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. 2023 Nov 8;19(7):1568–1574. doi: 10.4103/1673-5374.387979

Fibroblast growth factor 21 inhibits ferroptosis following spinal cord injury by regulating heme oxygenase-1

Qi Gu 1,2,#, Weiping Sha 1,2,#, Qun Huang 1,2, Jin Wang 1,2, Yi Zhu 1,2, Tianli Xu 1,2, Zhenhua Xu 3, Qiancheng Zhu 1,2, Jianfei Ge 1,2, Shoujin Tian 1,2,*, Xiaolong Lin 1,2,*
PMCID: PMC10883498  PMID: 38051901

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Key Words: ferroptosis, fibroblast growth factor 21, functional recovery, heme oxygenase-1, lipid peroxidation, neuron, reactive oxygen species, spinal cord injury

Abstract

Interfering with the ferroptosis pathway is a new strategy for the treatment of spinal cord injury. Fibroblast growth factor 21 can inhibit ferroptosis and promote neurofunctional recovery, while heme oxygenase-1 is a regulator of iron and reactive oxygen species homeostasis. The relationship between heme oxygenase-1 and ferroptosis remains controversial. In this study, we used a spinal cord injury rat model to show that the levels of fibroblast growth factor 21 in spinal cord tissue decreased after spinal cord injury. In addition, there was a significant aggravation of ferroptosis and a rapid increase in heme oxygenase-1 expression after spinal cord injury. Further, heme oxygenase-1 aggravated ferroptosis after spinal cord injury, while fibroblast growth factor 21 inhibited ferroptosis by downregulating heme oxygenase-1. Thus, the activation of fibroblast growth factor 21 may provide a potential treatment for spinal cord injury. These findings could provide a new potential mechanistic explanation for fibroblast growth factor 21 in the treatment of spinal cord injury.

Introduction

Spinal cord injury (SCI) is a serious traumatic injury to the central nervous system that can result in lower limb dysfunction below the damaged part of the cord and even cause death (Taccola et al., 2018; Li et al., 2019). There are primary and secondary pathophysiological manifestations in SCI. Primary injury is an irreversible mechanical injury, while secondary injury is reversible (Qian et al., 2020; Al-Sammarraie et al., 2023). Therefore, alleviating secondary injury and promoting residual neuronal survival may be crucial to protect neurological function after SCI. Autophagy, inflammation, apoptosis, oxidative stress, and ferroptosis are commonly associated with secondary injury. Inhibiting these pathological processes with certain drugs can reduce or even reverse secondary SCI (Ko et al., 2020; Vismara et al., 2020).

Ferroptosis is a cell death cascade caused by iron-dependent lipid peroxidation (Chen et al., 2022; Li and Jia, 2023). It is biochemically characterized by altered iron metabolism, active oxygen accumulation induced by iron, inhibition of glutathione peroxidase 4 (GPX4), and increased lipid peroxidation (Bersuker et al., 2019; Hirschhorn and Stockwell, 2019). After SCI, local hemorrhage during the acute phase increases the accumulation of iron and reactive oxygen species (ROS) in the injured area (Meng et al., 2017). Some studies have also shown that ferritin is expressed at high levels in spinal cord tissue, and is positively associated with the severity of SCI (Koszyca et al., 2002; Schonberg et al., 2012; Blissett et al., 2018). Furthermore, polyunsaturated fatty acids are abundant in neuronal membranes, and easily oxidized by ROS (Shichiri, 2014). Therefore, interfering with the ferroptosis pathway is a new strategy for the treatment of SCI. GPX4 is a critical regulator of ferroptosis that detoxifies cellular lipid peroxidation using glutathione as a cofactor. 4-Hydroxynonenal (4HNE) is a major by-product of lipid peroxidation and its elevation can indicate an exacerbation of ferroptosis. In our study, we selected GPX4 and 4HNE as markers of glutathione metabolism and lipoxidation to evaluate ferroptosis after SCI.

As a member of the fibroblast growth factor superfamily, fibroblast growth factor 21 (FGF21) participates in the regulation of both lipid and glucose homeostasis, and can prevent metabolic disorders (Keipert et al., 2020). This molecule is secreted mainly by the liver, pancreas, kidney, skeletal muscle, and adipose tissue (Gomez-Samano et al., 2017). In addition, FGF21 has potent neuroprotective effects after SCI, and exogenous administration of recombinant human FGF21 (rhFGF21) has been shown to inhibit neuronal death and promote axon elongation (Zhu et al., 2020).

Heme oxygenase-1 (HO-1) is an anti-inflammatory, antioxidant, and neuroprotective phase II enzyme. HO-1 is responsible for converting heme into biliverdin/bilirubin, ferrous iron (Fe2+), and carbon monoxide (CO). Wu et al., found that FGF21 inhibits ferroptosis in hepatocytes by promoting the ubiquitination and degradation of HO-1 (Wu et al., 2021). Currently, the role of FGF21 in ferroptosis after SCI is unclear, and whether FGF21 affects the ferroptotic process by regulating HO-1 requires further investigation.

To investigate this problem, we investigated expression trends of FGF21, HO-1, GPX4, and 4HNE after SCI. We also examined the effect of FGF21 on HO-1 and SCI-induced ferroptosis using rhFGF21 and hemin (a HO-1 agonist) as drug treatments after SCI in rats.

Methods

Animal and SCI model

Since menstruation in female rats can affect iron metabolism, male rats were used in our experiments to investigate ferroptosis. In addition, human SCI is more common in men (approximately 80%), therefore male rats are suitable experimental animals (Li et al., 2020). A total of 140 adult, male Sprague-Dawley rats (8–10 weeks old, 230–250 g) were used in this study. Eight died due to perioperative complications (e.g., infection, hemorrhage) during the experiments. Rats were all fed and watered freely under constant conditions of photoperiod (12-hour light and dark), temperature (22–25°C), and humidity (55–60%), with three rats per cage. All animal experimental protocols strictly adhered to China's animal welfare legislation on animal use and care. All procedures using experimental animals were performed under the supervision of the Institutional Review Board (IRB)/Independent Ethics Committee (IEC) of The Affiliated Zhangjiagang Hospital of Soochow University on December 20, 2021 (approval No. ZJGYYLL-2021-12-044). This study was reported according to the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines (Percie du Sert et al., 2020)

Rats were first anesthetized by inhalation with 3% isoflurane (R510-22-10, RWD Life Science, Shenzhen, China) for induction, and then 2% isoflurane (2–3 L/min) for maintenance. This was followed by complete exposure of the spinal process and vertebral plates of the thoracic (T)10 segment. After complete exposure of the spinal cord, SCI was induced using a precision impactor device (68099II, RWD Life Science). Parameters were set to a speed of 2 m/s, depth of 1 mm, and dwell time of 0.4 seconds to cause a moderate contusion injury (60 kdyn) according to the instruction manual. Hemorrhage and swelling of the spinal cord were immediately observed after modeling. The criteria for success of the SCI model were involuntary spasms of the lower extremities and tail twisting. In addition, loss of muscle strength and paralysis could be observed after awakening from anesthesia. Wounds were flushed with saline and sutured. Rats in the sham group had the same operation procedure except the spinal cord contusion. During surgery, body temperature was maintained on a heating pad at 37 ± 0.3°C. After modeling, all rats underwent manual urination twice a day until their voluntary urinary function returned to normal.

Experimental groups

Experiment 1: To examine the mRNA and protein expression trends of FGF21, HO-1, GPX4, and 4HNE at different time points after SCI, rats were randomly divided into seven groups (n = 6/group): Sham, 1, 2, 3, 7, 14, and 28 days.

Experiment 2: To investigate the effects of rhFGF21 and hemin on ferroptosis in the early days of SCI, rats were randomly divided into five groups, with ferrostatin-1 as a positive control (n = 6/group): Sham group (Sham), SCI group (SCI), SCI + ferrostatin-1-treated group (Fer-1), SCI + rhFGF21-treated group (rhFGF21), and SCI + rhFGF21 + hemin-treated group (rhFGF21+hemin).

Ferrostatin-1 (2 mg/kg per day, HY-100579, MCE, Monmouth Junction, NJ, USA) was diluted in 0.9% NaCl, and injected intraperitoneally into animals of the Fer-1 group as a positive control at 30 minutes after SCI and 24 hours later. rhFGF21 (1.5 mg/kg per day, Cat# ab133137, Abcam, Cambridge, UK) was dissolved in 0.9% NaCl, and injected intraperitoneally into animals of the rhFGF21 group and rhFGF21 + hemin group at 30 minutes after SCI and 24 hours later. In the rhFGF21 + hemin group, rats were injected intraperitoneally with hemin (5 mg/kg per day, HY-19424, MCE) and rhFGF21 (1.5 mg/kg per day) dissolved in 0.9% NaCl. Animals in both the Sham and SCI groups were injected with 0.9% NaCl at the same dose as drug-treated groups.

Behavioral tests

The Basso, Beattie, and Bresnahan (BBB) locomotor score is a common tool to evaluate behavioral outcomes after SCI in rats (Scheff et al., 2002). The range of scores is 0 to 21, with 0 indicating no motor function and 21 indicating a normal level of function. The rats in each group were allowed to walk freely for 5 minutes while movement of the hind limbs was closely observed. Motor recovery in all groups was assessed by two independent examiners using the BBB score on postoperative days 1, 2, 3, 7, 14, and 28.

The incline plate test is a commonly used neurological test for the assessment of behavioral outcomes (Wang and Zhang, 2012). A rubber tilt plate with a special shallow groove was used to measure the maximum angle at which the rat can hold its posture on the tilt plate for at least 5 seconds. Neurological recovery was measured on postoperative days 1, 2, 3, 7, 14, and 28.

Nissl staining

After fixation with 4% paraformaldehyde solution (BL539A, Biosharp, Beijing, China), spinal cord tissue was dehydrated by graded sucrose solution (15% and 30%). Dehydrated spinal cord tissue was embedded and then cut into 10 µm frozen axial sections using a cryostat microtome (CM1950, Leica, Weztlar, Germany). Frozen sections (10 μm) were thawed at room temperature. After soaking for 5 minutes in distilled water, sections were incubated with Nissl staining solution (Cat# C0117, Beyotime, Shanghai, China) for 15 minutes at room temperature. Slides were rinsed with distilled water and dehydrated with graded alcohol solutions: 75% alcohol and 95% alcohol for 10 minutes each. Xylene (Cat# 534056-500ML, Sigma-Aldrich, St. Louis, MO, USA) was used for clarification. Finally, the sections were sealed with neutral resin. Changes in the number of Nissl bodies within the spinal cord anterior horn at the same site were observed using a digital microscope (TCS SP8, Leica). Mean number of Nissl bodies per square millimeter (mm2) were counted using ImageJ software (v1.8.0, NIH, Bethesda, MD, USA; Schneider et al., 2012).

Perls staining

After fixation with 4% paraformaldehyde solution (Cat# BL539A, Biosharp), spinal cord tissue was dehydrated by graded sucrose solution (15% and 30%). Dehydrated spinal cord tissue was embedded and then cut into 10 µm frozen axial sections using a cryostat microtome (Cat# CM1950, Leica). Frozen sections (10 μm) were thawed at room temperature and soaked in distilled water for 5 minutes. Slides were soaked in Perls solution (1:1, potassium ferrocyanide solution and acid solution) (Cat# 60533ES60, Yeasen, Shanghai, China) for 60 minutes and then washed with distilled water for 5 minutes. Nuclear fast red solution (60533ES60, Yeasen) was used for 10 minutes. After rinsing with distilled water, the slides were dehydrated with graded alcohol: 75% alcohol and 95% alcohol for 10 minutes each. Xylene (Cat# 534056-500ML, Sigma-Aldrich) was used for clarification. Finally, the slides were sealed with neutral resin. Changes in the number of iron-positive cells within the spinal cord anterior horn at the same site were observed using a digital microscope (TCS SP8, Leica). Mean number of iron-positive cells per square millimeter (mm2) were counted using ImageJ software (v1.8.0).

Western blotting

The contusion site and surrounding tissue (about 5 mm) were collected from the spinal cord. In the presence of protease inhibitor cocktail (Cat# CW2200S, CWBIO, Taizhou, China), tissues were homogenized with precooled radioimmunoprecipitation assay (RIPA) lysis buffer (Cat# CW2333, CWBIO). Tissue solution was then centrifuged twice at 13,400 × g (20 minutes, 4°C). The supernatant was collected and the protein concentration of each sample determined using the bicinchoninic acid (BCA) method (P0010, Beyotime). Samples were boiled after the addition of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer (P001, Beyotime). Next, 30 μg of protein was separated by electrophoresis (10% SDS-PAGE gels) followed by transfer to nitrocellulose (NC) membranes (HATF00010, Millipore, Darmstadt, Germany). Skimmed milk (5%) was used to block non-specific sites on NC membranes. After 2 hours of blocking at room temperature on a shaker, NC membranes were incubated overnight at 4°C with primary antibodies. After 3 × 10 minute washes using Tris-buffered saline with Tween (TBST), goat anti-rabbit or anti-mouse secondary antibodies coupled with horseradish peroxidase (HRP) were incubated for an additional 1.5 hours at room temperature. After 3 × 10 minutes TBST washes, protein bands were visualized with chemiluminescent HRP substrate (WBKLS0500, Millipore) and a ChemiDocTM XRS+ Imaging System (BioRad, Hercules, CA, USA). Relative protein expression was determined using ImageJ software (v1.8.0) and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The antibodies used were: anti-FGF21 (rabbit monoclonal antibody [mAb], 1:1000, Cat# ab171941, Abcam), anti-HO-1 (rabbit mAb, 1:2000, Cat# ab189491, Abcam), anti-GPX4 (rabbit mAb, 1:1000, Cat# ab125066, Abcam), anti-4HNE (rabbit pAb, 1:3000, Cat# ab46545, Abcam), anti-GAPDH (mouse mAb, 1:20,000, Cat# 60004-1-lg, Proteintech, Chicago, IL, USA), goat pAb to rabbit lgG (HRP) (1:20,000, Cat# ab205718, Abcam), and goat pAb to mouse lgG (HRP) (1:5000, Cat# ab205719, Abcam).

Quantitative reverse transcription-polymerase chain reaction

The contusion site and surrounding tissue (about 5 mm) were collected from the spinal cord. Total RNA was extracted using TRIzol reagent (Cat# 15596026, Ambion, Austin, TX USA). RNA concentration was determined using the ultraviolet absorption method with a spectrophotometer (VL0000D0, Thermo Fisher Scientific, Waltham, MA, USA). cDNA was synthesized by reverse transcription with 2 μg of total RNA, followed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using SYBR Green (Cat# 4367659, Thermo Fisher Scientific). cDNA samples were amplified according to the following reaction protocol: initial denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, and annealing at 60°C for 30 seconds for 40 cycles. Both the primer sequences (forward and reverse) and their product sizes are shown in Table 1. mRNA levels were analyzed using the 2–ΔΔCt method after normalization to GAPDH (Livak and Schmittgen, 2001).

Table 1.

Primers used in the present study for quantitative reverse transcription-polymerase chain reaction

Forward primers (5'–3') Reverse primers (5'–3') Product size (bp)
GAPDH CAA CCC TCA ACA GGG ATG CT CGA TAC GGC CAA ATC CGT TC 105
FGF21 AGA TCA GGG AGG ACG GAA CA TCA GGA TCA AAG TGA GGC GAT 172
HO-1 ATC GTG CTC GCA TGA ACA CT CAG CTC CTC AAA CAG CTC AAT G 107
GPX4 GTT CCT GGG CTT GTG TGC AT CGG TTT TGC CTC ATT GCG AG 158
4HNE CTC ATC CTG CCT CGT CCT TG AGT TTC TGG CAC TCA GGG ATA CTA 135
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4HNE: 4-Hydroxynonenal; FGF21: fibroblast growth factor 21; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPX4: glutathione peroxidase 4; HO-1: heme oxygenase-1.

Iron, glutathione, and malondialdehyde measurement

The contusion site and surrounding tissue (about 5 mm) were homogenized and diluted 10-fold in phosphate buffered saline (PBS) (C0221A, Beyotime). Iron, glutathione (GSH), and malondialdehyde (MDA) levels were measured using enzyme-linked immunosorbent assays (ELISA) for Iron (Cat# ab83366, Abcam), GSH (Cat# S0053, Beyotime) and MDA (Cat# S0131S, Beyotime), respectively. ELISAs were performed in accordance with the manufacturer's instructions.

Transmission electron microscopy

Rats were first anesthetized by inhalation with 3% isoflurane (R510-22-10, RWD Life Science) for induction and then 2% isoflurane (2–3 L/min) for maintenance. Next, rats were transcardially perfused with 0.1 M sodium carbonate in 2% paraformaldehyde and 2% glutaraldehyde, and then fixed in 0.1 M sodium dimethyl arsonate with 2% osmium tetroxide and 1.6% potassium ferrocyanide. Tissue samples were cut into sections (70–90 nm). The sections were dehydrated in ethanol and embedded in ebonite. Sections were then deposited onto a copper channel grid. Finally, 2% uranyl acetate (SPI-02624, HEAD) and lead citrate (HD17810, HEAD) were used to stain the sections. Transmission electron microscopic images were obtained by a transmission electron microscope (Tecnai F20, FEI, Hillsboro, Oregon, USA).

Immunofluorescence staining

After fixation with 4% paraformaldehyde solution (BL539A, Biosharp), spinal cord tissue was dehydrated by graded sucrose solution (15% and 30%). Dehydrated spinal cord tissue was embedded and then cut into 10 µm frozen axial sections using a cryostat microtome (CM1950, Leica). Frozen sections (10 μm) were thawed at room temperature and fixed with 4% paraformaldehyde for 30 minutes. Next, 0.3% Triton X-100 (1139ML100, BioFroxx, Heidelberg, Germany) was added for 15 minutes, followed by incubation with 10% normal goat serum (AR0009, Boster, Wuhan, China) for 1 hour at room temperature. Sections were then incubated with primary antibodies overnight at 4°C. Sections were rinsed 3 times in PBS with Tween (PBST) and then incubated with fluorescein isothiocyanate and tetramethyl isothiocyanate-coupled secondary antibody for 1 hour at room temperature. After, sections were rinsed 3 times with PBST and then 4′,6-diamidino-2-phenylindole (DAPI) (36308ES20, Yeasen) staining was performed for 15 seconds. The expression of target proteins in spinal cord tissue was observed by a fluorescence microsystem (DMi8, Leica). ImageJ software (v1.8.0) was used to analyze the images. The antibodies used were: anti-HO-1 (rabbit mAb, 1:2000, Cat# ab189491, Abcam), anti-GPX4 (rabbit mAb, 1:1000, Cat# ab125066, Abcam), anti-NeuN (mouse mAb, 1:200, Cat# MAB377, Millipore), 488 donkey anti-rabbit lgG (H+L) (1:800, Cat# A21206, Invitrogen, Carlsbad, CA, USA), and 555 donkey anti-mouse lgG (H+L) (1:800, Cat# A31570, Invitrogen).

Statistical analysis

No statistical methods were used to estimate sample sizes; nevertheless, the sample sizes in our study are similar to those reported in previous articles (Chen et al., 2021b; Ge et al., 2022). All analyses were conducted by researchers blinded to the experimental conditions. GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA, www.graphpad.com) was used for data processing, statistical analysis, and statistical chart production. Measurement data are represented by box and whisker plots. Two-way analysis of variance followed by Tukey's post hoc test was performed to analyze BBB score and incline plate test data. Other data were analyzed by one-way analysis of variance followed by Dunnett's post hoc test. P < 0.05 was considered significant.

Results

Trends of FGF21, HO-1, and ferroptosis-related molecules after SCI

To explore the trends of FGF21 and HO-1 after SCI, we performed WB and qRT-PCR analyses of spinal cord tissue collected on days 1, 2, 3, 7, 14, and 28 after SCI (Figure 1). Protein expression of FGF21 decreased on the 1st day after SCI, reaching lowest levels on day 2 after SCI (P < 0.0001; Figure 1B). qRT-PCR analysis showed the same trend in FGF21 mRNA expression (P < 0.001; Figure 1F). In contrast, HO-1 mRNA and protein expression increased rapidly after SCI and peaked on day 2 after SCI (mRNA: P < 0.0001; protein: P < 0.0001; Figure 1C and G).

Figure 1.

Figure 1

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mRNA and protein expression trends of FGF21, HO-1, GPX4, and 4HNE in rats after SCI.

(A) Western blot bands of FGF21, HO-1, GPX4, 4HNE, and GAPDH. (B–E) Box and whiskers plots from A. n = 6. (F–I) mRNA expression trends of FGF21, HO-1, GPX4, and 4HNE detected by qRT-PCR were normalized to the Sham group. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, vs. Sham (one-way analysis of variance followed by Dunnett's post hoc test). 4HNE: 4-Hydroxynonenal; FGF21: fibroblast growth factor 21; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPX4: glutathione peroxidase 4; HO-1: heme oxygenase-1; NS: not significant; SCI: spinal cord injury.

To investigate the role of FGF21 in ferroptosis after SCI, GPX4 and 4HNE were used as ferroptosis-related markers. Protein and mRNA levels of GPX4 decreased rapidly on day 2 after SCI, increasing slowly after day 3 (protein: P < 0.0001, mRNA: P < 0.01; Figure 1D and H). Protein and mRNA expression of 4HNE reached a peak on day 2 after SCI (protein: P < 0.0001, mRNA: P < 0.0001; Figure 1E and I). The trends of GPX4 and 4HNE expression showed that the 2nd day after SCI was a suitable time point to examine the effect of drug treatment on SCI-induced ferroptosis.

Hemin attenuates the effect of FGF21 to inhibit ferroptosis after SCI

Based on the expression patterns of FGF21, HO-1, GPX4, and 4HNE, we chose the 2nd day after SCI as the time point for Experiment 2. The extent of neurodegeneration and iron accumulation were assessed by Nissl staining and Perls staining, respectively. The Nissl results revealed that normal neurons were round and darkly stained, while atrophied and mauve neurons appeared after SCI (P < 0.0001; Figure 2A and B). Both Fer-1 and rhFGF21 treatments improved the contracted morphology of neurons. In contrast, hemin treatment aggravated the morphological changes of neurons in the rhFGF21 + hemin group compared with the rhFGF21 group (Fer-1 vs. SCI: P < 0.0001; rhFGF21 vs. SCI: P < 0.0001; rhFGF21 + hemin vs. rhFGF21: P < 0.001; Figure 2A and B). Perls staining of spinal cord sections showed that the SCI group had significantly more iron-positive cells than the Sham group (P < 0.0001; Figure 2A and C). In the Fer-1 and rhFGF21 groups, iron-positive cells were significantly decreased, while the rhFGF21 + hemin group had more iron-positive cells than the rhFGF21 group (Fer-1 vs. SCI: P < 0.0001; rhFGF21 vs. SCI: P < 0.0001; rhFGF21 + hemin vs. rhFGF21: P < 0.0001; Figure 2A and C).

Figure 2.

Figure 2

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Hemin treatment attenuates the protective effect of rhFGF21 in rats after SCI.

(A) Images of Nissl staining (upper panels) and Perls staining (lower panels) in the spinal cord anterior horn. Arrows show iron-positive cells. Boxes show higher magnification views of low magnification images. Scale bars: 50 µm (Nissl staining and Perls staining) and 25 µm (higher magnification). (B, C) Box and whisker plots from A. n = 6. ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Dunnett's post hoc test). (D, E) BBB score and incline plate test on days 1, 2, 3, 7, 14, and 28 after SCI. n = 6. *P < 0.05, ***P < 0.001, ****P < 0.0001, SCI vs. rhFGF21; #P < 0.05, ###P < 0.001, ####P <0.0001, rhFGF21 vs. rhFGF21 + Hemin (two-way analysis of variance followed by Tukey's post hoc test). BBB: Basso, Beattie and Bresnahan; Fer-1: ferrostatin-1; rhFGF21: recombinant human fibroblast growth factor 21; SCI: spinal cord injury.

The BBB scores of the rhFGF21 group increased more rapidly than the SCI group on day 28 (P < 0.0001; Figure 2D). The rhFGF21 + hemin group had a lower BBB score than the rhFGF21 group on day 28 (P < 0.0001; Figure 2D). In the incline plate test, we observed that rats in the rhFGF21 group could hold their posture on the tilt plate at higher angles than the SCI group on day 28 (P < 0.0001; Figure 2E). The angles were lower in the rhFGF21 + hemin group than rhFGF21 group (P < 0.0001; Figure 2E).

We found that rhFGF21 treatment reduced iron-positive cells and attenuated pathological changes in neurons. Hemin treatment had the opposite effect and attenuated the protective effect of rhFGF21. Furthermore, the behavioral test results demonstrated that hemin treatment could attenuate the improved behavioral outcomes of rhFGF21 treatment.

FGF21 inhibits SCI-induced ferroptosis by downregulating HO-1

FGF21 protein expression significantly increased in the rhFGF21 group and rhFGF21 + hemin group compared with the SCI group (rhFGF21 vs. SCI: P < 0.001; rhFGF21+hemin vs. rhFGF21: P < 0.0001; Figure 3A and B). However, mRNA expression of FGF21 showed no significant differences after rhFGF21 treatment (rhFGF21 vs. SCI: P > 0.05; rhFGF21+hemin vs. rhFGF21: P > 0.05; Figure 3F). The expression of HO-1 and 4HNE significantly decreased in the rhFGF21 group compared with the SCI group (HO-1: P < 0.0001; 4HNE: P < 0.0001; Figure 3A, C, and E). Nevertheless, protein expression of HO-1 and 4HNE increased after hemin treatment in the rhFGF21 + hemin group compared with the rhFGF21 group (HO-1: P < 0.01; 4HNE: P < 0.0001; Figure 3A, C, and E), with the same trend in mRNA expression (HO-1: P < 0.0001; 4HNE: P < 0.001; Figure 3G and I). Protein expression of GPX4 significantly increased in the rhFGF21 group, while hemin treatment downregulated GPX4 protein expression in the rhFGF21 + hemin group compared with the rhFGF21 group (rhFGF21 vs. SCI: P < 0.0001; rhFGF21 + hemin vs. rhFGF21: P < 0.0001; Figure 3A and D). Moreover, mRNA expression of GPX4 showed consistent trends (rhFGF21 vs. SCI: P < 0.001; rhFGF21 + hemin vs. rhFGF21: P < 0.05; Figure 3H).

Figure 3.

Figure 3

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mRNA and protein expression levels of FGF21, HO-1, GPX4, and 4HNE in rats after treatment.

(A) Western blot bands of FGF21, HO-1, GPX4, 4HNE, and GAPDH. (B–E) Box and whisker plots from A. n = 6. (F–I) mRNA expression levels of FGF21, HO-1, GPX4, and 4HNE detected by qRT-PCR were normalized to the Sham group. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Dunnett's post hoc test). 4HNE: 4-Hydroxynonenal; Fer-1: ferrostatin-1; FGF21: fibroblast growth factor 21; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GPX4: glutathione peroxidase 4; HO-1: heme oxygenase-1; NS: not significant; rhFGF21: recombinant human fibroblast growth factor 21; SCI: spinal cord injury.

These results suggest that increased expression of FGF21 inhibited HO-1 and SCI-induced ferroptosis, while hemin treatment attenuated the inhibition of FGF21 to ferroptosis.

FGF21 reduces pathological changes in mitochondria by downregulating HO-1

For quantitative analysis of the ferroptotic process in spinal cord tissue of all groups, we measured the concentration of iron, reduced GSH, and lipid peroxide (MDA) (Figure 4). In the rhFGF21 group, iron and MDA content significantly decreased and GSH content increased compared with the SCI group (iron: P < 0.0001; MDA: P < 0.0001; GSH: P < 0.0001; Figure 4B–D). In contrast, iron and MDA content increased and GSH levels decreased in the rhFGF21 + hemin group compared with the rhFGF21 group (iron: P < 0.0001; MDA: P < 0.0001; GSH: P < 0.0001; Figure 4B–D). Transmission electron microscopic images revealed structural pathological changes in mitochondria of spinal cord neurons in the SCI group and rhFGF21 + hemin group. These changes included shrinkage of mitochondria and reduction or disappearance of mitochondrial cristae. Fer-1 and rhFGF21 treatments attenuated these pathological changes. The structure and morphology of mitochondria improved in the Fer-1 group and rhFGF21 group (Figure 4A). These results also indicate that FGF21 inhibited ferroptosis after SCI in rats, while HO-1 exacerbated it.

Figure 4.

Figure 4

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Mitochondrial pathological changes and ferroptotic process in rats after SCI.

(A) Transmission electron microscopy images. The morphology of mitochondria differs between groups. Arrows show mitochondria of spinal cord neurons. Boxes show high magnification views of low magnification images. Scale bars: 1 µm (upper panels) and 500 nm (lower panels). (B–D) Iron (B), GSH (C), and MDA (D) were measured by ELISA in spinal cord tissue. n = 6. ****P < 0.0001 (one-way analysis of variance followed by Dunnett's post hoc test). Fer-1: Ferrostatin-1; FGF21: fibroblast growth factor 21; GSH: glutathione; MDA: malondialdehyde; rhFGF21: recombinant human fibroblast growth factor 21; SCI: spinal cord injury.

FGF21 reduces HO-1 and increases GPX4 in the spinal cord anterior horn

We observed differences in HO-1 and GPX4 expression in the spinal cord anterior horn by immunofluorescence staining of spinal cord tissue sections from each group (Figure 5A). Similar to the WB and qRT-PCR results, HO-1 expression was decreased in the Fer-1 group and rhFGF21 group compared with the SCI group. Meanwhile, HO-1 expression was increased in the rhFGF21 + hemin group compared with the rhFGF21 group (Fer-1: P < 0.0001; rhFGF21: P < 0.0001; rhFGF21+hemin: P < 0.0001; Figure 5B and C). The change in GPX4 expression was opposite to that of HO-1, being increased in the Fer-1 group and rhFGF21 group and decreased in the rhFGF21 + hemin group (Fer-1: P < 0.0001; rhFGF21: P < 0.0001; rhFGF21 + hemin: P < 0.0001; Figure 5B and C). A cartoon schematic of the spinal cord anterior horn was showed in Figure 5D. These findings also indicate that FGF21 inhibited SCI-induced ferroptosis by suppressing HO-1 expression in the spinal cord anterior horn.

Figure 5.

Figure 5

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Immunofluorescence of HO-1 and GPX4 expression in the spinal cord anterior horn of rats after SCI.

(A) Fluorescence microscopy results of HO-1 and GPX4 with DAPI and NeuN. Green indicates HO-1 or GPX4 in neurons in the spinal cord anterior horn, red indicates NeuN-stained neurons, and blue indicates DAPI-stained nuclei. White boxes show higher magnification views of low magnification images. Scale bars: 50 µm (merge) and 25 µm (higher magnification). (B, C) Box and whisker plots from A. (D) A cartoon schematic of the spinal cord anterior horn (red box). n = 6. ****P < 0.0001 (one-way analysis of variance followed by Dunnett's post hoc test). DAPI: 4′,6-Diamidino-2-phenylindole; Fer-1: ferrostatin-1; FGF21: fibroblast growth factor 21; GPX4: glutathione peroxidase 4; HO-1: heme oxygenase-1; NeuN: neuron-specific nuclear protein; rhFGF21: recombinant human fibroblast growth factor 21; SCI: spinal cord injury.

Discussion

The findings of this study can be summarized as follows: (1) the levels of FGF21 in spinal cord tissue decreased after SCI with significant aggravation of ferroptosis; (2) the expression of HO-1 increased rapidly after SCI; (3) FGF21 inhibited, while HO-1 aggravated the process of ferroptosis after SCI; and (4) FGF21 inhibited ferroptosis by downregulating HO-1.

Several studies have shown that FGF21 is found in several brain regions, including the cortex, hippocampus, and striatum. Further, FGF21 is expressed in neurons to protect neurological function and alleviate glutamate excitotoxicity via Akt-1 activation and glycogen synthase kinase 3 inhibition (Makela et al., 2014; Leng et al., 2015). A study of a traumatic brain injury model reported that FGF21 expression was reduced by approximately 50% in brain tissue on day 28 after traumatic brain injury (Shahror et al., 2020). Consistently, we found a rapid decrease in FGF21 in spinal cord tissue in the early stages after SCI. Nevertheless, the specific mechanism by which FGF21 expression is decreased in spinal cord tissue after SCI remains to be elucidated.

Recent studies reported that SCI reduced the expression of serum FGF21 levels and hepatic FGF21 (Badman et al., 2007; Liu et al., 2021). The liver, the most important organ for FGF21 secretion, is at higher risk of diseases and metabolic disorders after SCI than the general population, which is associated with reduced physical activity and obesity (Gorgey et al., 2014; McMillan et al., 2021). It has been reported that SCI also decreased the levels and mRNA expression of two major adipokines, adiponectin and leptin, in serum and adipose tissue. Furthermore, SCI inhibited expression of the type 2 adiponectin receptor and initiation of peroxisome proliferator-activated receptor α (PPARα) in the liver. In addition, transcription of FGF21 was induced by the PPARα signaling pathway (Hui et al., 2016; Liu et al., 2021). The combined effect of these mechanisms may be responsible for the reduced expression of FGF21 in the liver, serum, and even spinal cord tissue after SCI.

Chen et al. (2018) found that rhFGF21 can cross the blood-brain barrier by simple diffusion, upregulating tight junction and adhesion junction proteins to preserve the blood-brain barrier. In our study, rhFGF21 was injected intraperitoneally into rats 30 minutes after modeling and reinjected 24 hours later. The WB results in Experiment 2 showed that rhFGF21 could cross the blood-spinal cord barrier and be detected in spinal cord tissue.

Both rhFGF21 and FGF21 overexpression significantly protect against iron overload-induced hepatocyte mitochondrial damage, liver injury, and fibrosis by inhibiting ferroptosis (Wu et al., 2021). In addition, a neuroprotective effect of FGF21 has been discovered in recent years. An in vivo study showed that toxin-induced demyelination in mice was improved by peripherally derived FGF21 (Kuroda et al., 2017). Kang et al. (2020) reported that FGF21 reduced neurodegeneration by attenuating neuroinflammation and oxidative stress. Furthermore, FGF21 increased the protective effect on neuronal mitochondria through regulation of the NF-κB and AMPKα/AKT signaling pathways. Functional recovery of neurons is supported by FGF21 via inhibition of SCI-induced autophagy, which suggests that systemic treatment of FGF21 could be useful as a beneficial treatment to repair SCI (Zhu et al., 2020). All the results of our study indicate that increasing FGF21 expression can inhibit SCI-induced ferroptosis by suppressing iron accumulation in spinal cord tissue, and thereby improve motor neuron survival and neurofunctional recovery. These findings could provide a new potential mechanistic explanation for the application of rhFGF21 in the treatment of SCI.

As a two-way regulatory factor of iron metabolism and ROS homeostasis (Abraham and Kappas, 2008), HO-1 was shown to play a critical role in ferroptosis (Chiang et al., 2018). It is well known that HO-1 metabolizes heme into biliverdin/bilirubin, ferrous iron (Fe2+), and carbon monoxide (CO). According to previous studies, HO-1 (through its metabolites, biliverdin/bilirubin and CO) provides cytoprotection against oxidative damage (Stockwell et al., 2017). However, iron is a possible toxicant that can lead to oxidative DNA damage and cause neurodegenerative diseases due to its pro-oxidant activity. Iron overload promotes the Fenton reaction and ROS production (Roemhild et al., 2021). Subsequently, a high level of ROS formation can result in oxidative damage to proteins, DNA, and lipids, eventually inducing ferroptosis (Chen et al., 2021a). Based on these contradictory results, it seems that HO-1 plays the role of a cytoprotective factor or ferroptosis promoter depending on the level of ROS production and subsequent oxidative damage, responding to signals of stimulation (Chiang et al., 2018). It has been shown that moderate activation of HO-1 has a cytoprotective effect, whereas excessive HO-1 activation exerts a cytotoxic effect because of an excessive increase in the labile iron pool (Hassannia et al., 2018).

An in vivo and in vitro study indicated that specific inhibition of HO-1 expression (by direct knockdown of HO-1 or using ZnPP, an inhibitor of HO-1) significantly blocked retinal pigment epithelium ferroptosis. This was regulated by the Nrf2/SLC7A11/HO-1 pathway (Tang et al., 2021). Chen et al. (2021c) identified a new role of Astragalus polysaccharide in preventing ferroptosis by inhibiting the Nrf2/HO-1 pathway in a murine model of experimental colitis and human Caco-2 cells. Han et al. (2022) found that luteolin induced ferroptosis in a clear cell renal cell carcinoma through excessive upregulation of HO-1 expression and activation of the labile iron pool. As reported, ferroptosis may enhance diabetic nephropathy and the damage of renal tubules via the HIF-1α/HO-1 pathway in a diabetic model (Feng et al., 2021).

Nevertheless, it has been found that HO-1 overexpression suppresses SCI-induced ferroptosis via the Nrf2/SLC7A11/HO-1 pathway. Ge et al. (2021) reported that zinc inhibited ferroptosis in neurons by reducing oxidative stress products and lipid peroxides via the Nrf2/HO-1 and GPX4 signaling pathways. Gong et al. (2022) indicated that trehalose provided neuroprotective effects by activating the Nrf2/HO-1 pathway, which inhibited ferroptosis and the associated inflammation.

In this study, HO-1 was significantly upregulated after SCI, while ferroptosis was enhanced after treatment with hemin, a HO-1 agonist. The continuous excessive increase in HO-1 and iron in spinal cord tissue after SCI might be responsible for the promotive effect of HO-1 on SCI-induced ferroptosis in the present experiments (Chang et al., 2018). Wu et al. (2021) indicated that the molecular mechanism by which FGF21 inhibits HO-1 expression is through promoting HO-1 ubiquitination, which thereby accelerates HO-1 degradation and reduces the conversion of heme to Fe2+, ultimately inhibiting ferroptosis. In our study, the WB, qRT-PCR, and immunofluorescence staining results demonstrated that FGF21 inhibited HO-1 expression after SCI. However, the molecular mechanism by which FGF21 induces the downregulation of HO-1 requires further investigation.

The limitations of our current study should be noted. Primarily, although mRNA and protein expression of FGF21 and HO-1 were estimated by qRT-PCR and WB, there was still a lack of intuitive quantification by immunohistochemistry. Secondly, even if the BBB locomotor score and incline plate test were observed by two blinded observers, a lack of video analysis of joint kinematics to assess functional recovery resulted in a rather subjective assessment. Due to these limitations, there is much future work to do.

In summary, the expression of FGF21 decreases in spinal cord tissue in the short term after SCI, while HO-1 levels excessively increase. FGF21 inhibits HO-1 and SCI-induced ferroptosis by rhFGF21 treatment. HO-1 promotes SCI-induced ferroptosis due to the continuous excessive increase in HO-1 and iron in spinal cord tissue after SCI.

Acknowledgments:

Imaging was performed with support of the Department of Translational Medicine Center of the Affiliated Zhangjiagang Hospital of Soochow University. We are also thankful for the technical support provided by Home for Researchers (www.home-for-researchers.com) in the mechanism diagram.

Funding Statement

Funding: This work was supported by grants from Jiangsu Commission of Health, No. Z2021086 (to XL); Science and Technology Program of Suzhou, Nos. SYSD2020008 (to XL), SKYD2022012 (to XL); Suzhou Municipal Health Commission, No. KJXW2020058 (to XL); and Science and Technology Program of Zhangjiagang, No. ZKS2018 (to XL).

Footnotes

Conflicts of interest: All authors declare no competing financial interests exist.

Data availability statement: No additional data are available.

C-Editors: Li JY, Zhao M; S-Editor: Li CH; L-Editors: Li CH, Song LP; T-Editor: Jia Y

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