SRT1720 ameliorates LPS-induced depressive-like behaviors in mice and activates Parkin-mediated mitophagy

Abstract

Background

Emerging evidence suggests a connection between mitophagy-a key mitochondrial quality control mechanism-and depression. Furthermore, sirtuin 1 (SIRT1), a NAD⁺-dependent deacetylase, has been implicated in the pathophysiology of depression, though its precise role remains elusive. This study aimed to investigate how SIRT1 modulates depressive-like behaviors in mice and to determine whether mitophagy mediates this process.

Methods

Male BALB/c mice were administered lipopolysaccharide (LPS) to mimic depressive-like behaviors. The treatment group received a pre-administration of SRT1720 (50 mg/kg, i.p.), a specific SIRT1 activator. Depressive-like behaviors were assessed by sucrose preference test (SPT) and forced swimming test (FST). Additionally, hippocampal neuronal and mitochondrial ultrastructure was detected via transmission electron microscopy (TEM), and mitophagy-related protein expression was examined by western blotting.

Results

Results demonstrated that activation of SIRT1 significantly mitigated LPS-induced depressive-like behaviors in mice. Moreover, it was observed that SIRT1 activation protected against LPS-induced neuronal and mitochondrial damage in the hippocampus. TEM analysis revealed a marked increase in hippocampal autophagosomes following SIRT1 activation, accompanied by significantly elevated expression of LC3II and Parkin, suggesting enhanced mitophagy. In vitro experiment using HT-22 cells provided additional evidence that SIRT1 activation ameliorated LPS-induced mitochondrial dysfunction and promoted mitophagy via Parkin-mediated pathway.

Conclusions

These findings suggested that activation of SIRT1 could alleviate depressive-like behaviors in mice following LPS challenge, potentially through a Parkin-dependent mitophagy mechanism.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12868-025-00968-2.

Introduction

Depression is a complex and detrimental psychiatric disorder, characterized by a persistent low mood, commonly accompanied by symptoms such as loss of interest or pleasure, fatigue or loss of energy, feelings of worthlessness or guilt, insomnia and suicidal ideation [1]. Data from the 2019 Global Burden of Disease (GBD) show that approximately 280 million individuals worldwide are affected by depression, with an estimated prevalence of 3.8% [2], a figure that continues to escalate. Despite significant strides in clinical and experimental advancements for depression treatment, over one-third of depressive patients [3], and potentially up to 50% [4], exhibit an insufficient or no response to standard antidepressant treatments, imposing a substantial economic and social burden globally [5]. Therefore, the identification of novel targets and the development of innovative therapeutic strategies for antidepressant treatment are still urgently needed.

Accumulating studies have identified a variety of biological mechanisms underlying depression, encompassing genetic and epigenetic factors, monoamines, neurotropins, stress, inflammation, mitochondrial dysfunction and oxidative stress [6]. Given the irreplaceable role of mitochondria in cellular bioenergetics and neural plasticity [7, 8], it has been proposed that mitochondrial dysfunction may be an early event in the pathogenesis of depression [9]. Mitophagy, referring to the selective removal of damaged or superfluous mitochondria through autophagy, is a crucial mechanism of mitochondrial quality control [10]. This process is essential for maintaining mitochondrial function and cellular homeostasis [11]. Recent studies have manifested the involvement of autophagy and mitophagy in the pathogenesis and treatment of depression, with potential mechanisms beginning to be elucidated [12–14]. It has been demonstrated that impaired mitophagy hinders the efficient clearance of damaged mitochondria, leading to the excessive generation of Reactive oxygen species (ROS) and the release of mitochondrial DNA (mtDNA), which can subsequently trigger oxidative stress and provoke inflammatory response [15, 16]. Furthermore, the accumulation of dysfunctional mitochondria can ultimately contribute to cell death [17]. Collectively, there is a considerable amount of evidence supporting a strong link between mitochondrial dysfunction, mitophagy defects and the development of depression [18–23], which provides the possibility of regulation of mitophagy as a promising therapeutic target for the treatment of depression.

Silent information regulator 2 homolog1 (SIRT1) is a class of NAD-dependent histone deacetylase that exerts regulatory control over a myriad of cellular processes, such as gene expression, DNA repair, metabolism, oxidative stress response, mitochondrial function, and other biological processes [24–26]. Previous studies have established the robust presence of SIRT1 in the adult brain, including the cortex, hippocampus, cerebellum and hypothalamus [27]. Furthermore, a wealth of evidence supports the protective effects of SIRT1 overexpression or activation in various neurodegenerative diseases and acute nervous system injuries [28–30]. Clinical studies have shown a downregulation of SIRT1 in the peripheral circulation of patients with depression [31, 32]. Besides, SIRT1 is recognized for its role in the regulation of autophagy and mitophagy [33]. However, the precise mechanisms by which SIRT1 regulates mitophagy in depression and its direct link to depression remain not yet fully elucidated. Therefore, the current study was designed to explore the behavioral and neurobiological effects of SIRT1 modulation and to examine the mechanistic contribution of mitophagy to these effects.

Materials and methods

Animals and treatments

Seven-week-old male BALB/c mice were procured from the Experimental Animal Center of Naval Medical University (Shanghai, China). Animals were maintained in groups of four to five per cage, with unrestricted access to food and water in a temperature-controlled environment (22 ± 2 ℃) with a 12-hour reversed light/dark cycle. After one week of environmental acclimation, these animals were randomly allocated to one of the following groups (n = 8 for each group): control, LPS and LPS + SRT1720 group. All procedures performed during the study were in accordance with the ethical standards of the Ethics Committee of Naval Medical University.

On the day of injection, lipopolysaccharide (LPS; L-3129, serotype 0127: B8; Sigma Aldrich, USA) and SRT1720 (HY-15145; MedChemExpress, USA) were freshly prepared by dissolving the compounds in sterile physiological saline. The model mice were injected intraperitoneally with LPS at a dose of 0.83 mg/kg to induce depressive-like behaviors, a method commonly employed to study the mechanisms underlying inflammation-related depression [34]. SRT1720, a specific activator of SIRT1 known to cross the blood-brain barrier [35], was administered intraperitoneally to the mice at a dose of 50 mg/kg, two hours prior to the LPS injection. The dosage of SRT1720 was determined based on previous research [36–38] and our pilot experiments. Mice in the control group received an equivalent volume of sterile saline.

To further verify the role of SIRT1 in depression and mitophagy, a specific inhibitor of SIRT1-Ex527 (HY-15452; MedChemExpress, USA) was utilized. Following environmental acclimation, mice were randomly assigned to one of the two groups: the control and Ex527 group (n = 8 for each group). Mice in the Ex527 group received daily peripheral injections of Ex527 at a dose of 10 mg/kg for a period of one week [39]. The control group was administered an equal volume of sterile saline.

Behavioral tests

All mice were subjected to behavioral tests 24 h after LPS administration, which were conducted during the dark phase (18:00–20:00) in a quiet environment.

Sucrose preference test (SPT)

Anhedonia, a key symptom of depression, was assessed using SPT based on a two-bottle free-choice paradigm [40]. Mice were first acclimated to a 48-hour sucrose exposure period prior to the test. Two bottles of 1% sucrose solution (weight/volume) were given to the mice for the initial 24 h, after which one bottle of sucrose solution was replaced with tap water for the subsequent 24 h, during which time the positions of the bottles were alternated every 12 h to prevent side bias. Following a 20-hour period of fasting and water deprivation, each mouse was provided with two bottles: one containing 1% fresh sucrose solution and the other containing tap water. The bottles were placed alternately to avoid side preference. The test lasted for 1 h, and the bottles were weighed at the beginning and end to document liquid consumption. The ratio of sucrose consumption to the total consumption of sucrose plus tap water was recognized as the measurement of sucrose preference [41].

Forced swimming test (FST)

The FST was performed as previously described [42]. Mice were placed individually in a transparent Plexiglas cylinder (10 cm in diameter, 25 cm in height) filled with clear water of room temperature (23–25℃) to a depth of 10 cm. Behavioral activity was tracked automatically by a video camera recording for 6 min, with the duration of immobility over the last 4 min being recorded. The mouse was considered immobile if it made slight movements only necessary to keep its head above water, which was indicative of behavioral despair [43].

Sacrifice and sample collection

After behavioral tests, mice were anesthetized with intraperitoneal injection of 1% pentobarbital sodium (50 mg/kg body weight) for tissue collection. A subset of three mice from each group was randomly selected for perfusion. The remaining were sacrificed immediately, then hippocampal tissues were rapidly dissected and kept at -80 ℃ freezer for further experimental procedures.

Transmission electron microscopy (TEM)

After anesthesia, mice were subjected to transcardiac perfusion with ice-cold saline followed by 4% paraformaldehyde. The brains were promptly dissected on ice to isolate hippocampal tissues, which were then trimmed into small blocks (no more than 1 mm³) in the fixative. Subsequently, the samples were post-fixed in 1% osmium tetroxide at room temperature for 2 h, dehydrated through a graded ethanol series (30%, 50%, 70%, 80%, 95% and 100%), and embedded in resin. Ultrathin sections were then cut, and the tissues were obtained with 150-mesh cuprum grids, followed by sequential staining with 2% uranyl acetate saturated alcohol solution and 2.6% lead citrate. For each section, five random fields of view were selected and the images were observed under a TEM (HT7700; Hitachi, Japan).

Cell culture and treatments

The mouse hippocampal neuronal cell line HT-22 was sourced from the American Type Culture Collection (ATCC; Rocville, MD, USA). HT-22 cells were cultured in DMEM supplemented with 10% fetal bovine serum (10099141; Gibco, USA), as well as 1% 100 unit/mL penicillin and 100 µg/mL streptomycin solution (15140122; Gibco, USA) in an incubator at 37 ℃ containing with 5% CO₂ (CCL-170B-8; ESCO, Singapore). When the cells reached approximately 80% confluence, they were digested with 0.25% trypsin (25200; Gibco, USA) and then collected for passaging or seeding. For all experiments, cells in the exponential phase of growth were used and allocated to control, LPS and LPS + SRT1720 groups. Cells were pre-treated with SRT1720 (10 µM) [44] for 2 h before being incubated with LPS (1 µg/mL) for an additional 12 h.

Measurement of mitochondrial membrane potential (MMP)

The fluorescent dye JC-1 (C2003S; Beyotime, Shanghai, China) was utilized to detect the changes in the MMP in HT-22 cells. According to the manufacturer’s protocol, cells were harvested and resuspended in culture medium. An appropriate amount of JC-1 staining solution was added, and the cells were incubated at 37 ℃ for 20 min for staining. Following the incubation, cells were washed twice and resuspended in 1 mL of JC-1 staining buffer for subsequent flow cytometry analysis (CytoFLEX S; Beckman, Germany).

Measurement of intracellular ROS level

The fluorescent probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) (S0033S; Beyotime, Shanghai, China) was used to measure the intracellular ROS levels in HT-22 cells. After digestion with 0.25% tryspin, cells were collected into 1.5 mL centrifuge tubes. Post-centrifugation, cells were resuspended in DCFH-DA solution diluted to a final concentration of 10 µM and incubated in the dark at 37 ℃ for 20 min. Afterwards, cells were washed three times with serum-free medium and resuspended in 0.5 mL of sterile PBS for flow cytometry analysis.

Measurement of adenosine triphosphate (ATP) content

The relative ATP content of HT-22 cells was determined using an ATP assay kit (S0026; Beyotime, Shanghai, China). Briefly, cells were lysed with an appropriate volume of ATP lysis buffer, followed by centrifugation (AG 22331 Hamburg; Eppendorf, Germany) at 4 °C at 12,000 g for 5 min. The supernatants were extracted and temporarily stored on ice for further analysis. Standard solutions with certain concentration gradients and working reagent were freshly prepared. Ultimately, the luminescence of both standard solutions and extracted supernatants of the samples was analyzed using a microplate reader (Synergy HTX; BioTek, America).

Western blotting

Hippocampal tissues and treated HT-22 cells were lysed by RIPA (P0013B, Beyotime, Shanghai, China) with protease (B14001; Bimake, USA) and phosphatase inhibitor (B15001; Bimake, USA) on the ice. Protein concentrations were determined by the Enhanced BCA Protein Assay Kit (P0010S; Beyotime, Shanghai, China). Then, total protein extracts were obtained from the supernatants after centrifugation at 12,000 rpm, 4 ℃ for 20 min, heated at 100 °C for 10 min and subsequently stored at -20 °C. For western blotting analysis, proteins were separated by 10% or 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by being transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were sealed with blocking buffer (NCM Biotech, Suzhou, Jiangsu, China) for 10 ~ 20 min and then incubated with specific, diluted primary antibodies at 4 ℃ overnight (anti-SIRT1: #ab189494, 1:1000, Abcam, USA; anti-LC3B: #381544, 1:1000, Zenbio, China; anti-Parkin: #2132S, 1:500, Cell Signaling Technology, USA; anti-GAPDH: #5174S, 1:1000, Cell Signaling Technology, USA). On the subsequent day, the membranes were incubated with IRDye conjugated secondary antibodies (1:5000; LI-COR, Inc., USA) at room temperature for 1 h, followed by visualization using the Odyssey Infrared Imaging System (LI-COR, Inc., USA). The quantification of protein expression levels was further performed by Image J Software (NIH, Bethesda, MD, USA).

Statistical analysis

All data were analyzed using GraphPad Prism 9.0 (GraphPad Software, Inc., La Jolla, CA, USA), with results presented as mean ± Standard error (SEM). One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was utilized to compare differences among multiple groups. Comparisons between two groups were made using two-tailed Student’s t-tests. Statistical significance was set at p < 0.05 for all analyses.

Results

SRT1720 alleviated LPS-induced depressive-like behaviors in mice

The decreased preference for sucrose in the SPT and the increased duration of immobility in the FST were regarded as indicative of anhedonia and behavioral despair in mice subjected to various treatments. As depicted in Fig. 1, significant differences existed among the groups in both the SPT (F = 17.230, p < 0.001) and the FST (F = 9.079, p = 0.001). Specifically, compared with the control group, LPS-treated mice displayed a notable reduction in sucrose preference in the SPT (p < 0.001), coupled with a significant increase in immobility time in the FST (p = 0.001). Conversely, administration of SRT1720 effectively retrieved these outcomes, as evidenced by an increase in sucrose preference (p = 0.004) and a decrease in immobility time (p = 0.025) compared to the LPS group.

Fig. 1.

SRT1720 alleviated LPS-induced depressive-like behaviors in mice. A The percentage of sucrose preference in the sucrose preference test. B The immobility time in the forced swimming test. N = 8 for each group. Data were presented as mean ± SEM. ** p < 0.01, *** p < 0.001, LPS vs. Control; # p < 0.05, ## p < 0.01, LPS + SRT1720 vs. LPS

SRT1720 alleviated the injury of hippocampal neurons induced by LPS

The hippocampal neuronal ultrastructure was examined by TEM. Figure 2 presents the representative ultrastructure of hippocampal neurons among different groups. The hippocampal neurons in the control group typically exhibited an intact and continuous cell membrane, homogeneous cytoplasm, a clear nucleus with evenly dispersed chromatin and a distinct double-layered nuclear membrane, along with a normal appearance of rough endoplasmic reticulum (RER), Golgi apparatus (Go) and mitochondria. In contrast, after LPS treatment, the hippocampal neurons sustained significant damage, characterized by an irregular cell shape, a blurred nuclear membrane, distended endoplasmic reticulum with degranulation, and an enlarged Golgi apparatus. Additionally, the mitochondria were severely damaged (described in detail below). However, administration of SRT1720 notably ameliorated LPS-induced alterations in hippocampal neuronal ultrastructure, restoring a more typical appearance. Furthermore, autophagosomes encapsulating cellular components were observed within the hippocampal neurons of mice that received SRT1720 treatment, a phenomenon that was relatively rare in both the control and LPS-treated groups.

Fig. 2.

SRT1720 alleviated the injury of hippocampal neurons induced by LPS. A Scale bar = 5 μm. B Scale bar = 2 μm. N = 3 for each group. Red arrows represent autophagosomes. N, nucleus; M, mitochondria; RER, rough endoplasmic reticulum; Go, Golgi apparatus

SRT1720 protected against the mitochondrial damage in hippocampal neurons induced by LPS

To delve deeper into the effect of SRT1720 on the mitochondria of hippocampal neurons, the mitochondrial ultrastructure was detected and the number of mitochondria was determined under TEM, as depicted in Fig. 3. Compared with the control group, the majority of mitochondria in the hippocampal neurons of the LPS group showed profound swelling with ruptured membranes, disintegrated matrix, and fractured or vanished cristae. Some severely damaged mitochondria even presented a vacuolar structure. However, when pre-treated with SRT1720, the mitochondrial morphology closely resembled that observed in the control group. Moreover, to quantitatively count the number of mitochondrial per neuron, two random images from each sample were selected (resulting in 6 images for each group). As illustrated in Fig. 3B and C, there was significant differences in the number of mitochondria among the groups (F = 8.909, p = 0.003). The LPS group showed a notable reduction in mitochondria number compared to the control group (p = 0.003), whereas a significant increase was observed in the LPS + SRT1720 group (LPS + SRT1720 vs. LPS, p = 0.026), indicating that SRT1720 might exert a protective effect on the mitochondria of hippocampal neurons.

Fig. 3.

SRT1720 protected against the mitochondrial damage in hippocampal neurons induced by LPS. A Scale bar = 1 μm. B Scale bar = 5 μm. N = 3 images for each group. M, mitochondria. C The average number of mitochondria per neuron (N = 6 images for each group). Data were presented as mean ± SEM. ** p < 0.01, LPS vs. Control; # p < 0.05, LPS + SRT1720 vs. LPS

SRT1720 activated mitophagy to eliminate damaged mitochondria in hippocampus

In addition to the detection of autophagosomes, western blotting was performed to measure the expression levels of mitophagy-related marker proteins, aiming to further examine the effect of SRT1720 on mitophagy in the hippocampus, with a focus on the E3 ubiquitin ligase Parkin-dependent mitophagy pathway. The expression of SIRT1 was also investigated in this experiment. As shown in Fig. 4A and B, exposure to LPS led to a significant decrease in SIRT1 expression (F = 15.880, p = 0.001; LPS vs. Control, p < 0.001). However, treatment with SRT1720 markedly upregulated SIRT1 expression in the hippocampus (p = 0.026). Furthermore, LPS treatment resulted in a decrease in LC3II protein levels (F = 6.769, p = 0.016; LPS vs. Control, p = 0.033) and an increase in Parkin levels (F = 14.930, p = 0.001; LPS vs. Control, p = 0.016) compared to the control group. Administration with SRT1720 significantly upregulated the expression of LC3II (p = 0.023) relative to the LPS-only group and further upregulated Parkin expression (LPS + SRT1720 vs. Control, p = 0.001). These results implied that the activation of Parkin-mediated mitophagy might be involved in the antidepressant effects of SRT1720.

Fig. 4.

SRT1720 upregulated the expression of SIRT1 and activated Parkin-mediated mitophagy pathway in the hippocampus. A The representant blot of SIRT1. B The relative expression of SIRT1. C The representant blots of Parkin and LC3II. D The relative expression of Parkin. E The relative expression of LC3II. N = 4 for each group. Data were presented as mean ± SEM. * p < 0.05, *** p < 0.001, LPS vs. Control; # p < 0.05, LPS + SRT1720 vs. LPS; ## p < 0.01, LPS + SRT1720 vs. Control

SRT1720 restored the depolarization of MMP induced by LPS in HT-22 cells

In healthy mitochondria with a normal MMP, the JC-1 dye penetrates and accumulates within the mitochondria, forming J-aggregates that emit red fluorescence. In contrast, when the mitochondrial membrane becomes depolarized, most JC-1 dye is unable to accumulate within the mitochondria and instead remains in the cytoplasm as a monomeric form, exhibiting green fluorescence (Sivandzade et al., 2019). The ratio of red to green fluorescence intensity is negatively related with the degree of mitochondrial depolarization. The results of flow cytometry analysis revealed a distinct decline in MMP in HT-22 cells following LPS treatment, as evidenced by the increased proportion of cells with depolarized mitochondria (Fig. 5A). Moreover, the ratio of red to green fluorescence intensity in the LPS group was significantly lower than that of the control group (F = 10.380, p = 0.011; LPS vs. Control, p = 0.015), indicating a higher level of mitochondrial depolarization. However, treatment with SRT1720 markedly increased this ratio (p = 0.022) (Fig. 5B), suggesting that SRT1720 promoted the restoration of MMP.

Fig. 5.

SRT1720 improved mitochondrial function of LPS-treated HT-22 cells. A and B Mitochondrial membrane potential of HT-22 cells was determined by flow cytometry with JC-1. C and D Intracellular ROS of HT-22 cells was determined by flow cytometry with DCFH-DA. E Relative ATP concentrations of HT-22 cells was determined by chemiluminescence assay. N = 3 for each group. Data were presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, LPS vs. Control; # p < 0.05, ## p < 0.01, LPS + SRT1720 vs. LPS

SRT1720 reduced the intracellular ROS induced by LPS in HT-22 cells

Intracellular ROS are primarily derived from mitochondria, and excessive accumulation of ROS can lead to cellular toxicity and oxidative stress [45, 46]. In this study, the production of total cellular ROS was measured using the DCFH-DA probe, with the level of ROS indicated by the intensity of DCF fluorescence detected by flow cytometry. As presented by Fig. 5C and D, the level of intracellular ROS was notably elevated in LPS-treated HT-22 cells (F = 21.450, p = 0.002; LPS vs. Control, p = 0.006). However, pretreatment with SRT1720 substantially attenuated ROS generation (p = 0.002).

SRT1720 restored the ATP content decreased by LPS in HT-22 cells

ATP serves as a crucial biomarker of mitochondrial function, given that mitochondria are the predominant producers of ATP that is pivotal for a wide array of cellular processes. In this study, the ATP content of HT-22 cells was measured by a luciferase-based luminescence assay, and the results are displayed in Fig. 5E. The results showed that LPS treatment contributed to a significant reduction in relative ATP concentrations in HT-22 cells (F = 42.490, p < 0.001; LPS vs. Control, p < 0.001). However, pretreatment with SRT1720 markedly restored the ATP concentrations (p = 0.002).

SRT1720 activated Parkin-mediated mitophagy in HT-22 cells treated by LPS

To substantiate that the protective effect of SRT1720 against LPS challenge was associated with the enhancement of mitophagy, the expression of mitophagy-related proteins in HT-22 cells was investigated, as shown in Fig. 6. Western blotting analysis presented that the expression levels of LC3II (p < 0.001) and Parkin (p < 0.001) were significantly upregulated in response to SRT1720 treatment when compared to the LPS-treated group.

Fig. 6.

SRT1720 activated Parkin-mediated mitophagy pathway in HT-22 cells treated by LPS. A The representant blots of Parkin and LC3II. B The relative expression of Parkin. C The relative expression of LC3II. N = 3 for each group. Data were presented as mean ± SEM. # p < 0.05, LPS vs. Control; ### p < 0.001, LPS + SRT1720 vs. LPS; ** p < 0.01, *** p < 0.001, LPS + SRT1720 vs. Control

Inhibition of SIRT1 induced depressive-like behaviors in mice and damaged mitophagy in hippocampus

Additionally, the activity of SIRT1 was inhibited by the intraperitoneal administration of Ex527. As illustrated by Fig. 7, mice treated with Ex527 showed a reduced preference for sucrose in the SPT (t = 9.125, p < 0.001), and a longer immobility time in the FST compared to the control group (t = 3.866, p = 0.002). Furthermore, the expression levels of SIRT1 and mitophagy-related proteins in the hippocampus were assessed by western blotting (Fig. 8). The findings revealed that inhibition of SIRT1 downregulated the protein level of SIRT1 (t = 2.732, p = 0.034), Parkin (t = 4.391, p = 0.005) and LC3II (t = 3.738, p = 0.010), indicating that SIRT1 inhibition impaired the process of Parkin-mediated mitophagy, which might lead to the induction of depressive-like behaviors in mice.

Fig. 7.

SRT1720 upregulated the expression of SIRT1 and activated Parkin-mediated mitophagy in the hippocampus. A The percentage of sucrose preference in the sucrose preference test. B The immobility time in the forced swimming test. N = 8 for each group. Data were presented as mean ± SEM. ** p < 0.01, *** p < 0.001

Fig. 8.

Inhibition of SIRT1 impaired Parkin-mediated mitophagy pathway. A The representant blot of SIRT1. B The relative expression of SIRT1. C The representant blots of Parkin and LC3II. D The relative expression of Parkin. E The relative expression of LC3II. N = 4 for each group. Data were presented as mean ± SEM. * p < 0.05, ** p < 0.01

Discussion

In the current study, acute activation of the peripheral innate immune system through LPS administration was utilized to induce depressive-like behaviors in mice [47], a method grounded in the well-established link between depressive symptoms and inflammation in patients with depression [48]. LPS is a potent stimulator, initiating the synthesis and release of a variety of cytokines, such as IL-1, IL-6 and TNF-α, in both peripheral and central cells within 2 to 6 h post-administration, with the level of cytokines returning to baseline by 24 h post-administration [49]. Importantly, the depressive-like behaviors induced by LPS at 24 h post-LPS are dissociated from the sickness behaviors observed at 6 h post-LPS [50]. Accordingly, the depressive-like behaviors in mice were judged by a reduction in sucrose preference in the SPT and an increase in immobility time in the FST at 24 h following LPS administration in this study. Nevertheless, the employment of SRT1720, a specific activator of SIRT1, significantly alleviated the depressive-like behaviors in mice and offered neuroprotection against the deleterious effects of LPS on hippocampal neurons and mitochondria. In addition, the expression levels of mitophagy-related proteins were found to be upregulated by the activation of SIRT1. Subsequent in vitro experiment further proved that activation of SIRT1 in HT-22 cells reversed LPS-induced depolarization of MMP, reduced elevated levels of intracellular ROS and restored ATP content, while also promoting mitophagy through Parkin-dependent pathway.

In line with previous studies, activation of SIRT1 through intraperitoneal injection of SRT1720 exhibited antidepressant-like effects in LPS-treated mice. As a potent activator of SIRT1, SRT1720 has demonstrated the capacity to alleviate neuronal injury and facilitate functional recovery across diverse neurological disorders [37, 51]. In this study, we also observed that inhibition of SIRT1 elicited depressive-like behaviors in mice, reinforcing the antidepressant role of SIRT1. Moreover, in our pilot study, mice were administered SRT1720 alone at a dose of 50 mg/kg weight. The results indicated that SRT1720, when given alone, did not significantly alter sucrose preference in the SPT or immobility time in the FST compared to the control group, suggesting that SRT1720 at this dosage was non-toxic to mice (Fig. S1). Therefore, SRT1720 was not administered alone to mice in subsequent experiments.

It is noteworthy that SIRT1 has been identified as a genetic variant that contributes to the risk of major depressive disorder in a large-scale genome-wide association study [52]. Moreover, SIRT1 gene expression in the periphery has been found to be significantly downregulated, by 37%, in patients with MDD compared to controls [31]. Consistent with this, Animal experiments reported that chronic stress decreased SIRT1 activity in the hippocampal dentate gyrus of mice, and the suppression of hippocampal SIRT1 function by pharmacologic or genetic approaches exacerbated depressive-like behaviors; by contrast, activation of SIRT1 inhibited the development of depression-related phenotypes and abnormal dendritic structures caused by chronic stress [53]. Similarly, specific activation of SIRT1 in the hippocampus was demonstrated to reverse depressive-like behaviors induced by chronic unpredictable mild stress (CUMS) in mice, potentially by modulating microglia polarization towards the M2 phenotype [54]. However, there are conflicting reports regarding the role of SIRT1 in depression. It was suggested that downregulation of SIRT1 expression in the basolateral amygdala of mice was protective against CUMS-induced depressive-like behaviors and synaptic abnormalities, whereas SIRT1 overexpression could be a direct cause of depressive-like symptoms [55]. The disparate actions of SIRT1 in depression might be attributed to various factors, including different origins and extents of stress, subcellular localizations, animal backgrounds, or administration methods. The potential implications of SIRT1 in the pathophysiology of depression warrant further investigation.

SIRT1 has been shown to be predominantly expressed in neurons within the brain [56, 57], especially under conditions of injury or stress [30]. In light of this, the ultrastructure changes of neuron and mitochondria in the hippocampus of mice were detected under TEM. The dilation of the endoplasmic reticulum and Golgi complex, which is indicative of the accumulation of dysfunctional proteins, is regarded as the highly characterized marker of cellular stress [58]. Our study observed obvious damage to hippocampal neurons following LPS treatment, with severely dilated and deformed mitochondria, as well as a significantly reduced mitochondrial count. However, administration of SRT1720 led to an apparent improvement in the ultrastructure of neurons and mitochondria compared to the LPS-treated group. Mitochondrial dysfunction and mitochondrial ultrastructural damage in the brains of mice exposed to chronic stress have been previously reported [59]. Besides, mitochondrial dysfunction or damage can trigger a range of responses, including the accumulation of ROS, aberrant calcium mobilization, reduction in cytoplasmic NAD⁺ levels and potassium efflux, which can initiate an inflammatory cascade, exacerbate mitochondrial damage and potentially result in cell death [60]. Due to the high demands for energy in the central nervous system, the maintenance of mitochondrial homeostasis is essential for preserving normal neural activity and function.

Mitophagy can dispose of damaged mitochondria, serving as a specific mechanism for mitochondrial quality control to maintain mitochondrial homeostasis, particularly in the face of stress [61]. Palikaras et al. have classified mitophagy into basal, stress-induced or programmed forms under physiological conditions [62], all of which exert crucial effects on mitochondrial quality control. From a molecular perspective, two major pathways have been well-established in mitophagy: ubiquitin-dependent and ubiquitin-independent pathway. Here, we focused on the Parkin-mediated ubiquitin pathway, which is the most extensively researched and is conserved across species, including Caenorhabditis elegans, Drosophila melanogaster and mammals [63]. Furthermore, two forms of microtubule-associated protein 1 light chain 3 (LC3) have been identified within cells (LC3I and LC3II), among which LC3II has emerged as an ideal marker for monitoring autophagosomes [64]. In this study, we found that SRT1720 promoted the formation of autophagosomes in hippocampal neurons and elevated the expression levels of mitophagy-related proteins (Parkin and LC3II), implying that SRT1720 activated Parkin-mediated mitophagy. Concurrently, we observed that administration of Ex527 led to a decrease in the expression levels of Parkin and LC3II, suggesting that inhibition of SIRT1 might impair Parkin-mediated mitophagy. Although LPS was found to upregulate the expression of Parkin, which might be the initiation of mitophagy in response to stress [65], the decreased expression of LC3II and the severe mitochondrial damage observed under TEM suggested the impairments in the mitophagy process, such as a failure to form autophagosomes. Notably, the concurrent improvement in depressive-like behaviors and the increase in autophagosomes in hippocampal neurons of SRT1720-treated mice implied that activation of SIRT1 might facilitate the elimination of damaged mitochondria through Parkin-mediated mitophagy, thereby preserving mitochondrial and neuronal physiological function. Beyond its role in mitophagy, Parkin also plays a crucial part in promoting mitochondrial biogenesis that generates new mitochondria, which acts in cooperation with mitophagy to maintain mitochondrial quality control [66].

The in vitro findings broadly matched those observed in in vivo experiments, further confirming the effect of SRT1720 on HT-22 cells and mitochondrial function challenged by LPS. The results showed that LPS induced the depolarization of MMP, elevated ROS levels and decreased ATP content in HT-22 cells, which are hallmarks of mitochondria dysfunction [67, 68]. It has been widely documented that mitochondrial dysfunction can lead to excessive production of ROS, and the accumulation of intracellular ROS can further impair mitochondrial function and neuronal homeostasis [69]. However, activation of SIRT1 was shown to reserve these effects by restoring MMP, reducing ROS levels and increasing ATP content in HT-22 cells, indicating the improvement of mitochondrial function. Furthermore, activation of SIRT1 upregulated the protein levels of LC3II and Parkin in HT-22 cells. Existing research has identified the loss of MMP as an early indicator of cellular apoptosis and a trigger signal for the initiation of mitophagy via the PINK1/Parkin pathway [70]. Therefore, it could be assumed that activation of SIRT1 could improve mitochondrial function via promoting Parkin-mediated mitophagy to eliminate damaged and dysfunctional mitochondria. In addition, it is noteworthy that no significant difference in Parkin expression was observed between the control and LPS groups in HT-22 cells, a finding that was inconsistent with the results from animal experiments. Discrepancies between in vivo and in vitro experiments and the varying time points at which detection was carried out might account for this inconsistent.

Although these findings provide meaningful insights, important methodological limitations must be considered. Mitophagy is a complex, multi-stage process involving mitochondrial membrane depolarization, autophagosome formation, lysosomal trafficking, and eventual degradation [71]. However, our current study only examined a single time point (24 h post-LPS in vivo and 12 h in vitro). In further experiments, we intend to measure the dynamic changes of mitophagy at multiple time points, such as 6, 12 and 48 h post LPS treatment, as well as across different animal models of depression. Importantly, employing a diverse array of approaches to monitor autophagic flux would provide a more comprehensive assessment of this process.

Conclusions

To sum up, this study demonstrated that SIRT1 activation exerted protective effects against depression by enhancing Parkin-mediated mitophagy. Our findings showed that SIRT1 inhibition induced depressive-like behaviors in mice, whereas SIRT1 activation alleviated these behaviors and protected against LPS-induced hippocampal neuronal and mitochondrial damage. Mechanistically, this protective effect occurred through the activation of Parkin-dependent mitophagy pathway. These findings strengthen the evidence linking SIRT1 and mitophagy to depression pathogenesis, highlighting their potential as therapeutic targets for depression treatment.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Luna Sun , Chaoran Li, Jianli Shi, Shunlun Wan and Yunxia Wang designed the study and wrote the protocols. Luna Sun , Chaoran Li, Wenfeng Zeng and Lingling Wu participated in performing the experiments. Luna Sun , Chaoran Li, Jianli Shi undertook the statistical analysis and wrote the manuscript, and then all authors participated in the revision. All authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81771301].

Data availability

The data analyzed during the current study are not publicly available but are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

All procedures performed during the study were in accordance with the ethical standards of the Ethics Committee of Naval Medical University. All animal experiments were carried out in strict accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 85–23) and the ARRIVE guidelines 2.0.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.