damage processes, including oxidative stress, neuroinflammation, scar formation, and ER stress.31 The survival of ECs and the maintenance of AJ and TJ are the key to the functional restoration of SCI. PHD, an essential modulator of HIF-1α, is involved in stabilizing HIF-1α. In the hypoxia condition, PHD was activated and hydrolyzed HIF-1α, causing cell death. Therefore, restriction of PHD may be the underlying way to block the apoptotic process.32 Besides, in emerging cases PHD inhibitors can stabilize HIF-1α to prevent apoptosis.21 As one of the most acknowledged PHD inhibitors, DMOG has been reported to be effective in stabilizing HIF-1α expression.33,34 So, it is hypothesized that DMOG prevents BSCB collapse and apoptosis in a HIF-1α-dependent manner. The observation of apoptotic proteins such as Bax and cleaved caspase3 is upregulated by SCI, but reversed by DMOG. The expression of Bcl2 is in the opposite. Finally, to figure out the role of HIF-1α in BSCB, we employed YC-1 (a HIF-1α inhibitor) and examined the expression of HIF-1α and proteins linking with AJ and TJ (ZO-1, P120, and β-catenin). DMOG increases the expression of HIF-1α, ZO-1, P120, and β-catenin, but this effect is inhibited by YC-1, indicating that DMOG enhances the repair of BSCB by HIF-1α stabilization.

The function of BSCB mainly depends on ECs and junction complexes. Junction complexes contain TJ and AJ.35 As a dynamic and highly regulated structure, TJ is composed of membrane proteins (claudin), closure proteins (occludin) linking-adhesion molecules, cytoplasmic accessory proteins (ZO-1, ZO-2, ZO-3), and bacterial ring proteins (cingulin). Calcium-dependent adhesive protein (cadherin) connected to the cytoskeleton through intermediate mediators such as catenin, forms intercellular AJ.4,36 P120-catenin, β-catenin, and calcium-dependent adhesive protein (VE-cadherin) in ECs are crucial for formation and stability of AJ.5 Therefore, the disruption of catenin and VE-cadherin complex induces the collapse of AJ and an increase in fluid accumulation in the intercellular space, leading to the unraveling of the integrity of BSCB.37 As a result, the changes in TJ and AJ proteins can reflect the damage or repair status of BSCB.38 Kumar et al.39 have proved that preventing BSCB in SCI favored the homeostasis and internal environment of CNS. The results of this work were consistent with the recognized findings. Herein, SCI led to a notable decrease in the expression of AJ and TJ proteins, indicating the disruption of BSCB. However, the application of DMOG reversed the reduction, reflecting the recovery of BSCB and ultimately promoting the functional recovery of SCI (Fig. 1, Fig. 2). This finding was further confirmed by the evaluation of apoptosis of ECs. As shown in Fig. 3, the expression of Bax and cleaved caspase3 was upregulated by SCI, whereas those were offset by DMOG. On the contrary, the expression of Bcl2 was remarkably reduced by SCI but the trend was inhibited by DMOG.

ER stress was proven to be involved in cell death in many models of diseases including Alzheimer's disease,40 kidney failure,41 and cancer.24 Evidence has proved that ER stress is one of the prominent mechanisms associated with the pathogenesis of SCI.42 Despite limited research on the relationship between ER stress and BSCB, studies have demonstrated that ER stress negatively impacts BSCB43 restoration and potentially induces cell apoptosis.44 Phenylbutyrate, an ER stress inhibitor, was reported to ameliorate the loss of AJ and TJ by reducing the ER stress level of ECs.45 While hyperglycemia activated ER stress to enhance the loss of AJ and TJ.46 Correspondingly, by evaluating the role of ER stress in AJ and TJ of BSCB and DMOG treatment in SCI, we found that ER stress induced by SCI favored the increase of GRP78, ATF6, and chop as well as the loss of ZO-1, P120, and β-catenin, but this phenomenon was suppressed by DMOG administration. Furthermore, the application of TM, an ER stress activator, reversed the positive effect of DMOG. However, TM did not show any influence on the expression of HIF-1α. Meanwhile, the treatment of YC-1, a HIF-1α inhibitor, inhibited the activation of ER stress, namely the increase of GRP78, ATF6, and chop, indicating that DMOG stabilizes the expression of HIF-1α to inhibit ER stress to promote the integrity of BSCB.

In summary, our study demonstrates that inhibition of PHD by DMOG maintains the integrity and permeability of BSCB and improves functional recovery after SCI (Fig. 6). This study also emphasizes that DMOG stabilizes HIF-1α to suppress apoptosis and ER stress. Thereby, it is a potential therapeutic approach to improve functional recovery after SCI that PHD inhibitor DMOG stabilizes HIF-1α expression.

Fig. 6.

Fig. 6

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A schematic diagram to show the potential molecular mechanism that DMOG decreases apoptosis and collapse of BSCB and promotes functional recovery after SCI by Hif-1α stabilization.

DMOG: dimethyloxalylglycine; ER: endoplasmic reticulum; BSCB: blood spinal cord barrier; SCI: spinal cord injury; Hif-1α: hypoxia inducible factor-1α.

CRediT authorship contribution statement

Wen Han: Conceptualization, Data curation, Formal analysis, Funding acquisition, Writing – original draft, Writing – review & editing. Chao-chao Ding: Data curation, Formal analysis, Investigation. Jie Wei: Data curation, Investigation, Project administration. Dan-Dan Dai: Project administration, Supervision, Validation. Nan Wang: Methodology, Project administration, Resources, Software. Jian-Min Ren: Investigation, Methodology. Hai-Lin Chen: Investigation, Methodology, Project administration, Supervision. Ling Xie: Conceptualization, Resources, Supervision, Validation, Visualization, Writing – review & editing.

Ethical statement

The care and use of animals were approved by the Laboratory Animal Ethics Committee of Wenzhou Medical University.

Funding

This work was supported by Natural Science Foundation of Ningbo Municipality(2021J260, 2021J033 and 2023J165).

Declaration of competing interest

The authors have no relevant financial or non-financial interests to disclose.

Acknowledgments

The work was completed with cooperation and assistance of students and staff in Cixi Biomedical Research Institute, Wenzhou Medical University, Zhejiang, China.

Footnotes

Peer review under responsibility of Chinese Medical Association.

Contributor Information

Wen Han, Email: 18840843748@163.com.

Ling Xie, Email: xieling0612@163.com.

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