Capsazepine Treatment Alleviates Depression-Like Behaviors via TRPV1-Mediated Modulation of Neuroinflammation and Neurogenesis

Capsazepine Treatment Alleviates Depression-Like Behaviors via TRPV1-Mediated Modulation of Neuroinflammation and Neurogenesis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Capsazepine Treatment Alleviates Depression-Like Behaviors via TRPV1-Mediated Modulation of Neuroinflammation and Neurogenesis Junjie Huang, Chen Li, Hailong Ge, Lan Wu, Ling Xiao, Yinping Xie, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6992017/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Molecular Neurobiology → Version 1 posted 15 You are reading this latest preprint version Abstract Depression is a common mental disorder with a currently unclear pathogenesis, and it often fails to repond to treatment. Transient receptor potential vanilloid 1 (TRPV1) is known to regulate glial activation and inflammatory responses, yet its specific role in stress-related depressive pathophysiology remains incompletely understood. In this study, we explored the effects of modulating TRPV1 in neuroinflammation and neurogenesis underlying depression-like behaviors. A chronic social defeat stress (CSDS) mice model, followed by behavioral tests, was used to evaluate the antidepressant potential of capsaicin (CAP) and capsazepine (CPZ). Then, western blotting, elisa, and immunofluorescence were employed to assess microglial activation, the pro-inflammatory cytokine levels, neurogenesis, and JAK2/STAT3 signaling pathway in hippocampus tissues. The JAK2 agonist coumarin A1 (CA1) was used to verify the involvement of the JAK2/STAT3 pathway. In CSDS-susceptible mice, the protein expression of TRPV1 and the levels of p-CaMKIIα were elevated, and CPZ downregulated the expression of these indicators. Also, CSDS reduced the migcroglial numbers and altered microglial morphology in the subregions of the hippocampus; these results were accompanied by proinflammatory cytokine production. In parallel, TRPV1 inhibition by CPZ suppressed promoted neurogenesis, which was testified by increased BrdU + and DCX + cells. Further, CPZ exerts its regulatory effects through inhibition of the JAK2/STAT3 signaling pathway. CA1 reversed the neuroprotective effects of CPZ, confirming the involvement of the JAK2/STAT3 pathway. The discovered microglia-mediated mechanism provides novel insight into how TRPV1 modulation ameliorate stress-induced neuroinflammation and impaired neurogenesis These findings identify microglial TRPV1 as a potential therapeutic target for depression. Depression TRPV1 Neuroinflammatory Neurogenesis JAK2/STAT3 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1.Introduction Depression is one of the most prevalent mood disorders, affecting over 350 million individuals worldwide[1]. Its onset and progression are the result of the interplay of genetic, environmental, and social factors[2]. Currently, fewer than one-third of patients with depression achieve clinical remission following six weeks of monotherapy at conventional dosages, while 20–50% exhibit inadequate treatment response[3]. This underscores the critical need to identify novel therapeutic targets to address these treatment limitations. While the precise etiology of depression remains unclear, increasing studies suggest a strong association between the endocannabinoid system (ECS) and the involvement of depression[4–6]. The cannabinoid receptors, as a primary elements of the ECS, modulate monoaminergic neurotransmitter, particularly serotonin and norepinephrine release [7]. Moreover, these receptors regulate fundamental neurobiological processes including hippocampal neurogenesis, synaptic plasticity, and functional connectivity within the basolateral amygdala-nucleus accumbens circuit[8]. These findings collectively position endocannabinoid receptors as promising therapeutic targets for major depressive disorder Recently, transient receptor potential vanilloid type 1 channel (TRPV1) is identified as a novel cannabinoid receptor which is highly expressed in the central nervous system (CNS)[9]. TRPV1 is a ligand-gated, non-selective cation channel that triggers influx of Ca 2+ , leading to an increase in intracellular ion concentrations[10]. These results in depolarization of both the plasma and organellar membranes, activating downstream Ca 2+ signaling pathways that initiate a series of intracellular biochemical reactions[11]. Previous studies have indicated that TRPV1 mediates depressive-like behaviors in animals. Pharmacological blockade of TRPV1 such as injection with capsazepine (CPZ, a TRPV1 antagonist) reversed the behavioral deficits caused by stress in rodent[12, 13]. From an epigenetic perspective, Wang et.al revealed that TRPV1 regulated emotional responses in mice by interacting with histone deacetylase 2, and TRPV1 deficiency increased mice’s resistance to chronic stress[14, 15]. Interestingly, low-dose capsaicin (CAP, a TRPV1 agonist), which activates the S4-S5 linker and C-terminal domain, was found to alleviate depression-like behaviors, as evidenced by reduced immobility in the forced swimming test[16]. Although these studies have established an association between TRPV1 and the modulation of depressive-like behaviors, the underlying mechanisms remain poorly understood and warrant further investigation[17]. In the present study, mice received intraperitoneal injection (i.p.) of CAP and CPZ separately to investigate the molecular mechanisms underlying TRPV1 channel modulation and its effects on depression-like behaviors. Neuroinflammation represents a critical component of the neurobiological mechanisms underlying depression[18]. The activated inflammatory signaling pathways, elevated oxidative stress, disrupted blood-brain barrier integrity, and peripheral immune cell infiltration constitute key mechanisms that exacerbate neuroinflammation and ultimately contribute to depressive-like behaviors[19]. Microglia, the resident immune cells of the brain, demonstrates altered activity patterns in depression pathology[20]. Its dysregulation drives neuroinflammatory responses that exacerbate depressive-like behaviors. TRPV1 channels are abundantly expressed on microglial membranes, where they might regulate microglial activity and contribute to neuroinflammatory processes[21]. Currently, there are two main perspectives on TRPV1’ role in neuroinflammation through microglial regulation. Some views argue that TRPV1 inhibition suppresses neuroinflammation, alleviating its effects[22], while others propose that TRPV1 activation exerts a protective effect in neuropsychiatric disorders by reducing neuroinflammation[23]. These findings indicate that TRPV1 plays paradoxical roles in neuroinflammation, and its precise molecular mechanisms remain to be fully elucidated[24]. Substantial evidences suggest that neuroinflammation contributes to depression pathogenesis through not only immune-mediated mechanisms but also impaired adult hippocampal neurogenesis (AHN), representing an additional pathway in depression development[25, 26]. Neuroinflammation can suppress neurogenesis via the neurotoxic effects of inflammatory cytokines, including IL-6, IL-1β, and TNF-α, or by disrupting the brain’s microenvironment. Conversely, impaired neurogenesis may exacerbate neuroinflammation by indirectly modulating the inflammatory state through altered neurotransmitter secretion and synaptic plasticity[27, 28]. Ultimately, excessive neuroinflammation and deficient neurogenesis form a self-reinforcing cycle, contributing to the pathogenesis of psychiatric disorders. The janus kinase2 (JAK2)/signal transducers and activators of transcription3 (STAT3) pathway plays a key role in the CNS by coordinating inflammatory responses, cell proliferation and gene expression[29]. Recent studies have shown that specific inhibition of the JAK2/STAT3 pathway not only reduces neuroinflammation but also improves depressive-like behaviors, which provides a potential target for the development of novel antidepressant drugs[30, 31]. A study showed that the JAK2/STAT3 pathway involved in TRPV1-mediated disorders of the nervous system, however, the mechanisms by which TRPV1 contributes to depression via neuroinflammation and impaired neurogenesis have not been fully elucidated. In the present study, the chronic social defeat stress (CSDS) paradigm was utilized in mice to model depression, and parallel interventions with CAP and CPZ were administered. Subsequently, we explored whether CSDS has influenced hippocampal TRPV1 expression. On this basis, to further investigate the changes of molecular markers associated with neuroinflammation and neurogenesis after treatment with CAP or CPZ, along with alterations in the JAK2/STAT3 signaling pathway. To further clarify the involvement of this pathway in the regulation of TRPV1, we administered Coumarin A1 (CA1), a selective JAK2/STAT3 inhibitor, and assessed its impact on the phenomena observed. This study investigates how TRPV1 regulation affects neuroinflammation and neurogenesis under chronic stress, providing mechanistic insights into depression and potential therapeutic targets. 2. Methods 2.1. Animals All animals used in this study were male C57BL/6 mice (20–25 g) of specific pathogen-free grade, purchased from the Laboratory Animal Center of Three Gorges University. All mice were housed in cages (21.5cm × 32cm ×17cm, six per cage) and maintained under controlled conditions at a temperature of 22–24°C and humidity of 60–65% in the animal facility of the First Clinical College of Wuhan University, which is free of specific pathogens. This experiment was reviewed and approved by the Ethics Committee of Renmin Hospital, Wuhan University, and was conducted in strict accordance with institutional and national regulations. 2.2. CSDS CSDS protocols were performed as described previously and shown in Fig. 1 A. C57BL/6 J mice were exposed to 10 min of physical aggression by an intruder male CD-1 mouse[32]. Following the defeat session, experimental mice were housed in the same cage as the CD-1 mice on the opposite side of a transparent and perforated plexiglass divider to maintain sensory contact for 24 h. This procedure was repeated for 10 consecutive days with a new CD-1 aggressor on each day. Control mice were pair housed in similar cages, separated from one another by a perforated plexiglass divider and rotated to a new one each day for 10 days. 2.3. Experimental design In experiment 1, mice were randomly divided into four groups: CON + PBS (n = 8), CSDS + PBS (n = 8), CSDS + CAP (n = 12), CSDS + CPZ (n = 12). CAP and CPZ were purchased from MedChemExpress (MCE). These compounds were dissolved in DMSO and prepared in PBS. The final concentrations were as follows: CPZ at 5 mg/kg/day [33, 34], CAP at 2.5 mg/kg/day [35, 36]. For drug administration, PBS, CPZ, and CAP were administered via intraperitoneal injection at the initiation of modeling and continued for 10 consecutive days. A flowchart of this procedure is shown in Figure. 1A. In experiment 2, mice were randomly divided into four groups: CON + PBS (n = 6), CSDS + PBS (n = 6), CSDS + CPZ (n = 6), CSDS + CPZ + CA1(n = 6). CA1 was purchased from MCE. The final concentration of CA1 was 100 µM [37]. For drug administration, PBS and CPZ were administered via intraperitoneal injection at the initiation of modeling and continued for 10 consecutive days. CA1 was administered via intraperitoneal injection during the last 3 days of the model. A flowchart of this procedure is shown in Figure. 1B. 2.4. Behavioral Testing Prior to the experiment, mice were acclimated to the room conditions for at least 1 hour. After each test, the equipment was thoroughly cleaned with 75% alcohol to eliminate the scent of the previous animal. 2.4.1. Social interaction test (SIT) The SIT was conducted 24 hours after the last CD-1 attack. The test consisted of two trials. In the first trial, the experimental mice were placed in a box (45 cm × 45 cm × 45 cm) for 2.5 minutes. A wire mesh frame (10 cm × 4.5 cm) was placed in the interaction area, and the time the test mice spent in the interaction area was recorded as "no target time"[38]. In the second trial, a stranger CD-1 mouse was placed in the wire mesh frame, and the experimental mice was returned to the box for an additional 2.5-minute test. The time spent in the interaction area during this trial was recorded as "target time" .Video tracking was performed using Maze software (Store Company, Wood Dale, IL, USA). The social interaction ratio was calculated by dividing target time by no target time[39]. 2.4.2. Sucrose preference test (SPT) Day 1: mice were individually housed and allowed to adapt to both 1% sucrose solution and tap water for 24 hours. Day 2:food and water were withheld for 24 hours. Day 3: the initial weight of the bottles was recorded, and two bottles were placed in the metal lid of the cage, with food positioned on the opposite side of the wire lid. At the halfway point of the experiment, the positions of the two bottles were switched to prevent location bias. After 24 hours, water consumption was assessed by weighing the bottles. Sucrose preference was calculated using the formula: Sucrose preference (%) = (Sucrose solution intake / (Sucrose solution intake + Water intake)) × 100%. 2.4.3. Tail suspension test (TST) In TST, mice were positioned head-down, maintaining a distance of approximately 30 cm from the bottom of the suspension box. The experiment lasted for a total of 6 minutes. Struggling behavior during the first 2 minutes (habituation period) was not recorded, while immobility time was recorded during the subsequent 4 minutes. 2.4.4. Forced swimming test (FST) FST was conducted using a cylindrical, translucent water tank (15 cm × 30 cm). Prior to the experiment, the tank was filled with tap water maintained at 25 ± 1°C. Each mouse was placed in the water and subjected to forced swimming for 6 minutes. Immobility time was recorded during the last 4 minutes of the test. 2.4.5. Open field test (OFT) Mice were placed in the central area of a semi-transparent, square plastic box (50 cm × 50 cm × 40 cm). Behavioral tracking was conducted using EthoVision XT 11.5 software (Noldus Information Technology, Netherlands), which automatically recorded the movement of the mice within the box. The total test duration was 5 minutes. After each trial, the box was cleaned with 75% ethanol to remove urine and feces, eliminating residual odors that could affect subsequent tests. 2.5. Western blotting (WB) Phosphatase and protease inhibitors were added to RIPA lysis buffer at a fixed ratio. Frozen hippocampal tissues from mice were homogenized in ice-cold RIPA buffer. Protein concentration was determined using the bicinchoninic acid (BCA) assay. The BCA assay kit was obtained from Beyotime Biotechnology. The protein samples were mixed with loading buffer and boiled at 100°C. Total extracted protein (20 µg/ per lane) was separated by 10–12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After transfer, the membrane was blocked with a rapid blocking solution for 15 minutes, followed by overnight incubation with the primary antibody at 4°C. The next day, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour, followed by chemiluminescent detection using an imaging system (Bio-Rad, USA). Band intensity was analyzed using ImageJ software (NIH, USA). The enhanced chemiluminescence (ECL) substrate was purchased from Vazyme Biotech. The specific antibody catalog numbers are shown in supplementary Table 1. 2.6. Immunofluorescence Staining Mice were anesthetized with 1% pentobarbital and perfused transcardially with ice-prechilled phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). The brains were extracted and post-fixed in 4% PFA overnight before being embedded in paraffin and sectioned. Coronal hippocampal sections were identified based on the mouse brain atlas and washed three times in PBS (7 minutes per wash). The sections were incubated overnight at 4°C with primary antibodies against ionized calcium-binding adapter molecule 1 (Iba-1), bromodeoxyuridine (BrdU), and doublecortin (DCX). The following day, sections were incubated with secondary antibodies at room temperature for 1 hour, followed by three PBST washes. Nuclei were counterstained with DAPI, and coverslips were mounted. Fluorescently labeled cells were visualized using a fluorescence microscope, and the number of positive cells was quantified using ImageJ software. The specific antibody catalog numbers are shown in supplementary Table 1. 2.7. Enzyme-Linked Immunosorbent Assay (ELISA) Hippocampal tissue was homogenized in PBS on ice using a glass homogenizer to prepare tissue lysates. The homogenates were centrifuged, and the supernatant was collected for ELISA. The concentrations of IL-6 (E-EL-M0044, Elabscience, China), IL-1β (E-EL-M0037, Elabscience, China), TNF-α (E-EL-M3063, Elabscience, China), and nitric oxide (NO: S0021s, Beyotime, China) were measured using ELISA kits. Optical density was measured at 450 nm using a microplate reader (EnSight; PerkinElmer) within 15 minutes. 2.8. Statistical analysis All data are presented as the mean ± SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) or Kruskal-Wallis test, followed by post hoc Bonferroni tests. GraphPad Prism 9 (GraphPad Software Inc., USA) was used for statistical analysis and data visualization. Statistical significance was defined as p-value < 0.05. 3. Results 3.1.CPZ ameliorates CSDS-induced depression-like behaviors in mice After a standard 10-day CSDS protocol with CD1 aggressors, we successfully recapitulated core depression-related behavioral deficits. Our results revealed statistically significant differences among four groups across the measured behavioral paradigms (H (SIT) =27.33, p < 0.0001; H (TST) =16.13, p = 0.001; H (FST) =14.86, p = 0.002; F (SPT:3,32) = 13.07, p < 0.0001; H (OFT) = 10.02, p = 0.0184). Compared to the CON + PBS group, the CSDS + PBS group exhibited significant social avoidance (SIR < 1, p = 0.012, r = 0.80, Figure. 2A), prolonged immobility time (TST: p =0.02, r =0.61; FST: p = 0.01, r = 0.64, Figure. 2B-C), decreased sucrose preference ( p = 0.002, Figure. 2D), and reduced total locomotion distance( p = 0.048, r = 0.47, Figure. 2E). However, these effects were reversed by CPZ (vs. CSDS + PBS, SIR > 1; p = 0.045, r = 0.67; TST: p = 0.01, r = 0.60; FST: p = 0.004, r = 0.68; SPT: p < 0.0001; OFT: p = 0.048, r = 0.47), whereas CAP showed no rescue effect (Figure. 2A-E). Our results demonstrated that stress-induced hypoactivity and anhedonia were partially rescued by CPZ treatment not CAP. 3.2. TRPV1 downregulation suppresses microglial activation Based on behavioral research, we supposed that chronic stress-induced depression-like behaviors might be related to the TRPV1. Therefore, we aimed to investigate how TRPV1 and their downstream phosphorylated Calcium/Calmodulin-dependent Protein Kinase II alpha (CaMKIIα) undergo changes. We evaluated hippocampal TRPV1 protein expression in four groups (TRPV1: F (3,8) = 12.85, p = 0.002); when compared with the CON+PBS group, CSDS + PBS exhibited significantly elevated TRPV1 protein expression in the hippocampus ( p = 0.002), the CSDS + CPZ group significantly attenuated stress-induced elevations of TRPV1(vs CSDS + PBS, TRPV1: p < 0.001, Figure 3A-B). As a selective TRPV1 antagonist, CPZ inhibits TRPV1-mediated calcium influx, to modulating cellular function. Given CaMKIIα's absolute dependence on calcium-calmodulin complex formation, serving as a dynamic cellular calcium sensor, we employed phospho-CaMKIIα (Thr286) levels as a sensitive readout of activity-dependent calcium signaling dynamics[40]. There was a significant difference in the levels of p-CaMKIIα among four groups (p-CaMKIIα/t- CaMKIIα: F (3,8) = 16.92, p < 0.001). Consistent with the expression pattern of TRPV1, the ratio of p-CaMKIIα to t-CaMKIIα was significantly increased in CSDS-susceptible mice ( p = 0.038). After CPZ administration, this ratio was markedly reduced ( p = 0.001, Figure. 3C); In contrast, CAP treatment failed to further increase hippocampal TRPV1 or p-CaMKIIα levels. Previous studies have established that depression-like behaviors in mice are frequently accompanied by neuroinflammation, which primarily originates from microglial and astrocytic activation in the CNS. Therefore, we quantified the expression of cluster of differentiation 68 (CD68, a microglial activation marker) and glial fibrillary acidic protein (GFAP, a astrocytic activation marker) across four groups(CD68: F (3,8) = 16.92, p < 0.001). In comparison to the CON + PBS group, the CD68 expression in CSDS + PBS group exhibited significantly increased ( p < 0.001, Figure. 2D); The CSDS + CPZ group significantly attenuated stress-induced elevations of CD68 in the hippocampus (vs CSDS + PBS, p = 0.049; Figure. 3D). These results demonstrated that the CSDS model successfully induced microglial activation, which could be effectively suppressed by administering CPZ(Figure. 3D). However, there were no significant differences in GFAP among the four groups (Supplementary Figure .1). Microglial activation in the hippocampus was evaluated through both quantitative and morphological analyses to assess neuroinflammatory responses. To determine the effect of CSDS on the number of microglia, we quantitatively analyzed the number of Iba1 positive cells in the hippocampus by immunofluorescence staining (DG: F (3,12) = 12.17, p < 0.001; CA1: F (3,12) = 6.62, p =0.005; CA3: F (3,12) = 5.18, p = 0.017). The result showed that the number of Iba1 positive cells in three subregions of the hippocampus were significantly increased in the CSDS + PBS group compared with the CON + PBS group (DG: p = 0.003; CA1: p = 0.045; CA3: p = 0.022); however, the CSDS + CPZ group had fewer Iba1 positive cells in DG area of the hippocampus than the CSDS + PBS group ( p = 0.013), but there were no significant differences Iba1 positive cells in CA1 or CA3 area between the CSDS + PBS and the CSDS + CAP group.(Figure. 3E-H). To further investigate the impact of CPZ on the morphology of microglia, we analyzed microglia morphology in confocal microscopy images using the skeleton analysis plugin of ImageJ (Figure. 3E). There were significant differences in the total branch length/cell, the number of microglial process endpoints/cell, and the maximum branch length among four groups ( F (3,20) = 11.33, p < 0.001; F (3,20) = 14.53, p < 0.0001; F (3,20) = 6.05, p = 0.004). Compared with the CON + PBS group, the total branch length/cell and the number of microglial process endpoints/cell were significantly decreased in the DG of the CSDS + PBS ( p = 0.024; p < 0.001); however, the CSDS + CPZ group had significantly increased total branch length/cell and the number of microglial process endpoints/cell in the DG of hippocampus relative to the CSDS + PBS group ( p = 0.002; p = 0.003), indicating that CPZ increased the hippocampal microglia branching complexity of CSDS model mice(Figure. 3I - K). In addition, the maximum branch length in the CSDS + PBS group was shorter after CSDS stimulation than in the CON + PBS group ( p = 0.008); compared with the CSDS + PBS group, the maximum branch length was longer in the CSDS + CPZ ( p = 0.035; Figure. 3L). 3.3. CPZ inhibits neuroinflammation and impaired neurogenesis via JAK2/STAT3 pathway Pro-inflammatory cytokines produced by microglia play a crucial role in the neuroinflammation of depression. We measured the levels of pro-inflammatory factors (IL-6, IL-1β, TNF-α, and NO) in hippocampal brain tissues( F (3, 20) = 14.27, p < 0.0001; F (3, 20) = 8.52, p < 0.001; F (3, 20) = 5.83, p = 0.039; F (3, 20) = 20.92, p < 0.0001). Compared to the CON +PBS group, the levels of IL-6, IL-1β, TNF-α, and NO were increased after stress exposure ( p = 0.001; p = 0.004; p = 0.030; p = 0.005); treatment with CPZ during stress decreased IL-6 and IL-1β levels compared with the CSDS + PBS group, while TNF-α and NO levels showed no significant changes(p = 0.019; p = 0.026; Figure. 4A-D). In contrast, treatment with CAP during stress resulted in no significant changes in IL-6, IL-1β, or TNF-α levels but significantly increased NO levels (p <0.0001). Considered the closely links between neuroinflammation and neurogenesis, we hypothesized that TRPV1 may influence neurogenesis while modulating neuroinflammation. Consistent with our hypothesis, the results demonstrated that, compared with the CON + PBS group, the CSDS + PBS group exhibited a significant reduction in the number of BrdU-positive cells in the hippocampal DG region ( p = 0.007); notably, the CSDS + CPZ group significantly increased the number of BrdU-positive cells in the DG ( p = 0.002), whereas the CSDS + CAP group did not exert a similar effect (Figure. 4E-F). To elucidate the specific signaling pathways involved, we conducted the levels of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) in the hippocampus ( F (3, 8) = 17.02, p < 0.001; F (3, 8) = 24.51, p < 0.001). Our findings showed that, compared with the CON + PBS group, the CSDS + PBS group significantly increased the levels of p-JAK2 at the Y1007-Y1008 sites and p-STAT3 at the Y705 site in hippocampal ( p = 0.003; p = 0.001), suggesting activation of the JAK2/STAT3 signaling pathway in hippocampus; importantly, the CSDS + CPZ group significantly reduced the levels of p-JAK2 (Y1007-Y1008), and p-STAT3 (Y705) compared with the CSDS + PBS group( p = 0.019; p = 0.024); in contrast, treatment with CAP did not significantly alter the level of p-JAK2 or p-STAT3 (Figure. 4G-I). Collectively, these results suggested that chronic stress induces significant activation of the JAK2/STAT3 signaling pathway in hippocampus contributing to neuroinflammation and impaired neurogenesis. However, whether the suppression of JAK2/STAT3 signaling is essential for the neurogenic effects of CPZ remained to be determined. To clarify this mechanism, we investigated the impact of direct JAK2 activation on the neuroprotective effects conferred by CPZ. 3.4 CA1 Reverses the alleviating effect of CPZ on depression-like behaviors To further validate the aforementioned findings, we employed CA1, a JAK2 agonist, to investigate whether the remission effect of depression-like behaviors was attributable to CPZ treatment via acting on JAK2/STAT3 pathway. Relative to the CON + PBS group, the CSDS + PBS exhibited significant social avoidance (SIR 1; p = 0.042), whereas the CSDS + CPZ +CA1 group showed blocked the rescue effect (vs. CSDS + CPZ, SIR < 1, p = 0.023, Figure. 5A). In the TST and SPT (H =16.36, p = 0.001; F (3,20) = 16.32, p < 0.0001), the CSDS + PBS group showed significantly prolonged immobility time and reduced sucrose preference compared with the CON + PBS group ( p =0.021; p = 0.002); these effects were attenuated by CPZ treatment (vs. CSDS + PBS; p = 0.009; Figure. 5B-C). However, the CSDS + CPZ + CA1 group significantly extended immobility time and decreased sucrose preference (vs. CSDS + CPZ, p = 0.035, p < 0.001, Figure. 5B-C). There were no significant differences in the total distance in the OFT between the four groups (Figure. 5D). 3.5. Activation of the JAK2/STAT3 pathway abrogates the neuroprotective effect of CPZ Consistent with our hypothesis, CA1 activation was found to counteract the antidepressant-like effects of CPZ treatment. We further investigated whether this reversal effect was associated with corresponding molecular changes. The results revealed that, within the CSDS + PBS group, administration of CA1 reversed the inhibitory effects of CPZ on the JAK2/STAT3 pathway ( F (3, 8) = 31.46, p < 0.0001; F (3, 8) = 30.16, p < 0.0001, Figure. 5A-G). CA1 blocked the protective effect of CPZ against CSDS-induced microglial activation and production of IL-6 ( F (3, 8) = 15.59, p = 0.001; F (3, 8) = 31.93, p < 0.0001, Figure. 6A-G). Consistently, neurogenesis showed significant differences among the four groups ( F (3, 20) = 9.80, p < 0.001). Immunofluorescence analysis demonstrated that in the CSDS model, the intensity of DCX immunoreactivity in the hippocampus was significantly diminished in the group receiving both CPZ and CA1 compared to the group treated with CPZ alone ( p = 0.008). Notably, DCX expression in the CPZ+CA1 group was reduced to a level comparable to that observed in the CSDS-only group, indicating that the neurogenic protective effect of CPZ was abolished by CA1 co-treatment ( p > 0.05, Figure. 6H-I). These findings provide compelling evidence that the neuroprotective effect of CPZ is mediated through inhibition of the JAK2/STAT3 signaling pathway. 4. Discussion This study demonstrated that the TRPV1 channel antagonist CPZ alleviates depression-like behaviors, exerting its potential antidepressant effects through attenuation of neuroinflammation and promotion of neurogenesis (Figure. 7). The expression of TRPV1 and phosphorylation level of CaMKIIα was upregulated in depression-like mice induced by CSDS, accompanied by microglial activation, evidenced by morphological and numerical changes, along with increased pro-inflammatory factor release. Similarly, hippocampal newborn neurons were reduced. These outcomes correlated with activation of the JAK2/STAT3 signaling pathway. Inhibition of TRPV1 mitigated microglial activation, reduced pro-inflammatory cytokine release, promoted the generation of newborn neurons, and suppressed JAK2/STAT3 signaling pathway activation. The social interaction ratio serves as a key behavioral indicator for evaluating whether CSDS model successfully induces depression-like phenotypes[38]. Our results demonstrated that mice exposed to aggressive encounters exhibited significant social avoidance and reduced interaction behaviors. Furthermore, mice exposed to aggressive encounters displayed pronounced despair-like behaviors, anhedonia, and reduced locomotor activity. Importantly, all these depression-liked behavioral deficits were effectively reversed by CPZ treatment. These findings are consistent with previous studies demonstrating that reduced TRPV1 expression ameliorates depression-like behaviors in mice[12, 41]. On the other hand, the treatment of CAP does not seem to exert an effect on depression-like behaviour in mice. Current opinion regarding the role of TRPV1 channel overactivation in depressive-like behaviors remain inconsistent, potentially due to unique desensitization properties of TRPV1[42]. TRPV1 channel opening in response to various stimuli triggers calcium ion influx, which may induce channel desensitization[43]. The desensitization mechanism of the TRPV1 channel exhibits distinct characteristics in comparison to other ligand-gated ion channels[44]. Specifically, following desensitization induced by a low concentration of CAP, the activity of the TRPV1 channel can be reinstated through the application of a higher concentration of the stimulating agent, CAP[45]. This bidirectional pharmacological action may explain the contradictory outcomes observed with CAP administration. Consequently, our subsequent experiments will systematically evaluate both the concentration and temporal dynamics of central CAP exposure to elucidate the precise role of TRPV1 channel activation in depression-like behaviors. Upon exposure to stress or pathogenic challenges, microglia and astrocytes within the CNS become activated, releasing a substantial amount of pro-inflammatory cytokines and metabolic byproducts that exert direct neurotoxic effects, thereby contributing to the pathophysiology of depression[46]. Previous studies have indicated that TRPV1 is closely associated with the onset of neuroinflammation[47]; however, its precise role, whether protective or detrimental, remains controversial[48, 49]. Our findings indicated that TRPV1 channel antagonist reduced the expression of microglial markers Iba1 and CD68, and decreased levels of pro-inflammatory cytokine IL-6 and IL-1β. Similar to our results, the downregulation of TRPV1 alleviated microglial hyperactivation induced by hyperthermia and lipopolysaccharide, restoring homeostasis in the inflammatory microenvironment of the brain[50]. Baban et al. demonstrated that aminoalkylindole cannabinoids, acting as TRPV1 channel antagonists, significantly attenuate microglial activation and reduce pro-inflammatory cytokine production[51]. A recent study inferred that TRPV1 deficiency or pharmacological blockade disrupts apoptosis-associated speck-like protein containing a card speck formation in microglia through the Ca²⁺-PP2A pathway, resulting in impairing inflammasome complex assembly, suppressing NOD-like receptor family pyrin domain-containing 3 inflammasomes activation, and mitigating neuroinflammatory responses[21]. Lin et al. reported that electroacupuncture effectively alleviates depressive symptoms, potentially through modulation of the TRPV1 pathway and suppression of pro-inflammatory cytokine production[43]. These results support that TRPV1 inhibition may alleviate neuroinflammation by suppressing microglial activation and reducing the release of specific pro-inflammatory cytokines[18, 52]. Furthermore, although TNF-α levels were elevated following chronic stress, modulation of TRPV1 did not alter its expression, suggesting that TNF-α secretion may be regulated by alternative intracellular pathways within microglia[19]. Previous studies demonstrated that excessive activation of the TRPV1 channel promotes microglial and astrocytic activation, accelerating their migration and chemotactic responses under stress conditions[53, 54]. In this study, we observed a marked activation of microglia in the hippocampus of mice subjected to CSDS, whereas astrocytes did not exhibit a similar response. This discrepancy may be attributed to the role of microglia as sensors of microenvironmental changes in the CNS, rendering them more sensitive to chronic and sustained stress[28]. Supporting this notion, research indicated that during the subacute phase of CNS disorders, both microglia and astrocytes become overactivated. However, microglial activation occurs approximately three hours earlier than that of astrocytes[55]. Consequently, in our study, significant changes were observed in the microglial markers Iba1 and CD68, whereas astrocytic markers remained largely unchanged. AHN contributes significantly to various neurocognitive and affective functions, including memory formation, memory extinction, stress resilience, and mood regulation[56]. Growing evidence indicates that this neurogenic process is fundamentally involved in both depression pathogenesis and the mechanisms underlying antidepressant efficacy[57]. These findings highlight hippocampal neurogenesis as a critical mediator of both depressive disorders and their treatment[58]. We found that the inhibition of the TRPV1 channel significantly promoted neurogenesis. Stock et al. demonstrated that TRPV1 channel expression in neural precursor cells decreases with aging, and TRPV1 knockout mice exhibited enhanced neural precursor cell proliferation compared to control littermates, suggesting that TRPV1 expression modulates activity-dependent neurogenesis during both postnatal development and adulthood[59]. Consequently, TRPV1 inhibition promotes neurogenesis and consequently alleviates depression-like behaviors. Through a comprehensive literature search, we speculated that TRPV1 may mediate the anti-inflammatory effects and promotes neurogenesis through modulation of the JAK2/STAT3 signaling pathway. Yang et al. reported that TRPV1 is essential for JAK2/STAT3 phosphorylation[28]. Within the CNS, TRPV1 activation has been demonstrated to promote pro-inflammatory STAT3 signaling, thereby amplifying neuroinflammatory responses. Previous study has confirmed that JAK2 inhibition, through suppression of STAT3 phosphorylation, effectively attenuates neuroinflammation. In alignment with these findings, our results indicated that TRPV1 mediates microglial activation and subsequent secretion of IL-6 and IL-1β via the JAK2/STAT3 pathway. Neural stem cells (NSCs) possess intrinsic self-renewal and proliferative capabilities, and their fate decisions are tightly regulated by phosphorylation of STAT3 at Tyr705[60]. Further evidence indicates that inhibition of the JAK2/STAT3 pathway restores Tet3-mediated DNA demethylation, facilitating neuronal differentiation[27]. Wang et al. reported that TRPV1 activation up-regulates HDAC2 and decreases acrtylation of histone H3, resulting in lessened expression of neurogenesis-related genes and the loss of NSCs[41]. Our results extended this line of research by identifying TRPV1 inhibition supports hippocampal neurogenesis. Despite the fact that we discovered CPZ treatment alleviates depression-like behaviors via TRPV1-mediated modulation of neuroinflammation and neurogenesis, there still remains some limitations. On the one hand, the activation of TRPV1 did not exert any effects in mice exhibiting depression-like behaviors. The observed outcome might result from the actual lack of involvement of TRPV1 in the pathogenic mechanisms of depression. Alternatively, it may stem from our inability to determine the concentration and effective duration of CAP within the CNS after intraperitoneal administration, potentially introducing confounding effects into the results. In future study, it is necessary to clarify the functional state of TRPV1 channels and determine their exact effects. On the other hand, existing evidence demonstrates that CAP can mediate anti-inflammatory effects through TRPV1-independent mechanisms. Potential intracellular targets of TRPV1 agonists beyond TRPV1 itself have yet to be fully deciphered. These alternative targets may modulate secondary or tertiary signaling cascades downstream of TRPV1, or potentially interact with other signaling receptors to trigger complex cross-talk mechanisms affecting neuroinflammatory pathways. Considering the intricacy of intracellular signaling networks, these hypotheses warrant further investigation. 5. Conclusion In conclusion, our findings suggest that CPZ produces significant antidepressant-like effects in CSDS-susceptible mice. This study provides novel mechanistic insights into TRPV1-mediated neuroinflammatory regulation and its impact on neurogenesis, suggesting that microglial TRPV1 might represent a promising therapeutic target for depression. Declarations Funding This study was supported by the National Natural Science Foundation of China (Nos. 81571325, 81871072, and 82071523) and the Medical Science Advancement Program of Wuhan University (No. TFLC2018001). The Key Research And Development Program Of Hubei Province (2020BCA064) also supported the design of this study. Authorship Contribution Statement Junjie Huang and Chen Li: Methodology, Software, Investigation, Writing-Original Draft. Hailong Ge: Conceptualization, Methodology. Lan Wu: Methodology. Ling Xiao: Writing- Review & Editing, Supervision, Project administration. Yinping Xie: Conceptualization, Writing-Review & Editing. Gaohua Wang: Resources, Supervision, Project administration, Funding acquisition. Availability of data and materials All the necessary data are included in the article. Further data will be shared by request. Ethics approval and consent to participate This work was carried out based on the Regulations of Experimental Animal Administmiceion issued by the State Committee of Science and Technology of the People’s Republic of China, with the approval of the Ethics Committee in Renmin Hospital of Wuhan University. Competing of Interest All authors declare that they have no competing interests. References Yang C-H, Lv J-J, Kong X-M, et al (2024) Global, regional and national burdens of depression in adolescents and young adults aged 10-24 years, from 1990 to 2019: findings from the 2019 Global Burden of Disease study. Br J Psychiatry 225:311–320. https://doi.org/10.1192/bjp.2024.69 Zhang R, Peng X, Song X, et al (2023) The prevalence and risk of developing major depression among individuals with subthreshold depression in the general population. 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(A) Mice were randomly divided into four groups: CON + PBS (n=8), CSDS+PBS (n=8), CSDS + CAP (n=12), CSDS + CPZ (n=12). PBS, CPZ, and CAP were administered via intraperitoneal injection at the initiation of modeling and continued for 10 consecutive days. (B) Mice were randomly divided into four groups: CON + PBS (n=6), CSDS + PBS (n=6), CSDS + CPZ (n=6), CSDS + CPZ+ CA1 (n=6). PBS, CPZ, and CA1 were administered via intraperitoneal injection at the initiation of modeling and continued for 10 consecutive days.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/7fa7fd0c699d121cc2ca0677.png"},{"id":93053733,"identity":"5efc0fa4-717d-4b66-b928-52e79fcb3040","added_by":"auto","created_at":"2025-10-08 14:34:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":824660,"visible":true,"origin":"","legend":"\u003cp\u003eCPZ alleviated depressive-like behaviors in mice exposed to CSDS. (A) The social interaction ratio of the mice in social interaction test (SIT). (B) The immobility time in the tail suspension test (TST) of the mice. (C) The immobility time in the force swim test (FST) of the mice. (D) Sucrose preferences of the mice. (E) The total distance in open field test (OFT) of the mice. The data was shown as the mean ± SD; n = 8-10 per group; * indicates significant difference as follows: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.001, ****\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/f66b0c7c44088ce52c73aaa7.png"},{"id":93053225,"identity":"396b4e3c-b0be-4aa5-942f-b15097d21842","added_by":"auto","created_at":"2025-10-08 14:26:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":10239845,"visible":true,"origin":"","legend":"\u003cp\u003eCPZ treatment decreased TRPV1 expression and ameliorated microglia activity in the hippocampus. (A)Representative western blot images of TRPV1, CD68, t-CaMK2α, and p-CaMK2α in the hippocampus. (B-D) Quantification of TRPV1, CD68, and p-CaMK2α/t-CaMK2α expression in the hippocampus, n=3. (E) Representative confocal images of Iba1 (green) and DAPI (blue) staining in the DG, CA1 and CA3 regions of hippocampus. Scale bar (white) = 100 μm. (F-H) Quantification analyses of the number of Iba1 positive cells in the DG, CA1 and CA3 regions of hippocampus. (I) Representative images of Iba1 positive cells (raw) that used for binary and skeletonization analysis as well as skeletonized cells with a circular soma or a soma with a single origin. Scale bar (red/black) = 50/25 μm. (J-L) Microglia morphology was quantified according to the number of microglia process endpoints per cell, total branch length per cell, and maximum branch length per cell in DG region. The data was shown as the mean ± SD. * indicates significant difference as follows: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/d0178be614952edc701a491c.png"},{"id":93053224,"identity":"dabc9c88-9fd7-4ab4-8650-7247426ee25d","added_by":"auto","created_at":"2025-10-08 14:26:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4336102,"visible":true,"origin":"","legend":"\u003cp\u003eCPZ inhibit neuroinflammation and impaired neurogenesis via JAK2/STAT3 pathway. (A-D) Quantification analyses of pro-inflammatory cytokines IL-6, IL-1β, TNF-α, and NO. (E) Representative confocal images of Brdu (red) and DAPI (blue) staining in the DG region of hippocampus. Scale bar (white) = 100 μm. (F) Quantification analyses of the number of Brdu positive cells in the DG of the hippocampus (G) Representative western blot images of t-JAK2, p-JAK2 (Y1007-1008), t-STAT3, and p-STAT3 (Y705) expression in the hippocampus, n=3. (H-I) Quantification of p-JAK2/t-JAK2 and p-STAT3/t-STAT3 expression levels in the hippocampus, n=3. The data was shown as the mean ± SD, n = 4-6 mice per group. * indicate significant difference as follows: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/97a4285001a472ba5d356c92.png"},{"id":93053223,"identity":"58de5787-9808-4841-adbd-91f1e439a39e","added_by":"auto","created_at":"2025-10-08 14:26:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":618082,"visible":true,"origin":"","legend":"\u003cp\u003eCA1 Reversed the alleviating effect of CPZ on depression-like behaviors. (A) Social interaction test (SIT) of the mice. (B) The immobility time in the tail suspension test of the mice. (C) Sucrose preferences of the mice. (D) The total distance in open field test of the mice.The data was shown as the mean ± SD; n = 6 per group; * indicates significant difference as follows:*\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, and ***\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/469fda00547e231b609d21f5.png"},{"id":93053230,"identity":"6843785d-74cf-4ef5-9463-f2d943f2e6fc","added_by":"auto","created_at":"2025-10-08 14:26:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":6029664,"visible":true,"origin":"","legend":"\u003cp\u003eActivated JAK2 blocks the effect of CPZ on ameliorating neuroinflammation and promoting neurogenesis. (A) Representative western blot images of TRPV1, t-CaMK2α, p-CaMK2α (T256), CD68, IL-6, t-JAK2, p-JAK2 (Y1007-1008), t-STAT3, and p-STAT3 (Y705) expression in the hippocampus, n=3. (B-G) Quantification of TRPV1, p-CaMK2α/t-CaMK2α, CD68, IL-6, p-JAK2/t-JAK2 and p-STAT3/t-STAT3 in the hippocampus, n=3. (H) Representative confocal images of DCX (red) and DAPI (blue) staining in the DG region of hippocampus, n = 4-6 mice per group. Scale bar (white) = 100μm. (I) Immunofluorescence intensity analysis of DCX in DG area, n = 4-6 mice per group. The data was shown as the mean ± SD. * indicates significant difference \u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u003cbr\u003e\n as follows: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.01, ***\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, and ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/d9dc1edeca09d3c49deec153.png"},{"id":93053734,"identity":"f0cc7bd0-2d5b-45f9-b30a-37cbc7a85130","added_by":"auto","created_at":"2025-10-08 14:34:11","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":2048485,"visible":true,"origin":"","legend":"\u003cp\u003eMechanism diagram of antidepressant effect of CPZ in the hippocampus. After chronic stress, the increased TRPV1 expression induces the microglia activity and then the damage of adult hippocampus neurogenesis in the hippocampus, leading to depressive-like behaviors of mice. Down-regulation of TRPV1 levels in the hippocampus inhibits microglia activity induced by CSDS, and alleviates hippocampus neurogenesis impairment, thereby exerting an antidepressant effect.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/75a0388c0aaa08cff97a647d.png"},{"id":103252005,"identity":"0f4ad3c3-06d0-4a33-8646-91e559a688b0","added_by":"auto","created_at":"2026-02-23 16:12:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":23831019,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/d83d4b98-2e4b-428e-ab78-882493d7572c.pdf"},{"id":93053227,"identity":"1160e0c8-3933-41ab-b987-5b185249d4ec","added_by":"auto","created_at":"2025-10-08 14:26:11","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":4178539,"visible":true,"origin":"","legend":"","description":"","filename":"supplementmaterials20250607.docx","url":"https://assets-eu.researchsquare.com/files/rs-6992017/v1/027bb0cdd50cc207613e6657.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Capsazepine Treatment Alleviates Depression-Like Behaviors via TRPV1-Mediated Modulation of Neuroinflammation and Neurogenesis","fulltext":[{"header":"1.Introduction","content":"\u003cp\u003eDepression is one of the most prevalent mood disorders, affecting over 350\u0026nbsp;million individuals worldwide[1]. Its onset and progression are the result of the interplay of genetic, environmental, and social factors[2]. Currently, fewer than one-third of patients with depression achieve clinical remission following six weeks of monotherapy at conventional dosages, while 20\u0026ndash;50% exhibit inadequate treatment response[3]. This underscores the critical need to identify novel therapeutic targets to address these treatment limitations. While the precise etiology of depression remains unclear, increasing studies suggest a strong association between the endocannabinoid system (ECS) and the involvement of depression[4\u0026ndash;6]. The cannabinoid receptors, as a primary elements of the ECS, modulate monoaminergic neurotransmitter, particularly serotonin and norepinephrine release [7]. Moreover, these receptors regulate fundamental neurobiological processes including hippocampal neurogenesis, synaptic plasticity, and functional connectivity within the basolateral amygdala-nucleus accumbens circuit[8]. These findings collectively position endocannabinoid receptors as promising therapeutic targets for major depressive disorder\u003c/p\u003e\u003cp\u003eRecently, transient receptor potential vanilloid type 1 channel (TRPV1) is identified as a novel cannabinoid receptor which is highly expressed in the central nervous system (CNS)[9]. TRPV1 is a ligand-gated, non-selective cation channel that triggers influx of Ca\u003csup\u003e2+\u003c/sup\u003e, leading to an increase in intracellular ion concentrations[10]. These results in depolarization of both the plasma and organellar membranes, activating downstream Ca\u003csup\u003e2+\u003c/sup\u003e signaling pathways that initiate a series of intracellular biochemical reactions[11]. Previous studies have indicated that TRPV1 mediates depressive-like behaviors in animals. Pharmacological blockade of TRPV1 such as injection with capsazepine (CPZ, a TRPV1 antagonist) reversed the behavioral deficits caused by stress in rodent[12, 13]. From an epigenetic perspective, Wang et.al revealed that TRPV1 regulated emotional responses in mice by interacting with histone deacetylase 2, and TRPV1 deficiency increased mice\u0026rsquo;s resistance to chronic stress[14, 15]. Interestingly, low-dose capsaicin (CAP, a TRPV1 agonist), which activates the S4-S5 linker and C-terminal domain, was found to alleviate depression-like behaviors, as evidenced by reduced immobility in the forced swimming test[16]. Although these studies have established an association between TRPV1 and the modulation of depressive-like behaviors, the underlying mechanisms remain poorly understood and warrant further investigation[17]. In the present study, mice received intraperitoneal injection (i.p.) of CAP and CPZ separately to investigate the molecular mechanisms underlying TRPV1 channel modulation and its effects on depression-like behaviors.\u003c/p\u003e\u003cp\u003eNeuroinflammation represents a critical component of the neurobiological mechanisms underlying depression[18]. The activated inflammatory signaling pathways, elevated oxidative stress, disrupted blood-brain barrier integrity, and peripheral immune cell infiltration constitute key mechanisms that exacerbate neuroinflammation and ultimately contribute to depressive-like behaviors[19]. Microglia, the resident immune cells of the brain, demonstrates altered activity patterns in depression pathology[20]. Its dysregulation drives neuroinflammatory responses that exacerbate depressive-like behaviors. TRPV1 channels are abundantly expressed on microglial membranes, where they might regulate microglial activity and contribute to neuroinflammatory processes[21]. Currently, there are two main perspectives on TRPV1\u0026rsquo; role in neuroinflammation through microglial regulation. Some views argue that TRPV1 inhibition suppresses neuroinflammation, alleviating its effects[22], while others propose that TRPV1 activation exerts a protective effect in neuropsychiatric disorders by reducing neuroinflammation[23]. These findings indicate that TRPV1 plays paradoxical roles in neuroinflammation, and its precise molecular mechanisms remain to be fully elucidated[24].\u003c/p\u003e\u003cp\u003eSubstantial evidences suggest that neuroinflammation contributes to depression pathogenesis through not only immune-mediated mechanisms but also impaired adult hippocampal neurogenesis (AHN), representing an additional pathway in depression development[25, 26]. Neuroinflammation can suppress neurogenesis via the neurotoxic effects of inflammatory cytokines, including IL-6, IL-1β, and TNF-α, or by disrupting the brain\u0026rsquo;s microenvironment. Conversely, impaired neurogenesis may exacerbate neuroinflammation by indirectly modulating the inflammatory state through altered neurotransmitter secretion and synaptic plasticity[27, 28]. Ultimately, excessive neuroinflammation and deficient neurogenesis form a self-reinforcing cycle, contributing to the pathogenesis of psychiatric disorders. The janus kinase2 (JAK2)/signal transducers and activators of transcription3 (STAT3) pathway plays a key role in the CNS by coordinating inflammatory responses, cell proliferation and gene expression[29]. Recent studies have shown that specific inhibition of the JAK2/STAT3 pathway not only reduces neuroinflammation but also improves depressive-like behaviors, which provides a potential target for the development of novel antidepressant drugs[30, 31]. A study showed that the JAK2/STAT3 pathway involved in TRPV1-mediated disorders of the nervous system, however, the mechanisms by which TRPV1 contributes to depression via neuroinflammation and impaired neurogenesis have not been fully elucidated.\u003c/p\u003e\u003cp\u003eIn the present study, the chronic social defeat stress (CSDS) paradigm was utilized in mice to model depression, and parallel interventions with CAP and CPZ were administered. Subsequently, we explored whether CSDS has influenced hippocampal TRPV1 expression. On this basis, to further investigate the changes of molecular markers associated with neuroinflammation and neurogenesis after treatment with CAP or CPZ, along with alterations in the JAK2/STAT3 signaling pathway. To further clarify the involvement of this pathway in the regulation of TRPV1, we administered Coumarin A1 (CA1), a selective JAK2/STAT3 inhibitor, and assessed its impact on the phenomena observed. This study investigates how TRPV1 regulation affects neuroinflammation and neurogenesis under chronic stress, providing mechanistic insights into depression and potential therapeutic targets.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Animals\u003c/h2\u003e\u003cp\u003eAll animals used in this study were male C57BL/6 mice (20\u0026ndash;25 g) of specific pathogen-free grade, purchased from the Laboratory Animal Center of Three Gorges University. All mice were housed in cages (21.5cm \u0026times; 32cm \u0026times;17cm, six per cage) and maintained under controlled conditions at a temperature of 22\u0026ndash;24\u0026deg;C and humidity of 60\u0026ndash;65% in the animal facility of the First Clinical College of Wuhan University, which is free of specific pathogens. This experiment was reviewed and approved by the Ethics Committee of Renmin Hospital, Wuhan University, and was conducted in strict accordance with institutional and national regulations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. CSDS\u003c/h2\u003e\u003cp\u003eCSDS protocols were performed as described previously and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA. C57BL/6 J mice were exposed to 10 min of physical aggression by an intruder male CD-1 mouse[32]. Following the defeat session, experimental mice were housed in the same cage as the CD-1 mice on the opposite side of a transparent and perforated plexiglass divider to maintain sensory contact for 24 h. This procedure was repeated for 10 consecutive days with a new CD-1 aggressor on each day. Control mice were pair housed in similar cages, separated from one another by a perforated plexiglass divider and rotated to a new one each day for 10 days.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Experimental design\u003c/h2\u003e\u003cp\u003eIn experiment 1, mice were randomly divided into four groups: CON\u0026thinsp;+\u0026thinsp;PBS (n\u0026thinsp;=\u0026thinsp;8), CSDS\u0026thinsp;+\u0026thinsp;PBS (n\u0026thinsp;=\u0026thinsp;8), CSDS\u0026thinsp;+\u0026thinsp;CAP (n\u0026thinsp;=\u0026thinsp;12), CSDS\u0026thinsp;+\u0026thinsp;CPZ (n\u0026thinsp;=\u0026thinsp;12). CAP and CPZ were purchased from MedChemExpress (MCE). These compounds were dissolved in DMSO and prepared in PBS. The final concentrations were as follows: CPZ at 5 mg/kg/day [33, 34], CAP at 2.5 mg/kg/day [35, 36]. For drug administration, PBS, CPZ, and CAP were administered via intraperitoneal injection at the initiation of modeling and continued for 10 consecutive days. A flowchart of this procedure is shown in Figure. 1A.\u003c/p\u003e\u003cp\u003eIn experiment 2, mice were randomly divided into four groups: CON\u0026thinsp;+\u0026thinsp;PBS (n\u0026thinsp;=\u0026thinsp;6), CSDS\u0026thinsp;+\u0026thinsp;PBS (n\u0026thinsp;=\u0026thinsp;6), CSDS\u0026thinsp;+\u0026thinsp;CPZ (n\u0026thinsp;=\u0026thinsp;6), CSDS\u0026thinsp;+\u0026thinsp;CPZ\u0026thinsp;+\u0026thinsp;CA1(n\u0026thinsp;=\u0026thinsp;6). CA1 was purchased from MCE. The final concentration of CA1 was 100 \u0026micro;M [37]. For drug administration, PBS and CPZ were administered via intraperitoneal injection at the initiation of modeling and continued for 10 consecutive days. CA1 was administered via intraperitoneal injection during the last 3 days of the model. A flowchart of this procedure is shown in Figure. 1B.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Behavioral Testing\u003c/h2\u003e\u003cp\u003ePrior to the experiment, mice were acclimated to the room conditions for at least 1 hour. After each test, the equipment was thoroughly cleaned with 75% alcohol to eliminate the scent of the previous animal.\u003c/p\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1. Social interaction test (SIT)\u003c/h2\u003e\u003cp\u003eThe SIT was conducted 24 hours after the last CD-1 attack. The test consisted of two trials. In the first trial, the experimental mice were placed in a box (45 cm \u0026times; 45 cm \u0026times; 45 cm) for 2.5 minutes. A wire mesh frame (10 cm \u0026times; 4.5 cm) was placed in the interaction area, and the time the test mice spent in the interaction area was recorded as \"no target time\"[38]. In the second trial, a stranger CD-1 mouse was placed in the wire mesh frame, and the experimental mice was returned to the box for an additional 2.5-minute test. The time spent in the interaction area during this trial was recorded as \"target time\" .Video tracking was performed using Maze software (Store Company, Wood Dale, IL, USA). The social interaction ratio was calculated by dividing target time by no target time[39].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2. Sucrose preference test (SPT)\u003c/h2\u003e\u003cp\u003eDay 1: mice were individually housed and allowed to adapt to both 1% sucrose solution and tap water for 24 hours. Day 2:food and water were withheld for 24 hours. Day 3: the initial weight of the bottles was recorded, and two bottles were placed in the metal lid of the cage, with food positioned on the opposite side of the wire lid. At the halfway point of the experiment, the positions of the two bottles were switched to prevent location bias. After 24 hours, water consumption was assessed by weighing the bottles. Sucrose preference was calculated using the formula: Sucrose preference (%) = (Sucrose solution intake / (Sucrose solution intake\u0026thinsp;+\u0026thinsp;Water intake)) \u0026times; 100%.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3. Tail suspension test (TST)\u003c/h2\u003e\u003cp\u003eIn TST, mice were positioned head-down, maintaining a distance of approximately 30 cm from the bottom of the suspension box. The experiment lasted for a total of 6 minutes. Struggling behavior during the first 2 minutes (habituation period) was not recorded, while immobility time was recorded during the subsequent 4 minutes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4. Forced swimming test (FST)\u003c/h2\u003e\u003cp\u003eFST was conducted using a cylindrical, translucent water tank (15 cm \u0026times; 30 cm). Prior to the experiment, the tank was filled with tap water maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. Each mouse was placed in the water and subjected to forced swimming for 6 minutes. Immobility time was recorded during the last 4 minutes of the test.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.4.5. Open field test (OFT)\u003c/h2\u003e\u003cp\u003eMice were placed in the central area of a semi-transparent, square plastic box (50 cm \u0026times; 50 cm \u0026times; 40 cm). Behavioral tracking was conducted using EthoVision XT 11.5 software (Noldus Information Technology, Netherlands), which automatically recorded the movement of the mice within the box. The total test duration was 5 minutes. After each trial, the box was cleaned with 75% ethanol to remove urine and feces, eliminating residual odors that could affect subsequent tests.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Western blotting (WB)\u003c/h2\u003e\u003cp\u003ePhosphatase and protease inhibitors were added to RIPA lysis buffer at a fixed ratio. Frozen hippocampal tissues from mice were homogenized in ice-cold RIPA buffer. Protein concentration was determined using the bicinchoninic acid (BCA) assay. The BCA assay kit was obtained from Beyotime Biotechnology. The protein samples were mixed with loading buffer and boiled at 100\u0026deg;C. Total extracted protein (20 \u0026micro;g/ per lane) was separated by 10\u0026ndash;12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After transfer, the membrane was blocked with a rapid blocking solution for 15 minutes, followed by overnight incubation with the primary antibody at 4\u0026deg;C. The next day, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 hour, followed by chemiluminescent detection using an imaging system (Bio-Rad, USA). Band intensity was analyzed using ImageJ software (NIH, USA). The enhanced chemiluminescence (ECL) substrate was purchased from Vazyme Biotech. The specific antibody catalog numbers are shown in supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.6. Immunofluorescence Staining\u003c/h2\u003e\u003cp\u003eMice were anesthetized with 1% pentobarbital and perfused transcardially with ice-prechilled phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). The brains were extracted and post-fixed in 4% PFA overnight before being embedded in paraffin and sectioned. Coronal hippocampal sections were identified based on the mouse brain atlas and washed three times in PBS (7 minutes per wash). The sections were incubated overnight at 4\u0026deg;C with primary antibodies against ionized calcium-binding adapter molecule 1 (Iba-1), bromodeoxyuridine (BrdU), and doublecortin (DCX). The following day, sections were incubated with secondary antibodies at room temperature for 1 hour, followed by three PBST washes. Nuclei were counterstained with DAPI, and coverslips were mounted. Fluorescently labeled cells were visualized using a fluorescence microscope, and the number of positive cells was quantified using ImageJ software. The specific antibody catalog numbers are shown in supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\u003c/h2\u003e\u003cp\u003eHippocampal tissue was homogenized in PBS on ice using a glass homogenizer to prepare tissue lysates. The homogenates were centrifuged, and the supernatant was collected for ELISA. The concentrations of IL-6 (E-EL-M0044, Elabscience, China), IL-1β (E-EL-M0037, Elabscience, China), TNF-α (E-EL-M3063, Elabscience, China), and nitric oxide (NO: S0021s, Beyotime, China) were measured using ELISA kits. Optical density was measured at 450 nm using a microplate reader (EnSight; PerkinElmer) within 15 minutes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Statistical analysis\u003c/h2\u003e\u003cp\u003eAll data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. Statistical analyses were performed using one-way analysis of variance (ANOVA) or Kruskal-Wallis test, followed by post hoc Bonferroni tests. GraphPad Prism 9 (GraphPad Software Inc., USA) was used for statistical analysis and data visualization. Statistical significance was defined as p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.1.CPZ ameliorates CSDS-induced depression-like behaviors in mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter a standard 10-day CSDS protocol with CD1 aggressors, we successfully recapitulated core depression-related behavioral deficits. Our results revealed statistically significant differences among four groups across the measured behavioral paradigms (H\u003csub\u003e(SIT)\u003c/sub\u003e=27.33, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; H\u003csub\u003e(TST)\u0026nbsp;\u003c/sub\u003e=16.13, \u003cem\u003ep\u003c/em\u003e = 0.001; H\u003csub\u003e(FST)\u0026nbsp;\u003c/sub\u003e=14.86, \u003cem\u003ep\u003c/em\u003e = 0.002;\u003cem\u003e\u0026nbsp;F\u003c/em\u003e\u003csub\u003e(SPT:3,32)\u0026nbsp;\u003c/sub\u003e= 13.07, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001; H\u003csub\u003e(OFT)\u0026nbsp;\u003c/sub\u003e= 10.02, \u003cem\u003ep\u003c/em\u003e = 0.0184). Compared to the CON + PBS group, the CSDS + PBS group exhibited significant social avoidance (SIR \u0026lt; 1, p = 0.012, r = 0.80, Figure. 2A), prolonged immobility time (TST: \u003cem\u003ep\u003c/em\u003e =0.02, r =0.61; FST: \u003cem\u003ep\u003c/em\u003e = 0.01, \u003cem\u003er\u003c/em\u003e = 0.64, Figure. 2B-C), decreased sucrose preference (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.002, Figure. 2D), and reduced total locomotion distance(\u003cem\u003ep\u003c/em\u003e = 0.048, \u003cem\u003er\u003c/em\u003e = 0.47, Figure. 2E). However, these effects were reversed by CPZ (vs. CSDS + PBS, SIR \u0026gt; 1; \u003cem\u003ep\u003c/em\u003e = 0.045, r = 0.67; TST: \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.01,\u003cem\u003e\u0026nbsp;r\u003c/em\u003e = 0.60; FST: \u003cem\u003ep\u003c/em\u003e = 0.004, \u003cem\u003er\u003c/em\u003e = 0.68; SPT: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; OFT:\u003cem\u003e\u0026nbsp;p\u003c/em\u003e = 0.048, \u003cem\u003er\u003c/em\u003e = 0.47), whereas CAP showed no rescue effect (Figure. 2A-E). Our results demonstrated that stress-induced hypoactivity and anhedonia were partially rescued by CPZ treatment not CAP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.2.\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eTRPV1 downregulation suppresses microglial activation\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on behavioral research, we supposed that chronic stress-induced depression-like behaviors might be related to the TRPV1. Therefore, we aimed to investigate how TRPV1 and their downstream phosphorylated Calcium/Calmodulin-dependent Protein Kinase II alpha (CaMKII\u0026alpha;) undergo changes. We evaluated hippocampal TRPV1 protein expression in four groups (TRPV1: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,8)\u003c/sub\u003e = 12.85, \u003cem\u003ep\u003c/em\u003e = 0.002); when compared with the CON+PBS group, CSDS + PBS exhibited significantly elevated TRPV1 protein expression in the hippocampus (\u003cem\u003ep\u003c/em\u003e = 0.002), the CSDS + CPZ group significantly attenuated stress-induced elevations of TRPV1(vs CSDS + PBS, TRPV1: \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Figure 3A-B). As a selective TRPV1 antagonist, CPZ inhibits TRPV1-mediated calcium influx, to modulating cellular function. Given CaMKII\u0026alpha;\u0026apos;s absolute dependence on calcium-calmodulin complex formation, serving as a dynamic cellular calcium sensor, we employed phospho-CaMKII\u0026alpha; (Thr286) levels as a sensitive readout of activity-dependent calcium signaling dynamics[40]. There was a significant difference in the levels of p-CaMKII\u0026alpha; among four groups (p-CaMKII\u0026alpha;/t- CaMKII\u0026alpha;:\u003cem\u003e\u0026nbsp;F\u003c/em\u003e\u003csub\u003e(3,8)\u003c/sub\u003e = 16.92, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Consistent with the expression pattern of TRPV1, the ratio of p-CaMKII\u0026alpha; to t-CaMKII\u0026alpha; was significantly increased in CSDS-susceptible mice (\u003cem\u003ep\u003c/em\u003e = 0.038). After CPZ administration, this ratio was markedly reduced (\u003cem\u003ep\u003c/em\u003e = 0.001, Figure. 3C); In contrast, CAP treatment failed to further increase hippocampal TRPV1 or p-CaMKII\u0026alpha; levels.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePrevious studies have established that depression-like behaviors in mice are frequently accompanied by neuroinflammation, which primarily originates from microglial and astrocytic activation in the CNS. Therefore, we quantified the expression of cluster of differentiation 68 (CD68, a microglial activation marker) and glial fibrillary acidic protein (GFAP, a astrocytic activation marker) across four groups(CD68:\u003cem\u003e\u0026nbsp;F\u003c/em\u003e\u003csub\u003e(3,8)\u003c/sub\u003e = 16.92,\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001). In comparison to the CON + PBS group, the CD68 expression in CSDS + PBS group exhibited significantly increased (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001, Figure. 2D); The CSDS + CPZ group significantly attenuated stress-induced elevations of CD68 in the hippocampus (vs CSDS + PBS, \u003cem\u003ep\u003c/em\u003e = 0.049; Figure. 3D). These results demonstrated that the CSDS model successfully induced microglial activation, which could be effectively suppressed by administering CPZ(Figure. 3D). However, there were no significant differences in GFAP among the four groups (Supplementary Figure .1).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMicroglial activation in the hippocampus was evaluated through both quantitative and morphological analyses to assess neuroinflammatory responses. To determine the effect of CSDS on the number of microglia, we quantitatively analyzed the number of Iba1 positive cells in the hippocampus by immunofluorescence staining (DG: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,12)\u003c/sub\u003e = 12.17,\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001; CA1: \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,12)\u003c/sub\u003e = 6.62, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e=0.005; CA3:\u003cem\u003e\u0026nbsp;F\u003c/em\u003e\u003csub\u003e(3,12)\u003c/sub\u003e = 5.18,\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003e= 0.017). The result showed that the number of Iba1 positive cells in three subregions of the hippocampus were significantly increased in the CSDS + PBS group compared with the CON + PBS group (DG: \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.003; CA1: \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.045;\u003cem\u003e\u0026nbsp;\u003c/em\u003eCA3: \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.022); however, the CSDS + CPZ group had fewer Iba1 positive cells in DG area of the hippocampus than the CSDS + PBS group (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.013), but there were no significant differences Iba1 positive cells in CA1 or CA3 area between the CSDS + PBS and the CSDS + CAP group.(Figure. 3E-H).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further investigate the impact of CPZ on the morphology of microglia, we analyzed microglia morphology in confocal microscopy images using the skeleton analysis plugin of ImageJ (Figure. 3E). There were significant differences in the total branch length/cell, the number of microglial process endpoints/cell, and the maximum branch length among four groups (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,20)\u003c/sub\u003e = 11.33, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001;\u003cem\u003e\u0026nbsp;F\u003c/em\u003e\u003csub\u003e(3,20)\u003c/sub\u003e = 14.53, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001; \u003cem\u003eF\u003c/em\u003e\u003csub\u003e(3,20)\u003c/sub\u003e = 6.05, \u003cem\u003ep\u003c/em\u003e = 0.004). Compared with the CON + PBS group, the total branch length/cell and the number of microglial process endpoints/cell were significantly decreased in the DG of the CSDS + PBS (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.024; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001); however, the CSDS + CPZ group had significantly increased total branch length/cell and the number of microglial process endpoints/cell in the DG of hippocampus relative to the CSDS + PBS group (\u003cem\u003ep\u003c/em\u003e = 0.002; \u003cem\u003ep\u003c/em\u003e = 0.003), indicating that CPZ increased the hippocampal microglia branching complexity of CSDS model mice(Figure. 3I - K). In addition, the maximum branch length in the CSDS + PBS group was shorter after CSDS stimulation than in the CON + PBS group (\u003cem\u003ep\u003c/em\u003e = 0.008); compared with the CSDS + PBS group, the maximum branch length was longer in the CSDS + CPZ (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.035; Figure. 3L).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.3. CPZ inhibits neuroinflammation and impaired neurogenesis via JAK2/STAT3 pathway\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePro-inflammatory cytokines produced by microglia play a crucial role in the neuroinflammation of depression. We measured the levels of pro-inflammatory factors (IL-6, IL-1\u0026beta;, TNF-\u0026alpha;, and NO) in hippocampal brain tissues(\u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 20)\u003c/sub\u003e = 14.27, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001; \u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 20)\u003c/sub\u003e = 8.52, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001; \u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 20)\u003c/sub\u003e = 5.83, \u003cem\u003ep\u003c/em\u003e = 0.039; \u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 20)\u003c/sub\u003e = 20.92, p \u0026lt; 0.0001). Compared to the CON +PBS group, the levels of IL-6, IL-1\u0026beta;, TNF-\u0026alpha;, and NO were increased after stress exposure (\u003cem\u003ep\u003c/em\u003e = 0.001; \u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.004; \u003cem\u003ep\u003c/em\u003e = 0.030; \u003cem\u003ep\u003c/em\u003e = 0.005); treatment with CPZ during stress decreased IL-6 and IL-1\u0026beta; levels compared with the CSDS + PBS group, while TNF-\u0026alpha; and NO levels showed no significant changes(p = 0.019; \u003cem\u003ep\u003c/em\u003e = 0.026; Figure. 4A-D). In contrast, treatment with CAP during stress resulted in no significant changes in IL-6, IL-1\u0026beta;, or TNF-\u0026alpha; levels but significantly increased NO levels (p \u0026lt;0.0001). Considered the closely links between neuroinflammation and neurogenesis, we hypothesized that TRPV1 may influence neurogenesis while modulating neuroinflammation. Consistent with our hypothesis, the results demonstrated that, compared with the CON + PBS group, the CSDS + PBS group exhibited a significant reduction in the number of BrdU-positive cells in the hippocampal DG region (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.007); notably, the CSDS + CPZ group significantly increased the number of BrdU-positive cells in the DG (\u003cem\u003ep\u003c/em\u003e = 0.002), whereas the CSDS + CAP group did not exert a similar effect (Figure. 4E-F).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo elucidate the specific signaling pathways involved, we conducted the levels of phosphorylated JAK2 (p-JAK2) and phosphorylated STAT3 (p-STAT3) in the hippocampus (\u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 8)\u003c/sub\u003e = 17.02, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001;\u003cem\u003e\u0026nbsp;F\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 8)\u003c/sub\u003e = 24.51, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Our findings showed that, compared with the CON + PBS group, the CSDS + PBS group significantly increased the levels of p-JAK2 at the Y1007-Y1008 sites and p-STAT3 at the Y705 site in hippocampal (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.003; \u003cem\u003ep\u003c/em\u003e = 0.001), suggesting activation of the JAK2/STAT3 signaling pathway in hippocampus; importantly, the CSDS + CPZ group significantly reduced the levels of p-JAK2 (Y1007-Y1008), and p-STAT3 (Y705) compared with the CSDS + PBS group(\u003cem\u003ep\u003c/em\u003e = 0.019; \u003cem\u003ep\u003c/em\u003e = 0.024); in contrast, treatment with CAP did not significantly alter the level of p-JAK2 or p-STAT3 (Figure. 4G-I).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCollectively, these results suggested that chronic stress induces significant activation of the JAK2/STAT3 signaling pathway in hippocampus contributing to neuroinflammation and impaired neurogenesis. However, whether the suppression of JAK2/STAT3 signaling is essential for the neurogenic effects of CPZ remained to be determined. To clarify this mechanism, we investigated the impact of direct JAK2 activation on the neuroprotective effects conferred by CPZ.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.4\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eCA1 Reverses the alleviating effect of CPZ on depression-like behaviors\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further validate the aforementioned findings, we employed CA1, a JAK2 agonist, to investigate whether the remission effect of depression-like behaviors was attributable to CPZ treatment via acting on JAK2/STAT3 pathway. Relative to the CON + PBS group, the CSDS + PBS exhibited significant social avoidance (SIR \u0026lt; 1) in the SIT (H =15.91, \u003cem\u003ep\u003c/em\u003e = 0.037). This effect was reversed by CPZ (vs. CSDS + PBS, SIR \u0026gt; 1; p = 0.042), whereas the CSDS + CPZ +CA1 group showed blocked the rescue effect (vs. CSDS + CPZ, SIR \u0026lt; 1, \u003cem\u003ep\u003c/em\u003e = 0.023, Figure. 5A). In the TST and SPT (H\u003csub\u003e\u0026nbsp;\u003c/sub\u003e=16.36, \u003cem\u003ep\u003c/em\u003e = 0.001;\u003cem\u003e\u0026nbsp;F\u003c/em\u003e\u003csub\u003e(3,20)\u0026nbsp;\u003c/sub\u003e= 16.32, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.0001), the CSDS + PBS group showed significantly prolonged immobility time and reduced sucrose preference compared with the CON + PBS group (\u003cem\u003ep\u003c/em\u003e =0.021;\u003cem\u003e\u0026nbsp;p\u0026nbsp;\u003c/em\u003e= 0.002); these effects were attenuated by CPZ treatment (vs. CSDS + PBS; p = 0.009; Figure. 5B-C). However, the CSDS + CPZ + CA1 group significantly extended immobility time and decreased sucrose preference (vs. CSDS + CPZ,\u003cem\u003e\u0026nbsp;p\u003c/em\u003e = 0.035, \u003cem\u003ep\u0026nbsp;\u003c/em\u003e\u0026lt; 0.001, Figure. 5B-C). There were no significant differences in the total distance in the OFT between the four groups (Figure. 5D).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003e3.5. Activation of the JAK2/STAT3 pathway abrogates the neuroprotective effect of CPZ\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConsistent with our hypothesis, CA1 activation was found to counteract the antidepressant-like effects of CPZ treatment. We further investigated whether this reversal effect was associated with corresponding molecular changes. The results revealed that, within the CSDS + PBS group, administration of CA1 reversed the inhibitory effects of CPZ on the JAK2/STAT3 pathway (\u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 8)\u003c/sub\u003e = 31.46, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001;\u003cem\u003e\u0026nbsp;F\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 8)\u003c/sub\u003e = 30.16, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, Figure. 5A-G). CA1 blocked the protective effect of CPZ against CSDS-induced microglial activation and production of IL-6 (\u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 8)\u003c/sub\u003e = 15.59, \u003cem\u003ep\u003c/em\u003e = 0.001; \u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 8)\u003c/sub\u003e = 31.93, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001, Figure. 6A-G). Consistently, neurogenesis showed significant differences among the four groups (\u003cem\u003eF\u0026nbsp;\u003c/em\u003e\u003csub\u003e(3, 20)\u003c/sub\u003e = 9.80, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). Immunofluorescence analysis demonstrated that in the CSDS model, the intensity of DCX immunoreactivity in the hippocampus was significantly diminished in the group receiving both CPZ and CA1 compared to the group treated with CPZ alone (\u003cem\u003ep\u0026nbsp;\u003c/em\u003e= 0.008). Notably, DCX expression in the CPZ+CA1 group was reduced to a level comparable to that observed in the CSDS-only group, indicating that the neurogenic protective effect of CPZ was abolished by CA1 co-treatment (\u003cem\u003ep\u003c/em\u003e \u0026gt; 0.05, Figure. 6H-I). These findings provide compelling evidence that the neuroprotective effect of CPZ is mediated through inhibition of the JAK2/STAT3 signaling pathway.\u0026nbsp;\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study demonstrated that the TRPV1 channel antagonist CPZ alleviates depression-like behaviors, exerting its potential antidepressant effects through attenuation of neuroinflammation and promotion of neurogenesis (Figure. 7). The expression of TRPV1 and phosphorylation level of CaMKIIα was upregulated in depression-like mice induced by CSDS, accompanied by microglial activation, evidenced by morphological and numerical changes, along with increased pro-inflammatory factor release. Similarly, hippocampal newborn neurons were reduced. These outcomes correlated with activation of the JAK2/STAT3 signaling pathway. Inhibition of TRPV1 mitigated microglial activation, reduced pro-inflammatory cytokine release, promoted the generation of newborn neurons, and suppressed JAK2/STAT3 signaling pathway activation.\u003c/p\u003e\u003cp\u003eThe social interaction ratio serves as a key behavioral indicator for evaluating whether CSDS model successfully induces depression-like phenotypes[38]. Our results demonstrated that mice exposed to aggressive encounters exhibited significant social avoidance and reduced interaction behaviors. Furthermore, mice exposed to aggressive encounters displayed pronounced despair-like behaviors, anhedonia, and reduced locomotor activity. Importantly, all these depression-liked behavioral deficits were effectively reversed by CPZ treatment. These findings are consistent with previous studies demonstrating that reduced TRPV1 expression ameliorates depression-like behaviors in mice[12, 41]. On the other hand, the treatment of CAP does not seem to exert an effect on depression-like behaviour in mice. Current opinion regarding the role of TRPV1 channel overactivation in depressive-like behaviors remain inconsistent, potentially due to unique desensitization properties of TRPV1[42]. TRPV1 channel opening in response to various stimuli triggers calcium ion influx, which may induce channel desensitization[43]. The desensitization mechanism of the TRPV1 channel exhibits distinct characteristics in comparison to other ligand-gated ion channels[44]. Specifically, following desensitization induced by a low concentration of CAP, the activity of the TRPV1 channel can be reinstated through the application of a higher concentration of the stimulating agent, CAP[45]. This bidirectional pharmacological action may explain the contradictory outcomes observed with CAP administration. Consequently, our subsequent experiments will systematically evaluate both the concentration and temporal dynamics of central CAP exposure to elucidate the precise role of TRPV1 channel activation in depression-like behaviors.\u003c/p\u003e\u003cp\u003eUpon exposure to stress or pathogenic challenges, microglia and astrocytes within the CNS become activated, releasing a substantial amount of pro-inflammatory cytokines and metabolic byproducts that exert direct neurotoxic effects, thereby contributing to the pathophysiology of depression[46]. Previous studies have indicated that TRPV1 is closely associated with the onset of neuroinflammation[47]; however, its precise role, whether protective or detrimental, remains controversial[48, 49]. Our findings indicated that TRPV1 channel antagonist reduced the expression of microglial markers Iba1 and CD68, and decreased levels of pro-inflammatory cytokine IL-6 and IL-1β. Similar to our results, the downregulation of TRPV1 alleviated microglial hyperactivation induced by hyperthermia and lipopolysaccharide, restoring homeostasis in the inflammatory microenvironment of the brain[50]. Baban et al. demonstrated that aminoalkylindole cannabinoids, acting as TRPV1 channel antagonists, significantly attenuate microglial activation and reduce pro-inflammatory cytokine production[51]. A recent study inferred that TRPV1 deficiency or pharmacological blockade disrupts apoptosis-associated speck-like protein containing a card speck formation in microglia through the Ca\u0026sup2;⁺-PP2A pathway, resulting in impairing inflammasome complex assembly, suppressing NOD-like receptor family pyrin domain-containing 3 inflammasomes activation, and mitigating neuroinflammatory responses[21]. Lin et al. reported that electroacupuncture effectively alleviates depressive symptoms, potentially through modulation of the TRPV1 pathway and suppression of pro-inflammatory cytokine production[43]. These results support that TRPV1 inhibition may alleviate neuroinflammation by suppressing microglial activation and reducing the release of specific pro-inflammatory cytokines[18, 52]. Furthermore, although TNF-α levels were elevated following chronic stress, modulation of TRPV1 did not alter its expression, suggesting that TNF-α secretion may be regulated by alternative intracellular pathways within microglia[19].\u003c/p\u003e\u003cp\u003ePrevious studies demonstrated that excessive activation of the TRPV1 channel promotes microglial and astrocytic activation, accelerating their migration and chemotactic responses under stress conditions[53, 54]. In this study, we observed a marked activation of microglia in the hippocampus of mice subjected to CSDS, whereas astrocytes did not exhibit a similar response. This discrepancy may be attributed to the role of microglia as sensors of microenvironmental changes in the CNS, rendering them more sensitive to chronic and sustained stress[28]. Supporting this notion, research indicated that during the subacute phase of CNS disorders, both microglia and astrocytes become overactivated. However, microglial activation occurs approximately three hours earlier than that of astrocytes[55]. Consequently, in our study, significant changes were observed in the microglial markers Iba1 and CD68, whereas astrocytic markers remained largely unchanged.\u003c/p\u003e\u003cp\u003eAHN contributes significantly to various neurocognitive and affective functions, including memory formation, memory extinction, stress resilience, and mood regulation[56]. Growing evidence indicates that this neurogenic process is fundamentally involved in both depression pathogenesis and the mechanisms underlying antidepressant efficacy[57]. These findings highlight hippocampal neurogenesis as a critical mediator of both depressive disorders and their treatment[58]. We found that the inhibition of the TRPV1 channel significantly promoted neurogenesis. Stock et al. demonstrated that TRPV1 channel expression in neural precursor cells decreases with aging, and TRPV1 knockout mice exhibited enhanced neural precursor cell proliferation compared to control littermates, suggesting that TRPV1 expression modulates activity-dependent neurogenesis during both postnatal development and adulthood[59]. Consequently, TRPV1 inhibition promotes neurogenesis and consequently alleviates depression-like behaviors.\u003c/p\u003e\u003cp\u003eThrough a comprehensive literature search, we speculated that TRPV1 may mediate the anti-inflammatory effects and promotes neurogenesis through modulation of the JAK2/STAT3 signaling pathway. Yang et al. reported that TRPV1 is essential for JAK2/STAT3 phosphorylation[28]. Within the CNS, TRPV1 activation has been demonstrated to promote pro-inflammatory STAT3 signaling, thereby amplifying neuroinflammatory responses. Previous study has confirmed that JAK2 inhibition, through suppression of STAT3 phosphorylation, effectively attenuates neuroinflammation. In alignment with these findings, our results indicated that TRPV1 mediates microglial activation and subsequent secretion of IL-6 and IL-1β via the JAK2/STAT3 pathway. Neural stem cells (NSCs) possess intrinsic self-renewal and proliferative capabilities, and their fate decisions are tightly regulated by phosphorylation of STAT3 at Tyr705[60]. Further evidence indicates that inhibition of the JAK2/STAT3 pathway restores Tet3-mediated DNA demethylation, facilitating neuronal differentiation[27]. Wang et al. reported that TRPV1 activation up-regulates HDAC2 and decreases acrtylation of histone H3, resulting in lessened expression of neurogenesis-related genes and the loss of NSCs[41]. Our results extended this line of research by identifying TRPV1 inhibition supports hippocampal neurogenesis.\u003c/p\u003e\u003cp\u003eDespite the fact that we discovered CPZ treatment alleviates depression-like behaviors via TRPV1-mediated modulation of neuroinflammation and neurogenesis, there still remains some limitations. On the one hand, the activation of TRPV1 did not exert any effects in mice exhibiting depression-like behaviors. The observed outcome might result from the actual lack of involvement of TRPV1 in the pathogenic mechanisms of depression. Alternatively, it may stem from our inability to determine the concentration and effective duration of CAP within the CNS after intraperitoneal administration, potentially introducing confounding effects into the results. In future study, it is necessary to clarify the functional state of TRPV1 channels and determine their exact effects. On the other hand, existing evidence demonstrates that CAP can mediate anti-inflammatory effects through TRPV1-independent mechanisms. Potential intracellular targets of TRPV1 agonists beyond TRPV1 itself have yet to be fully deciphered. These alternative targets may modulate secondary or tertiary signaling cascades downstream of TRPV1, or potentially interact with other signaling receptors to trigger complex cross-talk mechanisms affecting neuroinflammatory pathways. Considering the intricacy of intracellular signaling networks, these hypotheses warrant further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, our findings suggest that CPZ produces significant antidepressant-like effects in CSDS-susceptible mice. This study provides novel mechanistic insights into TRPV1-mediated neuroinflammatory regulation and its impact on neurogenesis, suggesting that microglial TRPV1 might represent a promising therapeutic target for depression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by the National Natural Science Foundation of China (Nos. 81571325, 81871072, and 82071523) and the Medical Science Advancement Program of Wuhan University (No. TFLC2018001). The Key Research And Development Program Of Hubei Province (2020BCA064) also supported the design of this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorship Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJunjie Huang and Chen Li: Methodology, Software, Investigation, Writing-Original Draft. Hailong Ge: Conceptualization, Methodology. Lan Wu: Methodology. Ling Xiao: Writing- Review \u0026amp; Editing, Supervision, Project administration. Yinping Xie: Conceptualization, Writing-Review \u0026amp; Editing. Gaohua Wang: Resources, Supervision, Project administration, Funding acquisition.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the necessary data are included in the article. Further data will be shared by request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was carried out based on the Regulations of Experimental Animal Administmiceion issued by the State Committee of Science and Technology of the People\u0026rsquo;s Republic of China, with the approval of the Ethics Committee in Renmin Hospital of Wuhan University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYang C-H, Lv J-J, Kong X-M, et al (2024) Global, regional and national burdens of depression in adolescents and young adults aged 10-24 years, from 1990 to 2019: findings from the 2019 Global Burden of Disease study. 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J Neuroinflammation 18:192. https://doi.org/10.1186/s12974-021-02242-8\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Depression, TRPV1, Neuroinflammatory, Neurogenesis, JAK2/STAT3","lastPublishedDoi":"10.21203/rs.3.rs-6992017/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6992017/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDepression is a common mental disorder with a currently unclear pathogenesis, and it often fails to repond to treatment. Transient receptor potential vanilloid 1 (TRPV1) is known to regulate glial activation and inflammatory responses, yet its specific role in stress-related depressive pathophysiology remains incompletely understood. In this study, we explored the effects of modulating TRPV1 in neuroinflammation and neurogenesis underlying depression-like behaviors. A chronic social defeat stress (CSDS) mice model, followed by behavioral tests, was used to evaluate the antidepressant potential of capsaicin (CAP) and capsazepine (CPZ). Then, western blotting, elisa, and immunofluorescence were employed to assess microglial activation, the pro-inflammatory cytokine levels, neurogenesis, and JAK2/STAT3 signaling pathway in hippocampus tissues. The JAK2 agonist coumarin A1 (CA1) was used to verify the involvement of the JAK2/STAT3 pathway. In CSDS-susceptible mice, the protein expression of TRPV1 and the levels of p-CaMKIIα were elevated, and CPZ downregulated the expression of these indicators. Also, CSDS reduced the migcroglial numbers and altered microglial morphology in the subregions of the hippocampus; these results were accompanied by proinflammatory cytokine production. In parallel, TRPV1 inhibition by CPZ suppressed promoted neurogenesis, which was testified by increased BrdU\u0026thinsp;+\u0026thinsp;and DCX\u0026thinsp;+\u0026thinsp;cells. Further, CPZ exerts its regulatory effects through inhibition of the JAK2/STAT3 signaling pathway. CA1 reversed the neuroprotective effects of CPZ, confirming the involvement of the JAK2/STAT3 pathway. The discovered microglia-mediated mechanism provides novel insight into how TRPV1 modulation ameliorate stress-induced neuroinflammation and impaired neurogenesis These findings identify microglial TRPV1 as a potential therapeutic target for depression.\u003c/p\u003e","manuscriptTitle":"Capsazepine Treatment Alleviates Depression-Like Behaviors via TRPV1-Mediated Modulation of Neuroinflammation and Neurogenesis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-08 14:26:06","doi":"10.21203/rs.3.rs-6992017/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-11T12:45:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T09:10:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-11T09:09:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-08T14:21:12+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-30T07:31:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"198174817932165678760323866577850792286","date":"2025-09-28T08:46:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"100169901497586965867031878941901709447","date":"2025-09-28T01:35:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"326253785007797592556946937169177505514","date":"2025-09-27T02:00:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"67779820392745616582677481123399648074","date":"2025-09-25T13:29:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"66207474462686709554406556521991729721","date":"2025-09-25T13:20:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"295639985641666420013969101552835736227","date":"2025-09-25T13:15:16+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-25T12:55:55+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-04T22:32:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-04T22:32:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Neurobiology","date":"2025-06-27T13:13:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-neurobiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"moln","sideBox":"Learn more about [Molecular Neurobiology](https://www.springer.com/journal/12035)","snPcode":"12035","submissionUrl":"https://submission.nature.com/new-submission/12035/3","title":"Molecular Neurobiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"25d57f3e-b603-4fdc-a1ac-b5a5c9fa325f","owner":[],"postedDate":"October 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-23T16:08:06+00:00","versionOfRecord":{"articleIdentity":"rs-6992017","link":"https://doi.org/10.1007/s12035-026-05733-y","journal":{"identity":"molecular-neurobiology","isVorOnly":false,"title":"Molecular Neurobiology"},"publishedOn":"2026-02-18 15:59:13","publishedOnDateReadable":"February 18th, 2026"},"versionCreatedAt":"2025-10-08 14:26:06","video":"","vorDoi":"10.1007/s12035-026-05733-y","vorDoiUrl":"https://doi.org/10.1007/s12035-026-05733-y","workflowStages":[]},"version":"v1","identity":"rs-6992017","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6992017","identity":"rs-6992017","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}