Neuroprotective Effects of 5-Aminolevulinic Acid Combined with Ferrous Iron on Chronic Stress- Induced Depressive-Like Behavior in Mice by Activating the HO-1/Nrf2 Signaling Pathway

Neuroprotective Effects of 5-Aminolevulinic Acid Combined with Ferrous Iron on Chronic Stress- Induced Depressive-Like Behavior in Mice by Activating the HO-1/Nrf2 Signaling Pathway | 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 Neuroprotective Effects of 5-Aminolevulinic Acid Combined with Ferrous Iron on Chronic Stress- Induced Depressive-Like Behavior in Mice by Activating the HO-1/Nrf2 Signaling Pathway Jiangang Hou, Haoran Chen, Daqian Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7949176/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Depression, classified as a mental disorder, exerts a profound negative impact on the overall well-being and life satisfaction of individuals affected. The aim of the current study was to investigate the role of 5-Aminolevulinic acid (5-ALA) in treating depression. Previous research has indicated that 5-ALA/ferrous iron (Fe 2+ ) possesses anti-oxidative and anti-inflammatory properties. However, the effectiveness of the combination of 5-ALA/Fe 2+ in treating depression remains inadequately characterized. In the mice model induced by 6 weeks chronic unpredictable mild stress, 5-ALA/Fe 2+ is tested to determine its effect on improving depression-like behavior. In the behavioral tests, 5-ALA/ Fe 2+ was found to have anxiolytic effects on mice behavior. The administration with 5-ALA/Fe 2+ exhibited significant advantages in terms of decreased oxidative stress and proinflammatory cytokines, and increased levels of neurotransmitters in the hippocampus. Additionally, the group treated with 5-ALA/ Fe 2+ exhibited a substantial decrease in the protein expression of IBA-1 and GFAP within the hippocampus of mice. The administration of 5-ALA/Fe 2+ promoted the expression of associated proteins within the activated HO-1/Nrf2 pathway, as well as the inhibition of the NLRP3 Inflammasome Activation induced by chronic stress. Consequently, 5-ALA/Fe 2+ exhibits promising potential as a therapeutic candidate for the treatment of depression. Depression Neuroinflammation 5-ALA Microglia Activation Astrocyte Activation NLRP3 Inflammasome HO-1/Nrf2 Signaling Pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Depression is widely acknowledged as a prevalent mental disorder characterized by a range of symptoms including sadness, guilt, diminished appetite, fatigue, impaired concentration, reduced self-worth, diminished interest or pleasure, and disrupted sleep patterns. The worldwide incidence of this condition is substantial, affecting an estimated 300 million individuals. Previous studies have demonstrated the involvement of neurotransmitters and inflammation in the etiology of depression, comprising a dearth of neurotransmitters such as serotonin (5-HT) and dopamine (DA) [ 1 – 3 ]. The presence of dysfunction in the electrical activity of dorsal raphe 5-HT neurons, coupled with reduced levels of extracellular 5-HT in the hippocampus, could potentially be linked to the manifestation of anxiety and depression. These symptoms serve as prominent and early indicators of depressive disorders [ 4 ]. Stressful experiences, particularly chronic and unintended stressors, are substantial risk factors that exert a pervasive influence on the development and exacerbation of a wide range of diseases. Chronic stress is associated with a neurobehavioral syndrome that exhibits characteristics indicative of depression, as well as various processes encompassing psychological disorders and activation of the inflammatory immune system [ 5 – 7 ]. Numerous studies have consistently demonstrated the connection between the psychological, behavioral, and neurobiological manifestations of depression and the impact of inflammatory cytokines [ 8 – 10 ]. Neuroinflammation serves as a protective response initiated by the central nervous system in reaction to cerebral irregularities. Its primary functions involve the elimination of damaged cell fragments through immune cells within the brain, as well as the regulation of neurotrophic factor secretion to uphold brain homeostasis and preserve normal physiological functioning [ 11 ]. The progressive development of chronic neuritis is associated with neurotoxicity caused by the substantial release of cytokines. The excessive accumulation of neurotoxic factors further stimulates the proliferation of glial cells within the nervous system, leading to secondary harm to the nervous system and impeding the regeneration of neuronal cells. Consequently, neuritis is considered a contributing factor to various neurodegenerative disorders [ 12 , 13 ]. In essence, uncontrolled or persistent neuroinflammation, whether as an initiator or as a cascading feedback response, has the potential to drive chronic and progressive neurodegenerative processes. Neuroinflammation has been identified as a prominent area of investigation in academic research, specifically in relation to its association with depression. Previous studies had demonstrated an augmented secretion of pro-inflammatory cytokines during the progression of neuroinflammation, such as IL-6, TNF-α and IL-1β [ 14 , 15 ]. Available clinical studies revealed that patients with depression exhibited elevated concentrations of IL-6, TNF-α and IL-1β, in both their cerebral spinal fluid and serum [ 16 ]. Neuroinflammation is involved in the excessive release of oxidative and neurotoxic molecules, impairing the physiological function of normal neurons and leading to synaptic dysfunction [ 10 , 18 ]. The manifestation of oxidative stress has provided evidence for the induction of neuronal degeneration and its contribution to the development of depression [ 19 , 20 ]. The existing literature has provided evidence that superoxide dismutase (SOD) serves as a crucial endogenous antioxidant enzyme and a fundamental component of the primary defense mechanism against reactive oxygen species (ROS) [ 21 , 22 ]. Malondialdehyde (MDA) is a byproduct resulting from the process of lipid peroxidation, and its concentration can serve as an indirect indicator of the extent of lipid peroxidation-induced damage. The NLRP3 inflammasome, a protein complex within the innate immune system, is responsible for detecting molecular patterns associated with pathogens and risks. According to current research, the activation of NLRP3 is associated with depression [ 23 ]. When excessively activated, it can culminate in the induction of pyroptosis, accompanied by the generation of proinflammatory cytokines such as IL-6, TNF-α and IL-1β, leading to the development of neuroinflammatory conditions [ 24 ]. Overall, NLRP3 has been extensively implicated in the modulation of neuroinflammatory processes [ 25 ]. Previous studies revealed that therapy can exert notable antidepressant-like effects, by regulating the activation of NLRP3 [ 26 ]. Moreover, microglia and astrocytes also assume a crucial role in neuroinflammatory [ 27 ]. They function as the primary defense mechanism in the brain, safeguarding the CNS. Microglia and astrocytes promptly undergo activation in response to diverse stimuli. Transient neuroinflammation serves a beneficial role in combating bacteria, and eliminating cell debris discharged from damaged cells. Nevertheless, prolonged overactivation and dysregulated neuroinflammation contribute to neuronal cell demise and exacerbate the progression of neurodegenerative disorders [ 28 ]. Microglia and astrocytes actively engage in the neuroinflammatory process through the secretion of various proinflammatory cytokines, including IL-6, TNF-α and IL-1β [ 29 ]. Therefore, the suppression of microglial activation and the safeguarding of neurons against neuroinflammation may present a viable alternative approach for the therapeutic intervention of depression. 5-Aminolevulinic acid (5-ALA) is an endogenous amino acid that is ubiquitously found in diverse organisms [ 30 ]. It is synthesized by the catalytic action of 5-ALA synthase, which involves the condensation of glycine and succinyl-CoA. In animal cells, 5-ALA plays a crucial role as a precursor in heme synthesis [ 31 ]. Heme oxygenase (HO)-1 enzymatically breaks down heme into biliverdin, carbon monoxide (CO), and ferrous iron (Fe 2+ ), which are utilized in conjunction with 5-ALA. Numerous studies have extensively demonstrated that the 5-ALA/Fe 2+ exerts anti-inflammatory and anti-oxidative effects by up-regulating the expression of HO-1 [ 32 – 35 ]. The HO-1, which governs the rate of heme metabolism and produces biliverdin, CO, and Fe2+, has been recognized as a pivotal factor in diverse anti-inflammatory, anti-apoptotic, and anti-oxidative mechanisms. [ 36 ]. Zinc protoporphyrin IX (Zinc protoporphyrin IX, ZnPPIX), an inhibitor of HO-1, can inhibit HO-1 expression, and has been used as an inhibitor to explore HO-1-related signaling pathways in previous studies. However, there is a lack of research investigating the impact of 5-ALA/Fe 2+ on the anti- neuroinflammation response in relation to depression. Therefore, the aim of our study was to evaluate the influence of 5-ALA/Fe 2+ on behavior, microglial activation, neuronal apoptosis, and neuroinflammatory responses in depressive mice, along with exploring potential anti-neuroinflammatory mechanisms. Results 1.The effect of 5-ALA/Fe 2+ on depressive-like behavior in chronic stress-induced depressive mice 1.1 Open Field Test The chronic stress-induced mice displayed a significantly depressed phenotype, as evidenced by their increased durations of immobility time during the Open Field Test (OFT), in comparison to the stress-free control mice (F(3, 20) = 3.016, p<0.001) (Fig. 1 B). Additionally, chronic stress decreased the total move distance, and lowers the time spent within center zone and the number of entries into center zone (F(3, 20) = 1.228, F(3, 20) = 3.016, F(3, 20) = 4.806, p<0.01) (Fig. 1 A, C, D). The mice belonging to the 5-ALA/Fe 2+ group demonstrated a notable reduction in immobility the duration in comparison to the chronic stress group. Moreover, the mice belonging to the 5-ALA/Fe 2+ group exhibited a noteworthy augmentation in the duration spent within the central area and the number of entries into center zone, in comparison to the chronic stress group (p<0.01) (Fig. 1 A-D). Moreover, treatment with ZnPPIX counteracted 5-ALA/Fe 2+ inhibitory effect on depressive-like behavior. 1.2 Forced Swim Test Our results demonstrated a significant difference in the durations of immobility observed in the forced swim test (FST) among various groups. Specifically, the chronic stress group exhibited significantly longer durations of immobility in the FST compared to the stress-free control group. Likewise, the chronic stress group displayed significantly elevated durations of immobility in the FST compared to the 5-ALA/Fe 2+ group (F=(3, 20)3.128, p<0.01) (Fig. 1 E). 1.3 Tail Suspension Test For tail suspension test (TST), the duration of immobility time exhibited a significant decrease in both the stress-free control group and the 5-ALA/Fe 2+ group, as compared to the chronic stress group (F(3, 20) = 0.2584, p<0.001) (Fig. 1 F). 2. The effect of 5-ALA/Fe 2+ on neurotransmitters concentration in chronic stress-induced depressive mice The measurement of 5-HT and DA levels in the hippocampus revealed a noteworthy elevation in the stress-free control group compared to the chronic stress group. Additionally, the 5-ALA/Fe 2+ group exhibited a significant increase in 5-HT and DA levels when compared to the chronic stress group (F(3, 20) = 1.118, F(3, 20) = 1.779, p<0.05) (Fig. 2 A, B). 3. The effect of 5-ALA/Fe 2+ on anti-oxidant ability in chronic stress-induced depressive mice The chronic stress group demonstrated a significant reduction in the activity of SOD compared to the control group. In contrast, the activity of SOD showed a significant increase in the 5-ALA/Fe 2+ group relative to the chronic stress group (F(3, 20) = 1.314, p<0.05) (Fig. 3 C). After exposure to prolonged stress, the hippocampal tissue of the group subjected to chronic stress exhibited a significant elevation in the level of MDA and ROS, as compared to the stress-free control group (p<0.05) (F(3, 20) = 0.9673, Fig. 3 A, B). Conversely, in the 5-ALA/Fe 2+ group, the alteration in MDA and ROS content was notably reduced in comparison to the chronic stress group, unlike the observed changes in SOD. However, the administration of ZnPPIX to the experiment led to the reversal of the aforementioned alterations (p<0.05) (Fig. 3 A-C). 4. The effect of 5-ALA/Fe 2+ on anti-inflammatory ability in chronic stress-induced depressive mice Our findings demonstrated a significant increase in the production of TNF-α, IL-1β, and IL-6 in the chronic stress group, which was effectively mitigated by pretreatment with 5-ALA/Fe 2+ . However, it was observed that the levels of IL-6, IL-1β, and TNF-α in serum were significantly elevated in the 5-ALA/Fe 2+ +ZnPPIX group in comparison to the 5-ALA/Fe 2+ group (p<0.01) (F=(3, 20)0.6697, F(3, 20) = 0.2157, F(3, 20) = 1.427, Fig. 4 A-C). Furthermore, the results demonstrate that the administration of 5-ALA/Fe 2+ significantly attenuated the expression of iNOS and COX-2 in brain hippocampus. But in the 5-ALA/Fe 2+ +ZnPPIX group, the expression of iNOS and COX-2 in brain hippocampus exhibited a statistically significant increase in comparison to the 5-ALA/Fe 2+ group (p<0.05) (F(3, 20) = 3.924, F(3, 20) = 2.428, Fig. 5 A-C). 5. The effect of 5-ALA/Fe 2+ on glial cell activation in chronic stress-induced depressive mice The expression of proteins related to activation of microglia and astrocyte in the brain hippocampus was investigated using Western blot techniques. Chronic stress resulted in elevated protein expression levels of IBA-1 and GFAP in the brain hippocampus. Conversely, treatment with 5-ALA/Fe 2+ reversed these alterations. Additionally in the 5-ALA/Fe 2+ +ZnPPIX group, the expression of IBA-1 and GFAP in brain hippocampus exhibited a statistically significant increase in comparison to the 5-ALA/Fe 2+ group (F(3, 20) = 1.509, F(3, 20) = 0.8686, p<0.05) (Fig. 6 A-C). 6.The effect of 5-ALA//Fe 2+ on NLRP3 Signaling Pathway in chronic stress-induced depressive mice We employed Western blotting to detect the expression of proteins related to the NLRP3 inflammasome. Our findings demonstrated that chronic stress enhanced the expression levels of NLRP3, ASC, IL-1β, IL-18 and caspase-1. However, these effects were counteracted by treatment with 5-ALA/Fe2+. Additionally, the inhibitory effect of 5-ALA/Fe2 + on activation of NLRP3 inflammasome was counteracted by treatment with ZnPPIX (F(3, 20) = 2.160, F(3, 20) = 1.372, F(3, 20) = 3.270, F(3, 20) = 1.011, F(3, 20) = 0.9422, p<0.05) (Fig. 7 A-F). 7.The effect of 5-ALA//Fe 2+ on HO-1/Nrf2 signaling pathway in in chronic stress-induced depressive mice hippocampus In comparison to the chronic stress group, the administration of 5-ALA/Fe 2+ tended to enhance the expression of Nrf2 and HO-1 in the chronic stress group with induced depression. Conversely, minimal expression of Nrf2 and HO-1 was observed in the 5-ALA/Fe 2+ + ZnPPIX group. In the present study, the administration of ZnPPIX resulted in a reduction in the expression of HO-1 in the brain hippocampus of chronic stress-induced mice (F(3, 20) = 0.7312, F(3, 20) = 1.601, p<0.005) (Fig. 8 A-C). Similarly, immunofluorescence staining of Nrf2 in the hippocampus showed the same result (Fig. 8 D). Discussion Chronic stress has been identified as a prominent factor in mental illness exacerbation. The findings of our study indicate that mice experiencing chronic stress-induced depression exhibited a notable elevation in anxiety levels during the OFT and increased immobility time during the FST and TST. These findings of Hiroi and Neumaier suggest that a decrease in anxiety-like behavior is evidenced by an increase in the time spent within center zone [ 37 ]. In OFT, the act of entering the center area serves as a dependable indicator of anxiety, as it exhibits sensitivity to anxiety states induced by chronic stress. The results of the OFT of this study demonstrate that the chronic stress groups, when compared to the stress-free control groups, exhibited a lower number of entries into the center zone and a shorter duration of time spent within center zone. And there was a significant alleviation of depressive behavior in mice after treatment by administration of 5-ALA/Fe 2+ . Multiple studies have provided evidence for the implication of the hippocampus in the pathological advancement of neurodegenerative disorders, and hippocampus is also closely related to the development of anxiety-depressive behaviors [ 38 ]. The hippocampus is a crucial element of the limbic system, positioned between the thalamus and the medial temporal lobe in the brain. Its primary functions include the storage, modification, and synchronization of temporary memories, as well as the acquisition of knowledge and regulation of emotional reactions [ 39 ]. To ascertain the neuroprotective efficacy of 5-ALA/Fe 2+ , we conducted an examination of various indicators, including neurotransmitters, proinflammatory factors and oxidative stress indicators, within the hippocampus of chronic stress-induced depressive mice. In this study, a significant reduction in 5-HT and DA levels within the hippocampus was observed in the mice with chronic stress-induced depression. The administration of 5-ALA/Fe 2+ significantly inhibited the decreased levels of 5-HT and DA induced by chronic stress. The increased levels of 5-HT and DA may be closely related to the antidepressant effects of 5-ALA/Fe 2+ . Much previous literature has confirmed, the neurotransmitter 5-HT has demonstrated a stabilizing effect on anti-depression activity in animal behavior tests [ 40 , 41 ]. Both experimental and clinical evidence consistently demonstrate that depression is associated with elevated levels of oxidative biomarkers and reduced levels of antioxidant defense biomarkers in the brain [ 42 ]. The activity of SOD and the levels of MDA and ROS were measured in the brain hippocampal tissue of chronic stress-induced mice exhibiting depressive symptoms. In the present study, the observed reduction in the activity of SOD and the concentrations of MDA in chronic stress-induced mice suggests a disruption in the antioxidant mechanisms within the brain. And we found that ROS levels increased in the hippocampal tissue of chronic stress-induced mice. However, this impairment was effectively reversed upon administration of 5-ALA/Fe 2+ treatment. The findings consistently demonstrated that the administration of 5-ALA/Fe 2+ significantly augmented the cerebral antioxidant capacity in depressive mice subjected to chronic stress. The findings of our study indicate that the administration of 5-ALA/Fe 2+ resulted in a significant decrease in depressive-related behavior in the behavioral experiments, accompanied by the regulation of neurotransmitters, specifically an increase in 5-HT and DA levels. Furthermore, we assessed the impact of 5-ALA/Fe 2+ on the activation of microglia and astrocyte. The inhibitory effect of 5-ALA/Fe 2+ on anti-activation of microglia and astrocyte was counteracted by treatment with ZnPPIX. The observed reduction in protein expression of IBA-1, GFAP and apoptosis-related proteins suggests that the anti-depressive effects of 5-ALA/Fe 2+ may be mediated through the regulation of microglial activation, potentially involving an indirect modulation of the monoamine neurotransmitter. Neuroinflammation is a key mechanism in the pathophysiology of depression. It is characterised by increased expression of inflammatory factors, activated microglia, astrocytes and NLRP3 inflammasomes in brain, and enhanced peripheral inflammation [ 43 ]. In our study, the activation of microglia and astrocyte was accomplished through the administration of chronic stress. The levels of proinflammatory cytokines (e.g., IL-6, TNF-α and IL-1β) released by activated glial cells exhibited a corresponding increase, leading to the development of neuroinflammation. The supplementation of 5-ALA/Fe 2+ effectively impeded the activation of microglia and astrocytes. These findings unequivocally indicate that 5-ALA/Fe 2+ effectively suppressed the neuroinflammatory response induced by chronic stress. It is widely acknowledged that NLRP3 inflammasome play a pivotal role in regulating neuroinflammation in various brain disorders. These molecules are responsible for transcribing and activating the aforementioned enzymes and proinflammatory cytokines, thereby instigating the onset of neuroinflammation [ 44 ]. To enhance comprehension of the regulation mechanism of neuroinflammation in depression and to identify potential therapeutic targets, an investigation was conducted to examine the involvement of the NLRP3 inflammasome. In our study, chronic stress promoted the activation of NLRP3 inflammasome in in brain hippocampus, and 5-ALA/Fe 2+ diminishes the chronic stress-induced activation of NLRP3 inflammasome. Furthermore, our findings substantiate the close association between NLRP3 inflammasome activation and the activation of microglia and astrocyte, as well as the progression of neuroinflammation in the hippocampus of chronic stress-induced depressive mice. This study demonstrates that the administration of 5-ALA/ Fe 2+ resulted in the enhancement of HO-1 expression in the hippocampus of chronic stress-induced depressive mice. ZnPPIX, which consists of protoporphyrin IX and metal Zn, serves as a heme structural analog. It is frequently employed as an inhibitor of HO-1 due to its capacity to competitively hinder HO activity by occupying the heme binding site [ 45 ]. In our study, the administration of ZnPPIX resulted in a decrease in the expression of HO-1 in the hippocampus of chronic stress-induced depressive mice. This counteracted the neuroprotective effects induced by 5-ALA/Fe 2+ , thereby demonstrating that the neuroprotective effects of 5-ALA/Fe 2+ are mediated through the enhancement of HO-1 expression. It was observed that 5-ALA/ Fe 2+ primarily modulates the HO-1/Nrf2 signaling pathway and NLRP3 inflammasome signaling pathways, leading to the suppression of proinflammatory mediators' synthesis and secretion. These findings demonstrate the anti-neuroinflammatory properties of 5-ALA/Fe 2+ . Conclusions In conclusion, the administration of 5-ALA/Fe 2+ demonstrated a neuroprotective effect on the hippocampus against neuroinflammation induced by chronic stress. Furthermore, 5-ALA/Fe 2+ alleviated the behavioral alterations observed in the OFT, FST and TST resulting from chronic stress. The underlying mechanism responsible for the observed neuroprotective effects of 5-ALA/Fe 2+ involved the enhancement of anti-inflammatory, anti-oxidative and anti-apoptotic effects within the hippocampal tissue. The findings of our study demonstrate that the administration of 5-ALA/Fe 2+ effectively mitigates neuroinflammation induced by chronic stress in an in vivo setting. This effect is achieved through the inhibition of the NLRP3 inflammasome and the activation of the HO-1/Nrf2 signaling pathway. Consequently, 5-ALA/Fe 2+ exhibits promising potential as a therapeutic candidate for the treatment of central nervous system disorders associated with neuroinflammation. Materials and Methods Animals Eighty male C57BL/6J mice (8–10 weeks, 24 ± 2g) were obtained from the Department of Laboratory Animal Science at Fudan University in Shanghai, China. The mice were housed under standard conditions and provided with rodent food and water ad libitum. All procedures involving the experimental animals were conducted in strict accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). The animal experiments were conducted in accordance with the protocols that were approved by the institutional review board of the Department of Laboratory Animal Science at Fudan University in Shanghai, China. Reagents As previously mentioned [ 46 ], 5-ALA/HCl from Merck Millipore in Billerica, USA, and Fe 2+ (sodium ferrous citrate) from Fanhai Biotechnology in Xi'an, China, were dissolved in distilled water. The molar ratio of 5-ALA to Fe 2+ was 1:0.5. Prior to administration, Fe2 + was diluted in distilled water. ZnPPIX (AbMole BioScience, Houston Tx, USA), a HO-1 inhibitor, was diluted in 100 mM NaOH to create a stock solution of 50 mM, which was subsequently stored at -80°C until required. The 5-aminolevulinic acid (5-ALA)/ Fe 2+ complex was orally administered in a volume of 0.3 ml distilled water, while the ZnPPIX solution was administered intraperitoneally in the same volume of 0.3 ml. Efforts were made to minimize light exposure. The dosage administered of 5-ALA/Fe 2 was 100mg/kg, and the dosage administered of ZnPPIX was 5 mg/kg. Study design The mice were subjected to random allocation into four distinct groups, each consisting of six individuals. These groups were designated as follows: the stress-free control group, which received saline for a duration of 6 weeks without chronic stress; the chronic stress group, which received saline for 6 weeks and was subjected to chronic stress; the chronic stress + 5-ALA/Fe 2+ (100mg/kg) group, mice were subjected to daily oral administration of 5-ALA/Fe 2+ for a duration of 6 weeks; and the chronic stress + 5-ALA/Fe 2+ (100 mg/kg) + ZnPPIX (5 mg/kg) group, mice were subjected to daily oral administration of 5-ALA/Fe 2+ for a duration of 6 weeks, while concurrently receiving simultaneous administration of ZnPPIX. On a subsequent day following the administration of the behavioral tests, the mice were euthanized through decapitation. The brain hippocampus were subsequently collected and stored at − 80°C. Chronic immobilization stress procedure As previously mentioned [ 47 , 48 ], the mice were exposed to a range of stressors over a period of six weeks. Each week, the stress regimen included periods of food or water deprivation lasting 16 hours, a tilted cage position of 45° for 12 hours, a soiled cage with 200 mL of water spilled onto the bedding for 8 hours, and stroboscopic lighting with 400 flashes per minute in the dark for 8 hours. The mice experienced one of these stressors each day of the week, with no repetition of the same stressor on consecutive days. Open‑Field Test The OFT was conducted in accordance with previously established protocols [ 49 ]. Prior to the test, mice were acclimated to the behavior room while exposed to background white noise for a minimum of 30 minutes. Subsequently, individual mice were introduced into a 35 cm x 35 cm open field arena by an experimenter who was unaware of the experimental conditions. The mice were then allowed to freely explore the arena for a duration of 20 minutes. To quantify the mice's behavior, video tracking software (TopScan, CleverSys) was utilized to measure the total distance traveled, velocity, and time spent in the center of the arena (which measured 22 cm x 22 cm). Forced Swim Test The FST was conducted according to the previously established protocol [ 50 ]. Each mouse was individually positioned within a glass cylindrical vessel measuring 21 cm in height and 12 cm in diameter, with a total volume of approximately 1000 mL. The vessel was filled with water at a temperature of 22 ± 1°C, reaching a depth of 12 cm. During the experiment, all mice were subjected to a forced swimming task lasting 6 minutes, wherein the duration of immobility was meticulously observed and quantified over the concluding 4-minute period. Immobility time was defined as the duration in which the mouse remained buoyant in the water without exhibiting any signs of struggle, while solely engaging in the essential movements required to maintain its head above the water surface. Tail Suspension Test The TST was conducted following the established protocol [ 51 ]. The mice, with a medical tape positioned 1 cm from the tail tip, was suspended in an inverted position for a duration of 6 minutes using the TST instrument holder. The immobility duration of each mouse during the final 4 minutes was subjected to statistical analysis using the ANY-MAZE software. Serum and brain hippocampus samples collection Following the completion of behavioral tests, mice were euthanized through decapitation. Blood samples were subsequently obtained for the purpose of serum separation, and then plasma was obtained via centrifuging at 2000 rpm for 10 min, which were then stored at a temperature of -20°C until further analysis. Whole brains were extracted and the hippocampus was meticulously dissected and stored at a temperature of -80°C until assay. Prior to formal measurement, the hippocampus tissues were pulverized using liquid nitrogen and subsequently aliquoted into tubes. Measurement of Monoamine Neurotransmitters in Hippocampus The levels of 5-HT and DA in the brain were measured using HPLC chromatography methods. The centrifuge tube, containing the homogenized hippocampus tissue, was subjected to mixing with 5 mL/g of distilled water to achieve tissue sample homogenization. Subsequently, the samples were centrifuged at a speed of 5000 g for a duration of 10 min at a temperature of 4°C, resulting in the collection of the supernatant. Following centrifugation, the supernatant was diluted by a factor of 100, and an additional 1:5 ratio of the 1 mmol/L d9-Ach was introduced. The samples were subjected to a second round of centrifugation at a speed of 10,000 revolutions per minute (rpm) for a duration of 10 minutes. Subsequently, 200 µL of brain tissue homogenate was combined with 600 µL of acetonitrile that had been pre-cooled by Beijing dicoma Technology Co., Ltd. (Catalog number 50101). This mixture was then centrifuged at a speed of 14,000 rpm for 10 minutes at a temperature of 4°C. As a result, 400 µL of the resulting supernatant was collected and dried using nitrogen. The dried residue was then reconstituted by mixing it with 75 µL of a 0.1% formic acid solution (MERCK, CAS number 64-18-6). Finally, the obtained supernatant was utilized for the identification of neurotransmitters through the utilization of high-performance liquid chromatography (1200, Agilent, USA). Oxidative stress assay The measurement of SOD activity and MDA content in the hippocampus of mice was conducted using commercial kits (Jiancheng Biotechnology, Nanjing, China) in accordance with the instructions provided by the manufacturers. The activities of SOD were quantified as unit per milligram of protein in the tissue, while the levels of MDA were quantified as nanomole per milligram of protein in the tissue. Frozen hippocampus tissue was homogenized in ice-cold Nacl solution. The homogenate was centrifuged at 5000 g for 15 min at 4°C, and the protein concentration of the super natant was analyzed using a BCA kit (Beyotime, Shanghai, China). SOD activity and MDA content in mice hippocampus were measured using commercial kits (Jiancheng Biotechnology, Nanjing, China) according to the manufacturers’ instructions. In brief, SOD in the hippocampus inhibited WST-1 reduction by catalyzing the dismutation of superoxide ions generated from the xanthine/xanthine oxidase system into molecular oxygen and hydrogen peroxide, which could be measured spectrophotometrically at 450 nm. The total SOD activity was determined by measuring the absorbance at 450 nm and expressed as U/mg. By calculating the absorb ance (405 nm) of the complex generated by the reaction between glutathione and 5, 5-dithiobis-(2-nitrobenzoic), MDA content (expressed as nmol/mg) was determined by detecting the absorbance at 532 nm. Measurement of ROS Levels and Immunofluorescence of NRF2 in Hippocampus The levels of ROS in the brain hippocampus were assessed using the fluorescent indicator DCFH-DA (DCFH-DA, MAK143,Sigma, United States) in combination with fluorescence microscopy, following the methodology described by Lau et al [52]. DCFH-DA is a trusted fluorescent probe that can be oxidized by endogenous hydrogen peroxide upon entry into cells and converted to the intensely fluorescent 2',7'-dichlorofluorescein (DCF). Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Staining: frozen sections were incubated with ROS staining solution (1:1000 dilution of DCFH-DA to a final concentration of 10µM) for 30min at 37° in a thermostat protected from light; DAPI re-staining of cell nuclei: the slide was placed in PBS (PH7.4) on a decolourising shaker and washed 3 times, each time for 5 min. the slides were shaken and dried, then DAPI staining solution (DAPI working solution 5mg/ml: Dilute the DAPI reservoir with PBS to a final concentration of 0.1ug/ml) was added dropwise inside the circle, and the slides were incubated at room temperature, protected from light, for 10 min; Seal: The slides were washed in PBS (PH7.4) on a decolourising shaker with shaking for 3 times, each time for 5 min, and the slices were shaken dry slightly; Then seal the slides with anti-fluorescence quenching sealer; Microscopic examination and photographing: the sections were observed under a fluorescence microscope and images were collected. In brief, cryostat sections (5 µm) of brain hippocampus were incubated with DCFH-DA in the absence of light. Subsequently, the cryostat sections were rinsed with PBS three times at a temperature of 4°C, and fluorescence images were captured using an excitation wavelength of 488 nm and an emission wavelength of 525 nm. Similarly, the brains were sliced into 5-µm slices in a freezing microtome (Leica, CM1900, Wetzlar, Germany). Cells were first fixed with 4% paraformaldehyde.. The treatment was carried out with 0.25% Triton-100 for 20–30 min and PBS immersion was performed three times for 2 min each time; Closure was done with PBST containing 10% donkey serum for 1 hour. The primary antibody was diluted in PBST and added dropwise to the sample to completely cover the sample tissue. Place in a wet box and incubate overnight at 4°C; PBST washed 3 times for 2 min each; Incubate the secondary antibody, add the appropriate secondary antibody configured according to the primary antibody dropwise:Incubate at room temperature away from light for 2-3h; Wash with PBST 3 times, 5min each time; The nuclei were stained with DAPI (DAPI working solution 5mg/ml: Dilute the DAPI reservoir with PBS to a final concentration of 0.1ug/ml.) for 5 min and washed 3 times with PBST for 2 min each time. Fluorescently labeled samples were imaged with a fluorescence microscope. Enzyme-linked immunosorbent assay (ELISA) Serum concentrations of TNF-α, IL-1β, and IL-6 were quantified utilizing standard ELISA kits (Elabscience biotechnology Co., Ltd., Wuhan, China) in accordance with the manufacturer's guidelines. Western blot analysis The brain hippocampus tissues were homogenized in RIPA Lysis Buffer (Beyotime, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime). The homogenate was then centrifuged at 12,000 × g for 10 minutes at 4 ℃, and the resulting supernatant was collected. The total protein concentrations of the supernatant were determined using a BCA protein assay kit (Beyotime). Subsequently, equal amounts of proteins (20 µg) were separated by 4–20% sodium dodecyl sulfate polyacrylamide gels (SDSPAGE) and transferred onto polyvinylidene difuoride (PVDF) membranes. The membranes were obstructed using a 5% non-fat milk powder solution in Tris-buffered saline (TBS) containing 0.1% Tween-20 for a duration of 2 hours. Subsequently, they were subjected to an overnight incubation at a temperature of 4°C with primary antibodies: β-Actin (1:5000; SAB #21338, USA), iNOS (1:1000; CST #13120, USA), COX-2 (1:1000; CST #12282, USA), IBA-1 (1:1000; CST #17198, USA), GFAP (1:1000; CST #2956, USA), Bax (1:1000; CST #2722, USA), Bcl-2 (1:1000; CST #3498, USA), caspase-3 (1:1000; CST #9662, USA), Cl-caspase-3 (1:5000; ab #214430, UK), HO-1 (1:1000; CST #70081s, USA), Nrf2 (1:1000; CST #12721, USA), NLRP3 (1:1000; CST #15101, USA), caspase-1 (1:1000; CST #24232, USA), IL-18 (1:1000; CST #57058, USA), IL-1β (1:1000; CST #31202s, USA), ASC (1:1000; CST #67824, USA). Following incubation with secondary antibodies for one hour at room temperature, the bands were visualized using Emitter Coupled Logic (ECL) substrate from Merck, Germany, and the intensities of the bands were evaluated using Image J software. Statistical analysis The data were presented as means ± standard deviation (SD) and were subjected to statistical analysis using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Statistical significance was determined at P-values < 0.05. The data were analyzed using GraphPad Prism 9.0.0 (GraphPad Software, USA). The images were analyzed using Fiji (ImageJ) software. Declarations Institutional Review Board Statement This study was approved by the Ethical Committee of recommendations of Department of Laboratory Animal Science Fudan University (NO. 2017 1325 A253). Informed Consent Statement : Not applicable. Conflicts of Interest : The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results Funding: This research was funded by a grant-in-aid from the national natural science foundation of China (81771683) and the Program to Correct Highly Deficient Areas in Shanghai's Healthcare System-Psychosomatic medicine (Grant No.2019ZB0203). There are no financial conflicts of interest to disclose. Author Contribution Conceptualization, HRC and ZJW; methodology, HRC, ZJW and YL; validation, PG, JMZ, ZJF, JGH and DQZ; formal analysis, HRC and ZJW; investigation, HRC, YL and JGH; data curation, HRC, YL and JGH; writing—original draft preparation, HRC; writing—review and editing, JGH and DQZ; visualization, HRC; supervision, YL; project administration, YL. All authors have read and agreed to the published version of the manuscript. 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Suppressing the Na(+)/H(+) exchanger 1: a new sight to treat depression. CELL DEATH DIS. 2019;10(5):370. Lau YS, Tian XY, Mustafa MR, Murugan D, Liu J, Zhang Y, Lau CW, Huang Y. Boldine improves endothelial function in diabetic db/db mice through inhibition of angiotensin II-mediated BMP4-oxidative stress cascade. BRIT J PHARMACOL. 2013;170(6):1190–8. Additional Declarations No competing interests reported. Supplementary Files OriginalpictureROS2.zip OriginalpictureNrf2Immunofluorescence1.zip OriginalpictureWB1.zip Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7949176","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542203323,"identity":"ae9679f3-c7c2-4d4e-b2e5-34361db0708c","order_by":0,"name":"Jiangang Hou","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Jiangang","middleName":"","lastName":"Hou","suffix":""},{"id":542203324,"identity":"987940c6-1ff8-432e-afa6-3490b7f2894d","order_by":1,"name":"Haoran Chen","email":"","orcid":"","institution":"Fudan 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16:22:08","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":25108,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/81a05b65e2268f6eba68a0c1.png"},{"id":95638725,"identity":"62761139-9b19-414d-847b-734d6c1b310d","added_by":"auto","created_at":"2025-11-11 13:02:16","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":28762,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/91365440dfc69a19de7696a1.png"},{"id":95638722,"identity":"9ce805b3-5a6a-4210-9818-ceb6033467f4","added_by":"auto","created_at":"2025-11-11 13:02:16","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":29342,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/c1272efdc4194097d534dc60.png"},{"id":95657021,"identity":"04145eab-c902-45bc-8696-208c3afb1cd1","added_by":"auto","created_at":"2025-11-11 16:19:56","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":41711,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/44b331142ff6689a22952261.png"},{"id":95638718,"identity":"d38db5d8-cfeb-489d-b769-d46feb059f29","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114973,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/7fc100f84a036ebcf739af8c.png"},{"id":95638728,"identity":"07787579-7417-4cac-82c4-836116fbd0d1","added_by":"auto","created_at":"2025-11-11 13:02:16","extension":"xml","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140882,"visible":true,"origin":"","legend":"","description":"","filename":"860213c745a145cb8e29628b0f5308b31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/a1d785701d039b54a86ef695.xml"},{"id":95656940,"identity":"804cf107-603f-4c2e-928d-7e897595fa92","added_by":"auto","created_at":"2025-11-11 16:19:45","extension":"html","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":147721,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/2369d83178b5af01fea2dd31.html"},{"id":95638697,"identity":"b94a9921-0ded-4e87-8541-ea91a4d2f4da","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":126669,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on depressive-like behavior in chronic stress-induced mice. (A-D) Number of entries into the center (Control:18.17±2.441, Chronic:10.17±1.901, 5-ALA:14.67±2.233, 5-ALA+ZnPPIX:13±2.349) (A), immobility time \u0026nbsp;( Control:58.5±7.07, Chronic:148±7.72, 5-ALA:100.8±14.29, 5-ALA+ZnPPIX:118.3±8.14) (B), total move distance (Control:4500±223, Chronic:3800±163, 5-ALA:4017±117, 5-ALA+ZnPPIX:3883±175) (C) and time spent in the center (Control:26.83±6.565, Chronic:12.17±1.472, 5-ALA:20.33±2.582, 5-ALA+ZnPPIX:16.17±3.545) (D) in open field test. \u0026nbsp;(E) ( Control:98.33±7.638, Chronic:155.8±27.64, 5-ALA:119.2±25.45, 5-ALA+ZnPPIX:104.2±22.3) Immobility time in forced swimming test. \u0026nbsp;(F) (Control:100±9, Chronic:212±10, 5-ALA:148±9, 5-ALA+ZnPPIX:177±15) Immobility time in tail suspension test. Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/e26d2a2632fb0a4209d97009.jpg"},{"id":95638698,"identity":"1a918418-b3ae-407a-941b-b4806e370f0e","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55925,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on neurotransmitters concentration in brain hippocampus of chronic stress-induced mice. (A) (Control:0.344±0.065, Chronic:0.126±0.069, 5-ALA:0.245±0.061, 5-ALA+ZnPPIX:0.224±0.046) The concentrations of DA in hippocampus. (B) (Control:0.498±0.073, Chronic:0.166±0.04, 5-ALA:0.358±0.02, 5-ALA+ZnPPIX:0.246±0.098) The concentrations of 5-HT in hippocampus. Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/703420611eb9dead008d560a.jpg"},{"id":95638700,"identity":"ee285ae7-e3e6-44d5-917c-7b85c6bb4e40","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":108639,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on oxidative stress parameters in brain hippocampus of chronic stress-induced mice. (A) Representative images showing ROS staining in hippocampus. Scale bar: 20 μm, magnification: ×40.0 (B) (Control:6.58±1.16, Chronic:13.17±2.32, 5-ALA:9.5±1.9, 5-ALA+ZnPPIX:12.25±1.22) The level of MDA in hippocampus. (C) (Control:168.6±4.554, Chronic:105.8±5.215, 5-ALA:145.5±4.958, 5-ALA+ZnPPIX:122.5±4.354) The activity of SOD in hippocampus. Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/775390cdda5fd900ea18711c.jpg"},{"id":95638699,"identity":"13b64678-be76-40a0-943f-3b161b9e0f39","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":74697,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on pro-inflammatory factor in serum of chronic stress-induced mice. (A) (Control:566.7±57.15, Chronic:833.3±110.50, 5-ALA:581.7±52.69, 5-ALA+ZnPPIX:692.0±97.6) The levels of IL-1β in serum. (B) (Control:1833±133.3, Chronic:4517±127.2, 5-ALA:3450±187.6, 5-ALA+ZnPPIX:3850±177.3) The levels of TNF-α in serum. (C) (Control:503.3±16.33, Chronic:833.3±92.9, 5-ALA:608.3±32.6, 5-ALA+ZnPPIX:658.3±37.04) The levels of IL-6 in serum. Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/551bd262d9c0d8928c2790ec.jpg"},{"id":95656720,"identity":"32b17db7-e99c-44bc-bd15-f0b1d5bdd6b9","added_by":"auto","created_at":"2025-11-11 16:19:26","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":81684,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on pro-inflammatory factor in brain hippocampus of chronic stress-induced mice. (A) The protein expression levels of iNOS and COX-2 were detected by Western blotting. (B-C) Semi-quantification of iNOS (Control:1.000±0.04082, Chronic:6.576±0.4823, 5-ALA:3.956±0.2821, 5-ALA+ZnPPIX:4.976±0.3685) and COX-2 (Control:1.000±0.04082, Chronic:3.540±0.3964, 5-ALA:2.008±0.1461, 5-ALA+ZnPPIX:3.180±0.3564) . Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/c161293dcbc220dd49be1494.jpg"},{"id":95638702,"identity":"25b9deb3-3258-48d9-9e5e-304e336f07b8","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":83178,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on activation of microglia and astrocyte in brain hippocampus of chronic stress-induced mice. (A) The protein expression levels of IBA-1 and GFAP were detected by Western blotting. (B-C) Semi-quantification of IBA-1 (Control:1.000±0.04082, Chronic:1.948±0.1576, 5-ALA:1.372±0.1026, 5-ALA+ZnPPIX:1.624±0.04037) and GFP (Control:1.000±0.04082, Chronic:1.597±0.07054, 5-ALA:1.234±0.07599, 5-ALA+ZnPPIX:1.410±0.08972) . Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/e15ed2a03ad679f5c6cdd207.jpg"},{"id":95657898,"identity":"9017acad-b480-4c3d-8897-f019430dfb3d","added_by":"auto","created_at":"2025-11-11 16:22:21","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":115588,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on NLRP3 inflammasome in brain hippocampus of chronic stress-induced mice. (A) The protein expression levels of NLRP3, caspase-1, ASC, IL-1β, and IL-18 were detected by Western blotting. (B-F) Semi-quantification of NLRP3 (Control:1.000±0.04082, Chronic:5.640±0.2677, 5-ALA:1.564±0.1083, 5-ALA+ZnPPIX:2.574±0.4454) , caspase-1 (Control:1.000±0.09000, Chronic:2.152±0.1792, 5-ALA:1.230±0.06162, 5-ALA+ZnPPIX:1.318±0.2193) , ASC Control:1.000±0.05000, Chronic:3.067±0.2433, 5-ALA:1.089±0.0632, 5-ALA+ZnPPIX:1.711±0.0768) , IL-18β (Control:1.000±0.05000, Chronic:2.406±0.1576, 5-ALA:1.258±0.0968, 5-ALA+ZnPPIX:1.843±0.1592) , and IL-1β (Control:1.000±0.08000, Chronic:4.027±0.1425, 5-ALA:2.132±0.0744, 5-ALA+ZnPPIX:3.218±0.1048) . Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/55948ecdb1fa4b72e95f3375.jpg"},{"id":95638715,"identity":"f05255b5-2a4f-4248-9f30-fb396d2b966a","added_by":"auto","created_at":"2025-11-11 13:02:15","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":93695,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment on HO-1/Nrf2 signaling pathway in brain hippocampus of chronic stress-induced mice. (A) The protein expression levels of Nrf2 (Control:1.000±0.07638, Chronic:1.470±0.07899, 5-ALA:1.767±0.0896, 5-ALA+ZnPPIX:1.468±0.0850)\u0026nbsp; and HO-1 ( Control:1.010±0.1574, Chronic:1.470±0.1151, 5-ALA:1.922±0.2117, 5-ALA+ZnPPIX:1.183±0.07416) \u0026nbsp;were detected by Western blotting. (B-C) Semi-quantification of Nrf2 and HO-1. (D) Immunofluorescence staining of Nrf2 in the hippocampus of mice among four groups. Data are expressed as mean±SD (n=6 in each group). * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.005 ，**** p \u0026lt; 0.001, ns: no significance.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/4168d49158c3365b7d260e8e.jpg"},{"id":97893507,"identity":"e9c32143-ee2d-4545-aaba-5d63a07aecbe","added_by":"auto","created_at":"2025-12-10 15:30:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1767065,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/bde0cdd1-8e81-47b2-a7bc-df77bfd9babe.pdf"},{"id":95638729,"identity":"e546c8ad-0ce7-460f-9233-3b55fa3e84ef","added_by":"auto","created_at":"2025-11-11 13:02:16","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":16510061,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalpictureROS2.zip","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/8afde2bf4b4cab8ac11014dd.zip"},{"id":95658187,"identity":"ea54a6cd-0756-46b5-80da-8076f21f0179","added_by":"auto","created_at":"2025-11-11 16:23:35","extension":"zip","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29102250,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalpictureNrf2Immunofluorescence1.zip","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/8c6f6642ee96794536a54126.zip"},{"id":95638730,"identity":"bf8b3cc9-41c6-48c3-ab72-4f935de03157","added_by":"auto","created_at":"2025-11-11 13:02:16","extension":"zip","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":38037700,"visible":true,"origin":"","legend":"","description":"","filename":"OriginalpictureWB1.zip","url":"https://assets-eu.researchsquare.com/files/rs-7949176/v1/95d79a737d13ab3999cb2054.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"Neuroprotective Effects of 5-Aminolevulinic Acid Combined with Ferrous Iron on Chronic Stress- Induced Depressive-Like Behavior in Mice by Activating the HO-1/Nrf2 Signaling Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eDepression is widely acknowledged as a prevalent mental disorder characterized by a range of symptoms including sadness, guilt, diminished appetite, fatigue, impaired concentration, reduced self-worth, diminished interest or pleasure, and disrupted sleep patterns. The worldwide incidence of this condition is substantial, affecting an estimated 300\u0026nbsp;million individuals.\u003c/p\u003e\u003cp\u003ePrevious studies have demonstrated the involvement of neurotransmitters and inflammation in the etiology of depression, comprising a dearth of neurotransmitters such as serotonin (5-HT) and dopamine (DA) [\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The presence of dysfunction in the electrical activity of dorsal raphe 5-HT neurons, coupled with reduced levels of extracellular 5-HT in the hippocampus, could potentially be linked to the manifestation of anxiety and depression. These symptoms serve as prominent and early indicators of depressive disorders [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eStressful experiences, particularly chronic and unintended stressors, are substantial risk factors that exert a pervasive influence on the development and exacerbation of a wide range of diseases. Chronic stress is associated with a neurobehavioral syndrome that exhibits characteristics indicative of depression, as well as various processes encompassing psychological disorders and activation of the inflammatory immune system [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Numerous studies have consistently demonstrated the connection between the psychological, behavioral, and neurobiological manifestations of depression and the impact of inflammatory cytokines [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNeuroinflammation serves as a protective response initiated by the central nervous system in reaction to cerebral irregularities. Its primary functions involve the elimination of damaged cell fragments through immune cells within the brain, as well as the regulation of neurotrophic factor secretion to uphold brain homeostasis and preserve normal physiological functioning [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. The progressive development of chronic neuritis is associated with neurotoxicity caused by the substantial release of cytokines. The excessive accumulation of neurotoxic factors further stimulates the proliferation of glial cells within the nervous system, leading to secondary harm to the nervous system and impeding the regeneration of neuronal cells. Consequently, neuritis is considered a contributing factor to various neurodegenerative disorders [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn essence, uncontrolled or persistent neuroinflammation, whether as an initiator or as a cascading feedback response, has the potential to drive chronic and progressive neurodegenerative processes. Neuroinflammation has been identified as a prominent area of investigation in academic research, specifically in relation to its association with depression. Previous studies had demonstrated an augmented secretion of pro-inflammatory cytokines during the progression of neuroinflammation, such as IL-6, TNF-α and IL-1β [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Available clinical studies revealed that patients with depression exhibited elevated concentrations of IL-6, TNF-α and IL-1β, in both their cerebral spinal fluid and serum [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eNeuroinflammation is involved in the excessive release of oxidative and neurotoxic molecules, impairing the physiological function of normal neurons and leading to synaptic dysfunction [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The manifestation of oxidative stress has provided evidence for the induction of neuronal degeneration and its contribution to the development of depression [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The existing literature has provided evidence that superoxide dismutase (SOD) serves as a crucial endogenous antioxidant enzyme and a fundamental component of the primary defense mechanism against reactive oxygen species (ROS) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Malondialdehyde (MDA) is a byproduct resulting from the process of lipid peroxidation, and its concentration can serve as an indirect indicator of the extent of lipid peroxidation-induced damage.\u003c/p\u003e\u003cp\u003eThe NLRP3 inflammasome, a protein complex within the innate immune system, is responsible for detecting molecular patterns associated with pathogens and risks. According to current research, the activation of NLRP3 is associated with depression [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. When excessively activated, it can culminate in the induction of pyroptosis, accompanied by the generation of proinflammatory cytokines such as IL-6, TNF-α and IL-1β, leading to the development of neuroinflammatory conditions [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Overall, NLRP3 has been extensively implicated in the modulation of neuroinflammatory processes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Previous studies revealed that therapy can exert notable antidepressant-like effects, by regulating the activation of NLRP3 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMoreover, microglia and astrocytes also assume a crucial role in neuroinflammatory [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. They function as the primary defense mechanism in the brain, safeguarding the CNS. Microglia and astrocytes promptly undergo activation in response to diverse stimuli. Transient neuroinflammation serves a beneficial role in combating bacteria, and eliminating cell debris discharged from damaged cells. Nevertheless, prolonged overactivation and dysregulated neuroinflammation contribute to neuronal cell demise and exacerbate the progression of neurodegenerative disorders [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Microglia and astrocytes actively engage in the neuroinflammatory process through the secretion of various proinflammatory cytokines, including IL-6, TNF-α and IL-1β [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, the suppression of microglial activation and the safeguarding of neurons against neuroinflammation may present a viable alternative approach for the therapeutic intervention of depression.\u003c/p\u003e\u003cp\u003e5-Aminolevulinic acid (5-ALA) is an endogenous amino acid that is ubiquitously found in diverse organisms [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. It is synthesized by the catalytic action of 5-ALA synthase, which involves the condensation of glycine and succinyl-CoA. In animal cells, 5-ALA plays a crucial role as a precursor in heme synthesis [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Heme oxygenase (HO)-1 enzymatically breaks down heme into biliverdin, carbon monoxide (CO), and ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e), which are utilized in conjunction with 5-ALA. Numerous studies have extensively demonstrated that the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e exerts anti-inflammatory and anti-oxidative effects by up-regulating the expression of HO-1 [\u003cspan additionalcitationids=\"CR33 CR34\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The HO-1, which governs the rate of heme metabolism and produces biliverdin, CO, and Fe2+, has been recognized as a pivotal factor in diverse anti-inflammatory, anti-apoptotic, and anti-oxidative mechanisms. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Zinc protoporphyrin IX (Zinc protoporphyrin IX, ZnPPIX), an inhibitor of HO-1, can inhibit HO-1 expression, and has been used as an inhibitor to explore HO-1-related signaling pathways in previous studies. However, there is a lack of research investigating the impact of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e on the anti- neuroinflammation response in relation to depression.\u003c/p\u003e\u003cp\u003eTherefore, the aim of our study was to evaluate the influence of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e on behavior, microglial activation, neuronal apoptosis, and neuroinflammatory responses in depressive mice, along with exploring potential anti-neuroinflammatory mechanisms.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003e1.The effect of 5-ALA/Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon depressive-like behavior in chronic stress-induced depressive mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e1.1 Open Field Test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chronic stress-induced mice displayed a significantly depressed phenotype, as evidenced by their increased durations of immobility time during the Open Field Test (OFT), in comparison to the stress-free control mice (F(3, 20)\u0026thinsp;=\u0026thinsp;3.016, p\u0026lt;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Additionally, chronic stress decreased the total move distance, and lowers the time spent within center zone and the number of entries into center zone (F(3, 20)\u0026thinsp;=\u0026thinsp;1.228, F(3, 20)\u0026thinsp;=\u0026thinsp;3.016, F(3, 20)\u0026thinsp;=\u0026thinsp;4.806, p\u0026lt;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, C, D). The mice belonging to the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group demonstrated a notable reduction in immobility the duration in comparison to the chronic stress group. Moreover, the mice belonging to the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group exhibited a noteworthy augmentation in the duration spent within the central area and the number of entries into center zone, in comparison to the chronic stress group (p\u0026lt;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). Moreover, treatment with ZnPPIX counteracted 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e inhibitory effect on depressive-like behavior.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e1.2 Forced Swim Test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur results demonstrated a significant difference in the durations of immobility observed in the forced swim test (FST) among various groups. Specifically, the chronic stress group exhibited significantly longer durations of immobility in the FST compared to the stress-free control group. Likewise, the chronic stress group displayed significantly elevated durations of immobility in the FST compared to the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group (F=(3, 20)3.128, p\u0026lt;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003cb\u003e1.3 Tail Suspension Test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor tail suspension test (TST), the duration of immobility time exhibited a significant decrease in both the stress-free control group and the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group, as compared to the chronic stress group (F(3, 20)\u0026thinsp;=\u0026thinsp;0.2584, p\u0026lt;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003e\u003cb\u003e2. The effect of 5-ALA/Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon neurotransmitters concentration in chronic stress-induced depressive mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe measurement of 5-HT and DA levels in the hippocampus revealed a noteworthy elevation in the stress-free control group compared to the chronic stress group. Additionally, the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group exhibited a significant increase in 5-HT and DA levels when compared to the chronic stress group (F(3, 20)\u0026thinsp;=\u0026thinsp;1.118, F(3, 20)\u0026thinsp;=\u0026thinsp;1.779, p\u0026lt;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e3. The effect of 5-ALA/Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon anti-oxidant ability in chronic stress-induced depressive mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe chronic stress group demonstrated a significant reduction in the activity of SOD compared to the control group. In contrast, the activity of SOD showed a significant increase in the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group relative to the chronic stress group (F(3, 20)\u0026thinsp;=\u0026thinsp;1.314, p\u0026lt;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). After exposure to prolonged stress, the hippocampal tissue of the group subjected to chronic stress exhibited a significant elevation in the level of MDA and ROS, as compared to the stress-free control group (p\u0026lt;0.05) (F(3, 20)\u0026thinsp;=\u0026thinsp;0.9673, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Conversely, in the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group, the alteration in MDA and ROS content was notably reduced in comparison to the chronic stress group, unlike the observed changes in SOD. However, the administration of ZnPPIX to the experiment led to the reversal of the aforementioned alterations (p\u0026lt;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e4. The effect of 5-ALA/Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon anti-inflammatory ability in chronic stress-induced depressive mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOur findings demonstrated a significant increase in the production of TNF-α, IL-1β, and IL-6 in the chronic stress group, which was effectively mitigated by pretreatment with 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e. However, it was observed that the levels of IL-6, IL-1β, and TNF-α in serum were significantly elevated in the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e+ZnPPIX group in comparison to the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group (p\u0026lt;0.01) (F=(3, 20)0.6697, F(3, 20)\u0026thinsp;=\u0026thinsp;0.2157, F(3, 20)\u0026thinsp;=\u0026thinsp;1.427, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFurthermore, the results demonstrate that the administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e significantly attenuated the expression of iNOS and COX-2 in brain hippocampus. But in the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e+ZnPPIX group, the expression of iNOS and COX-2 in brain hippocampus exhibited a statistically significant increase in comparison to the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group (p\u0026lt;0.05) (F(3, 20)\u0026thinsp;=\u0026thinsp;3.924, F(3, 20)\u0026thinsp;=\u0026thinsp;2.428, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e5. The effect of 5-ALA/Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon glial cell activation in chronic stress-induced depressive mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe expression of proteins related to activation of microglia and astrocyte in the brain hippocampus was investigated using Western blot techniques. Chronic stress resulted in elevated protein expression levels of IBA-1 and GFAP in the brain hippocampus. Conversely, treatment with 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e reversed these alterations. Additionally in the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e+ZnPPIX group, the expression of IBA-1 and GFAP in brain hippocampus exhibited a statistically significant increase in comparison to the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e group (F(3, 20)\u0026thinsp;=\u0026thinsp;1.509, F(3, 20)\u0026thinsp;=\u0026thinsp;0.8686, p\u0026lt;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-C).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e6.The effect of 5-ALA//Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon NLRP3 Signaling Pathway in chronic stress-induced depressive mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe employed Western blotting to detect the expression of proteins related to the NLRP3 inflammasome. Our findings demonstrated that chronic stress enhanced the expression levels of NLRP3, ASC, IL-1β, IL-18 and caspase-1. However, these effects were counteracted by treatment with 5-ALA/Fe2+. Additionally, the inhibitory effect of 5-ALA/Fe2\u0026thinsp;+\u0026thinsp;on activation of NLRP3 inflammasome was counteracted by treatment with ZnPPIX (F(3, 20)\u0026thinsp;=\u0026thinsp;2.160, F(3, 20)\u0026thinsp;=\u0026thinsp;1.372, F(3, 20)\u0026thinsp;=\u0026thinsp;3.270, F(3, 20)\u0026thinsp;=\u0026thinsp;1.011, F(3, 20)\u0026thinsp;=\u0026thinsp;0.9422, p\u0026lt;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003e7.The effect of 5-ALA//Fe\u003c/b\u003e\u003csup\u003e\u003cb\u003e2+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003eon HO-1/Nrf2 signaling pathway in in chronic stress-induced depressive mice hippocampus\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn comparison to the chronic stress group, the administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e tended to enhance the expression of Nrf2 and HO-1 in the chronic stress group with induced depression. Conversely, minimal expression of Nrf2 and HO-1 was observed in the 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e + ZnPPIX group. In the present study, the administration of ZnPPIX resulted in a reduction in the expression of HO-1 in the brain hippocampus of chronic stress-induced mice (F(3, 20)\u0026thinsp;=\u0026thinsp;0.7312, F(3, 20)\u0026thinsp;=\u0026thinsp;1.601, p\u0026lt;0.005) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-C). Similarly, immunofluorescence staining of Nrf2 in the hippocampus showed the same result (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eChronic stress has been identified as a prominent factor in mental illness exacerbation. The findings of our study indicate that mice experiencing chronic stress-induced depression exhibited a notable elevation in anxiety levels during the OFT and increased immobility time during the FST and TST. These findings of Hiroi and Neumaier suggest that a decrease in anxiety-like behavior is evidenced by an increase in the time spent within center zone [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In OFT, the act of entering the center area serves as a dependable indicator of anxiety, as it exhibits sensitivity to anxiety states induced by chronic stress. The results of the OFT of this study demonstrate that the chronic stress groups, when compared to the stress-free control groups, exhibited a lower number of entries into the center zone and a shorter duration of time spent within center zone. And there was a significant alleviation of depressive behavior in mice after treatment by administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eMultiple studies have provided evidence for the implication of the hippocampus in the pathological advancement of neurodegenerative disorders, and hippocampus is also closely related to the development of anxiety-depressive behaviors [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The hippocampus is a crucial element of the limbic system, positioned between the thalamus and the medial temporal lobe in the brain. Its primary functions include the storage, modification, and synchronization of temporary memories, as well as the acquisition of knowledge and regulation of emotional reactions [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. To ascertain the neuroprotective efficacy of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e, we conducted an examination of various indicators, including neurotransmitters, proinflammatory factors and oxidative stress indicators, within the hippocampus of chronic stress-induced depressive mice.\u003c/p\u003e\u003cp\u003eIn this study, a significant reduction in 5-HT and DA levels within the hippocampus was observed in the mice with chronic stress-induced depression. The administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e significantly inhibited the decreased levels of 5-HT and DA induced by chronic stress. The increased levels of 5-HT and DA may be closely related to the antidepressant effects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e. Much previous literature has confirmed, the neurotransmitter 5-HT has demonstrated a stabilizing effect on anti-depression activity in animal behavior tests [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eBoth experimental and clinical evidence consistently demonstrate that depression is associated with elevated levels of oxidative biomarkers and reduced levels of antioxidant defense biomarkers in the brain [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The activity of SOD and the levels of MDA and ROS were measured in the brain hippocampal tissue of chronic stress-induced mice exhibiting depressive symptoms. In the present study, the observed reduction in the activity of SOD and the concentrations of MDA in chronic stress-induced mice suggests a disruption in the antioxidant mechanisms within the brain. And we found that ROS levels increased in the hippocampal tissue of chronic stress-induced mice. However, this impairment was effectively reversed upon administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e treatment. The findings consistently demonstrated that the administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e significantly augmented the cerebral antioxidant capacity in depressive mice subjected to chronic stress.\u003c/p\u003e\u003cp\u003eThe findings of our study indicate that the administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e resulted in a significant decrease in depressive-related behavior in the behavioral experiments, accompanied by the regulation of neurotransmitters, specifically an increase in 5-HT and DA levels. Furthermore, we assessed the impact of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e on the activation of microglia and astrocyte. The inhibitory effect of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e on anti-activation of microglia and astrocyte was counteracted by treatment with ZnPPIX. The observed reduction in protein expression of IBA-1, GFAP and apoptosis-related proteins suggests that the anti-depressive effects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e may be mediated through the regulation of microglial activation, potentially involving an indirect modulation of the monoamine neurotransmitter.\u003c/p\u003e\u003cp\u003eNeuroinflammation is a key mechanism in the pathophysiology of depression. It is characterised by increased expression of inflammatory factors, activated microglia, astrocytes and NLRP3 inflammasomes in brain, and enhanced peripheral inflammation [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In our study, the activation of microglia and astrocyte was accomplished through the administration of chronic stress. The levels of proinflammatory cytokines (e.g., IL-6, TNF-α and IL-1β) released by activated glial cells exhibited a corresponding increase, leading to the development of neuroinflammation. The supplementation of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e effectively impeded the activation of microglia and astrocytes. These findings unequivocally indicate that 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e effectively suppressed the neuroinflammatory response induced by chronic stress.\u003c/p\u003e\u003cp\u003eIt is widely acknowledged that NLRP3 inflammasome play a pivotal role in regulating neuroinflammation in various brain disorders. These molecules are responsible for transcribing and activating the aforementioned enzymes and proinflammatory cytokines, thereby instigating the onset of neuroinflammation [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. To enhance comprehension of the regulation mechanism of neuroinflammation in depression and to identify potential therapeutic targets, an investigation was conducted to examine the involvement of the NLRP3 inflammasome. In our study, chronic stress promoted the activation of NLRP3 inflammasome in in brain hippocampus, and 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e diminishes the chronic stress-induced activation of NLRP3 inflammasome. Furthermore, our findings substantiate the close association between NLRP3 inflammasome activation and the activation of microglia and astrocyte, as well as the progression of neuroinflammation in the hippocampus of chronic stress-induced depressive mice.\u003c/p\u003e\u003cp\u003eThis study demonstrates that the administration of 5-ALA/ Fe\u003csup\u003e2+\u003c/sup\u003e resulted in the enhancement of HO-1 expression in the hippocampus of chronic stress-induced depressive mice. ZnPPIX, which consists of protoporphyrin IX and metal Zn, serves as a heme structural analog. It is frequently employed as an inhibitor of HO-1 due to its capacity to competitively hinder HO activity by occupying the heme binding site [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In our study, the administration of ZnPPIX resulted in a decrease in the expression of HO-1 in the hippocampus of chronic stress-induced depressive mice. This counteracted the neuroprotective effects induced by 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e, thereby demonstrating that the neuroprotective effects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e are mediated through the enhancement of HO-1 expression. It was observed that 5-ALA/ Fe\u003csup\u003e2+\u003c/sup\u003e primarily modulates the HO-1/Nrf2 signaling pathway and NLRP3 inflammasome signaling pathways, leading to the suppression of proinflammatory mediators' synthesis and secretion. These findings demonstrate the anti-neuroinflammatory properties of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn conclusion, the administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e demonstrated a neuroprotective effect on the hippocampus against neuroinflammation induced by chronic stress. Furthermore, 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e alleviated the behavioral alterations observed in the OFT, FST and TST resulting from chronic stress. The underlying mechanism responsible for the observed neuroprotective effects of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e involved the enhancement of anti-inflammatory, anti-oxidative and anti-apoptotic effects within the hippocampal tissue. The findings of our study demonstrate that the administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e effectively mitigates neuroinflammation induced by chronic stress in an in vivo setting. This effect is achieved through the inhibition of the NLRP3 inflammasome and the activation of the HO-1/Nrf2 signaling pathway. Consequently, 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e exhibits promising potential as a therapeutic candidate for the treatment of central nervous system disorders associated with neuroinflammation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003eEighty male C57BL/6J mice (8\u0026ndash;10 weeks, 24\u0026thinsp;\u0026plusmn;\u0026thinsp;2g) were obtained from the Department of Laboratory Animal Science at Fudan University in Shanghai, China. The mice were housed under standard conditions and provided with rodent food and water ad libitum. All procedures involving the experimental animals were conducted in strict accordance with the guidelines outlined in the Guide for the Care and Use of Laboratory Animals (Ministry of Science and Technology of China, 2006). The animal experiments were conducted in accordance with the protocols that were approved by the institutional review board of the Department of Laboratory Animal Science at Fudan University in Shanghai, China.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eAs previously mentioned [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], 5-ALA/HCl from Merck Millipore in Billerica, USA, and Fe\u003csup\u003e2+\u003c/sup\u003e (sodium ferrous citrate) from Fanhai Biotechnology in Xi'an, China, were dissolved in distilled water. The molar ratio of 5-ALA to Fe\u003csup\u003e2+\u003c/sup\u003e was 1:0.5. Prior to administration, Fe2\u0026thinsp;+\u0026thinsp;was diluted in distilled water. ZnPPIX (AbMole BioScience, Houston Tx, USA), a HO-1 inhibitor, was diluted in 100 mM NaOH to create a stock solution of 50 mM, which was subsequently stored at -80\u0026deg;C until required. The 5-aminolevulinic acid (5-ALA)/ Fe\u003csup\u003e2+\u003c/sup\u003e complex was orally administered in a volume of 0.3 ml distilled water, while the ZnPPIX solution was administered intraperitoneally in the same volume of 0.3 ml. Efforts were made to minimize light exposure. The dosage administered of 5-ALA/Fe\u003csup\u003e2\u003c/sup\u003e was 100mg/kg, and the dosage administered of ZnPPIX was 5 mg/kg.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eStudy design\u003c/h2\u003e\u003cp\u003eThe mice were subjected to random allocation into four distinct groups, each consisting of six individuals. These groups were designated as follows: the stress-free control group, which received saline for a duration of 6 weeks without chronic stress; the chronic stress group, which received saline for 6 weeks and was subjected to chronic stress; the chronic stress\u0026thinsp;+\u0026thinsp;5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e (100mg/kg) group, mice were subjected to daily oral administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e for a duration of 6 weeks; and the chronic stress\u0026thinsp;+\u0026thinsp;5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e (100 mg/kg)\u0026thinsp;+\u0026thinsp;ZnPPIX (5 mg/kg) group, mice were subjected to daily oral administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e for a duration of 6 weeks, while concurrently receiving simultaneous administration of ZnPPIX. On a subsequent day following the administration of the behavioral tests, the mice were euthanized through decapitation. The brain hippocampus were subsequently collected and stored at \u0026minus;\u0026thinsp;80\u0026deg;C.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eChronic immobilization stress procedure\u003c/h3\u003e\n\u003cp\u003eAs previously mentioned [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], the mice were exposed to a range of stressors over a period of six weeks. Each week, the stress regimen included periods of food or water deprivation lasting 16 hours, a tilted cage position of 45\u0026deg; for 12 hours, a soiled cage with 200 mL of water spilled onto the bedding for 8 hours, and stroboscopic lighting with 400 flashes per minute in the dark for 8 hours. The mice experienced one of these stressors each day of the week, with no repetition of the same stressor on consecutive days.\u003c/p\u003e\n\u003ch3\u003eOpen‑Field Test\u003c/h3\u003e\n\u003cp\u003eThe OFT was conducted in accordance with previously established protocols [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Prior to the test, mice were acclimated to the behavior room while exposed to background white noise for a minimum of 30 minutes. Subsequently, individual mice were introduced into a 35 cm x 35 cm open field arena by an experimenter who was unaware of the experimental conditions. The mice were then allowed to freely explore the arena for a duration of 20 minutes. To quantify the mice's behavior, video tracking software (TopScan, CleverSys) was utilized to measure the total distance traveled, velocity, and time spent in the center of the arena (which measured 22 cm x 22 cm).\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eForced Swim Test\u003c/h2\u003e\u003cp\u003eThe FST was conducted according to the previously established protocol [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Each mouse was individually positioned within a glass cylindrical vessel measuring 21 cm in height and 12 cm in diameter, with a total volume of approximately 1000 mL. The vessel was filled with water at a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, reaching a depth of 12 cm. During the experiment, all mice were subjected to a forced swimming task lasting 6 minutes, wherein the duration of immobility was meticulously observed and quantified over the concluding 4-minute period. Immobility time was defined as the duration in which the mouse remained buoyant in the water without exhibiting any signs of struggle, while solely engaging in the essential movements required to maintain its head above the water surface.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eTail Suspension Test\u003c/h2\u003e\u003cp\u003eThe TST was conducted following the established protocol [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. The mice, with a medical tape positioned 1 cm from the tail tip, was suspended in an inverted position for a duration of 6 minutes using the TST instrument holder. The immobility duration of each mouse during the final 4 minutes was subjected to statistical analysis using the ANY-MAZE software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eSerum and brain hippocampus samples collection\u003c/h2\u003e\u003cp\u003eFollowing the completion of behavioral tests, mice were euthanized through decapitation. Blood samples were subsequently obtained for the purpose of serum separation, and then plasma was obtained via centrifuging at 2000 rpm for 10 min, which were then stored at a temperature of -20\u0026deg;C until further analysis. Whole brains were extracted and the hippocampus was meticulously dissected and stored at a temperature of -80\u0026deg;C until assay. Prior to formal measurement, the hippocampus tissues were pulverized using liquid nitrogen and subsequently aliquoted into tubes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of Monoamine Neurotransmitters in Hippocampus\u003c/h2\u003e\u003cp\u003eThe levels of 5-HT and DA in the brain were measured using HPLC chromatography methods.\u003c/p\u003e\u003cp\u003eThe centrifuge tube, containing the homogenized hippocampus tissue, was subjected to mixing with 5 mL/g of distilled water to achieve tissue sample homogenization. Subsequently, the samples were centrifuged at a speed of 5000 g for a duration of 10 min at a temperature of 4\u0026deg;C, resulting in the collection of the supernatant. Following centrifugation, the supernatant was diluted by a factor of 100, and an additional 1:5 ratio of the 1 mmol/L d9-Ach was introduced. The samples were subjected to a second round of centrifugation at a speed of 10,000 revolutions per minute (rpm) for a duration of 10 minutes. Subsequently, 200 \u0026micro;L of brain tissue homogenate was combined with 600 \u0026micro;L of acetonitrile that had been pre-cooled by Beijing dicoma Technology Co., Ltd. (Catalog number 50101). This mixture was then centrifuged at a speed of 14,000 rpm for 10 minutes at a temperature of 4\u0026deg;C. As a result, 400 \u0026micro;L of the resulting supernatant was collected and dried using nitrogen. The dried residue was then reconstituted by mixing it with 75 \u0026micro;L of a 0.1% formic acid solution (MERCK, CAS number 64-18-6). Finally, the obtained supernatant was utilized for the identification of neurotransmitters through the utilization of high-performance liquid chromatography (1200, Agilent, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eOxidative stress assay\u003c/h2\u003e\u003cp\u003eThe measurement of SOD activity and MDA content in the hippocampus of mice was conducted using commercial kits (Jiancheng Biotechnology, Nanjing, China) in accordance with the instructions provided by the manufacturers. The activities of SOD were quantified as unit per milligram of protein in the tissue, while the levels of MDA were quantified as nanomole per milligram of protein in the tissue.\u003c/p\u003e\u003cp\u003eFrozen hippocampus tissue was homogenized in ice-cold Nacl solution. The homogenate was centrifuged at 5000 g for 15 min at 4\u0026deg;C, and the protein concentration of the super natant was analyzed using a BCA kit (Beyotime, Shanghai, China). SOD activity and MDA content in mice hippocampus were measured using commercial kits (Jiancheng Biotechnology, Nanjing, China) according to the manufacturers\u0026rsquo; instructions. In brief, SOD in the hippocampus inhibited WST-1 reduction by catalyzing the dismutation of superoxide ions generated from the xanthine/xanthine oxidase system into molecular oxygen and hydrogen peroxide, which could be measured spectrophotometrically at 450 nm. The total SOD activity was determined by measuring the absorbance at 450 nm and expressed as U/mg. By calculating the absorb ance (405 nm) of the complex generated by the reaction between glutathione and 5, 5-dithiobis-(2-nitrobenzoic), MDA content (expressed as nmol/mg) was determined by detecting the absorbance at 532 nm.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eMeasurement of ROS Levels and Immunofluorescence of NRF2 in Hippocampus\u003c/h2\u003e\u003cp\u003eThe levels of ROS in the brain hippocampus were assessed using the fluorescent indicator DCFH-DA (DCFH-DA, MAK143,Sigma, United States) in combination with fluorescence microscopy, following the methodology described by Lau et al [52]. DCFH-DA is a trusted fluorescent probe that can be oxidized by endogenous hydrogen peroxide upon entry into cells and converted to the intensely fluorescent 2',7'-dichlorofluorescein (DCF). Nuclei were stained with 4\u0026prime;,6-diamidino-2-phenylindole (DAPI). Staining: frozen sections were incubated with ROS staining solution (1:1000 dilution of DCFH-DA to a final concentration of 10\u0026micro;M) for 30min at 37\u0026deg; in a thermostat protected from light; DAPI re-staining of cell nuclei: the slide was placed in PBS (PH7.4) on a decolourising shaker and washed 3 times, each time for 5 min. the slides were shaken and dried, then DAPI staining solution (DAPI working solution 5mg/ml: Dilute the DAPI reservoir with PBS to a final concentration of 0.1ug/ml) was added dropwise inside the circle, and the slides were incubated at room temperature, protected from light, for 10 min; Seal: The slides were washed in PBS (PH7.4) on a decolourising shaker with shaking for 3 times, each time for 5 min, and the slices were shaken dry slightly; Then seal the slides with anti-fluorescence quenching sealer; Microscopic examination and photographing: the sections were observed under a fluorescence microscope and images were collected. In brief, cryostat sections (5 \u0026micro;m) of brain hippocampus were incubated with DCFH-DA in the absence of light. Subsequently, the cryostat sections were rinsed with PBS three times at a temperature of 4\u0026deg;C, and fluorescence images were captured using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.\u003c/p\u003e\u003cp\u003eSimilarly, the brains were sliced into 5-\u0026micro;m slices in a freezing microtome (Leica, CM1900, Wetzlar, Germany). Cells were first fixed with 4% paraformaldehyde.. The treatment was carried out with 0.25% Triton-100 for 20\u0026ndash;30 min and PBS immersion was performed three times for 2 min each time; Closure was done with PBST containing 10% donkey serum for 1 hour.\u003c/p\u003e\u003cp\u003eThe primary antibody was diluted in PBST and added dropwise to the sample to completely cover the sample tissue. Place in a wet box and incubate overnight at 4\u0026deg;C; PBST washed 3 times for 2 min each; Incubate the secondary antibody, add the appropriate secondary antibody configured according to the primary antibody dropwise:Incubate at room temperature away from light for 2-3h; Wash with PBST 3 times, 5min each time; The nuclei were stained with DAPI (DAPI working solution 5mg/ml: Dilute the DAPI reservoir with PBS to a final concentration of 0.1ug/ml.) for 5 min and washed 3 times with PBST for 2 min each time. Fluorescently labeled samples were imaged with a fluorescence microscope.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e\u003cp\u003eSerum concentrations of TNF-α, IL-1β, and IL-6 were quantified utilizing standard ELISA kits (Elabscience biotechnology Co., Ltd., Wuhan, China) in accordance with the manufacturer's guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\u003cp\u003eThe brain hippocampus tissues were homogenized in RIPA Lysis Buffer (Beyotime, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF, Beyotime). The homogenate was then centrifuged at 12,000 \u0026times; g for 10 minutes at 4 ℃, and the resulting supernatant was collected. The total protein concentrations of the supernatant were determined using a BCA protein assay kit (Beyotime). Subsequently, equal amounts of proteins (20 \u0026micro;g) were separated by 4\u0026ndash;20% sodium dodecyl sulfate polyacrylamide gels (SDSPAGE) and transferred onto polyvinylidene difuoride (PVDF) membranes. The membranes were obstructed using a 5% non-fat milk powder solution in Tris-buffered saline (TBS) containing 0.1% Tween-20 for a duration of 2 hours. Subsequently, they were subjected to an overnight incubation at a temperature of 4\u0026deg;C with primary antibodies:\u003c/p\u003e\u003cp\u003eβ-Actin (1:5000; SAB #21338, USA), iNOS (1:1000; CST #13120, USA), COX-2 (1:1000; CST #12282, USA), IBA-1 (1:1000; CST #17198, USA), GFAP (1:1000; CST #2956, USA), Bax (1:1000; CST #2722, USA), Bcl-2 (1:1000; CST #3498, USA), caspase-3 (1:1000; CST #9662, USA), Cl-caspase-3 (1:5000; ab #214430, UK), HO-1 (1:1000; CST #70081s, USA), Nrf2 (1:1000; CST #12721, USA), NLRP3 (1:1000; CST #15101, USA), caspase-1 (1:1000; CST #24232, USA), IL-18 (1:1000; CST #57058, USA), IL-1β (1:1000; CST #31202s, USA), ASC (1:1000; CST #67824, USA). Following incubation with secondary antibodies for one hour at room temperature, the bands were visualized using Emitter Coupled Logic (ECL) substrate from Merck, Germany, and the intensities of the bands were evaluated using Image J software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eThe data were presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) and were subjected to statistical analysis using a one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison test. Statistical significance was determined at P-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The data were analyzed using GraphPad Prism 9.0.0 (GraphPad Software, USA). The images were analyzed using Fiji (ImageJ) software.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eInstitutional Review Board Statement\u003c/h2\u003e\u003cp\u003eThis study was approved by the Ethical Committee of recommendations of Department of Laboratory Animal Science Fudan University (NO. 2017 1325 A253).\u003c/p\u003e\u003cp\u003e\u003cb\u003eInformed Consent Statement\u003c/b\u003e: Not applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003e\u003cb\u003eConflicts of Interest\u003c/b\u003e:\u003c/h2\u003e\u003cp\u003eThe funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e\u003cp\u003eThis research was funded by a grant-in-aid from the national natural science foundation of China (81771683) and the Program to Correct Highly Deficient Areas in Shanghai's Healthcare System-Psychosomatic medicine (Grant No.2019ZB0203). There are no financial conflicts of interest to disclose.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization, HRC and ZJW; methodology, HRC, ZJW and YL; validation, PG, JMZ, ZJF, JGH and DQZ; formal analysis, HRC and ZJW; investigation, HRC, YL and JGH; data curation, HRC, YL and JGH; writing\u0026mdash;original draft preparation, HRC; writing\u0026mdash;review and editing, JGH and DQZ; visualization, HRC; supervision, YL; project administration, YL. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability Statement:\u003c/h2\u003e\u003cp\u003eThe data presented in this study are available upon request from the corresponding authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLeonard BE. Impact of inflammation on neurotransmitter changes in major depression: an insight into the action of antidepressants. PROG NEURO-PSYCHOPH. 2014;48:261\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCastren E. Is mood chemistry? NAT REV NEUROSCI. 2005;6(3):241\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHamon M, Blier P. Monoamine neurocircuitry in depression and strategies for new treatments. PROG NEURO-PSYCHOPH. 2013;45:54\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eZemdegs J, Martin H, Pintana H, Bullich S, Manta S, Marques MA, Moro C, Laye S, Ducrocq F, Chattipakorn N, et al. Metformin Promotes Anxiolytic and Antidepressant-Like Responses in Insulin-Resistant Mice by Decreasing Circulating Branched-Chain Amino Acids. J NEUROSCI. 2019;39(30):5935\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eIrwin MR, Miller AH. Depressive disorders and immunity: 20 years of progress and discovery. 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BRIT J PHARMACOL. 2013;170(6):1190\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Depression, Neuroinflammation, 5-ALA, Microglia Activation, Astrocyte Activation, NLRP3 Inflammasome, HO-1/Nrf2 Signaling Pathway","lastPublishedDoi":"10.21203/rs.3.rs-7949176/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7949176/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eDepression, classified as a mental disorder, exerts a profound negative impact on the overall well-being and life satisfaction of individuals affected. The aim of the current study was to investigate the role of 5-Aminolevulinic acid (5-ALA) in treating depression. Previous research has indicated that 5-ALA/ferrous iron (Fe\u003csup\u003e2+\u003c/sup\u003e) possesses anti-oxidative and anti-inflammatory properties. However, the effectiveness of the combination of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e in treating depression remains inadequately characterized. In the mice model induced by 6 weeks chronic unpredictable mild stress, 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e is tested to determine its effect on improving depression-like behavior. In the behavioral tests, 5-ALA/ Fe\u003csup\u003e2+\u003c/sup\u003e was found to have anxiolytic effects on mice behavior. The administration with 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e exhibited significant advantages in terms of decreased oxidative stress and proinflammatory cytokines, and increased levels of neurotransmitters in the hippocampus. Additionally, the group treated with 5-ALA/ Fe\u003csup\u003e2+\u003c/sup\u003e exhibited a substantial decrease in the protein expression of IBA-1 and GFAP within the hippocampus of mice. The administration of 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e promoted the expression of associated proteins within the activated HO-1/Nrf2 pathway, as well as the inhibition of the NLRP3 Inflammasome Activation induced by chronic stress. Consequently, 5-ALA/Fe\u003csup\u003e2+\u003c/sup\u003e exhibits promising potential as a therapeutic candidate for the treatment of depression.\u003c/p\u003e","manuscriptTitle":"Neuroprotective Effects of 5-Aminolevulinic Acid Combined with Ferrous Iron on Chronic Stress- Induced Depressive-Like Behavior in Mice by Activating the HO-1/Nrf2 Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 13:02:10","doi":"10.21203/rs.3.rs-7949176/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"aadc8b4a-11a6-4d8e-8289-444624017549","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-08T14:23:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 13:02:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7949176","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7949176","identity":"rs-7949176","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}