UAMC-3203 improves the inhibitory microenvironment to repair spinal cord injury by inhibiting ferroptosis

doi:10.21203/rs.3.rs-2189023/v1

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UAMC-3203 improves the inhibitory microenvironment to repair spinal cord injury by inhibiting ferroptosis

https://doi.org/10.21203/rs.3.rs-2189023/v1

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Background: Spinal cord injury (SCI) is a type of trauma to the nervous system that can lead to severe dysfunction of the motor and sensory systems. The inhibitory microenvironment is the basis of secondary injury after SCI, and ferroptosis may aggravate secondary SCI. In this study, the role of ferroptosis inhibitor UAMC-3203 in the recovery of SCI was explored by examining the tissue structure, biochemical indexes, and motor function of rats after SCI.

Methods: A model of SCI was established using Alice's method. The protective effect of UAMC-3203 on spinal cord neurons and the recovery of motor function were studied by Basso, Beattie, Bresnahan (BBB) score, incline plate test, Hematoxylin-eosin (HE) staining and Luxol Fast Blue (LFB) staining. Immunofluorescence and Elisa analysis were used to study the effect of UAMC-3203 on the microenvironment. Western blot analysis was used to measure the level of ferroptosis markers. Reactive oxygen species (ROS) and malondialdehyde (MDA) were detected to reflect the level of oxidation products.

Results: We found that in the UAMC-3203 group, the loss and demyelination of neurons was reduced, the activation and proliferation of astrocytes and microglia and the secretion of related inflammatory cytokines was restrained, and motor function recovery was faster and better compared with those in the SCI group.

Conclusions: UAMC-3203 has played an active role in the recovery process of SCI, providing evidence that post-SCI microenvironment imbalances are associated with ferroptosis.

Spinal cord injury

UAMC-3203

ferroptosis

inhibitory microenvironment

Inflammation

Spinal cord injury (SCI) is a serious traumatic central nervous system disease characterized by high disability and mortality, which can lead to severe and permanent motor, sensory, and autonomic dysfunction, and faces great challenges in clinical treatment and basic research[1]. Treatment of SCI has long been a question of great interest in a wide range of fields. SCI can be divided into direct injury and indirect injury according to the time of onset[2]. The pathophysiological process caused by SCI can form inhibitory microenvironment[3] and lead to secondary injury, which is not conducive to regeneration and repair after SCI. Therefore, the inhibitory microenvironment is the basis of secondary injury after SCI[4].

Some studies have shown that ferroptosis may occur after SCI[5] which is a new form of regulated cell death, and ferroptosis may aggravating secondary SCI[6]. However, the mechanism of ferroptosis in SCI has not been clearly elucidated. Most of these studies were limited to analyzing the relationship between ferroptosis and SCI, however, the effect of ferroptosis on the microenvironment of SCI has not been elucidated.

Ferrostatin-1 (Fer-1) is a ferroptosis inhibitor[7, 8]. As a result of Fer-1's poor metabolic stability, its use in vivo is limited[9]. Therefore, it is imperative to modify the structure of ferroptosis scaffold to synthesize better ferroptosis inhibitors. Compared with other Fer-1 analogues, UAMC-3203 showed higher stability and solubility, and showed better protection against multiple organ damage in mice, as well as effective inhibition of ferroptosis[9]. It has been reported that UAMC-3203 can improve myocardial function and microcirculation and improve myocardial dysfunction after resuscitation by inhibiting ferroptosis in a cardiac arrest rat model[10]. This research has two key aims. Firstly, by comparing the local ferroptosis markers of SCI rats of each group, the existence of ferroptosis in secondary SCI was confirmed; secondly, to investigate the effects of UAMC-3203 on inhibitory microenvironment, tissue repair and nerve function after SCI, and to provide a new therapeutic target for SCI repair.

2.1 Animals and treatments

Forty-five 230g-240g female Wistar rats were purchased from the Chinese Academy of Medical Science Institute of Radiation Medicine. Female rats were chosen because they were less likely to die from urinary tract infections. Humidity and temperature were controlled in a 12-hour diurnal cycle. A random sample of rats was divided into three groups: sham group, only laminectomy without SCI; SCI group, injury and saline; and UAMC-3203 group, injury and 5 mg/kg UAMC-3203. The intraperitoneal injection of UAMC-3203 started immediately following surgery and continued daily until the 7th day. This study was conducted under the permission of a protocol which is approved by the Tianjin Union Medical Center Ethics Review Committee. All the animals were taken care of humanely. If necessary, data supporting this study may be obtained from the appropriate authors upon reasonable request.

Intraperitoneal injection of pentobarbital anesthetizes rats. After anesthesia, the rats were fixed in prone position, the back incision was clipped, disinfected, and covered with cloth. By touching the ribs, the longitudinal skin incision of about 4cm in length was made with T11 as the center, and the subcutaneous tissue of each layer was cut successively to expose the T10 spinous process, and the thoracic spinal cord was exposed. After that, the iron bar weighing 20g was dropped freely from a height of 10cm using Stereotaxic Instruments (D01611-002, RWD Life Science Company). The dural sac was impacted, causing an injury to the spinal cord at T10, and then the muscles, fascia, and skin were sutured successively. Rats with SCI should meet the following characteristics to ensure that all models are at similar basal levels, including T10-segment congestive edema, caudal hindlimb twitching, and neurogenic bladder. After acute SCI, rats will have a temporary loss of voluntary urination function, which can be manually urinated by pressing the bladder under the abdomen. Until the spontaneous urination function of rats recovered. In the UAMC-3203 group, the rats were given UAMC-3203 (5 mg/kg, MedChemExpress, CAS:2271358-65-5) intraperitoneal injection 30min after modeling and then for 7 consecutive days. Three spine samples were taken from each group and frozen on the 14th day, and all the spine sample were taken on the 28th day, 3 samples/group were fixed and 6 samples/group were frozen.

2.2 Histological analysis

The spinal cord of T10 segment was dehydrated, transparent, immersed in wax, embedded in wax, sliced, baked, and dewaxed. The spinal cord was stained with hematoxylin and Solvent Blue. After immersion, the spinal cord was air-dried in a fume hood, sealed with neutral gum, and observed by microscope. Hematoxylin-eosin (HE) staining was used to show the tissue structure of spine cord in 10th thoracic vertebra. Luxol Fast Blue (LFB) staining was used to show the myelin sheath of neurons in 10th thoracic vertebra.

2.3 Western blotting

Place a small amount of tissue blocks in the spherical part of a 1 ~ 2mL homogenizer, and cut the tissue blocks as far as possible with clean scissors. Add lysis (including PMSF) to the homogenizer for homogenization, and then put on ice. After lysis for 30 min, the lysate could be transferred to a 1.5ml centrifuge tube with a pipette, and then centrifuged at 12000rpm for 5min at 4℃. The supernatant was divided into a 0.5ml centrifuge tube and stored at -20℃. Subsequently, the protein concentration was determined with the BCA kit according to the instructions, and the prepared protein samples and maker were added to the sample wells with a micropipette, separated by SDS-PAGE gel electrophoresis, and transferred to polyvinylidene fluoride (PVDF) membrane. The membranes were blocked with 1% bovine serum albumin (BSA) and then incubated overnight at 4°C with the following corresponding primary antibodies: Rabbit Anti-CCBR1/ xCT antibody (1:1000, bs-6883R), Rabbit Anti-glutathione peroxidase 4 antibody (1:1000, bs-3884R). After incubation with matched secondary antibodies (1:5000), ECL reagent was used to form bands and ImageJ was used to obtain protein densities.

2.4 Enzyme-linked immunosorbent assay (ELISA) Analysis

ELISA for assessing secretion of IL-1β, TNF-α, IL-4 and IL-10 from the Inflammatory microenvironment. Based on the manufacturer’s instructions, the corresponding protein concentrations were determined using Rat ELISA Kit (Fankewei, F3071-B) of IL-1β, TNF-α, IL-4, and IL-10.

2.5 Reactive oxygen species (ROS)

The frozen sections were stained with the working solution prepared by ROS fluorescent probe Dihydroethidium (DHE), and observed by fluorescence microscope at the excitation wavelength of 535nm and emission wavelength of 610nm. DHE is a fluorescent probe of cell membrane permeability that is specifically oxidized by ROS to a red fluorescent substance whose intensity is proportional to the level of ROS.

2.6 Malondialdehyde (MDA)

Based on the manufacturer’s instructions, the corresponding MDA concentrations of spine cord tissue were determined using Lipid Peroxidation MDA Assay Kit (Beyotime Biotechnology, S0131S).

2.7 Immunofluorescence

In the culture plate, soak the slides with PBS. Antigen repair was performed by microwave when the repair solution (0.01M citrate buffer, pH6.0) was reduced to room temperature, the slides were removed and rinsed with PBS (PH7.4) three times, 3min each time. The slides were sealed after adding goat blood. Absorbent paper was used to suck up the sealing solution, and each slide was dropped with adequate diluted primary antibody Anti-Myelin Basic (1:1000, CST, 78896), Anti-Iba1 (1:1000, Proteintech Group, Inc, 10904-1-AP), and incubate overnight at 4°C in a humidified chamber. Drop the diluted fluorescent secondary antibody, wet box 20–37℃ incubation for 1h, from the addition of fluorescent secondary antibody, all subsequent steps should be carried out in the dark as far as possible. The liquid on the slipper was sucked dry with absorbent paper, and the slipper was sealed with sealing solution containing anti-fluorescence quencher, and then images were observed and collected under a fluorescence microscope.

2.8 Basso, Beattie, Bresnahan (BBB) score

BBB score was performed to assess lower limb motor function. The rats were placed in an open field and observed by three examiners independent who are blinds to the groups. The tests were conducted at days 0, 1, 3, 7, 14, 21, 28 post-SCI surgery.

2.9 Incline plate test

The recovery of nerve function in rats was appraised using an incline plate test, and the tests were conducted at days 0, 1, 3, 7, 14, 21, 28 post-SCI surgery. The experimental animals were placed on an inclined plate, and the maximum Angle value of 5s was obtained by adjusting the Angle of the inclined plate after SCI. Three independent examiners who are blinds to the group information performed assessments.

2.10 Statistical analysis

All data was expressed as mean ± standard error of mean (SEM). One-way ANOVA was conducted to compare multiple groups, and unpaired Student’s t test was conducted to compare two groups by using SPSS Statistics 25.0 (SPSS Inc., Chicago, IL, USA). A P value < 0.05 was considered statistically significant.

3.1 UAMC-3203 can improve the behavior after SCI

For purpose of evaluating the effect of UAMC-3203 on the behavioral function recovery of rats after SCI, BBB score and inclined plate test were utilized on postoperative day 1, 3, 7, 14, 21 and 28. Within 1 days after SCI, the scores dropped to 0 immediately in each group and began to increase as time goes on, but remained lower compared to those in the sham group (Fig. 1A). The findings showed that compared with the SCI group, the UAMC-3203 group showed greater recovery of behavioral function. Especially on the 7th day after SCI, the UAMC-3203 group had a marked improvement in BBB score. The inclined plate test also confirmed that the UAMC-3203 group had significant higher motor scores than the SCI group, with a significant difference on and after the 7th day after SCI (Fig. 1B).

3.2 UAMC-3203 narrowed the damaged area and alleviated demyelination after SCI

To explore the effect of UAMC-3203 on SCI recovery, histological analysis of spinal cord tissue, including HE staining and LFB staining, was performed. Spinal cord tissue was taken from the SCI group, the UAMC-3203 group, and the sham surgery group. HE staining showed that compared with the UAMC-3203 group, the injury group had a larger area of the injury area, in which cells were sparser, and more irregular arrangement (Fig. 2A). To evaluate the effect of UAMC-3203 on the demyelination post SCI, LFB staining was performed. The LFB staining results showed that the UAMC-3203 group had a larger positive volume than the SCI group, indicating that the UAMC-3203 group had less myelinating loss, and proved that UAMC-3203 could alleviate demyelination post SCI (Fig. 2B). Similarly, immunofluorescence analysis showed that the fluorescence intensity of myelin Basic protein (MBP) compared with that in the sham group, decreased significantly in the SCI group, and UAMC-3203 group showed less MBP immunofluorescence intensity decrease (Fig. 2C).

3.3 UAMC-3203 can reduce the ferroptosis markers of damaged parts after SCI

To explore whether ferroptosis is involved, western blotting was performed on the 28th day post SCI. As expected, xCT and GPX4 showed a noticeable decrease in the SCI group (Fig. 3). Furthermore, the expression of xCT and GPX4 was upregulated in the UAMC-3203 group compared with the SCI group. Therefore, we can come to the conclusion that neurons in spinal cord tissue occur ferroptosis indeed after SCI according to the expression of ferroptosis markers above, and UAMC-3203 inhibits ferroptosis in spinal cord tissue.

3.4 UAMC-3203 inhibits the production of ROS and lipid peroxides after SCI

Hemorrhage of the spinal cord leads to increased iron concentration at the injury site, then iron-mediated Fenton reaction generate more ROS, which further leads to more serious lipid peroxidation. In order to determine if UAMC-3203 could reduce the oxidative damage of cells, DHE staining was performed to detect the production of ROS post SCI. Immunofluorescence intensity of ROS increased significantly after SCI, and decreased significantly after UAMC-3203 treatment (Fig. 4A-B). In addition, lipid peroxide detection showed similarly result that MDA in the UAMC-3203 group was dramatically lower than that in the SCI group (Fig. 4C).

3.5 UAMC-3203 reduced the expression of proinflammatory cytokines and increase the expression of anti-inflammatory cytokine after SCI

To verify the effect of UAMC-3203 on inflammatory factors, ELISA was performed to detect the releases of IL-1β, TNF-α, IL-4, and IL-10 in spinal cord tissues. Compared with the SCI group, the levels of TNF-α and IL-1β decreased significantly in the UAMC-3203 group, and the important anti-inflammatory cytokine, IL-4 and IL-10 increased markedly (Fig. 5). Collectively, UAMC-3203 indeed alleviated inflammatory response post SCI. Compared with the SCI group, the levels of IL-4 and IL-10 in the sham group were not statistically different. We speculated that the levels of IL-4 and IL-10 in the SCI group had decreased to normal levels at the time of measurement.

3.6 UAMC-3203 suppressed the accumulation of astrocytes and macrophages/microglia after SCI

GFAP and Ionized calcium binding adaptor molecule 1 (Iba1) are critical markers of astrocytes and macrophages/microglia. The immunofluorescence analysis results showed that the fluorescence intensity of GFAP and Iba1 markedly increased 28 days post injury, and that in UAMC-3203 group was markedly lower compared to the SCI group (Fig. 6A-B). The results demonstrated that UAMC-3203 suppressed the accumulation of astrocytes and macrophages/microglia after SCI.

SCI is accompanied with non-reversible tissue injury and neurological dysfunction[11]. After SCI, neurons, glial cells, and blood vessels suffer mechanical damage immediately[12]. Hours to days after that, secondary injury emerges[3].

Ferroptosis is a special kind of regulated cell death (RCD)[13], which was first studied as a potential target for anti-cancer therapy[14]. Iron-dependent membrane lipid peroxidation is an important feature of ferroptosis. Cysteine transport and glutathione synthesis is an important target of its development process. Ferroptosis can occur through a decrease in glutathione peroxidase 4 (GPX4) function[15], meanwhile can also be inhibited by inhibitors such as Fer-1. When the antioxidant capacity of cells goes down, ROS will accumulate in the cells and finally experience oxidative cellular death. Ferroptosis has been found playing an important role in many diseases[16], such as brain stroke, cardiac infarction, bone metabolism, and ischemia-reperfusion injury. Our studies suggest that ferroptosis may be involved in secondary injury after SCI by detecting markers of ferroptosis at the traumatic site after SCI.

One study[17] showed that Fer-1 could reverse acrylamide induced dorsal root ganglion neuron damage by inhibiting ferroptosis. Fer-1 can directly inhibit lipid peroxidation by capturing chain-carrying radicals, especially in the phospholipid bilayer, thereby preventing cell death caused by GPX4 inhibition, GPX4 deletion, or GSH depletion[18]. Inhibitors derived from the Fer-1 scaffold inhibited ferroptosis potently but suffered from solubility issues. Compared with Fer-1, UAMC-3203 is more stable and easier to dissolve, and has a better protective effect against multiple organ injury in mice.

HE staining showed that the quantity of necrotic neurons increased significantly post SCI. Treatment with UAMC-3203 can significantly reduce the quantity of necrotic neurons, suggesting that UAMC-3203 may play a neuroprotective role by inhibiting ferroptosis of neurons. A series vivo experiments have indeed confirmed, which is similar to our research, that UAMC-3203 can reduce ROS and lipid peroxides production post SCI. A western blot analysis of ferroptosis markers (xCT and GPX4) ulteriorly validated our findings. In summary, UAMC-3203 can inhibit the increase of lipid peroxides and ROS, reduce neuron degeneration, and facilitate the motor function recovery by inhibiting ferroptosis after SCI.

To further explain the mechanism by which UAMC-3203 inhibits ferroptosis and promotes spinal nerve recovery, we tested the effect of UAMC-3203 on the inhibitory microenvironment after SCI. As a result of the contusion, the spinal microenvironment is dysregulated, which results in a sequence of pathophysiological changes, with the downregulation of beneficial factors and the upregulation of harmful factors. As a result of this study, UAMC-3203 has been proved for the first time to facilitate spinal nerve recovery after SCI by improving the inhibitory microenvironment by inhibiting ferroptosis.

Three levels at different times and sites contribute to the microenvironment imbalance: tissues, cells, and molecules. In tissue level, we observed hemorrhage and ischemia as well as demyelination at the injury site and surrounding area by HE and LFB slice staining after SCI. In cellular level, we observed the activation of astrocytes and microglia, and demyelination changes after SCI. It has been revealed that ferroptosis accelerates prostaglandin-endoperoxide synthase (PTGS2) expression and release. PTGS2 could accelerate the metabolism of arachidonic acid and facilitate the release of inflammatory signaling molecules, while ferroptosis can promote the expression of PTGS2[19]. In molecular level, we observed increased expression of IL-1β, TNF-α after SCI. With the treatment of UAMC-3203, all above three aspects pathological changes were reversed.

Inhibitory factors existed in the microenvironment after SCI may affect endogenous stem cell differentiation[20], including astrocytes and microglia. Microglia are resident macrophages that normally remain static in the central nervous system[21]. After SCI, microglia and astrocytes are rapidly activated by various pathological factors. Then activated microglia release IL-1β, IL-6 and TNF-α, causing a large number of nerve cell death, axonal degeneration, demyelination[22]. Glial scars include both fibrous and glial components, and astrocytes and microglia are involved in glial formation[23]. The formation of Glial scar is an important part of SCI pathology, which hinders the repair of the spinal cord after SCI[24]. In addition, microglia/macrophages can release inflammatory mediators and aggravate secondary tissue damage after SCI, which is an important component of neuroinflammation[25]. Inflammatory cytokines like IL-1 family have been shown to be extremely important to initiate inflammatory process, and induces cell activation, which aggravates SCI[26].

After the treatment of UAMC-3203, activation of astrocytes and microglia were inhibited, and the secretion of proinflammatory factors was reduced, which limits the further deterioration of the inhibitory microenvironment. In our study, we observed UAMC-3203, the specificity of ferroptosis inhibitors, reversed the above three aspects, namely the pathological changes of the tissue, cells, and molecules, including decreasing bleeding, reducing the loss of neurons, demyelination, activation and proliferation of astrocyte and microglia, and improving motor function recovery. Comparing BBB score, angle of incline, and the number of neural cells between the SCI group and UAMC-3203 group, it is not difficult to see that UAMC-3203 has a certain positive effect on the repair of SCI. Combined with the influence of UAMC-3203 on the microenvironment after SCI, we conclude that UAMC-3203 plays a protective role in SCI by improving the inhibitory microenvironment. However, the molecular mechanism by which UAMC-3203 inhibits ferroptosis has not been fully determined, and we plan to gradually explore the mechanism in the following studies.

In conclusion, our research suggests that neuroprotective effect of UAMC-3203 is through the inhibition of ferroptosis and inflammation associated with ferroptosis in the local area of SCI, thereby improving the inhibitory microenvironment and promoting neuron survival. Our results enrich the insights of UAMC-3203 in SCI recovery and provide new evidence that inhibition of ferroptosis promotes SCI recovery.

SCI: spinal cord injury

BBB: Basso, Beattie, Bresnahan

ROS: Reactive oxygen species

MDA: malondialdehyde

Fer-1: Ferrostatin-1

PVDF: polyvinylidene fluoride

BSA: bovine serum albumin

DHE: Dihydroethidium

TNF-α: tumor necrosis factor-α

IL-1β: interleukin-1β

IL-4: interleukin-4

IL-10: interleukin-10

xCT/ SLC7A11: cystine/glutamate antiporter solute carrier family 7member11

GPX4: glutathione peroxidase 4

HE: haematoxylin and eosin

LFB: Luxol Fast Blue

GFAP: glial fibrillary acidic protein

RCD: regulated cell death

Iba1: Ionized calcium binding adaptor molecule 1

MEP: motor evoked potentials

ELISA: Enzyme-linked immunosorbent assay

Data Availability

The data generated during the current study are available from the corresponding author upon request.

Availability of data and materials

The data and materials generated during the current study are available from the corresponding author upon request.

Funding

This work was funded by Tianjin Union Medical Center (2019JZPY01,

2019JZPY07, 2018YJ010), Tianjin Health Research Project (ZC20225, KJ20061),

Tianjin Key Medical Discipline (Specialty) Construction Project (TJYXZDXK-064B)

Author information

Authors and Affiliations

Department of Spine Surgery, Tianjin Union Medical Center, Tianjin, China

Shunli Kan, Xinyan Zhao, Chengjiang Liu, Haoqiang Zhu, Sa Feng, Boyuan Ma, Mengmeng Zhou, Xuanhao Fu, Wei Hu& Rusen Zhu

Contributions

Shunli Kan and Rusen Zhu conceived and designed the study; Xinyan Zhao and Chengjiang Liu performed the experiments; Xinyan Zhao drafted the manuscript; Boyuan Ma, Mengmeng Zhou and Xinyan Zhao contributed to acquisition of data or analysis data; Haoqiang Zhu and Sa Feng. were responsible for editing; Xuanhao Fu and Wei Hu reviewed the manuscript. All authors approved the final manuscript.

Corresponding authors

Correspondence to Rusen Zhu

Ethics declarations

Ethics Approval

The animal experiments were approved by the Tianjin Union Medical Center Ethics Review Committee.

Consent for Publication

All authors agreed to publication.

Consent to Participate

Not applicable.

Conflict of Interest

The authors declare no competing interest.

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No competing interests reported.

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UAMC-3203 improves the inhibitory microenvironment to repair spinal cord injury by inhibiting ferroptosis

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Abstract

Figures

1. Introduction

2. Methods

2.1 Animals and treatments

2.2 Histological analysis

2.3 Western blotting

2.4 Enzyme-linked immunosorbent assay (ELISA) Analysis

2.5 Reactive oxygen species (ROS)

2.6 Malondialdehyde (MDA)

2.7 Immunofluorescence

2.8 Basso, Beattie, Bresnahan (BBB) score

2.9 Incline plate test

2.10 Statistical analysis

3. Results

3.1 UAMC-3203 can improve the behavior after SCI

3.2 UAMC-3203 narrowed the damaged area and alleviated demyelination after SCI

3.3 UAMC-3203 can reduce the ferroptosis markers of damaged parts after SCI

3.4 UAMC-3203 inhibits the production of ROS and lipid peroxides after SCI

3.5 UAMC-3203 reduced the expression of proinflammatory cytokines and increase the expression of anti-inflammatory cytokine after SCI

3.6 UAMC-3203 suppressed the accumulation of astrocytes and macrophages/microglia after SCI

4. Discussion

5. Conclusions

Abbreviations

Declarations

References

Additional Declarations

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