Twenty-eight male C57BL/6 mice (6–8 weeks old), purchased from Southern Medical University Experimental Animal Center (Guangdong, China; license No. SYXK (Yue) 2016-0167), were housed in an SPF facility (temperature 24 ± 2°C, humidity 50%-60%, light/dark cycle 12h/12h) with free access to water and food. After seven days of adaptation, the mice were randomly divided into a CUMS model group (CM, n=20) and a control group (NC, n=8), using a random number generator of SPSS software (IBM, USA), and the CUMS modeling process began. The Southern Medical University Experimental Animal Ethics Committee approved the study protocol (No. L2017178), in accordance with the United States National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1986).

The CUMS model is commonly used to simulate the onset and symptoms of depressive conditions (22). All animals in the CUMS model group (CM, n=20) underwent a 28-day CUMS procedure (23). The mice were randomly exposed to stressors, including noise (24 h), water deprivation (24 h), food deprivation (24 h), inversion light/dark cycle (12/12 h), strobe illumination (12 h), restraint (12 h), and level shaking (5 min). These stressors were administered randomly, with 1–2 stressors per day, and the same stressor was not used on consecutive days to prevent animals from predicting the occurrence of stimulation. After the modeling, if sucrose preference rate in sucrose preference test (SPT) is higher than 80%, the animal is considered failed the modeling and were excluded (24). Approximately 80% of the CUMS model mice were successfully induced, and the four mice that failed to develop depressive behavior were excluded. Ultimately, 16 CUMS model animals were successfully established.

Sixteen successfully modeled mice were randomly assigned to either the CUMS group (n=8, sham electroacupuncture) or the EA group (n=8, EA interventions) by a random number generator of SPSS software (IBM, USA), with 10 mice in each group. The NC group (n=8) continued with free access to water and food.

The SPT and tail suspension test (TST) were used to examine depressive behaviors in the animals at the end of the intervention. The animals were euthanized the day after the behavioral experiments (Figure 1A).

After intraperitoneal injection with sodium pentobarbital (60 mg/kg), the bilateral ventromedial prefrontal cortex (vmPFC) tissues were isolated. Mice were randomly assigned to different tissue acquisition groups. For 3 animals/group, the left brain, used for Nissl staining and immunofluorescence staining, was fixed with 4% paraformaldehyde for 48 h and routinely embedded in optimal cutting temperature (OCT) compound. The right brain (3 animals/groups), intended for transmission electron microscopy (TEM), was immediately cut into 1 mm³ pieces and preserved in 2.5% glutaraldehyde (G5882, Sigma-Aldrich, Massachusetts, USA) until TEM processing. For samples used in immunoblotting (3 animals/groups), PFC tissues were washed at 4°C in phosphate-buffered saline (PBS) to remove excess blood and stored at -80°C until needed. All samples were labeled with initials and numbers to minimize bias.

Tissues were routinely embedded in OCT and sectioned in a freezing microtome (CM1850, Leica, Hessen, Germany) to produce 30 μm-thick slides. After Nissl staining, dehydration, transparency, and sealing, neurons were observed under a microscope at 400× magnification. Six randomly selected, non-overlapping fields of view were photographed for each animal. The average optical density (AOD) values of the captured images was analyzed using ImageJ software.

Tissues were immersed in 1% osmium tetroxide for 1 h and then rinsed with PBS three times for 15 min each. Next, the samples were soaked in a gradient of acetone (50%, 70%, and 90% each for 15 min; 100%, 3x, for 15 min). The tissues were soaked in a mixture of acetone and Spurr’s resin (acetone:Spurr’s resin = 1:1 for 1 h; acetone: Spurr’s resin = 1:2 for 2 h), then immediately placed in 100% Spurr’s resin overnight and embedded in Spurr’s resin in coffin molds, which were cured for 8 h in an oven at 70°C. PFC tissues were sliced at 60 nm using an ultramicrotome and counterstained with saturated aqueous uranyl acetate and Reynolds lead citrate (3x for 5 min). Sections were examined using TEM (H-7500, Hitachi, Tokyo, Japan) and photographed at 3,000g and 8,000graphededsred3A8 Four neurons were randomly selected per animal and photographed using ImageJ to analyze the average area and diameter of mitochondria.

Tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer at 4°C for 30 min. Following centrifugation at 12,000 rpm for 15 min, protein content was determined using the bicinchoninic acid (BCA) assay. Samples containing 20 μg of protein were loaded onto a 10% SDS-PAGE gel and transferred onto PVDF membranes. After blocking with BSA/TBST at room temperature for 3 h, the membranes were incubated with primary antibodies, including DRP1 (1:1500, 12957-1-Ap, Proteintech, Chicago, USA), MFF (1:4000, 66527-1-Ig, Proteintech), FIS1 (1:1000, SAB2702049, Sigma-Aldrich), SUMO2/3 (1:1500, 11251-1-Ap, Proteintech), SENP3 (1:1000, 17659-1-Ap, Proteintech), and GAPDH (1:3000, HRP-60004, Proteintech), overnight at 4°C. Secondary antibodies were incubated at room temperature for 1 h (1:3000, anti-rabbit SA00001-2, Proteintech; 1:3000, anti-mouse SA00001-1, Proteintech). WB images were captured using enhanced chemiluminescence (ECL) and a gel imaging system (UVP Company, Upland, California, USA). Band intensities were analyzed using ImageJ software.

Tissues were embedded in OCT and sectioned in a freezing microtome to produce 30 μm-thick slides. Slices were rinsed with PBS, and incubated with FIS1 (1:400, 66635-1-Ig, Proteintech) and SENP3 (1:400, 17659-1-Ap, Proteintech) antibodies overnight at 4°C. After rinsing with PBS, the sections were incubated with secondary antibodies (1:300, green, RS3211, Immunoway, California, USA; 1:300, red, RS3608, Immunoway) for 2 h at room temperature and blocked with a blocking solution containing DAPI. Images of FIS1 and SENP3 were obtained using a laser confocal microscope (63x oil lens, LSM 800, ZEISS, Baden-Württemberg, Germany).

Required sample sizes were estimated based on other similar experiments (24, 27). For normally distributed data, results are expressed as mean ± SEM. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s post hoc multiple comparisons test. For non-normally distributed data, values are presented as medians with interquartile ranges. Nonparametric analyses, including Kruskal-Wallis one-way ANOVA, were employed, followed by Dunn’s post hoc correction. All analyses were conducted using GraphPad Prism 10 (GraphPad, California, USA), and all statistical tests were two-sided. Statistical significance was set at P < 0.05.

After 14 days of EA intervention, the SPT results showed that the sucrose preference rate was significantly higher in the EA group compared with the CUMS group (P < 0.05, Figure 1C). Moreover, the TST showed a significant reduction in immobility time in the EA group compared with the CUMS group (P < 0.01, Figure 1D). These findings suggested that EA at GV20 and GV29 alleviated the primary symptoms of depression in CUMS mice, particularly anhedonia and desperate behaviors.

Given the PFC’s crucial role in the pathogenesis of depression, Nissl staining of the PFC was performed to assess neuronal pathological changes. Staining results demonstrated a significant increase in AOD in the EA group compared with the CUMS group (P < 0.05, Figures 1E, F). Additionally, neurons in the EA group appeared more orderly arranged. These observations suggested that EA ameliorated neuronal lesions in the PFC of depressed mice.

TEM results revealed pronounced mitochondrial fragmentation in PFC neurons of the CUMS group, characterized by swelling, cristae disorganization, and reduced mitochondrial diameter. Conversely, PFC neurons in the EA group exhibited normal mitochondrial morphology, intact cristae, and significantly longer diameters and larger regions compared with the CUMS group (P < 0.05, P < 0.05). Furthermore, autophagy lysosomes in PFC neurons were significantly reduced in the EA group compared to the CUMS group (Figures 2A–C). These findings indicated that EA alleviated depression in part by decreasing mitochondrial fragmentation in PFC neurons.

Although mitochondrial fragmentation is typically mediated by DRP1-regulated mitochondrial division, WB did not show a significant difference in DRP1 levels in the PFC between the EA and CUMS groups (P > 0.05; Figures 2D, G). However, further examination of mitochondrial DRP1 ligands MFF and FIS1 revealed that while MFF levels were not significantly different between the groups (P > 0.05; Figures 2E, G), FIS1 expression was significantly elevated in the EA group (P < 0.05; Figures 2D, G). These results suggested that EA might mitigate FIS1-induced mitochondrial fragmentation in depressive PFC.

SUMO is a post-translational modification of FIS1 and is also involved in the pathogenesis of depression (19). Further analysis of SUMO modification in the PFC also showed that EA led to a significant increase in overall SUMO levels (P < 0.01, Figures 3A, B), indicating that EA’s efficacy is associated with enhanced SUMO modification within the PFC. Previous research has demonstrated that FIS1 activity is modulated by sentrin-specific protease 3 (SENP3), a SUMO-modified FIS1-specific hydrolase that deSUMOylates FIS1, thus augmenting its pro-fission activity (20). Our analysis revealed that EA significantly reduced SENP3 levels in the PFC of depressed animals (P < 0.01; Figures 3A, C). Furthermore, IF co-staining of FIS1 and SENP3 indicated that EA significantly decreased their co-localization (Figure 3D). These findings suggested that EA suppressed FIS1 and mitochondrial fragmentation, possibly by reducing SENP3-regulated deSUMOylation of FIS1, which enhancing the SUMOylation of FIS1 in depressed PFC.