Zinc defends against Parthanatos and promotes functional recovery after spinal cord injury through SIRT3‐mediated anti‐oxidative stress and mitophagy

Abstract Introduction Spinal cord injury (SCI) is a central nervous system injury that is primarily traumatic and manifests as motor, sensory, and autonomic dysfunction below the level of damage. Our previous studies confirmed the ability of zinc to protect mitochondria, protect neurons and promote spinal cord recovery. However, the role of zinc in Parthanatos is unknown. Aim We investigated the effects of zinc in Parthanatos from oxidative stress and mitophagy. We elucidated the role of SIRT3 in providing new ideas for treating spinal cord injury. The Results Zinc protected SCI mice by regulating Parthanatos. On the one hand, zinc eliminated ROS directly through SIRT3 deacetylation targeting SOD2 to alleviate Parthanatos. On the other hand, zinc eliminated ROS indirectly through SIRT3‐mediated promotion of mitophagy to alleviate Parthanatos. Conclusion Zinc defends against Parthanatos and promotes functional recovery after spinal cord injury through SIRT3‐mediated anti‐oxidative stress and mitophagy.

Parthanatos is a poly ADP-ribose polymerase-1 (PARP-1) -dependent programmed death. 6,7 That is important in neurological diseases such as neurodegenerative diseases, cerebrovascular diseases, spinal cord injuries, and gliomas. 8 Mechanistically, the DNA damage sensor PARP-1 is hyper activated and produces large amounts of poly ADP-ribose (PAR) released into the cytoplasm, inducing depolarization of mitochondria and promoting the release of apoptosis-inducing factor (AIF) from mitochondria into the cytoplasm. 9,10 AIF binds to macrophage migration inhibitory factor (MIF) in the cytoplasm and enters the nucleus, causing chromatin and DNA breaks, thereby accelerating cell death. 8,11 Also, extensive studies have shown that pathological changes after spinal cord injury eventually lead to accumulating reactive oxygen species (ROS), which can damage DNA and activate Parthanatos. 12 Mitochondria play an essential role in cellular activity as the leading site for maintaining cellular energy metabolism and mitochondrial homeostasis. 13 Increasing evidence suggests that the secondary injury phase after spinal cord injury is closely associated with mitochondrial damage and ROS accumulation. [14][15][16] Mitophagy, selective autophagic degradation of damaged mitochondria, 17 can reduce ROS accumulation. 18,19 SIRT3 is a member of the Sirtuins family of proteins localized in mitochondria and characterized as a deacetylase that deacetylates associated with acetylated proteins in mitochondria. 20 In addition, it has been shown that SIRT3 is involved in maintaining mitochondrial function and regulating ROS. 20,21 A promising strategy is to intervene with Parthanatos to treat spinal cord injury by protecting mitochondria, scavenging reactive oxygen species, or reducing the accumulation of reactive substances.
Zinc is essential for vital activity and binds with numerous proteins (enzymes and transcription factors). 22,23 Approximately 10% of human proteins are zinc proteins, and most human zinc-binding proteins regulate gene expression. 24 Previous literature suggests that Sirtuin catalyzes the characteristic sequence motifs corresponding to the core structural domain, including the zinc-binding site. [25][26][27] This feature determines that zinc affects almost all aspects of cell biology and is essential for maintaining cellular homeostasis. 28 Studies have shown that zinc is involved in oxidative stress, inflammatory response, and immune regulation. 23,29,30 Our previous studies confirmed the ability of zinc to protect mitochondria, protect neurons and promote spinal cord recovery. 31,32 However, the role of zinc in Parthanatos is unknown.
In this study, we investigated the effects of zinc in Parthanatos from oxidative stress and mitophagy. We elucidated the role of SIRT3 in providing new ideas for treating spinal cord injury. The animals were randomly assigned to the Sham, SCI, ZnG, and 3-TYP groups. Mice were extensively anesthetized by intrapulmonary injection with 1% sodium pentobarbital (50 mg/kg, P-010, Sigma-Aldrich). Mice were deeply anesthetized by intrapulmonary injection with 1% sodium pentobarbital (50 mg/kg, P-010, Sigma-Aldrich). Mice were profoundly anesthetized by intrapulmonary injection with 1% sodium pentobarbital (50 mg/kg, P-010, Sigma-Aldrich). Mice were deeply anesthetized by intrapulmonary injection with 1% sodium pentobarbital (50 mg/kg, P-010, Sigma-Aldrich).

| Animal and drug treatment
Mice were profoundly anesthetized by intrapulmonary injection with 1% sodium pentobarbital (50 mg/kg, P-010, Sigma-Aldrich). 33 Partial laminectomy was performed on the T9/T10 vertebral body to expose the T9/T10 segment. An altered impactor (diameter: 2 mm. Weight: 12.5 g. Height: 1.5 cm) was used to cause a spinal cord contusion. The ZnG group was given an intraperitoneal injection of 30 mg/kg ZnG 2 h postoperatively, 1 time/day until day 3. The 3-TYP group was assigned 50 mg/kg 3-TYP intraperitoneally 1 week before surgery, 1 day every other day, three times, 33 and postoperative treatment was the same as the ZnG group. The SCI group was given an intraperitoneal isotonic glucose injection 2 h postoperatively, 1 time/day until day 3.
Only the laminae were removed in the Sham group, and postoperative isotonic glucose injection was administered intraperitoneally.

| Behavioral assessment
The BMS score was used to assess motor function in the hind limbs of mice. 34 Professionally trained operators were unaware of the experimental conditions and repeated the evaluation of the mice on three occasions. These mice were evaluated 1 h before surgery, and 1, 3, 7, 14, 21 and 28 days after surgery.

| Ultrasonic imaging test
The urinary bladder volumes of mice in each group were measured at 28 days after surgery using an ultrasound diagnostic system (Ex. No. 9362, ESAOTE S.p.A Esaote Group) and a color Doppler ultrasound diagnostic instrument (Model No. DC-6EII, Shenzhen Myriad Biomedical Electronics Co., Ltd.), and the volume calculations were performed on the ultrasound images. 35 Specifically, mice were anesthetized using the intrapulmonary anesthesia injection with 1% sodium pentobarbital (50 mg/kg, P-010, Sigma-Aldrich). After fixing the mice in the prone position, the abdomen was first abdominally shaved, then cleaned with 70% ethanol and soap. A medical ultrasound coupling agent was applied to facilitate the propagation from the transducer to the skin. While maintaining slight pressure, the transducer was placed to visualize and digitally capture the maximum cross-sectional area of the bladder, sequentially capturing the length, depth, and width, with the hypertonic bladder appearing as a black oval structure with the hyperbolic surrounding tissue. Ultrasound imaging system software automatically calculates the bladder volume.

| Testing tissue preparation
At the designed time, mice were anesthetized and sequentially perfused with 0.9% NaCl and 4% paraformaldehyde for cardiac perfusion. A 0.5 cm spinal cord specimen was taken and fixed in 4% PFA for 48 h and then dehydrated sequentially using 4% paraformaldehyde solution containing 10%, 20%, and 30% sucrose. The area of the spinal cord 3 mm above and below the point of injury was cut with a frozen cut section to make frozen sections of 10 μm, stored at −80°C. The tissue was fixed for 48 h, dehydrated in gradient, soaked in xylene, and then cut into 7 μm paraffin sections after paraffin embedding and stored at room temperature.

| QRT-PCR
After the euthanasia of mice, 1.5 cm of spinal cord tissue was extracted with the TRIzol reagent (Ambion) for total RNA. CDNA Co., Ltd.) used in the study are listed above in Table 1 (n = 3 mice/ group).
Cell drug toxicity tests were tested with the CCK-8 kit (#CA1210, Solarbio). The cells were inoculated into 96-well plates at 5000 cell/ well density and incubated at 37°C in a CO 2 incubator for 24 h. The cells were processed with various concentrations of ZnG, H 2 O 2 , Mdivi-1, and 3-TYP and placed at other times, followed by incubation with 10 μ CCK8 per well for 4 h. Finally, absorbance (DO) was measured at 450 nm with an enzymatic marker. (80 μmol/L) for 3 h, followed by ZnG (100 μmol/L, # Z820656-100 g, Macklin) for 24 h. The Mdivi-1 group was treated with the mitophagy inhibitor Mdivi-1 (25 μmol/L, # ab144589, Abcam) for 3 h before the treatment with the addition of H 2 O 2 , and the rest of the treatment was the same as the ZnG group. 38 The 3-TYP group was treated with the SIRT3 inhibitor 3-TYP (50 μmol/L, # IT1960, Solarbio) before adding H 2 O 2 for 3 h, and the rest of the treatment was the same as that of the ZnG group. 36 The Vehicle group was cultured in DMEM, and other treatment factors were identical.

| Western blotting
We used the same method as before to perform Western blotting. 37 Before Western blotting, we used a total protein extraction kit (BC3710, Solarbio), cellular mitochondrial isolation kit (C3601, Beyotime) or tissue mitochondrial isolation kit (C3606, Beyotime), and nuclear protein extraction kit (R0050, Solarbio) to extract total protein, mitochondrial protein or de-mitochondrial cytoplasmic protein, and nuclear protein, respectively. The pri- visualized on a Tanon 2500R gel imaging system (Tanon) using a developer (Tanon), and the band intensity was quantified using ImageJ 1.39V software.

| Transmission electron microscopy
Transmission electron microscopy (TEM) was used to monitor mitochondrial injury in the spinal cord, as described previously. 39

| Mitochondrial membrane potential detection
Mitochondrial membrane potential was detected using the JC-1 kit

| Reactive oxygen detection
Cells were inoculated in 24-well plates and treated accordingly.

| 8-OH-dG detection
The 8-OH-dG content of each mouse spinal cord tissue group and VSC 4.1 cells was measured by ELISA assay kit (#YX-E20120, Wuhan Yipu Biotechnology Co., Ltd.), respectively. The absorbance (OD value) was measured at 450 nm wavelength using an enzyme marker, and the sample concentrations were calculated.

| Determination of manganese superoxide dismutase (Mn-SOD) enzyme activity
Mn-SOD activity assay kit (#S0103, Beyotime Institute of Biotechnology) was used to detect Mn-SOD enzyme activity in spinal cord tissue and VSC4.1 cells of each group of mice, respectively.

| Statistical analysis
Data were analyzed using SPSS Software, version 25.0. The Shapiro-Wilk test was used to assess the data distribution. Normally distribution date is expressed as mean ± standard deviation. In cases with more than two groups, we used a one-way analysis of variance

| Zinc can inhibit spinal cord neurons' Parthanatos after SCI
To clarify whether PARP-1-dependent Parthanatos occurred after SCI. First, we examined the protein expression of PARP-1 by western blotting and immunofluorescent staining, respectively.
Western blotting results showed a significantly higher PARP-1 expression in the SCI group than in the Sham group. At the same time, it decreased significantly in the ZnG group compared with the SCI group ( Figure 1A,B). Similar results were obtained by im- probe widely used to detect mitochondrial membrane potential in isolated mouse spinal cord tissue. Our results showed that the membrane potential was significantly reduced in the SCI group compared to Sham. At the same time, it was restored to the ZnG group, suggesting that mitochondrial depolarization occurred after SCI, while ZnG therapy significantly weakened mitochondrial depolarization ( Figure 1E). Nuclear translocation of AIF was another feature of Parthanatos, with reduced Mito-AIF expression and increased Cyto-AIF and Nucleo-AIF after SCI compared to the Sham group, and ZnG treatment suppressed these expressions ( Figure 1F-K). Overall, activation of PARP-1 after SCI decreased the membrane potential and accelerated nuclear translocation, features that are consistent with the manifestation of Parthanatos, confirming the occurrence of Parthanatos after SCI, which the administration of ZnG treatment significantly inhibited.

| Zinc can promote mitophagy in spinal cord neurons after SCI
Mitophagy is a specific type of autophagy that maintains mito- In summary, zinc can promote Parkin and PINK1 pathways for mitophagy.

| Inhibits mitophagy, increases the accumulation of ROS, and reverses the effect of zinc on Parthanatos
In vitro, to clarify whether zinc can regulate mitophagy, and sec- In summary, zinc can reduce Parthanatos by favoring mitophagy by reducing the production of ROS.

| Zinc mediates anti-oxidative stress via SIRT3
It has been shown that SIRT3 can target SOD2 to regulate ROS,

| Zinc can mediate mitophagy via SIRT3
It has been shown that SIRT3 is involved in regulating mitophagy. Also, we further explored the effect of SIRT3 on the promotion of mitophagy by ZnG. Western blotting results suggested that when 3-TYP treatment was given, the OD values of PINK1 and Mito-Parkin were significantly reduced compared with the ZnG treatment group, and the OD values of Cyto-Parkin group were significantly increased compared with the ZnG group ( Figure 6A-D), suggesting that 3-TYP reversed the ZnG treatment effect. In addition, we detected LC3B using immunocytofluorescence, and LC3B expression was significantly reduced in the 3-TYP group ( Figure 6E,F). In summary, zinc promotes mitophagy via SIRT3.

| Inhibition of SIRT3 reverses the effect of Zinc on Parthanatos
We further clarified the role of SIRT3 in regulating Parthanatos by zinc.
In vitro, we assayed parthanatos-related indicators. Western blotting suggested that PARP-1 expression was significantly increased in the 3-TYP group compared with the ZnG group, implying that 3-TYP could reverse the therapeutic effect of ZnG ( Figure 7A,B). Meanwhile, we also detected the change of AIF in vitro using Western blotting, and the results showed that the 3-TYP group could reverse the nuclear translocation of AIF in the ZnG group ( Figure 7A,C-E). In addition, we used a JC-1 fluorescent probe to detect the mitochondrial membrane The data represent means ± SD, *p < 0.05, **p < 0.01, ***p < 0.001. potential, and the 3-TYP group showed a significant increase compared to the ZnG JC-1 monomer, suggesting that 3-TYP reverses the therapeutic effect of ZnG ( Figure 7F,G). In summary, SIRT3 is involved in the regulation of Parthanatos by zinc.

| Inhibition of SIRT3 reverses the effect of zinc in promoting recovery of function after spinal cord injury
BMS scores were used to assess zinc's effect on motor function recovery after SCI. The results showed that mice in the ZnG group scored better than the SCI group at 28 and 35 days after SCI, and the 3-TYP group on postoperative days 28 and 35 were significantly lower than those of the ZnG group ( Figure 8A). We performed HE staining on the spinal cord of each group 28 days after SCI to observe the changes in the injury site. The results showed that the area of spinal cord injury in the ZnG group was significantly reduced compared with the SCI group and more prominent in the 3-TYP group than in the ZnG group ( Figure 8B,C). The degree of recovery of bladder function after SCI indirectly reflected the recovery of the spinal cord. Therefore, we assessed the bladder of mice 28 days after SCI using ultrasound imaging urinary retention.
The results showed that urinary retention was more advanced

| DISCUSS ION
Traumatic spinal cord injury is the most common type of spinal cord injury, resulting in immediate or delayed paraplegia in approximately 11%-40% of patients, which causes long-term morbidity and high medical costs for patients worldwide. 40 Zinc is essential for life activities, and studies have shown that zinc is involved in oxidative stress, inflammatory responses, and immune regulation. 23,29,30 Our previous studies confirmed that zinc protects mitochondria, protects neurons, and promotes spinal cord recovery. 31 Studies have shown that mitophagy can indirectly reduce ROS accumulation and prevent further neuronal damage. 43 Thus, we explored the hypothesis of whether enhanced mitophagy after zinc treatment is the underlying mechanism of zinc inhibi-

| CON CLUS ION
In summary, we demonstrate that zinc protects SCI mice by regulating Parthanatos. The protective mechanism is closely related to mitochondria, and the regulation of Parthanatos of zinc is multifaceted.
On the one hand, zinc eliminates ROS through SIRT3 deacetylation targeting SOD2 to alleviate Parthanatos; on the other hand, zinc indirectly eliminates ROS through SIRT3-mediated promotion of mitophagy to alleviate Parthanatos. Therefore, zinc therapy is a promising and effective treatment strategy for SCI.

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that they have no competing interests.

DATA AVA I L A B I L I T Y S TAT E M E N T
The raw data of experiments used to support the findings of this study are available from the corresponding author upon request.