Vitamin E Alleviates Oxidative Damage and Attenuates Ferroptosis Caused by Prolonged Contraction of the Ciliary Muscle by Activating ACOT7.

The reagents and materials used are provided in Supplementary Table S1 . We obtained 4-week-old male guinea pigs from Chongqing Medical University's Experimental Animal Center. The animals were housed under controlled conditions in Chongqing Medical University's Experimental Animal Center and maintained in an environment with a stable temperature of 25 ± 2°C and a 12-hour alternating light–dark schedule with free access to food and water. All animal experimental protocols were approved by the Animal Ethics Committee of Chongqing Medical University and conducted in accordance with the principles of the Declaration of Helsinki (Approval NO. IACUC-CQMU-2023-0323). This study adhered to the statement of ARVO regarding the use of animals in ophthalmic and visual research. Four-week-old male wild-type tricolor guinea pigs were randomly divided into four groups ( n = 9 per group): control (saline), 2% carbachol (Car), Car + VitE (2% Car + VitE 2 µM), and VitE (2 µM). To induce ciliary muscle contraction, animals received 20 µL of 2% Car (dissolved in saline, C838452, MACKLIN) via eye drops five times daily from 09:00 to 17:00, once every 2 hours for 2 weeks (w). The control group was treated with the equal volume of saline. Ten minutes after Car eye drops, 2 µM VitE (dissolved in saline, HY-N0683, MCE) was applied again in the same way. The VitE group received VitE eye drops alone at the same dose and frequency. After the end of the experiment, the animals were anesthetized with 3% sodium pentobarbital, asphyxiated with a high concentration of CO 2 , and their eyeballs carefully removed for further experimentation. The carcasses of the guinea pigs were subsequently stored in a designated refrigerator at the Animal Center for unified harmless treatment. The enucleated eyeballs were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5-µm-thick slices for histological examination. The refraction and pupil diameter (PD) of the animals were measured by Photorefractor (Striatech, Tübingen, Germany). Axial length (AL) was assessed by AL Scan (NIDEK, Gamagori, Aichi, Japan). These parameters were recorded at baseline, 1 week and 2 weeks after administration. Measurements were first obtained before administration, followed by the application of two consecutive 20-µL drops of 2% Car solution, with a 5-minute interval between doses. Subsequent measurements were then repeated immediately. The difference between the two measurements was used to represent the amplitude of accommodation (AMP). The eyeballs were immediately removed and dissected. The ciliary body annulus, a 4-mm diameter ring located 2 to 3 mm posterior to the corneal limbus, serves as the source of CMCs. Blunt separation of vitreous body, exposure of lens and ciliary body tissue, careful disconnection of suspensory ligament, removal of lens, circular cutting of iris, and ciliary epithelium allowed for the identification of the grayish–white ciliary muscle tissue under high magnification microscopy, then blunt separation with a toothless forceps induced ciliary body detachment. Subsequently, the isolated tissues were minced into 1-mm² fragments and transferred into T25 cell culture flasks. CMCs was cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 20% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Gibco), maintained at 37°C in a humidified atmosphere of 5% CO 2 . Primary cells were cultured for 10 to 14 days, and passaged upon reaching 80% confluence. The CMCs were segregated into several distinct groups: control, Car (500 µg/mL Car), Car + Fer-1 (500 µg/mL Car + Fer-1 2 µM, dissolved in DMSO), Car + VitE (500 µg/mL Car + VitE 2 µM, dissolved in DMSO), VitE and Car + VitE + si-NC, and Car + VitE + si-ACOT7. After 6 hours of pretreatment with Fer-1 or VitE, cells were exposed to Car (500 µg/mL, dissolved in PBS) for 24 hours. For transfection, a mixture of small interfering RNA (siRNA) and Lipofectamine 2000 Transfection Reagent (11668019, Thermo Fisher Scientific, Waltham, MA, USA) was added to CMCs at growth density of 70%. Replace the normal culture medium at 4 hours. After a transfection period of 48 hours, the expression levels of the target gene and corresponding protein were assessed. The siRNAs were purchased from Gene Create (Beijing, China), and used at a final concentration of 50 nM. The siRNAs target sequences were provided in Supplementary Table S1 . ACOT7 overexpression was achieved by infecting CMCs with adenovirus constructs (multiplicity of infection = 30) purchased from the Gene Create. After 72 hours of co-culture, the expression levels of target gene and protein were detected. Cell viability was assessed using the Cell Counting Kit-8 (C0037, Beyotime, Shanghai, China). CMCs (1 × 10 4 cells per well) were subjected to different concentrations of Car and VitE. We added Car at concentrations of 0, 100, 250, 500, 750, 1000, and 1500 µg/mL to 1 well in 96 culture plates and incubated for 6 or 24 hours. We pretreated 0, 1, 2, 5, 10, 20, and 50 µM of VitE in another 96 well plate for 6 hours, followed by incubation with 500 µg/mL Car for 24 hours. Data were repeated six times for each concentration. We added 10 µL of CCK-8 reagent and incubated the mixture for 1 hour. The absorbance (OD) was then measured at 450 nm using a microplate reader (Thermo Fisher Scientific). CMCs (4 × 10 5 cells per well) were cultured in 5% low-serum medium and a scratch was made vertically on the plate from top to bottom. After that, CMCs were treated by adding the drugs as described previously and incubated in a 37°C, 5% CO 2 incubator. Photographs were taken by a light microscope (Leica, Wetzlar, Germany) to record the state of cell fusion at 0, 6, and 24 hours. Image J software was used to analyze the scratch area: Wound width = (6 hour or 24 hour width – 0 hour width)/0 hour width. The migratory ability of CMCs (1 × 10 4 cells per well) was assessed using Transwell chambers (pore size, 8 µm wells; Millipore, Burlington, MA, USA). CMCs were grown on the upper chamber surface with 5% FBS and the lower chamber with 20% fetal bovine serum to induce migration. Cells were stimulated with or without drugs in the upper chamber. Then, the upper chamber was immersed in 4% paraformaldehyde and fixed for 20 minutes. After washing, the crystal violet dyeing solution was added to the upper chamber for staining. Finally, we removed the upper chamber, placed it on a slide, and observed and photographed it using an inverted microscope. Image J was used to count the stained cells to compare the migration abilities of different treated cells. CMCs were counted and cultured with 3 × 10 4 cells per well in a six-well plate or dissect tissues according to the above method. Based on the instructions provided by the manufacturer, MDA, GSH, Fe 3+ , Fe 2+ , and antioxidant enzyme activity levels were measured using the assay kits. Those were measured using a microplate reader (Thermo Fisher Scientific). Cell samples and eye sections were incubated overnight with primary antibody at 4°C. Afterward, we treated them with secondary antibodies from multiple immunofluorescence staining kits (AFIHC033, AiFang Biological, Changsha, China) at 25°C for 1 hour. The samples were counterstained using DAPI (C0060, Solarbio, Beijing, China) and encapsulated in an aqueous medium (VectaShield, Burlingame, CA, USA), and fluorescence signals were collected using a fluorescence microscope (Leica). We measured the relative fluorescence intensity through image J. Paraffin sections of guinea pig eyeballs were used as the materials for immunohistochemistry. The sections were dewaxed, rehydrated, and placed in a container with a repair solution, which was then heated to 96°C. After natural cooling, endogenous peroxidase inactivation was performed on the sections, followed by blocking with 10% goat serum blocking solution. A primary antibody was then added, and the sections were incubated overnight at 4°C, after which a secondary antibody was added. All sections were stained with a 3,3'-diaminobenzidine solution and counterstained with hematoxylin. After dehydration and mounting with neutral balsam, the sections were observed and photographed under a light microscope. Intracellular oxidative stress was determined with ROS assay kit. Incubate CMCs with DCFH-MDA (1:1000) in a cell culture incubator at 37°C for 30 minutes. Then, we washed the cells three times with serum-free cell culture medium to completely remove the DCFH-MDA that has not entered the cells. Finally, we used a 488-nm excitation wavelength and a 525-nm emission wavelength to detect the fluorescence intensity in real time or flow cytometry analysis was conducted on a CytoFLEX flow cytometer (Beckman Coulter, Southfield, MI, USA) to assess ROS level. The resulting data were analyzed using FlowJo software (version 10.0.7). We used an Assay Kit with tetramethylrhodamine ethyl ester (TMRE) (Beyotime) for mitochondrial membrane potential detection. CMCs underwent two washes with PBS followed by the addition of TMRE staining working solution and incubation at 37°C for 30 minutes. Subsequently, the cells were washed twice with staining buffer. The fluorescence was observed using a fluorescence microscope. Total cell RNA was obtained by TRIzol (Invitrogen, Waltham, MA, USA). With the Universal SYBR Green Fast qPCR Mix (RK21203, ABclonal, Wuhan, China), mRNA expression was detected on ABI PRISM 7700 (Applied Biosystems, Tokyo, Japan). The relative gene expression levels were calculated with the 2 −△△CT formula with Actin as the internal reference. The primer sequences were provided in Supplementary Table S2 . The protein concentration was determined using the BCA Protein Assay (Beyotime). The proteins were separated by 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene membranes. After blocking in 5% nonfat milk in Tris-buffered saline-tween 20 for 1 hour at room temperature, the membranes were incubated with specific primary antibodies overnight at 4°C. Subsequently, the membranes were washed three times with Tris-buffered saline-tween 20 and incubated with the indicated secondary antibodies for 1h at room temperature. The ECL Kit (Beyotime) was used for detecting immunoreactive proteins. By comparing gray values of target proteins to β-tubulin, we calculated relative protein expression using Image J. We prepared ultrathin sections of ciliary muscle tissue and stain them with dioxane acetate and lead citrate. Observe the ultrastructural changes of cells through transmission electron microscopy (Olympus, Tokyo, Japan). The experiments were conducted in triplicate for each independent investigation, with data presented as mean ± standard deviation. All data referenced above were analyzed by GraphPad Prism 9.5.1 statistical software. Differences between the two groups were assessed by an unpaired Student t test. For multiple group comparisons, one-way ANOVA, followed by Tukey's post hoc test, was applied. Significant differences were considered at P values of less than 0.05.

The ciliary muscle contraction of guinea pigs was induced by Car according to the method described in prior studies ( Fig. 1 A). 10 Refraction, AL, and PD measurements were measured at baseline, 1 week, and 2 weeks after administration. As shown in  Figures 1 B–D, no significant differences were observed in refraction, AL, and PD between the Car group and the control group at baseline and 1 week. However, at 2 weeks, the Car group exhibited lower refraction (2.91 ± 0.49 vs. 3.44 ± 0.57; P = 0.0003), longer AL (8.20 ± 0.24 vs. 8.04 ± 0.35; P = 0.4560), and smaller PD (5.19 ± 0.10 vs. 5.29 ± 0.11; P = 0.0003) compared with the control group. Then, we further explored the influence of prolonged contraction of the ciliary muscle on ocular AMP. We measured these parameters before and after pharmacologically induced contraction, and the difference between the two measurements was calculated to represent the AMP as an indicator of accommodative capacity. At baseline, there were no significant differences in the changes of each ocular parameters between the two groups. At 1 week, the change of refractive in the Car group was significantly lower than that in the control group ( Fig. 1 E) (1.46 ± 0.15 vs. 1.57 ± 0.19; P = 0.0108). At 2 weeks, both the change of refractive ( Fig. 1 E) (1.19 ± 0.33 vs. 1.46 ± 0.27; P = 0.0010) and PD ( Fig. 1 G) (0.54 ± 0.23 vs. 0.71 ± 0.41; P = 0.0453) in the Car group were lower than those in the control group, whereas no significant difference was observed in the change of AL ( Fig. 1 F). Furthermore, hematoxylin and eosin staining of guinea pig eye slices revealed no apparent morphological or structural alterations in the ciliary muscle tissue between the two groups ( Fig. 1 H). In conclusion, prolonged contraction of the ciliary muscle leads to impaired ocular accommodation and a reduction in AMP in guinea pigs. Effects of Car treatment on refraction, AL, and PD in guinea pigs. ( A ) Schematic illustration of the animal experiments. ( B–D ) The refraction ( B ), AL ( C ), and PD ( D ) of the two groups at each time point after administration ( n = 30). ( E–G ) Amplitude of change of refraction ( E ), AL ( F ), and PD ( G ) of the two groups at each time point (△ Refraction = refraction at baseline − refraction after inducing contraction) ( n = 30). ( H ) Hematoxylin and eosin (H&E) staining of two groups of guinea pig eyeball slices. Scale bar , 100 µm. Data are shown as means ± SD, t test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. Primary CMCs were isolated and cultured from guinea pigs. After the cultivation period, the cells exhibited the characteristic spindle shaped morphology of smooth muscle cells ( Supplementary Fig. S1 A). The smooth muscle cell identity of the extracted cells was confirmed by positive expression of α-smooth muscle actin (α-SMA), a well-established marker for smooth muscle cells by Western blot analysis. Conversely, the absence of BMP5, a myofibroblast-associated marker protein, effectively excluded the presence of fibroblasts ( Supplementary Fig. S1 B). These findings were further validated through immunofluorescence analysis ( Supplementary Fig. S1 C). To investigate the effects of prolonged contraction of CMCs on cellular function, the cells were exposed to varying concentrations of Car to simulate a contracted cellular state. Taking 6 hours as a physiological short-term contraction and 24 hours as a long-term contraction, the CCK-8 assay was used to select 500 µg/mL Car at which the cell survival rate began to show differences for the in vitro treatment of CMCs ( Fig. 2 A). After 24 hours of exposure to Car, CMCs exhibited obvious contraction, accompanied by increased intercellular space ( Fig. 2 B). Then, we evaluated the impact of prolonged contraction on CMCs migration. Wound healing assays ( Fig. 2 C) revealed no significant differences between the Car and control groups at 6 hours, whereas the 24 hours long-term contraction significantly reduced the migration width of CMCs in the Car group. Transwell cell migration assays ( Fig. 2 D) further confirmed a decreased number of migrating cells in the Car group. Intracellular Ca 2+ levels were determined using fluorescence microscopy ( Fig. 2 E), revealing an increase in Ca 2+ concentration after Car exposure. We also detected the intracellular adenosine triphosphate (ATP) content, which increased during short-term contraction but declined during long-term contraction, as depicted in  Fig. 2 F. Immunofluorescence staining showed a significant upregulation of α-SMA in the Car group compared with the control ( Figs. 2 G,  2 H). Consistent results were obtained by Western blot analysis ( Figs. 2 I,  2 J), demonstrating increased expression of contraction-related proteins after long-term contraction, including α-SMA, CNN1, and SM22α. The elevated expression of ATP2A2 in the Car group supports the observed Ca 2+ accumulation and suggests enhanced ATP consumption. In conclusion, prolonged contraction of the ciliary muscle impairs the function of primary CMCs, leading to Ca 2+ accumulation and ATP depletion. Changes in cell function and contractile marker proteins. ( A ) CCK­8 assay with Car treatment for 6 or 24 hours ( n = 4). ( B ) Morphological changes of CMCs. ( C, D ) The capacity of cell migration in CMSs was analyzed by wound healing assay ( C ) and transwell assay ( D ) ( n = 3). ( E ) The concentration of intracellular Ca 2+ in CMCs ( n = 3). ( F ) Intracellular ATP content ( n = 6). ( G ) Immunofluorescence images of α-SMA. ( H ) Relative fluorescence intensity quantification ( n = 4). ( I ) Car regulates contractile marker proteins in CMCs. ( J ) Analysis of the protein relative expression quantification ( n = 3). Scale bar , 100 µm. Data are shown as means ± SD, t test. * p < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. Next, ciliary muscle tissues were harvested from guinea pigs for Western blot analysis after 2 weeks of modeling. The expression levels of contraction-related proteins in the Car group were significantly increased, consistent with the in vitro findings from previous experiments ( Fig. 3 A). Immunofluorescence staining for α-SMA corroborated the findings of cell experiments, showing a significant increase in α-SMA protein levels in the Car group compared with the control ( Fig. 3 B), indicating that Car effectively induces ciliary muscle contraction in guinea pigs. Mitochondria serve as central organelles for energy production, calcium homeostasis, and iron metabolism. 15 Transmission electron microscopy was used to assess mitochondrial morphology in ciliary muscle tissues from two groups. Strikingly, as shown in  Fig. 3 C, after prolonged contraction of the ciliary muscle, the mitochondria of CMCs in the Car group were smaller than normal mitochondria with darker-stained mitochondrial membranes and disorganized mitochondrial crista compared with the control group, exhibiting characteristic of mitochondrial damage associated with ferroptosis. Therefore, we investigated the impact of long-term contraction of the ciliary muscle on oxidative stress by measuring malondialdehyde (MDA), a marker of lipid peroxidation, ROS levels and antioxidant enzyme activities, as indicators of redox status. Results showed that prolonged contraction of the ciliary muscle increased MDA content in ciliary muscle tissue and CMCs ( Figs. 3 D,  3 F), and decreased the activities of GSH peroxidase, superoxide dismutase, and catalase, which represent antioxidant capacity. Notably, pretreatment of CMCs with ferrostatin-1 (Fer-1), a specific inhibitor of ferroptosis, markedly attenuated these alterations ( Figs. 3 E,  3 G). DCFH-DA staining further confirmed that Fer-1 treatment suppressed the intracellular ROS accumulation induced by Car ( Figs. 3 H,  3 I). Accumulating evidence indicates that dysregulated ROS production can trigger oxidative stress and cell death through activating the mitochondrial–permeability transition pore and inducing mitochondrial dysfunction. 16 Accordingly, mitochondrial membrane potential was detected using a TMRE fluorescent probe, which revealed that Fer-1 reversed the mitochondrial membrane potential collapse induced by prolonged contraction of the ciliary muscle ( Figs. 3 J,  3 K). Taken together, these findings demonstrate that prolonged contraction of the ciliary muscle induces oxidative stress and mitochondrial dysfunction, and Fer-1 can mitigate these detrimental effects on CMCs. Prolonged contraction of the ciliary muscle induced oxidative stress and mitochondrial dysfunction. ( A ) Car regulates contractile marker proteins in ciliary muscle tissues of guinea pigs ( n = 3). ( B ) Immunofluorescence images of α-SMA of guinea pigs’ eyeball slices ( n = 5). ( C ) Mitochondrial morphology on transmission electron microscopy. ( D , F ) MDA content in ciliary muscle tissues of guinea pigs ( D ) and CMCs ( F ) ( n = 9). ( E , G ) Antioxidant enzymes expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in ciliary muscle tissues of guinea pigs ( E ) and CMCs ( G ) ( n = 9). ( H ) Intracellular ROS generation and relative fluorescence analyses was assessed by DCFH-DA staining ( n = 3). ( J ) Mitochondrial membrane potential was analyzed with a TMRE probe. ( I ) The ROS level was detected by flow cytometry. ( K ) Relative fluorescence analyses of TMRE ( n = 3). White scale bar , 100 µm; black scale bar , 2 µm. Data are shown as means ± SD, t test and one-way ANOVA followed by Tukey's multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. In light of the mitochondrial morphological alterations observed by transmission electron microscopy, we pretreated CMCs with Fer-1 to evaluate cellular iron metabolism. The CCK-8 assay indicated that cell viability ( Fig. 4 A) and GSH content ( Fig. 4 B), an important intracellular antioxidant, were reduced after Car intervention. In contrast, intracellular Fe 3+ ( Fig. 4 C) and Fe 2+ ( Fig. 4 D) levels were markedly elevated. As expected, Fer-1 supplementation alleviated the Car-induced reductions in cell viability and GSH, and notably attenuated the deposition of both Fe 3+ and Fe 2+ . Furthermore, we examined changes in the expression of ferroptosis-related molecules by RT-qPCR and Western blotting. After prolonged contraction of CMCs inducing by Car, mRNA levels of ACSL4, PTSG2, and TFR were upregulated, whereas those of FTH1, GPX4, and xCT were downregulated. Pretreatment with Fer-1 apparently reversed these transcriptional changes ( Fig. 4 E). Western blot analysis displayed that protein expression changes were consistent with the RT-qPCR results ( Fig. 4 F). Additionally, we used immunofluorescence staining to detect the expression of xCT, a system X C − cystine/glutamate antiporter, and GPX4, a selenoprotein, in both CMCs and guinea pig ciliary muscle tissues. Previous studies have demonstrated that inhibition of either protein can trigger ferroptosis. 17 We observed that Fer-1 pretreatment obviously reversed the Car-induced downregulation of xCT and GPX4 in CMCs ( Figs. 4 G–I), and similar results were obtained in eyeball slice of guinea pigs ( Figs. 4 J,  4 K) Quantitative analysis of immunofluorescence are presented in Supplementary Fig. S2 A. Altogether, we reasoned that prolonged contraction of the ciliary muscle disrupts iron homeostasis and induces ferroptosis in CMCs. Effects of prolonged contraction of the ciliary muscle on iron metabolism and ferroptosis-related molecules. ( A ) CCK­8 assay with Car or Fer-1 treatment ( n = 6). ( B – D ) The concentrations of GSH ( B ), Fe 3+ ( C ), and Fe 2+ ( D ) were analyzed in CMCs ( n = 9). ( E ) Ferroptosis-related genes expression were analyzed by RT-qPCR ( n = 6). ( F ) Western blotting and quantitative analysis of the expression of ferroptosis-related proteins, using β-tubulin as the loading control ( n = 3). ( G – I ) Immunofluorescence images ( G ) and quantitative analysis of xCT ( H ) and GPX4 ( I ) in CMCs ( n = 3). ( J , K ) Immunofluorescence images of xCT ( J ) and GPX4 ( K ) of guinea pigs’ eyeball slices ( n = 3). Scale bar , 100 µm. Data are shown as means ± SD, one-way ANOVA followed by Tukey's multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. VitE possesses the ability to scavenge free radicals, productively preventing the emergence of lipid peroxidation. 11 A key question then arises: Can VitE inhibit ferroptosis induced by prolonged contraction of CMCs? To address this, we used the CCK-8 assay to determine an optimal concentration of 2 µM for the VitE analogues (D-α-tocopherol), which effectively rescued cell viability after Car intervention, and applied this concentration in subsequent experiments ( Fig. 5 A). We observed a significant decline in lipid peroxidation levels of MDA ( Fig. 5 B) and ROS ( Figs. 5 D,  5 F, Supplementary Fig. S2 B), as well as a marked increase in intracellular antioxidant enzyme activity ( Fig. 5 C) and mitochondrial membrane potential ( Fig. 5 E, Supplementary Fig. S2 C) in CMCs with VitE pretreatment. Given the substantial physiological differences between in vitro and in vivo environments, we further investigated whether VitE exerts comparable protective effects in mitigating oxidative stress and inhibiting ferroptosis under biological conditions. Prolonged contraction of the ciliary muscle in guinea pigs was induced by Car, and 2 µM VitE was administered via eye drop. We carefully measured MDA levels and antioxidant enzyme activities in ciliary muscle tissue. As expected, VitE treatment reduced MDA content ( Fig. 5 G) and restored antioxidant enzyme activities ( Supplementary Fig. S2 D), consistent with the in vitro findings. Additionally, a significant increase in GSH was observed ( Fig. 5 H), along with marked reductions in Fe 3+ ( Fig. 5 I) and Fe 2+ ( Fig. 5 J). Remarkably, Western blot analysis showed VitE reversed the Car-induced downregulation of GPX4 and xCT protein levels, and led to a significant decrease in TFR and ACSL4 expression following treatment ( Fig. 5 K). Quantitative analysis of relative protein expression are presented in Supplementary Fig. S2 E. Finally, we used α-SMA to label ciliary muscle, the results of immunofluorescence ( Fig. 5 L, Supplementary Fig. S4 B) and immunohistochemistry ( Fig. 5 M, Supplementary Fig. S4 C) were consistent with Western blot analysis. The ocular safety of VitE was verified by hematoxylin and eosin staining of eyeball sections ( Supplementary Fig. S3 A, S3 B) and panoramic OCT of the fundus ( Supplementary Fig. S3 C). These compelling results firmly indicated that VitE not only directly suppresses lipid peroxidation and alleviates oxidative stress, but also effectively mitigates ferroptosis in CMCs without adverse effects on other ocular tissues. VitE inhibited ferroptosis and improved oxidative stress in vivo and in vitro. ( A ) CCK­8 assay with VitE treatment for 24 hours ( n = 6). ( B , G ) MDA content in CMCs ( B ) and ciliary muscle tissues of guinea pigs ( G ) ( n = 9). ( C ) Antioxidant enzymes expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in CMCs ( n = 9). ( D ) Intracellular ROS generation was assessed by DCFH-DA staining ( n = 3). ( E ) Mitochondrial membrane potential was analyzed with TMRE probe ( n = 3). ( F ) The ROS level was detected by flow cytometry. ( H – J ) The concentrations of GSH ( H ), Fe 3+ ( I ), and Fe 2+ ( J ) were analyzed in ciliary muscle tissues of guinea pigs ( n = 9). ( K ) Western blotting of Ferroptosis-related proteins in ciliary muscle tissues ( n = 3). ( L ) Immunofluorescence images of xCT of guinea pigs’ eyeball slices ( n = 3). ( M ) Immunohistochemical detection of protein expression in guinea pig ciliary muscle tissue. Scale bar , 100 µm. Data are shown as means ± SD, one-way ANOVA followed by Tukey's multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. Whole-genome RNA sequencing analysis was performed to investigate the molecular mechanism underlying the protective effect of VitE against Car-induced prolonged contraction in CMCs. The heatmap showed significant differences in gene expression between the Car group and the control group, as well as between the Car + VitE group and the Car group ( Fig. 6 A). We used a volcano plot to visualize differentially expressed genes (DEGs) across the three groups, and plotted the overall distribution of DEGs. A total of 382 DEGs were identified when comparing control cells with Car-treated cells. In comparison with the Car group, VitE treatment resulted in the identification of 954 DEGs, including 533 downregulated and 421 upregulated. We took an intersection and discovered 143 overlapping DEGs altered both by Car and VitE, and we annotated part of them in a volcano plot, indicating that these genes might play key roles in the protective effect of VitE in Car-induced prolonged contraction of the ciliary muscle ( Fig. 6 B). Moreover, Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses revealed that Car upregulated genes associated with the cell cycle, cellular senescence, HIF-1 signaling pathway, and ferroptosis, consistent with our results ( Fig. 6 C). Meanwhile, we noticed that the genes related to oxidative phosphorylation, thermogenesis, and GSH metabolism were upregulated after VitE treatment ( Fig. 6 D). Gene set enrichment analysis was subsequently performed to evaluate the association between VitE treatment and signaling pathways derived from the RNA array results. Gene set enrichment analysis indicated significant differences between Car + VitE cells and Car cells in multiple metabolism-related pathways, including oxidative phosphorylation, tricarboxylic acid cycle, fatty acid degradation, and GSH metabolism, which were all related to ferroptosis ( Fig. 6 E). Next, we conducted RT-qPCR to assess several DEGs identified by the volcano plot. Interestingly, among the DEGs we annotated, we found that ACOT7 exhibited the most pronounced and reliable changes among all tested candidates ( Fig. 6 F). It has been reported to play an important role in fatty acid metabolism, participating in the tricarboxylic acid cycle, β-oxidation, and other metabolic pathways, connecting our results. To further explore the functional role of ACOT7, Western blotting was conducted and the results demonstrated pretreatment with VitE obviously reversed the Car-induced downregulation of ACOT7 in CMCs ( Fig. 6 G). To determine the functional contribution of ACOT7 to the protective effects of VitE, siRNA was used to knock down ACOT7 expression in CMCs. For the assessment of in vitro transfection efficiency, CMCs were transfected using lipofectamine as the transfection reagent encapsulating three distinct siRNA sequences. After transfection, the superior gene knockdown effect of si-ACOT7-2 was jointly confirmed by RT-qPCR ( Fig. 6 H) and Western blot analysis ( Fig. 6 I) for subsequent experiments. Effect of VitE on transcript profiling of Car-induced CMCs. ( A ) Heatmaps displayed the DEGs between the control and Car groups, as well as the Car and Car + VitE groups ( n = 3). ( B ) Volcano plot displayed DEGs and tagged common DEGs. ( C , D ) Kyoto Encyclopedia of Genes and Genomes enrichment revealed the regulated pathways between the control and Car groups ( C ), as well as the Car and Car + VitE groups ( D ). ( E ) Gene set enrichment analysis (GSEA) from RNA-seq between the Car and Car + VitE groups. ( F ) qRT-PCR was used to detect the DEGs expression in the volcano plot ( n = 6). ( G ) Western blotting and quantitative analysis of the expression of ACOT7 in CMCs ( n = 3). ( H ) ACOT7 expression was analyzed by RT-qPCR ( n = 3). ( I ) Western blotting and quantitative analysis of the expression of ACOT7 after transfection with three sequences ( n = 3). Data are shown as means ± SD, one-way ANOVA followed by Tukey's multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. To determine the role of ACOT7 in the protective effect of VitE, we used siRNA to knock down the ACOT7 gene in CMCs. In vitro results, including cell viability ( Fig. 7 A), MDA ( Fig. 7 B), antioxidant enzyme activity ( Fig. 7 C), ROS ( Figs. 7 D,  7 F), and TMRE ( Fig. 7 E) displayed that the knockdown of ACOT7 dramatically impeded the protective effect of VitE against Car-induced mitochondrial dysfunction and oxidative damage. The results also showed that ACOT7 knockdown exacerbated ferroptosis triggered by prolonged contraction of CMCs, as evidenced by reduced protein levels of xCT and GPX4 ( Fig. 7 K, Supplementary Fig. S4 A) and decreased GSH content ( Fig. 7 I), and along with elevated intracellular Fe 3+ ( Fig. 7 G) and Fe 2+ ( Fig. 7 H). Finally, we verified the effect on cell function after knockdown of ACOT7, which partially attenuated the recovery of ATP content ( Fig. 7 J) and cell migration function ( Fig. 7 L) by VitE. These experimental findings underscores the pivotal role of ACOT7 in mediating the ability of VitE to suppress ferroptosis in CMCs under long-term contraction. Knockdown of ACOT7 attenuated the beneficial effect of VitE on oxidative stress and ferroptosis. ( A ) CCK-8 assay with siRNA transfection ( n = 6). ( B ) MDA content in CMCs ( n = 9). ( C ) Antioxidant enzymes expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT) in CMCs ( n = 9). ( D ) Intracellular ROS images and relative fluorescence analysis ( n = 3). ( E ) Mitochondrial membrane potential TMRE staining and relative fluorescence quantification ( n = 3). ( F ) The ROS level was detected by flow cytometry. ( G – J ) The concentration of Fe 3+ ( G ), Fe 2+ ( H ), GSH ( I ), and ATP ( J ) were analyzed in CMCs ( n = 9). ( K ) Western blotting after transfection ( n = 3). ( L ) The capacity of cell migration in CMSs was analyzed by transwell assay ( n = 3). Scale bar , 100 µm. Data are shown as means ± SD, one-way ANOVA followed by Tukey's multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. In vitro, we overexpressed ACOT7 in CMCs to further validate its vital mechanism in mediating VitE's regulation of antioxidant defense and anti-ferroptotic effects. The efficiency of ACOT7 overexpression was evaluated using RT-qPCR ( Fig. 8 A) and Western blotting ( Fig. 8 B). The activity of cellular antioxidant enzymes ( Fig. 8 C, Supplementary Fig. S5 A) and GSH levels ( Supplementary Fig. S5 B) increased to some extent after ACOT7 overexpression, suggesting that ACOT7 may possess intrinsic antioxidant capacity. Subsequently, Car and VitE were added to the overexpressed cells. The in vitro oxidative stress–related experiments, including MDA ( Fig. 8 D), antioxidant enzyme activity ( Fig. 8 E), GSH ( Fig. 8 F), and ROS ( Fig. 8 I) demonstrated that, after Car-induced prolonged contraction of CMCs, the Car + oeACOT7 group partially restored cellular antioxidant capacity compared with the Car group. Meanwhile, key indicators of ferroptosis also exhibited a certain degree of reversal ( Figs. 8 G,  8 H,  8 J). However, the salvage effect of oeACOT7 on CMCs was limited. The regulatory amplitudes of antioxidant enzymes, GSH levels, and ferroptosis-related proteins in the Car + oeACOT7 group were lower than those observed in the Car + VitE group. Given that VitE itself possesses broad-spectrum antioxidant properties and can directly scavenge intracellular free radicals and suppress lipid peroxidation chain reactions, we speculate that VitE exerts a basal antioxidant effect independent of ACOT7, which may act in concert with ACOT7 to modulate CMCs’ contractive function. Overexpression of ACOT7 alleviated oxidative stress and ferroptosis in CMCs. ( A ) ACOT7 expression were analyzed by RT-qPCR ( n = 3). ( B ) Western blotting and quantitative analysis of the expression of ACOT7 in CMCs ( n = 3). ( C ) Antioxidant enzymes expression of catalase (CAT) in CMCs ( n = 3). ( D ) MDA content in CMCs ( n = 3). ( E ) Antioxidant enzymes expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), CAT in CMCs ( n = 3). ( F – H ) The concentrations of GSH ( F ), Fe 3+ ( G ), and Fe 2+ ( H ) were analyzed in CMCs ( n = 3). ( I ) The ROS level was detected by flow cytometry. ( J ) Western blotting and quantitative analysis of the expression of ferroptosis-related proteins ( n = 3). ( K ) Antioxidant enzymes expression of GSH-Px, SOD, and CAT in CMCs ( n = 3). ( L ) The concentration of GSH ( n = 3). Data are shown as means ± SD, one-way ANOVA followed by Tukey's multiple comparison test. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; ns, not significant. α = 0.05. To support this hypothesis, we reevaluated the antioxidant enzyme activities and GSH levels in the Car + VitE, Car + si-ACOT7, and Car + VitE + si-ACOT7 groups. We found that, compared with the Car + si-ACOT7 group, the Car + VitE + si-ACOT7 group maintained a certain level of antioxidant activity ( Figs. 8 K,  8 L). Nevertheless, the overall effect did not reach that of the Car + VitE group, suggesting that, although VitE can exert a baseline antioxidant effect even when ACOT7 expression is suppressed, this effect is relatively modest. The majority of VitE's protective effects appear to depend on activation of the ACOT7. Our findings confirm that ACOT7 plays a significant role in mediating the protective effects of VitE on CMCs.

The ciliary muscle, a crucial intraocular smooth muscle, plays a key role in ocular accommodation by governing the position and shape of the lens. 2 Prior studies have noted that acetylcholine initiates contraction of isolated rabbit ciliary muscle tissue, and prolonged stimulation decreases the amplitude of contraction, which may potentially indicate muscle fatigue. 18 Driven by environmental changes and social requirements, near-work activities for professional and recreational purposes have escalated among all age groups. Individuals engaged in prolonged near vision tasks may suffer from long-term excessive ciliary muscle tension, leading to accommodative spasm and a range of visual fatigue symptoms. Ciliary muscle dysfunction stands out as a prevalent etiological factor contributing to visual impairments in clinical ophthalmology. 19 However, the potential detrimental effects of prolonged contraction on ciliary muscle regulatory capacity and the precise mechanism underlying the cell damage remain elusive. This study constituted the inaugural investigation into the interplay among prolonged contraction, oxidative stress, and ferroptosis in the ciliary muscle, with the objective of improving adverse effects of prolonged contraction through pharmacological intervention. Notably, the effects of VitE, a novel therapeutic agent, on the ciliary muscle have not been documented previously. Collectively, our results suggested that prolonged contraction of the ciliary muscle may lead to impaired accommodation function, intracellular ATP depletion, decreased mitochondrial membrane potential, and ROS accumulation. Moreover, sustained cell contraction upregulated specific contractile proteins such as α-SMA, while simultaneously reducing key intracellular antioxidant enzymes, including GSH peroxidase, superoxide dismutase, and catalase. This imbalance led to oxidative stress and mitochondrial dysfunction, which may ultimately result in impaired cellular migration and ferroptosis. To evaluate potential rescue strategies, we used the fat-soluble antioxidant VitE in our experiments. Our findings demonstrated that pretreatment with VitE effectively suppressed lipid peroxidation, alleviated oxidative stress, and mitigated ferroptosis in CMCs. Additionally, knocking down ACOT7 attenuated the beneficial effects of VitE, suggesting a functional link between ACOT7 and VitE-mediated cytoprotection. Our study underscored the detrimental consequences of ciliary muscle long-term contraction and highlighted the therapeutic potential of VitE in counteracting these effects, offering novel insights into the molecular mechanisms underlying accommodative spasm. Furthermore, these discoveries suggested that ACOT7 may serve as a promising therapeutic target, particularly through the modulation of lipid metabolism pathways, to prevent ferroptosis in CMCs. As is well-known, near work induces contraction of the ciliary muscle. 20 , 21 Our study used topical administration of Car, an M-receptor agonist, to stimulate ciliary muscle contraction. 18 Refraction, AL, and PD were measured at baseline, 1 week, and 2 weeks to assess the impact of Car-induced group compared with the saline-treated control group. The results indicated that the refraction decreased and AL increased progressively over time between the two groups, which is consistent with previous findings, indicating that the ocular development pattern in guinea pigs resembles that in humans. 22 There were no significant differences in refraction, AL, and PD between the Car group and control group at baseline or 1 week. However, the Car group exhibited lower refraction, longer AL, and smaller PD compared with the control group at 2 weeks. These changes align with the established mechanism of axial myopia and may further support the role of ciliary muscle prolonged contraction in the onset and progression of myopia from another perspective. 23 – 25 Ocular AMP was also considered in our analysis. Initially, no significant difference in AMP was detected between the groups. As the experiment progressed, the AMP of refractive and PD in the Car group were lower than those in the control group. This observation suggested that prolonged contraction may have an impact on the contractility of the ciliary muscle, leading to diminished accommodative function. Additionally, hematoxylin and eosin staining suggested that no apparent differences in the morphology or structure of ciliary muscle tissue between the two groups. This finding contrasts with earlier reports, 5 potentially owing to the relatively short duration of our intervention. In other words, extending the experimental period by a couple of months might yield more pronounced structural alterations. To further validate the aforementioned experimental findings in vivo, primary CMCs were isolated from guinea pigs. The smooth muscle cell identity of the extracted cells was confirmed by positive α-SMA expression detected on Western blot analysis and immunofluorescence analysis. The CCK-8 assay indicated that differences in cell viability were observed at different intervention times at a concentration of 500 µg/mL of Car. After an intervention with 500 µg/mL Car, the morphology of CMCs, as well as cell function and contraction-related protein levels, were assessed. Our results suggested that Car treatment induced a distinct contracted morphology in CMCs, accompanied by increased intercellular space. Furthermore, prolonged contraction over 24 hours significantly affected the migratory function of CMCs, which may subsequently impact refractive regulation in guinea pigs, whereas no significant effect was observed at 6 hours. These results implied that persistent contraction of the ciliary muscle may cause no injuries to CMCs within a short time. Western blotting and immunofluorescence staining were used to evaluate the expression of contraction-related proteins. It is worth noting that the expression level of α-SMA increased in the Car group. Although some studies suggest that increased α-SMA expression signifies enhanced contractile function, 26 our current findings do not support this view, possibly owing to the fact that the contractile intensity of the ciliary muscle, a specialized smooth muscle, does not directly correlate with its accommodative function. Given that α-SMA is widely recognized as a biomarker of tissue fibrosis, 27 its elevation may indicate a tendency toward fibrotic transformation after prolonged contraction, which could contribute to impaired cellular function. ATP2A2, a key protein involved in calcium reuptake during smooth muscle relaxation, was also upregulated, suggesting increased energy demand in CMCs under sustained contractile activity. Additionally, compared with the 6-hour short-term contraction, the 24-hour long-term contraction actually exhibited a decrease in ATP levels, which may indicate dysfunction of mitochondria, the intracellular energy supply factory, and a compensatory increase in ATP2A2. To provide a comprehensive understanding of this phenomenon, we examined these protein levels in vivo, yielding results consistent with those from cell experiments. Excitingly, transmission electron microscopy revealed mitochondrial features characteristic of ferroptosis in the Car group. Therefore, we have embarked on further investigation using the ferroptosis inhibitor Fer-1. Our scrutiny has centered on elucidating the oxidative stress during the contraction of CMCs. Our findings revealed that CMCs undergoing long-term contraction exhibited significantly heightened levels of MDA and ROS, as well as decreased antioxidant enzymes. Additionally, a notable decrease in mitochondrial membrane potential also suggested mitochondrial dysfunction, and Fer-1 could attenuate the detrimental effects on CMCs. Subsequently, we detected intracellular GSH, iron levels, as well as the expression of ferroptosis-related molecules at both the mRNA and protein levels. Immunofluorescence staining was used to process paraffin sections of the animals. Specifically, α-SMA was used as a marker protein for smooth muscle fibers, aiding in the precise localization of the ciliary muscle. Compelling evidence demonstrated that prolonged contraction of CMCs disrupted iron homeostasis and induced ferroptosis. Prior research has confirmed that mitochondrial dysfunction and damage promote oxidative stress. Unfortunately, oxidative stress and ROS accumulation exacerbate mitochondrial impairment, creating a vicious cycle that ultimately drives ferroptosis. 28 – 30 VitE is a well-established fat-soluble antioxidant. Accumulating evidence has demonstrated that VitE exerts therapeutic effects in various conditions, including necrotizing enterocolitis, 12 lung injury, 31 and acute kidney injury, 32 primarily by inhibiting ferroptosis. In this study, we aimed to investigate whether VitE supplementation could ameliorate adverse effects induced by prolonged ciliary muscle contraction. Notably, the potential effects of VitE on ciliary muscle function have not been previously documented in the scientific literature. To address this knowledge gap, we replicated these experiments both in vivo and in vitro. Our findings revealed that VitE significantly attenuated oxidative stress and effectively mitigated ferroptosis in CMCs. Transcriptome sequencing was performed to investigate the protective mechanism of VitE against prolonged contraction of the ciliary muscle. We noticed that genes involved in oxidative phosphorylation and GSH metabolism were upregulated, suggesting that VitE can alleviate the detrimental effects of CMCs’ prolonged contraction by enhancing mitochondrial energy production and cellular antioxidant capacity. We focused on ACOT7 among the DEGs. We were surprised to find that the knockdown of ACOT7 attenuated the beneficial effects of VitE against oxidative stress and ferroptosis in vitro. Previous studies have established that ACOT7 plays a critical role in both energy and fatty acid metabolism. 14 As previously reported, ACOT7 is implicated in the regulation of ferroptosis. Zhang et al. 33 demonstrated that ACOT7 protects epidermal stem cells from iron-dependent lipid peroxidation. Wang et al. 34 reported that ACOT7 modulates ferroptosis in non-small cell lung cancer cells. Furthermore, Liu et al. 35 revealed that the NAT10–ACOT7 axis modulates fatty acid metabolism in cancer cells and promotes tumor progression by suppressing ferroptosis. Our study has uncovered that prolonged contraction of CMCs leaded to the downregulation of ACOT7, and the interruption of its metabolite CoASH supply may conduct to mitochondrial dysfunction and oxidative stress. In contrast, the accumulation of polyunsaturated fatty acids induced lipid peroxidation, eventually leading to ferroptosis. The fundamental antioxidant properties of VitE arise from the hydrogen atom-donating capacity of the phenolic hydroxyl groups in its molecular structure, enabling it to directly scavenge intracellular ROS and inhibit lipid peroxidation chain reactions. 11 This finding aligns with previous findings demonstrating the nonspecific antioxidant effects of VitE in cataracts and glaucoma. 36 – 38 Moreover oxidative stress and ferroptosis pathways have been implicated in the pathogenesis of various ocular conditions, including corneal injury and AMD, further substantiating the biological plausibility of VitE's ophthalmic use. 39 In our study, we observed that compared with the Car + si-ACOT7 group, the antioxidant activity in the Car + VitE + si-ACOT7 group was moderately enhanced, yet remained significantly lower than that in the Car + VitE group. These results suggest that ACOT7 is essential for the antioxidant protective effects of VitE in CMCs. The activation of ACOT7 may potentiate the antioxidant efficacy of VitE by modulating lipid metabolic homeostasis and regulating the expression or activity of antioxidant enzymes. Our research team has undertaken pioneering investigations into the role of VitE in the regulation of ferroptosis, cell migration, and contraction in the ciliary muscle. Our findings not only elucidate a novel mechanism underlying VitE therapeutic potential, but also highlight ACOT7 as a critical mediator in the pathogenesis of CMCs prolonged contraction. Consequently, this discovery may hold potential as a therapeutic target for clinical diagnosis and treatment. However, the present study has some limitations. First, the intervention time in our in vivo experiments is relatively short. Therefore, future studies with longer expected outcomes may provide deeper insights into the observed phenomena. Second, VitE has no targeted effect on the ciliary muscle and may affect other tissues in the anterior segment of the eye. In addition, the inherent interspecies differences in ocular anatomy (such as corneal permeability and aqueous humor turnover rate) and in pharmacokinetic profiles (including drug absorption efficiency and tissue distribution), which may reflect the uncertainty of the efficiency of VitE penetrating the anterior segment of the eye in the ciliary muscle of humans and guinea pigs. To address this limitation, our future research will focus on developing targeted delivery systems for VitE and seek to further enhance the understanding of the mechanism by using primary human ciliated muscle cells. Finally, we confirmed in vitro through knockdown and overexpression experiments that ACOT7 plays a core role in the protective effects of VitE on CMCs. However, we have not yet conducted protein interaction experiments between VitE and ACOT7, and thus cannot directly prove the direct regulation of ACOT7 by VitE. These aspects will be the key points of our continuous research. Based on the results presented herein, we systematically showed that prolonged contraction of the ciliary muscle reduces the AMP, impairs cell function, induces oxidative stress, and triggers ferroptosis. Furthermore, our findings provide mechanistic insights into the protective role of VitE mediated through ACOT7.