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ORIGINAL RESEARCH published: 07 May 2021 doi: 10.3389/fmolb.2021.660072

Cox4i2 Triggers an Increase in Reactive Species, Leading to Ferroptosis and Apoptosis in HHV7 Infected Schwann Cells

Bowen Chang1†, Haochen Guan2†, Xueyi Wang1, Zheng Chen1, Wanchun Zhu1, Xiangyu Wei1 and Shiting Li1*

1 Department of Neurosurgery, School of Medicine, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai, China, 2 Department of Nephrology, School of Medicine, Shanghai General Hospital, Shanghai Jiao Tong University, Shanghai, China

Emerging evidence suggests that reactive oxygen species (ROS) play a significant role in the pathogenesis of peripheral nerve damage. Our previous study indicated that human herpesvirus 7 (HHV7) induces Bell’s palsy. However, the specific mechanism underlying Edited by: the effects of ROS in HHV7 infection-induced facial nerve damage is unknown. In Fernando Antunes, this study, we established a rat FN model by inoculating an HHV7 virus solution. University of Lisbon, Portugal The facial grading score and LuxolFastBlue (LFB) staining were used to assess the Reviewed by: Ana Cipak Gasparovic, success of the model. Using mRNA-sequencing analysis, we found that the expression Rudjer Boskovic Institute, Croatia of Complex IV Subunit 4 Isoform 2 (Cox4i2) increased in infected Schwann cells (SCs). Ana Sofia Fernandes, CBIOS, Universidade Lusófona Cox4i2 was suggested to increase COX activity, thereby promoting ROS production. Research Center for Biosciences The changes in the endogenous oxidant and antioxidant system were assessed, and and Health Technologies, Portugal the results showed that oxidative stress increased after HHV7 infection in vivo and *Correspondence: in vitro. However, we found that oxidative injury was relieved after the transfection Shiting Li [email protected] of shCox4i2 in HHV7-treated SCs by evaluating cell death, cell proliferation, and the †These authors have contributed ROS level as well as the levels of malondialdehyde (MDA), (SOD), equally to this work and glutathione (GSH). Furthermore, we hypothesised that Cox4i2 loss would attenuate HHV7-induced ferroptosis and apoptosis, which are closely related to ROS in SCs. Our Specialty section: This article was submitted to research illustrated that the knockdown of Cox4i2 suppresses HHV7-induced RSC96 Cellular Biochemistry, cell ferroptosis as well as apoptosis via the ERK signalling pathway. Overall, several a section of the journal Frontiers in Molecular Biosciences in vitro and in vivo methods were adopted in this study to reveal the new mechanism of Received: 28 January 2021 ROS-induced and Cox4i2-mediated apoptosis and ferroptosis in HHV7 infected SCs. Accepted: 14 April 2021 Keywords: COX4I2, HHV7, Schwann cells, ferroptosis, apoptosis Published: 07 May 2021 Citation: Chang B, Guan H, Wang X, INTRODUCTION Chen Z, Zhu W, Wei X and Li S (2021) Cox4i2 Triggers an Increase Bell’s palsy is characterised by acute facial nerve paralysis, which frequently leads to peripheral in Reactive Oxygen Species, Leading to Ferroptosis and Apoptosis in HHV7 facial palsy, with a morbidity of 11.5–40.2/100 000 persons every year (Eviston et al., 2015). Infected Schwann Cells. Previous studies have suggested that human herpesvirus 7 (HHV7) infection may contribute to Front. Mol. Biosci. 8:660072. peripheral nerve damage (Wakisaka et al., 2001; Kanerva et al., 2008). In our previous study, doi: 10.3389/fmolb.2021.660072 HHV-7 was found in the epineurium of the facial nerve in patients with Bell’s palsy using

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metagenomic next-generation sequencing (Chang et al., 2020). no significant difference in bilateral vibrissae movement; 1, the Typically, the reactivated hidden HHV7 infection within the vibrissae movement for the affected side was weaker than that facial nerve has been recognised as the cause of Bell’s palsy. The of the contralateral side; 2, the vibrissae movement disappeared). study of the pathogenesis of HHV7-induced peripheral nerve The position of the apex nasi was observed and scored on a 0– injury is particularly important for the treatment and prevention 1 scale (0, the position of the apex nasi is in the middle; 1, the of Bell’s facial paralysis. position of the apex nasi is tilted to the opposite side). Facial nerve Human herpesvirus 7 belongs to the Herpesviridae family, palsy was represented by a total score of 3 or 4. the Betaherpesvirinae superfamily, and the Roseolovirus genus. Similar to other human herpesviruses, HHV-7 is omnipresent, Cell Culture and HHV7 Infection which leads to a persistent hidden infection that may trigger The Rat Schwann cell line RSC96 was obtained from the additional reactivation or reinfection (Pohl-Koppe et al., American Type Culture Collection (ATCC). The cells were 2001; Agut et al., 2016). Several questions about HHV7 cultured in Dulbecco’s modified Eagle’s medium (DMEM) remain unanswered, especially the mechanism underlying its (Gibco, United States) supplemented with 10% foetal bovine disease causation. serum (Gibco, United States) and 1% penicillin/streptomycin Due to lack of research on HHV7 in peripheral nerve disease, (Sigma-Aldrich, United States) in an incubator at 37◦C in a the pathogenesis of HHV7 in peripheral nerve damage remains humidified 5% CO2 atmosphere. The cells were cultured in six- largely unknown. Peripheral facial paralysis usually results from well plates at a density of 5 × 105 per well. A normal culture damage to the myelin sheath of the facial nerve, which has various medium was used for the control group. For viral infections, causes. Schwann cells (SCs), the main components of the myelin HHV7 was added at a multiplicity of infection (MOI) of 10 until sheath, have been considered principal targets of viruses. In this obvious lesions appeared. All the cells were collected after 72 h study, we established for the first time an animal model of facial for subsequent experiments. paralysis caused by HHV7 and investigated the effects of HHV7 infection on rat SCs. HHV7 mediated increased production LuxolFastBlue Staining of reactive oxygen species (ROS) by upregulating Complex IV LuxolFastBlue (LFB) staining was used to observe the Subunit 4 Isoform 2 (Cox4i2), which resulted in SCs apoptosis demyelination of the nerve tracts, and it was performed and ferroptosis and led to myelin injury of the facial nerve. according to standard techniques (Fujiwara et al., 2017). Briefly, paraffin sections were immersed in a 0.1% LFB reagent for 8–16 h at 60◦C. After this, 0.05% lithium carbonate aqueous solution MATERIALS AND METHODS and 70% alcohol, 0.25% tar violet solution, and glacial acetic acid dyeing solution were used to redye all specimens for 10 min. Animals and Virus Inoculation After rinsing, the sections were observed as dehydrated and Twenty three-weeks-old female Wistar rats weighing 60–80 g transparent under a light microscope. were randomly divided into control and experimental groups after adaptive feeding for 7 days. The rats were treated by Immunofluorescent Staining of Facial scratching the unilateral auricular surface with needle 27 after Nerve Nuclei 3% halothane anaesthesia. The scratching side of auricular On the seventh day after the inoculation of HHV7 infection, surface was inoculated with 0.1 mL HHV7 virus solution [titer the brainstem of rats were collected to evaluate the facial of 1.0 × 106 plaque-forming units (PFU) per mL] in the nerve nucleus. Immunofluorescent staining of the brainstem experimental group, whereas PBS was used in the control group. was performed as described previously (Yokoyama et al., 2006). The blink reflex and vibrissae movement were evaluated after The brain sections were incubated with the primary antibodies 7 days of inoculation. The rats were sacrificed at the end of against anti-Neun (#ab177487, Abcam, United States) and the seventh day. All animal procedures in this study were Anti-CD11b (#66519-1-Ig, Proteintech, United States) at 4◦C reviewed and approved by the Institutional Animal Care and Use overnight followed by incubation with DyLight 488-and DyLight Committee of Xinhua Hospital Affiliated to Shanghai Jiao Tong 649-labelled secondary antibodies. NeuN, which is a marker University School of Medicine. for neurons, was labelled with DyLight 488 to label the facial The Evaluation of Facial Nerve Palsy nucleus. CD11b, a microglial marker that is also expressed by leukocytes, including monocytes, neutrophils, natural killer The model was evaluated by scores of the blink reflex, vibrissae cells, and granulocytes, was labelled with DyLight 649; 40,6- movement, and the position of the apex nasi after the virus diamidino-2-phenylindole (DAPI, Beyotime, China) was used for inoculation. Blink reflex was performed using a 5-mL syringe nuclear staining. equipped with an 18G needle to blow air into the eye at a distance of 2 cm. We observed the movement and closure of the rats’ RNA Extraction, Library Preparation, and eyelids and the degree was graded on a 0–2 scale (0, no significant difference between the two sides; 1, the blink reflex was delayed Quantitative Real-Time Polymerase compared to the unaffected side; 2, the eyelid of the affected Chain Reaction side cannot be closed). Counting the vibrissae movement on Total RNA of rats’ facial nerve tissues and RSC96 cells were both sides of the rats within 30 s and scored on a 0–2 scale (0, extracted per the manufacturer’s instructions (Life Science,

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United States). The libraries were prepared from cDNA, which of 1 × 105 cells per well. When the cell density reached was established using a cDNA Synthesis Kit (Takara, Japan) approximately 70–80%, the plasmids were transfected into cells and sequenced on an Illumina Hiseq4000 (Illumina Inc., using Lipofectamine 3,000 (Invitrogen, United States) according United States). Transcriptome sequencing and analysis were to the manufacturer’s instructions. After different treatments of performed by Majorbio Co., Ltd., (Shanghai, China). P-values the cells, the knockdown efficiency was detected by RT-qPCR and of <0.05 and a fold change of >2 or <0.5 denoted a statistically WB as mentioned above. significant difference in the expression of . RT-qPCR was performed on an ABI 7700 sequence detector (Applied Lipid Peroxidation Assays Biosystems, United States) using the TaKaRa RT-PCR kit (Takara, Lipid peroxidation was determined using a malondialdehyde Japan) according to the manufacturer’s instructions. The relative (MDA) detection kit (Nanjing Jiancheng, China) and used to mRNA levels of the target genes were calculated using the cycle detect the MDA levels of rats’ facial nerve and RSC96 cells threshold (Ct) values and the 2−11Ct method. GAPDH was used according to the manufacturer’s instructions. In brief, the samples as an internal control for the normalisation of genes. Sequences ◦ were homogenised in an MDA lysis buffer and incubated at 95 C of the primer pairs are listed in the supplementary materials. for 40 min. Each sample was cooled to room temperature and the Western Blotting absorbance at 532 nm was measured. Homogenates from extracted RSC96 cells and rats’ facial nerve tissues using RIPA solution (Thermo Fisher Scientific, Superoxide Dismutase Measurement United States) were prepared. The supernatant was Superoxide Dismutase (SOD) activity of rats’ facial nerve and collected after centrifugation at 12,000×g for 15 min. Equal RSC96 cells were assessed with the SOD assay kit (Nanjing amounts of were loaded for 6–12% sodium dodecyl Jiancheng, China) according to the method. ◦ sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and After 40 min of reaction time at 37 C, the fully mixed samples transferred to a polyvinylidene difluoride (PVDF) membrane and reagents were placed at room temperature for 10 min. The (GE Healthcare, United States). The membrane was inoculated absorbance at 550 nm was determined. The activity of SOD was with a primary antibody against Anti-SLC7A11 (ab175186, measured as units per milligramme of protein (U/mg protein). abcam, United States), Anti-SLC3A2 (#13180, Cell Signaling Technology, United States), Anti-GPX4 (ab125066, abcam, Activity of Lactate Dehydrogenase United States), anti-FTH1 (#4393, Cell Signaling Technology, To determine the LDH activity of RSC96 cells in each treatment United States), anti-TFRC (ab269513, Abcam, United States), group, an LDH assay kit (Nanjing Jiancheng, China) was used Anti-caspase3 (ab13847, Abcam, United States), Anti-IREB2 according to the manufacturer’s instructions. LDH is a stable (ab181153, abcam, United States), ERK (#9102, Cell Signaling protein that exists in the cytoplasm of normal cells and is released Technology, United States), p-ERK (#4370, Cell Signaling into the extracellular space after damage to the cell membrane. Technology, United States), and anti-GAPDH (#5174, Cell After 15 min of reaction with the matrix buffer and 2,4- ◦ Signaling Technology, United States) at 4 C overnight. dinitrophenylhydrazine at 37◦C, the sample was assessed with Subsequently, the membrane was washed with TBST and a spectrophotometer. The absorbance at 450 nm was measured. incubated with goat anti-rabbit secondary antibody (#7074, LDH activity was expressed as units per litre (U/L). Cell Signaling Technology, United States) and goat anti- mouse secondary antibody (ab6789, Abcam, United States) Intracellular Reduced Glutathione Level at room temperature for 2 h. Finally, the protein expression The intracellular concentrations of GSH in RSC96 cells were was visualised using enhanced chemiluminescence (Millipore, determined using a commercial GSH kit (Nanjing Jiancheng, United States) and a chemiluminescence detection system (GE China). After mixing the samples with the homogenate reagent Healthcare, United States). for approximately 5 min at room temperature, the yellow colour Transfection of shRNA of the resulting reaction could be observed. The absorbance at 405 nm was measured using a spectrophotometer. The The target was knocked down using short hairpin RNA intracellular concentration of GSH was expressed as µmol per (shRNA). Based on the design principle of shRNA, the sequence gram of protein (µmol/g protein). of the Cox4i2 gene was obtained from GenBank (NM_053472.1). Three RNA interference sites targeting the Cox4i2 gene were designed and synthesised by Genepharma (Genepharma, China) Measurement of Reactive Oxygen with a non-specific sequence as a negative control. The Species sequences of the four shRNAs were as follows: shRNA-negative The intracellular concentrations of ROS were determined using control: 50-CAGUACUUUUGUGUAGUACAA-30; shRNA-Cox 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime, 4i2-1: 50-CCCGGAGUCUGGUAAUGAATT-30; shRNA-Cox4 China) according to the manufacturer’s instructions. Briefly, the i2-2: 50-GGGUCUAUGUGUUCCCUAATT-30; shRNA-Cox4i2- cells were digested by typsin, and then loaded with a 10 µM 3: 50-CCCACUGGGAUUACGAGAATT-30. The shRNA was probe and incubated at 37◦C for 30 min. The ROS levels were inserted into a pSIREN-RetroQ-TetH vector (TaKaRa, Japan). detected by flow cytometry after washing the cells three times Next, the cells were seeded in six-well plates at a concentration with a serum-free cell culture medium.

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EdU Flow Cytometry Assay unto it, and the nose shifted to the right. We judged the facial The dissociated cells were exposed to a 50-µM 5-ethynyl- nerve palsy by evaluating the facial grading, which significantly 20-deoxyuridine (EdU, RiboBio, China) solution for 2 h at increased in HHV7 infected rats (P < 0.01; Figure 1B). We 37◦C. They were washed with 1% BSA dissolved in PBS. After also detected the demyelination of the facial nerve on LFB centrifugation, the cells were suspended in 4% paraformaldehyde staining, and the facially paralysed rats showed demyelination and incubated at room temperature for 15 min without light. with degenerate fibres scattered across the intact nerve fibres as After permeabilisation with 0.5% Triton-X for 20 min, a seen in Figure 1C. Next, immunohistochemistry was performed 1 × Apollo reaction cocktail was added to each sample and to assess the facial nerve nuclei. Figure 1D shows that CD11b- incubated at room temperature for 30 min. Subsequently, flow positive microglia and Neun-positive facial nerve nuclei were cytometry was used for the detection after suspending the observed on the intact side. On the facially paralysed side, the cells with 1% BSA. Neun-positive cells decreased and the CD11b-positive cells were integrated. We concluded that the decrease in the facial nerve Phen Green SK Staining nuclei and inflammatory cell infiltration may also be important causes of HHV7-induced facial paralysis. To investigate the Cellular free was demonstrated using the phen green SK changes in the endogenous oxidant and antioxidant system (PGSK, Cayman Chemical, United States). After washing with after the HHV7 inoculation, the levels of MDA and SOD were Hanks’ buffered salt solution (HBSS, pH 7.3), the cells were ◦ evaluated (Figure 1E). The concentration of MDA is an index loaded with 10µM PGSK at 37 C for 10 min. The nuclei of lipid peroxidation, which increased significantly in the HHV7 were stained with Hoechst 33342 for 10 min. Laser scanning inoculated rats compared with the healthy rats (P < 0.01). The microscopy, at excitation of 507 nm and emission of 532 nm, was activities of SOD, an antioxidant product, exhibited opposite used to evaluate the fluorescence of the images. The intensity of changes that decreased after infection with HHV7 (P < 0.01). fluorescence was quantified by using GraphPad Prism 5.0. Taken together, these data proved that HHV7 could induce facial Terminal Deoxynucleotidyl palsy and oxidative facial nerve injury in rats. dUTP Nick-End Labelling Staining RNA-Sequencing Analysis of Normal The extent of apoptosis was measured by Transferase dUTP RSC96 Cells and HHV7 Infected RSC96 Nick-End Labelling (TUNEL) staining (Roche, United States) Cells according to the manufacturer’s instructions. Briefly, RSC96 cells To explore the key genes responding to HHV7 infection in and rats’ facial nerve were fixed with 4% paraformaldehyde for RSC96 cells, transcriptome sequencing was performed in the 15–24 min and rinsed with PBS twice. The specimens were present study. There were 1,403 differentially expressed genes, successively incubated with the TUNEL reaction mixture for including 962 up-regulated genes and 441 down-regulated genes 60 min at 37◦C without light, and DAPI (4, 6-Diamidino-2- (Figure 2A). The significantly enriched (GO) Phenylindole, dihydrochloride) was used to stain the nucleic acid. analysis and the Kyoto Encyclopaedia of Genes and Genomes The analysis of apoptosis (ratio of TUNEL-positive cells to all (KEGG) pathway are illustrated in Figures 2B,C, respectively. cells) was conducted using images of randomly selected fields The GO analysis revealed that the differentially expressed genes obtained with a fluorescence microscope. in the normal cells and HHV7-infected cells were involved in the biological response to oxygen levels, response to , and Statistics response to decreased oxygen levels. The KEGG pathway analysis The experiments were performed in triplicate and independently indicated that the differentially expressed genes were mainly repeated at least three times. All data are expressed as the enriched by the HIF-1 signalling pathway and the metabolism ± mean standard deviation from at least three independent of xenobiotics by the cytochrome P450 and FoxO signalling experiments. The results were statistically analysed using one- pathways. Furthermore, a heatmap of the differentially expressed way analysis of variance (ANOVA) among multiple groups or gene analysis demonstrated that some genes were related to Student’s t-test between two groups. P < 0.05 was considered to oxidative stress and the cell respiratory chain (Figure 2D). To indicate statistical significance. The graph visualisations and data verify the differentially expressed genes in the RNA-sequencing analyses were performed using GraphPad Prism 5.0. data, we performed western blotting and RT-qPCR to detect the protein and mRNA expression of three genes, including Cox4i2, Map2k6, and Csf1r. The results showed that the protein and RESULTS mRNA expressions of Cox4i2, Map2k6, and Csf1r signifificantly increased after HHV7 infection compared with that in the control Human Herpesvirus 7 Infection Induces group (P < 0.01) (Figures 2E,F). Facial Palsy and Increased Oxidative Stress in Rats Knockdown of Cox4i2 Alleviated First, we established an animal model to determine whether Oxidative Stress and Cell Cytotoxicity in HHV7 infection caused facial nerve injury. Figure 1A shows the HHV7-Infected RSC96 Cells paralysed face of a rat 7 days after HHV7 inoculation. The left eye, Our research has shown that HHV7 infection may lead on the HHV7 infected side, could not close when air was blown to oxidative injury in vivo, and we estimated the specific

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FIGURE 1 | Human herpesvirus 7 (HHV7) infection induces facial paralysis and increased oxidative stress in rats. (A) Representative photo of rats. On day 7 after the HHV7 inoculation of the auricle. The infected side was paralyzed and the nose shifted to the infected side. (B) Analysis of facial grading. (C) Images of LFB staining for normal control rats and HHV7-inoculated rats. (D) Images of facial nerve sections from normal control rats and HHV7-inoculated rats stained with an anti-CD11b and Neun antibody. (E) MDA and SOD were detected to determine the level of oxidative stress. Data are presented as the mean ± standard deviation, n = 10. Statistical significance was assessed with the Student’s t-test. **P < 0.01.

mechanism underlying HHV7-induced changes in cell death, cell after HHV7 infection compared with that in the control group proliferation, and ROS levels in RSC96 cells. The Cox4i2 levels (P < 0.01). In contrast, the transfection of shCox4i2 significantly were altered in the RSC96 cells by cell transfection and verified decreased the Cox4i2 expression (P < 0.01). The increased by western blotting and RT-qPCR (Figures 3A,B). The results levels of MDA and the decreased levels of SOD and GSH showed that the expression of Cox4i2 significantly increased were observed after the HHV7 infection, reflecting the lipid

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FIGURE 2 | RNA-sequencing analysis of normal RSC96 cells and HHV7-infected RSC96 cells. (A) Volcano plot of differentially expressed genes revealed by high-throughput sequencing. Red points represent upregulated genes, grey points represent the insignificantly changed genes, and the green points represent. (B,C) GO enrichment analysis and KEGG enrichment analysis of normal RSC96 cells and HHV7-infected RSC96 cells. (D) Heatmap of differentially expressed genes in normal RSC96 cells and HHV7-infected RSC96 cells. (E,F) The verification of three differentially expressed genes by Western Blotting and RT-qPCR. Data are presented as the mean ± standard deviation, n = 3. Statistical significance was assessed with the Student’s t-test. **P < 0.01; *P < 0.05.

peroxidation and the weakening of the antioxidant capacity. concentrations (P < 0.01; Figure 3G). HHV7 infection increased However, the oxidative injury was relieved by transfection of the LDH release, whereas the knockdown of Cox4i2 suppressed shCox4i2 in HHV7-treated RSC96 cells (P < 0.01 and P < 0.05; LDH release from RSC96 cells (P < 0.01; Figure 3F), suggesting Figures 3C–E). The same trend was observed for the ROS that Cox4i2 may be a target for inhibiting RSC96 cell death. As

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shown in Figure 3H, the cell proliferation significantly decreased regulatory mechanism of Cox4i2 in HHV7-induced RSC96 cell after HHV7 infection (P < 0.01), whereas a reversed change was apoptosis, we further evaluated the role of ERK activation in induced by transfection with shCox4i2 (P < 0.01). These results HHV7-induced cell death. A western blot analysis showed that indicate that shCox4i2 can protect RSC96 cells from oxidative HHV7 treatment increased ERK phosphorylation, which was injury induced by HHV7 infection. decreased in the presence of shCox4i2 (P < 0.01; Figure 5F). Similar results were also found for the protein and mRNA Complex IV Subunit 4 Isoform 2 expressions of caspase3 (P < 0.01; Figures 5E,F). These data Depletion Attenuated HHV7-Induced showed that the depletion of Cox4i2 relieved apoptosis in HHV7- Ferroptosis treated RSC96 cells through the ERK signalling pathway. Ferroptosis, a new form of programmed cell death, is driven by an iron-dependent increase in ROS. Thus, we explored several differentially expressed genes related to the ferroptosis signalling DISCUSSION pathway, such as iron-related genes heavy chain (FTH1, The facial nerve plays a vital role in expression and reflected the ability of intracellular iron storage), transferrin communication, and its impaired function may greatly impact receptor 1 (TFRC, a necessary receptor for cellular iron uptake), the quality of life (Lindsay et al., 2014). Bell’s palsy accounts for iron-responsive element-binding protein 2 (IREB2), as well as the frequently observed cranial neuropathy that induces acute the genes of the solute carrier family 7 member 11 (SLC7A11), unilateral lower motor neuron facial paralysis. Generally, viral solute carrier family 3 member 2 (SLC3A2), and glutathione infections have been identified as contributors to the occurrence peroxidase 4 (GPX4) related to redox homeostasis. Our results of Bell’s palsy; however, the underlying mechanism is still showed that the TFRC protein and mRNA of rats’ facial nerve unknown (Eviston et al., 2015; Zhang et al., 2020). According to tissue significantly increased, whereas the others significantly our prior work, HHV7 induces Bell’s palsy (Chang et al., 2020). decreased in HHV-inoculated rats (P < 0.01 and P < 0.05; Therefore, the present work aimed to examine the mechanism by Figures 4A,B). Further experiments were conducted in vitro. which HHV7 causes facial nerve injury. SCs are the major glial We observed similar patterns in HHV7-infected RSC96 cells: the cell types within the peripheral nervous system (PNS), and they knockdown of Cox4i2 significantly suppressed and play vital roles in regulating nerve regeneration and myelination relieved redox imbalance (P < 0.01and P < 0.05; Figures 4C,D). (Hall, 1978). Therefore, it is particularly important to study the Intracellular free iron levels were measured using PGSK, which influence of HHV7 on SCs. is quenched after binding to iron; therefore, the fluorescence In this study, microarray analysis revealed that the HHV7 levels are negatively correlated with the free iron levels. Our infection group had increased Cox4i2 expression compared with data demonstrated that HHV7 infection induced a lower green the control group. Specifically, Cox4i2 is the nuclear-encoded fluorescence than the control group, which was consistent regulatory subunit in the mitochondrial with the higher free iron levels. Conversely, the depletion of terminal complex, and it plays a vital role in inflammatory Cox4i2 restored the fluorescence levels and reduced the free iron response and oxidative stress (OS) (Misiak et al., 2010; Hwang levels (P < 0.05; Figure 4E). Taken together, our data suggest et al., 2015). Recently, hypoxia has been suggested to increase that Cox4i2 promotes HHV7-induced facial nerve injury by the levels of Cox4i2 in the and liver, and this changes the modulating ferroptosis. activity of (CcO), the secretion of ROS, Knockdown of Cox4i2 Suppresses HHV7 and the ATP content (Hüttemann et al., 2012; Aras et al., 2013; Horsch et al., 2015). In the present study, our results showed Induced RSC96 Cells Apoptosis via ERK that the increased expression of Cox4i2 in SCs infected with Signalling Pathway HHV7 promoted the production of ROS. On the contrary, the Since ROS function as signal transduction intermediates to knockdown of Cox4i2 expression in the SCs infected with HHV7, activate transcriptors, which may induce cell apoptosis, we the levels of ROS decreased relatively. Consistent with the effect investigated the extent of apoptosis induced by HHV7 infection of Cox4i2 on OS, the upregulation of Cox4i2 in SCs promoted in vivo and in vitro. First, a significantly higher number of the production of ROS in cells (Roemgens et al., 2011; Reguera TUNEL-positive cells was observed in the HHV7-inoculated et al., 2020). In addition, the aggravation of OS in facial nerves rats than in the control rats (P < 0.01; Figure 5A). The infected with HHV7 was found in animal models. Some previous caspase3 protein expression of rats’ facial nerve was higher in studies have shown that the down-regulation of Nf1 gene and the rats inoculated with HHV7; similar results were found for the up-regulation of nuclear factor-E2-related factor 2 (Nrf2) can its mRNA expression (P < 0.01; Figures 5B,C). To determine lead to the increase of ROS in SCs (Zhang et al., 2017; Xu et al., whether Cox4i2 is involved in HHV7-induced apoptosis, the 2020). This seems to be the reason why the ROS level of SCs did TUNEL staining indicated that the HHV7 infection significantly not revise to normal after Cox4i2 knockdown, which still needs promoted cell apoptosis, and the knockdown of Cox4i2 further study to verify. suppressed apoptosis in HHV7-treated RSC96 cells (P < 0.01; Reactive oxygen species are produced through normal Figure 5D). It has been reported that the ERK pathway is physiological processes, which play an important role in cell involved in the activation of caspase signalling, which initiates signal transduction and tissue homeostasis (Ferreira et al., apoptotic processes after oxidant injury. To determine the 2018). However, excessive free radicals can cause undesirable

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FIGURE 3 | Knockdown of Cox4i2 alleviated oxidative stress and cell cytotoxicity in HHV7-infected RSC96 cells. (A,B) Protein expressions and relative mRNA expressions of Cox4i2 detected by Western Blotting and RT-qPCR in treated RSC96 cells. (C–E) The extent of oxidative stress evaluated by SOD, MDA, and GSH. (F) LDH assay release reflecting cell death. (G) The ROS concentrations were determined by flow cytometry. (H) EdU Flow Cytometry Assay was used to detect cell proliferation. Data are presented as the mean ± standard deviation, n = 3. Statistical significance was assessed with the one-way ANOVA. **P < 0.01; *P < 0.05.

modifications of cellular components and facilitate various polyunsaturated fatty acids (PUFAs). In recent years, lipid pathogeneses underlying damages to the lipids, proteins, and peroxidation has been suggested to induce ferroptosis, a DNA, among others (Kerkweg et al., 2007). Generally, (organelle) novel programmed cell death (Que et al., 2018). Ferroptosis membranes are vulnerable to ROS damage, through what plays a vital role in cell proliferation, differentiation, and is called lipid peroxidation, because of the high content of senescence (Stockwell et al., 2017). It may occur as a result

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FIGURE 4 | Complex IV subunit 4 isoform 2 (Cox4i2) depletion attenuated HHV7-induced ferroptosis. (A,B) Protein and relative mRNA levels of FTH, TFRC, SLC7A11, SLC3A2, and GPX4 were determined by Western Blotting and RT-qPCR in vivo. (C,D) Protein and relative mRNA levels of FTH, TFRC, IREB2, SLC7A11, SLC3A2, and GPX4 were determined by Western Blotting and RT-qPCR in HHV7-treated RSC96 cells with or without shCoc4i2. (E) Image of PGSK staining of RSC96 cells. Data are presented as the mean ± standard deviation, n = 3. Statistical significance was assessed with Student’s t-test and one-way ANOVA. **P < 0.01; *P < 0.05.

of the elevated ROS production induced by the increased iron critically participate in neurological disorders, such as stroke, content within the cells and the depleted glutathione (GSH), neurodegeneration, or neurotrauma (Ren et al., 2020). Generally causing lipid peroxidation and ultimately cell death (Masaldan speaking, neurodegenerative disorders involve the complicated et al., 2018). Ferroptosis has been previously identified to and versatile mechanisms of cell death and various pathways, and

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FIGURE 5 | Knockdown of Cox4i2 suppresses HHV7-induced RSC96 cell apoptosis via ERK signalling pathway. (A) The apoptotic cells appear green, and the DAPI staining for the nuclei is blue. Representative TUNEL staining image of a mouse section and the percentage of TUNEL positive cells. (B,C) Protein and relative mRNA expressions of caspase3 were analysed by Western Blotting and RT-qPCR in vivo. (D) Representative TUNEL staining image of HHV7-infected RSC96 cells with or without shCox4i2 and the percentage of TUNEL positive cells. (E) Relative mRNA expression of caspase3 was analysed by RT-qPCR in RSC96 cells. (F) Protein expressions of pERK, ERK, and caspase3 were analysed by Western Blotting in RSC96 cells. Data are presented as the mean ± standard deviation, n = 3. Statistical significance was assessed with Student’s t-test and one-way ANOVA. **P < 0.01.

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FIGURE 6 | The proposed model of Cox4i2 mediates an increase in ROS, ferroptosis, and apoptosis in HHV7-induced RSC96 cells. HHV7 infection increases the concentrations of the ROS, increasing oxidative stress via the activation of Cox4i2. On the one hand, Cox4i2 facilitates the intracellular iron overload and redox imbalance and induces ferroptosis. On the other hand, the upregulation of oxidative stress induced by Cox4i2 activates caspase3 by regulating the ERK signalling pathway and promotes apoptosis. The arrows indicate a positive action.

they are related to lipid peroxidation and excessive accumulation (Zhong et al., 2017). Furthermore, Jun N-terminal kinase (JNK), of iron within diverse cerebral regions (Weiland et al., 2019). p38, and extracellular signal-regulated kinase (ERK) can induce Nutritional and metabolic coupling between neurons and glial the activation of mitogen-activated protein kinases (MAPKs) cells (oligodendrocytes, microglia, and astroglial cells) are vital in different contexts, and this has an impact on apoptosis or characteristics of nervous system dysfunction, and it may cytoprotection signalling (Lee et al., 2016). Lipid peroxidation result in neuronal death, in particular ferroptosis. An identical products are reported to produce adducts with JNK, p38, and phenomenon in the PNS was observed in this study. SCs are ERK to activate MAPKs, which, in turn, activates the caspase the main glial cells, which constitute the myelin sheaths, in signal to initiate apoptosis (Forman et al., 2003; Mcelhanon et al., the PNS; they provide neurons with nutrients and support (Xia 2013). In this study, significantly higher numbers of apoptotic et al., 2020). Therefore, the increased concentrations of ROS in cells were observed in the HHV7-inoculated rats than in the SCs may induce ferroptosis, leading to myelin damage. In this control rats. The protein expression of caspase3 was upregulated study, our results showed that the increased expression of Cox4i2 in the rats with HHV7 inoculation, and similar results were led to an increase in ROS production in HHV7 infected SCs, obtained for its mRNA expression. The knockdown of Cox4i2 which induced ferroptosis. Conversely, ferroptosis was inhibited suppressed the apoptosis of HHV7-induced SCs. This study when Cox4i2 was knocked down in HHV7-infected SCs. HHV7 showed that the depletion of Cox4i2 relieved the apoptosis infection in the facial nerve tissue has also been shown to cause of HHV7-treated SCs through the ERK signalling pathway. ferroptosis in animal models. Interestingly, in our study, it was found that the apoptosis On the other hand, lipid peroxidation plays a vital role level of SCs did not completely revise to normal after Cox4i2 in apoptosis. Lipid peroxidation products can interact with knockdown. We hypothesize that other targets may be involved transcription factors and membrane receptors to induce in the apoptotic process of SCs. Some previous studies have apoptosis. In addition, it activates the extrinsic and intrinsic reported that down-regulation of Calreticulin (CRT) and CDK6 apoptosis-related signal transduction pathways (Pistritto et al., lead to apoptosis of SCs, respectively (Huang et al., 2016; Ma et al., 2016; Zhong et al., 2017). ROS can potentially result in the 2018). The effect of HHV7 on these targets needs further study. peroxidation of cardiolipin, which is the inner mitochondrial In summary, several in vitro and in vivo methods were adopted membrane phospholipid; in addition, the subsequently formed in this study to reveal the new mechanism of ROS-induced and lipid peroxidation products can activate intrinsic apoptosis Cox4i2-mediated apoptosis and ferroptosis in HHV7-infected

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SCs (Figure 6). The results of this study show the mechanism help in the experiment. SL took overall control of the whole underlying the effect of Cox4i2 on nerve injury caused by study. All authors contributed to the article and approved the HHV7 infection, and support the application of cell therapy, submitted version. gene therapy, or additional fundamental biological approaches in treating Bell’s palsy in the future. FUNDING

DATA AVAILABILITY STATEMENT This work was supported by the National Natural Science Foundation of China (81974186). The data presented in the study are deposited in the (https:// www.ebi.ac.uk/fg/annotare/login/) repository, accession number (fgsubs#498064). ACKNOWLEDGMENTS

We would like to express my gratitude to my lover Haochen Guan ETHICS STATEMENT for her help in the whole research and her care for me in life. We hope you can accept my proposal when the article is published. The animal study was reviewed and approved by Committee Love you forever. Meanwhile, we would like to thank Editage for of Xinhua Hospital Affiliated to Shanghai Jiao Tong University English language editing. School of Medicine. SUPPLEMENTARY MATERIAL AUTHOR CONTRIBUTIONS The Supplementary Material for this article can be found BC and HG jointly completed the experiment and the writing, online at: https://www.frontiersin.org/articles/10.3389/fmolb. XW and ZC assisted in the writing. XW and WZ provided 2021.660072/full#supplementary-material

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