Depression, a complex heterogeneous and a widespread psychiatric disorder characterized by main symptoms of low mood and anhedonia, which can lead to severe impairment in daily life and with a lifetime prevalence of 15-20%. It is often accompanied by considerable morbidity and comorbidity, excess mortality, increased risk of suicide and substantial costs worldwide (Fox and Lobo, 2019; Liu et al., 2023a; Shuang et al., 2024). During the past decades, numbers of achievements were gained in study on anti-depressant study. Synthesized agents of antidepressants including selective 5-HT reuptake inhibitors (SSRIs), tricyclic antidepressants (TCAs), MAO inhibitors and atypical antidepressants, which have been widely used in depressed patients. Indeed, despite their efficacy, antidepressant drugs often come with undesirable side effects and can be associated with high rates of clinical recurrence. (Monroe and Harkness, 2022; Liu et al., 2023c; Mrozek et al., 2023). Up to now, depression was still considered as a serious public health issue. Its pathogenesis is complex and not fully understood, with many factors still under investigation.

Multiple hypotheses have been developed to unravel the pathophysiology of depression. Previous studies have indicated that the inflammatory response of bodies is associated with the pathophysiology of depression, and the levels of pro-inflammatory cytokines such as IL-1β and TNF-α have been observed in many depressed patients (Li et al., 2022a; Novakovic et al., 2023). Inflammatory response could cause depression through multiple pathways. Depression, though primarily a neurological disorder, is influenced not only by central nervous system alterations but also by peripheral inflammatory responses, highlighting the complexity of its etiology. (Al-Hakeim et al., 2023). Persistent peripheral neuroinflammation can further exacerbate the neuropathology of depression by affecting brain immunity, such as interfering with the normal function of microglia which is a resident neuroimmune cell and plays an important role in the pathological process of depression by targeting inflammatory signals and dysfunctional phagocytosis (Bai et al., 2022). In addition, chronic inflammation increases tryptophan metabolism along the kynurenine pathway (KP), and the generation of neuroactive kynurenine metabolites contributes to subsequent depressive-like behaviors. For instance, inflammatory cytokines can activate the activity of indoleamine 2,3 dioxygenase (IDO) through the KP leading to depression (Achtyes et al., 2020; Brown et al., 2021). The KP is a potential causative mechanism in depression development, which also associated with gut microbiota in the pathological process of depression.

Gut microbiota refers to a complex ecosystem composed of bacteria, fungi, protozoa, and viruses that reside in intestines, has attracted increasing attention for its pivotal role in sustaining health and its implications in disease. Notably, changes in the abundance of various microbial taxa in depressed people compared to healthy controls. Studies have showed that the gut of depressed patients is often characterized by extra growth of pro-inflammatory bacteria instead of the beneficial anti-inflammatory species (Wiedlocha et al., 2021). Additionally, the disorder of kynurenine metabolism in patients can also induce alterations in gut microbiota composition. Conversely, the gut microbiota influences the KP via both peripheral and central inflammatory mechanisms (Chevalier et al., 2020; Li et al., 2023a; Zhou et al., 2023).

To date, a series of natural compounds from phytomedicine, such as lipophilic terpenoids, flavonoids, and polyphenols, have attracted considerable research interest due to their antidepression potentials (Jahanabadi et al., 2023). The anti-depressive effects of many natural ingredients have been validated through animal experiments and clinical trials (Dai et al., 2022). Meanwhile, natural flavonoids have shown diversified physiological activities in promoting human health (Gui et al., 2023). Isorhamnetin (ISO), a flavonoid compound, can be found in the leaves, flowers and fruits of Hemerocallis citrina Baroni (H. citrina) and other plants possessing a high medicinal value (Gong et al., 2020; Ma et al., 2023). Studies have showed that ISO exhibits a broad spectrum of pharmacological benefits, including the promotion of neural function, enhancement of cognitive and memory capacities, as well as showing therapeutic potential for neurodegenerative diseases (Xu et al., 2016; Jamali-Raeufy et al., 2019). Its mechanisms mainly involve anti-inflammation, antioxidation, along with regulation of immunity through pathways such as PI3K/AKT and NF-κB. While ISO is recognized as one of the key active ingredients in H. citrina, a plant known for its potential in treating depression via multiple pathways, the antidepressant effects of ISO have been rarely reported. In this paper, the antidepressant effect of ISO and its possible mechanism were explored to advance the understanding of ISO’s neuroprotective effects and provide potential candidate drug for the treatment of depression.

Materials

Isorhamnetin (ISO, 98% purity) and Fluoxetine Hydrochloride (Flu) were provided by Solarbio Biotechnology Co., LTD (Beijing, China). ISO was dissolved in DMSO (Servicebio Biotechnology Co., Ltd., Wuhan, China) to prepare a stock solution with a concentration of 200 mg/mL, and then dilute the stock solution to 2 mg/mL with physiological saline. Fluoxetine Hydrochloride (Flu) were dissolved in physiological saline at a stock concentration of 2 mg/mL directly. LPS purchased from Servicebio Biotechnology Co., Ltd. was dissolved in physiological saline to a stock concentration of 25 μg/μL.

Animals

Male C57BL/6 mice weighing 18-20 g were provided by the Central Animal House of Shanxi Medical University (Taiyuan, China). Mice were housed (temperature was 22 ± 2°C; relative humidity was 50 ± 5%) with food/water ad libitum in a light-dark cycle (lights on at 07:00 and off at 19:00). Animals were allowed to habituate for at least one week before the onset of experiments. All experiments were conducted in accordance with the protocol approved by the Animal Care Committee of the Animal Center at Shenyang Pharmaceutical University (Animal Ethics Committee of Shenyang Pharmaceutical University, No. SYPU-IACOC-2024-0311-079) and the principles outlined in the National Institute of Health Guide for the Care and Use of Laboratory Animals.

As shown in Fig. 1A, animals were randomly divided into control (Con) group, CUMS (Mod) group, ISO group (ISO) and fluoxetine (Flu) group with 10 mice per group. Mouse in ISO group received irrigation (i.g.) administered with ISO at 20 mg/kg for 28 consecutive days based on previous studies and pre-experiment. Mouse in Flu group were received intraperitoneal injection (i.p.) administered with Flu at 14 mg/kg and the mice in the Con and Mod were given 0.5% CMC-Na solution for 28 days. For the establishment of the CUMS model, animals except for the Con group were exposed to chronic unpredictable mild stressors (CUMS) for 28 days, including fasting and water prohibition (24 h), tilted cage (12 h), damp bedding (24 h), restraint (2 h), cold water stimulation, day and night reversal, clamp the tail (2 min) and white noise (2 h). The stimulation was continued for 28 days and generated randomly to avoid the adaptation of mice.

Figure 1. ISO improved the depression-like behaviors and histopathological changes in CUMS mice. The schedule of the experiments (A). Result of Sucrose preference (B) and total liquid intake (C) in SPT. Result of Immobility time (D) and Climbing time (E) of FST. The histopathological changes in hippocampus of mice (F), and arrow points to nuclear retraction. n=10/group; *p<0.05 vs. Con group; **p<0.01 vs. Con group; ^#p<0.05 vs. Mod group; ^##p<0.01 vs Mod group.

Sucrose preference test (SPT)

The SPT was used to assess the pleasure loss of mice (Liu et al., 2023b). Two bottles (100 mL) were placed in each cage. On the first day, both bottles were filled with a 0.5% sucrose solution. On the second day, replace one of the bottles with drinking water. Exchange the positions of the two bottles after 12 h. Then, mice were deprived of food and water for 24 h. On the last day, the mice were placed one per cage which with a bottle filled with 0.5% sucrose solution and another bottle filled with drinking water. After two hours, the consumption of sucrose solution and drinking water was measured. Sucrose preference was calculated as 100%×sucrose solution consumption (mL) / total fluid consumption (mL).

Forced swim test (FST)

FST is a test for behavioral despair assessment, which is one of the main methods for screening the efficacy of antidepressant drugs (Qin et al., 2023). The mice were first familiarized with the environment (a quiet test room) for 1 h to make them adapt. Then the mice were placed in a 5000 mL cylindrical device (water depth of 20 cm, water temperature of 24°C) for 6 min, the immobility time and climbing time of mice within last the 4 min were recorded. Immobility time is recorded when mice stop struggling and move slowly to keep their heads floating on the water, and climbing time means vertical movement of the forepaws directed towards the sides of the chamber. After test, dry the mice and place them back in the cage.

Cell culture and viability assay

The murine BV2 microglial cells were obtained from Servicebio Biotechnology Co., Ltd., and cultured in DMEM supplemented with 10% FBS and 100 units/mL penicillin-streptomycin at 37°C in 5% CO₂.

BV2 microglial cells were seeded into 96-well plates at a density of 8×10³ cells per well overnight. Then, cells were cultured with or without ISO (3.125, 6.25, 12.5, 25, 50 and 100 μM) for 2 h and treated with or without 1 μg/mL of LPS for 24 h at 37°C. Cell viability was test by CCK-8 kit according to the instruction. In short, cells were exposed to CCK-8 (10 μL) solution per well for 1 h. The absorbance was detected at 450 nm using a microplate reader (Shi et al., 2024).

Histology assay

Hematoxylin-eosin (H&E) stain were used for histopathology (Ge et al., 2023). Briefly, brain tissue fixed in 4% paraformaldehyde was dehydrated, transparent and embedded in paraffin, then cut into 4-μm-thick sections. Paraffin sections were dewaxed in xylene, dehydrated by gradient elution in ethanol, then stained with H&E. Seal with neutral gum and observed under an optical microscope (Leica Microsystems Trading Ltd, Shanghai, China).

ELISA assay

The concentrations of IL-1β in serum and hippocampus, and proteins in KP include tryptophan (TRP), kynurenine (KYN), quinolinic acid (QA), kynurenic acid (KYNA) and 5-hydroxytryptamine (5-HT) in the feces, serum, and hippocampus of mice, and inflammatory factors include iNOS both in cell supernatant were measured through ELISA assays based on the instruction book supplied by the manufacturer (Mishra and Chopra, 2022) (ELISA kit, MEIMIAN, Jiangsu, China).

16S rRNA sequencing

Microbiome profiling was assessed by amplifying the bacterial 16S rRNA (Li et al., 2023b). Genomic DNA of the gut microbiota was extracted from the contents of the cecum using the QIAamp DNA Mini Kit, then used for 16S rRNA sequencing.

Western blot

Tissues of hippocampus and cells were lysed in RIPA (Servicebio Biotechnology Co., Ltd.) containing 1% PMSF (Servicebio Biotechnology Co., Ltd.). Kept homogenate on ice for 30 min, then centrifuged at 4°C for 20 min. Supernatant was collected, and total protein were measured by BCA assay kit (Dalian Melonepharma, Dalian, China), diluting the total protein of different samples to the same concentration. Stored samples at –80°C until use. Diluted with an equal volume of loading buffer (Beyotime Biotech Co., Shanghai, China, Chinaer2448), and boiled it at 100°C for 5 min. 15 μg of total protein was loaded in each well and separated in 10% SDS-PAGE gels. Then, the proteins bands were transferred onto nitrocellulose membranes. Blocked the membranes in TBST at 37°C with 5% fat-free powdered milk for 2 h, then incubated overnight at 4°C in one of the following primary antibodies for detection of target proteinsPI3K (1:1000, Abcam, Cambridge, UK), p-PI3K (1:1000, HUABIO, Hangzhou, China, ET1608-70), AKT1/2 3 (1:1000, HUABIO, ET1609-5), p-AKT (S473) (1:1000, HUABIO, ET1607-73) and β-actin Rabbit mAb (1:1000, HUABIO, R1207-1). Diluted the primary antibodies in 5% fat-free powdered milk in TBST: After three times washed for 5 min in TBST at room temperature, the membranes were incubated for 1.5 h in HRP-conjugated antibodies which were diluted in 5% fat-free powdered milk in TBST: Anti-rabbit IgG (1:3000; HUABIO, HA1001). After three times washed for 5 min in TBST, we detected immunolabeled protein bands via ECL western blot detection kit (Beyotime Biotech Co.). Graphs of blots were obtained in the linear range of detection (Liu et al., 2023b).

Immunofluorescence staining

The brains of mice were perfused transcardially with phosphate buffered saline (50 mM PBS with pH 7.4) followed by 4% paraformaldehyde in PBS. Embedded the brains in paraffin, then cut into 3 μm transverse sections. Kept brains with a constant temperature of 63°C for 2 h. Dewaxed brains by xylene for 20 min and 3 times. Then, washed the slices with distilled and ethanol water. The activity of endogenous peroxidase was blocked by placed section into 100 μL of 3% H2O2 and incubated for 10 min at 24°C. The slices were preincubated with normal goat serum to block nonspecific binding for 30 min. Slides were then incubated with primary antibodies for Iba-1 (1:1000; Abcam), and p65 (1:1000; Abcam) overnight at 4 °C. Then, sections were washed in PBS 3 times, then incubated with secondary antibody FITC anti rabbit (1:2000; Abcam) for 1 h at room temperature. Finally, sections were washed three times in PBS and covered with DAPI/Fluorescence quenching agent (Beyotime Biotech Co.). Viewed the slides via a fluorescence microscope (Leica Microsystems Trading Ltd). The cells were washed with PBS, fixed with 4% polyformaldehyde PBS for 30 min, then infiltrated with 0.1% Triton X-100 at room temperature for 10 min.

Blocked cells with 3% BSA and incubated with primary antibodies anti-p65 overnight. The second antibody FITC anti-rabbit (1:2000; Abcam) and CY3 anti-rabbit (1:2000; Abcam) immunoglobulin were incubated for 1 h at room temperature. Washed cells with PBS for 3 times. Collected the images by fluorescence microscopy (Leica Microsystems Trading Ltd). IOD were analyzed by image J and image-pro plus 6.0 (Bai et al., 2022).

Molecular docking

To evaluate the binding modes and interactions between ISO and its predicted targets, the molecular docking simulation was carried out by AutoDock Vina 1.1.2 (The Scripps Research Institute, CA, USA). The two-dimensional (2D) structure of ISO was downloaded from the PubChem database (https://pubchem.ncbi.nlm.nih.gov/). The three-dimensional (3D) structure of targets was obtained from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (http://www.rcsb.org/pdb/). Then, Chem3D 19.0 software (Cambridge Technology, MA, USA) was applied to optimize the mechanical structure to minimize energy. AutoDockTools was used to generate the docking input files. Selected the best-scoring pose based on the Vina docking score, and visually analyzed were performed using PyMoL 1.7.6 software (https://pymol.org/) (Fan et al., 2024).

Statistical analysis

The data were analyzed using Graphpad Prism 7.0 (GraphPad Software, San Diego, CA, USA). Differences were regarded as significant when p<0.05. Data are expressed as means ± SEM. Tests data included one-way or two-way ANOVA and unpaired Student’s tests. Data heterogenity was tested and, where variance was significant, appropriate non-parametric tests were used. Corrections for multiple comparisons were performed using appropriate post-hoc tests.

ISO improved the depression-like behaviors and histopathological changes in CUMS mice

To evaluate the antidepressant effect of ISO, the SPT and FST were performed to evaluate the depression-like behaviors in mice. As shown in Fig. 1B and 1C, the sucrose preference was significantly reduced in the CUMS group compared with the Con group, and total liquid intake remained unchanged in all groups. However, ISO or Flu treatment could increase the sucrose preference decreased by CUMS. In addition, in FST, CUMS markedly prolonged the immobility time, which significantly decreased by treatment with ISO or Flu. However, ISO did not reduce the climbing times increased by CUMS (Fig. 1D, 1E).

H&E staining was used to determine changes in the pathological and physiological characteristics of the mice. As shown in Fig. 1F, the neurons of the hippocampus appeared intact and ordered in mice of Con, and shrinkage, degeneration and necrosis of the nuclei were not observed. The mice in Mod group had neuronal deformities, cell loss, and shrinkage of nuclei, swollen and dispersed neuronal bodies. After administeration of ISO, the mice were normalized and tightly arranged, and the number of necrosis was decreased.

ISO inhibited CUMS-induced peripheral and neuroinflammatory activation in CUMS mice

Previous studies have shown that ISO has a positive effect on immunoregulation, the levels of IL-1β were assessed to detect ISO’s effects on peripheral and neuroinflammation caused by CUMS. As shown in Fig. 2A and 2D, ISO treatment could decrease the concentration of IL-1β both in the serum and hippocampus. Further more, the correlations among behavioral and IL-1β level variables showed that a cluster of serum IL-1β was significantly correlated with sucrose preference (%) (R²=0.516; p=0.008), immobility time (R²=0.469; p=0.017) (Fig. 2B, 2C); the decreased of IL-1β level in hippocampus were strongly associated with the increase of sucrose preference (%) (R²=0.6046; p=0.001), immobility time (R²=0.4687; p=0.017) (Fig. 2E, 2F). Microglia play an important role in the immunopathological process of depression. Therefore, the activation status of microglia was investigated after ISO administration. The increased levels of Iba-1 indicate the sustained activation of microglia. As shown in Fig. 2G and 2H, CUMS significantly increased Iba-1 expression in hippocampus. Whereas, the activation of microglia cells tends to normalize after treatment with ISO.

Figure 2. ISO inhibited CUMS-induced peripheral and neuroinflammatory activation. The levels of IL-1β in serum (A). Correlation analysis between IL-1β level in serum and immobility time (B), and Sucrose preference (C). The levels of IL-1β in hippocampus (D). Correlation analysis between IL-1β level in hippocampus and immobility time (E), and Sucrose preference (F). Activation of microglia in the hippocampu (G), the expression of Iba-1 detected by immunofluorescence staining with anti-Iba-1 antibody (green) and DAPI (nuclei, blue), and quantification of the expression of Iba-1 (H). n=10/group; **p<0.01 vs. Con group; ***p<0.001 vs. Con group; ^#p<0.05 vs. Mod group.

To further confirm the effect of ISO on microglia under inflammatory conditions, the effect of ISO were detected at different doses (6.25-100 μg/mL) on the BV2 cell (Fig. 3A-3D). The cell viability were tested by CCK8 kit, and the concentration of 12.5 and 50 μg/mL were chosen for the next test. Similarly, the increased level of Iba-1 induced by LPS decreased after co-culture with ISO. In addition, the level of NO in the supernatant was significantly reduced.

Figure 3. ISO inhibited inflammatory activation in BV2 cells induced by LPS. The cell viability tested by CCK-8 kit (A). The expression of Iba-1 detected by immunofluorescence staining with anti-Iba-1 antibody (green) and DAPI (nuclei, blue) (B), and quantification of the expression of Iba-1 (C). The secretion of NO was tested by Nitric Oxide Assay Kit (D). n=3/group; *p<0.05 vs. Con group; **p<0.01 vs. Con group; ^#p<0.05 vs. Mod group; ^##p<0.01 vs. Mod group.

ISO ameliorated metabolism of kynurenine pathway

The KP is one of the key mood-related signals related to immunity. The levels of KP metabolites including TRP, KYN, 5-HT, 5-HIAA, QA, and KYNA of hippocampal, serum and feces were measured by ELISA KIT. As shown in Fig. 4, the levels of KYN and QA were significantly increased and the level of 5-HT, HIAA, TRP and KYAN were significantly decrease in the hippocampal and serum of Mod mice compared with Con mice. Treatment with ISO considerably restored these indicators. In addition, learning and the speed of processing in patients with major depressive disorder are associated with the ratio of KYN/TRP. The results of animal experiments showed that the ratio of KYN/TRP was significantly increased in mice under CUMS, which could be alleviated by ISO administration.

Figure 4. ISO ameliorated metabolism of kynurenine pathway. The concentration of 5-HT, KYNA, QA, HIAA, KYN, TRP and the ratio of KYN/TRP in the hippocampus (A) and serum (B). Correlation analysis between IL-1β level and KYNA level in hippocampus (C). Correlation analysis between IL-1β level and KYNA level in serum (D). n=10/group; *p<0.05 vs. Con group; **p<0.01 vs. Con group; ***p<0.001 vs. Con group; ****p<0.0001 vs. Con group; ^#p<0.05 vs. Mod group; ^##p<0.01 vs. Mod group; ^###p<0.001 vs. Mod group.

The data obtained from 30 mice (10 per group) were pooled for correlation analysis. KYNA and QA, which play the role of neuroprotection and neurotoxicity in KP respectively, were selected for correlation analysis. As shown in Fig. 4C and 4D, a, a cluster of KYNA and QA was significantly correlated with the levels of IL-1β in the hippocampus and serum.

ISO altered the distribution of gut microbiota in CUMS mice

Alpha diversity includes the Shannon, Simpson, Chao and Ace indices. The Shannon and Simpson are related to the material richness and species homogeneity in the sample’s intestinal bacterial community which indices represent the diversity of the community. While the Ace and Chao indices reflect the number of species in the community which could indicate the community richness of the sample. The result showed that the Ace index, Chao index, and Simpson index of mice in Mod group were reduced compared with the Con group, which indicates that the abundance and diversity of the intestinal community of mice were reduced after exposed to CUMS. While, the diversity was restored after the administration of ISO (Fig. 5A-5D). PCA analysis showed that Mod group drove different gut bacteria from that in control group and ISO treatment altered the intestinal bacteria (Fig. 5E). We analyzed the data at the phylum level, a significant reduction and enrichment in the abundances of the phylum Firmicutes and Bacteroidetes were observed in Con and ISO group compared with Mod group (Fig. 5F). To identify intestinal bacteria regulated by ISO, LEfSe analysis was applied to identify significantly altered taxa. The dendrogram (Fig. 5G) demonstrates that, compared with the control group, the CUMS and ISO groups showed significant changes in gut bacteria in different taxonomic groups (arranged in phylum order), and the LDA score histogram (Fig. 5H) clearly showed the changed bacteria in each group. Among the 25 biomarkers listed, c-Clostridia, o-Oscillospirales, s-unclassified-Lachnospiraleae and f-Rikenellaceae were specific in the ISO group, which may indicate an association with its anti-depression-like behaviors in mice.

Figure 5. ISO altered the distribution of gut microbiota in CUMS mice. ACE index (A), Chao index (B), Shannon index (C) and Simpson index (D) among groups. PCA analysis was performed to show the changes in gut microbiota diversity among groups (E). Relative abundances of selected phylum with significant differences among each group (F). LDA score histogram (H) and Cladogram (G) among groups in LEfSe analysis was used to analyze the biomarker bacteria of each group. n=6/group; *p<0.05 vs. Con group; ^#p<0.05 vs. Mod group; ^##p<0.01 vs. Mod group.

ISO exerts antidepressant effects by activating the PI3K/AKT pathway

Previous studies have shown that PI3K/AKT is one of the key pathways of ISO to exert neuroprotective effect, which is also a key pathway in the pathological process of depression. As shown in Fig. 6, the up-regulation of p-PI3K/PI3K and p-AKT/AKT in CUMS-exposed mice indicates activation of the PI3K/AKT pathway. However, the expression of these proteins was markedly decreased after ISO treatment, suggesting an inhibitory effect of ISO on the PI3K/AKT pathway in vivo. NF-κB is an important downstream of the PI3K/Akt pathway which also act key activator of in immunoregulation. As shown in the result of immunofluorescence staining, the level of NF-κB in the nucleus increased significantly in Mod group compared with that in Con. ISO treatment could reverse the nuclear translocation of NF-κB (Fig. 6B).

Figure 6. ISO exerts antidepressant effects by activating the PI3K/AKT pathway in vivo. Representative bands of PI3K, p-PI3K, AKT, p-AKT and their control protein β-actin in the hippocampus of mice, and Relative levels of p-PI3K to PI3K, p-AKT to AKT (A). The nuclear translocation of NF-κB in the hippocampus of mice (B), the expression of NF-κB detected by immunofluorescence staining with anti-NF-κB antibody (red) and DAPI (nuclei, blue). n=3/group.

The effect of ISO on the PI3K/AKT/NF-κB pathway has also been validated in vitro experiments. The results were consistent with the results of the hippocampal in mice. As shown in Fig. 7, LPS stimulation significantly induced phosphorylation of PI3K and AKT, and promoted the nuclear translocation of NF-κB. These changes could be prevented by ISO’s protection.

Figure 7. ISO exerts antidepressant effects by activating the PI3K/AKT pathway in BV2 cells. Representative bands of PI3K, p-PI3K, AKT, p-AKT and their control protein β-actin in the BV2 cells treated with LPS and ISO of different concentrations, and Relative levels of p-PI3K to PI3K, p-AKT to AKT (A). The nuclear translocation of NF-κB in BV2 cells treated with LPS and ISO of different concentrations (B), the expression of NF-κB detected by immunofluorescence staining with anti-NF-κB antibody (green) and DAPI (nuclei, blue). n=3/group; *p<0.05 vs. Con group; **p<0.01 vs. Con group; ^#p<0.05 vs. Mod group.

Molecular docking patterns of ISO with core targets in PI3K/AKT pathway

Docking score between these 8 targets and ISO was shown in Table 1 and Fig. 8. The smaller the binding energy, the greater the docking activity and the greater the possibility of action. A binding energy below –5 kcal/mol is indicative of strong protein-ligand binding. This is indicative of a high likelihood of spontaneously binding eight core targets between ISO, with the docking scores ranging from –8.7 to –5.8. ISO and PI3KR1 could develop H bonds with GLU-849 (2.4Å) and VAL-851 (2.4Å, 2.5Å and 2.1Å), ISO and PIK3CG could develop H bonds with ARG-277 (2.1Å and 2.9Å) and HIS-304 (2.2Å and 2.3Å), ISO and EGFR could develop H bonds with MET-769 (2.1Å), ISO and IGF1R could develop H bonds with HIS-1130 (2.6Å) and THR-1154 (2.5Å), ISO and MET could develop H bonds with ASP-207 (1.8Å) and ALA-170 (2.7Å),ISO and KDR could develop H bonds with CYS-919 (1.9Å), ISO and AKT1 could develop H bonds with LYS-39 (2.4Å) and ALA-50 (2.2Å), ISO and PKT2 could develop H bonds with ASP-207 (1.8Å) and ALA-170 (2.7Å).

Table 1 Binding energy and interacted residues

Core target	Binding energy (kcal/mol)	Interacted residues
PIK3R1	–8.7	LYS-39 (2.4 Å), ALA-50 (2.2 Å)
PIK3CG	–8.4	VAL-851 (2.1 Å, 2.4Å, 2.5Å), GLU-849 (2.4Å)
EGFR	–7.9	MET-769 (2.1 Å)
IGF1R	–7.7	HIS-1130 (2.6 Å), THR-1154 (2.5 Å)
MET	–7.4	ASP-207 (1.8 Å), ALA-170 (2.7 Å)
KDR	–6.8	CYS-919 (1.9Å)
AKT1	–6.1	ALA-50 (2.2 Å), LYS-39 (2.4Å)
PKT2	–5.8	GIU-984 (2.6Å)

Figure 8. Molecular docking patterns of ISO with core targets in PI3K/AKT pathway.

At present, there have been numerous investigations on ISO which display medicinal properties include cardiovascular and cerebrovascular protection, anti-tumor, anti-inflammatory, anti-oxidation, organ protection, prevention of obesity, and thus ISO has a high value of development and application (Cai et al., 2020; Kalai et al., 2022; Li et al., 2022b). In our previous study, ISO was confirmed as one of the important components in flower buds of H. citrina that exhibited pharmacological effects. H. citrina is distributed widely from Europe to Asia, and its flower buds are regarded as a useful meal. Its benefits, such as improving sleep and curing depression, were initially recorded in the book Bencao Gangmu Shiyi. Moreover, modern pharmacology study confirmed the antidepressant activity of H. citrina (Yi et al., 2012; Li et al., 2017; Ma et al., 2022). In this paper, the antidepressant activity of ISO was investigated. Interest in rewarding stimuli was assessed via sucrose preference tests, while despair-like behaviors in mice were quantified using the forced swimming test. These behavioral pharmacology experiment revealed that long-term administration of ISO alleviate the depression behavior of mice to a notable extent. Considering that the dosage of ISO used to exert antidepressant effects was much higher than that in H. citrina, the antidepressant effect of ISO was not compared with that of H. citrina.This also revealed that ISO is not the only effective component in the H. citrina. Additionally, The mechanism was further investigated, with findings suggesting that the antidepressant effect of ISO is based on improving inflammation.

Given LPS can only minimally or cannot penetrate blood-brain barrier BBB, and Chronic unpredictable mild stress (CUMS) is a classic mouse model of depression. It was reported that CUMS increases the expression of proinflammatory cytokines and reduces the expression of anti-inflammatory cytokines in animals,and accompanied by the activation of glial cells. Hence, some studies which focus on the inflammatory response in the pathological process of depression also created depressed animals through CUMS. Microglial cells play a central role in host defence response to external stimulation. Various stimuli induce the activation of microglia, including LPS. Excessive activation of microglia gives rise to the production of inflammatory factors, including IL-1β, IL-6, and TNF-α.Therefore, in this study, CUMS induced depressed mice were used for in vivo,and LPS cultured BV2 cells were used for in vitro. Inflammatory processes have been implicated in the pathophysiology of depression. Microglia are the predominant resident immune cells in the brain which play a pivotal role in the induction of neuroinflammation (Rodriguez-Zas et al., 2018). Microglia can transform into reactive states in response to inflammatory insults (Li et al., 2022a). Activated microglia undergo rapid changes in morphology and function, including cell enlargement, thickening, and shortening of branches to migrate towards lesions for phagocytosis. They also secrete numerous inflammatory cytokines simultaneously. Recent studies have suggested that depression can be regarded as a microglial disease (Miron and Priller, 2020). On the one hand, massive evidence has suggested that microglia-mediated neuroinflammation interacts with three theories correlated with depression include monoamine neurotransmitter depletion hypothesis, neuroplasticity hypothesis, and hypothalamus-pituitary-adrenal (HPA) axis hypothesis (Garrison et al., 2018; Sugama and Kakinuma, 2020). PI3K/Akt signaling pathway is a classical pathway for cell metabolism, survival, apoptosis, and other physiological activities, which regulates neurotoxicity and mediates neuronal survival (Yang et al., 2023). Accumulating evidences has indicated that the PI3K/AKT signaling pathway can effectively regulate production of pro-inflammatory cytokines and microglial polarization. Our results showed that ISO could decrease the levels of pro-inflammatory-M2-microglia, accompanied with inhibition of activation of PI3K/AKT pathway, while the molecular docking suggested that PI3K as a key pathway of ISO. Moreover, ISO further reduced nuclear translocation of NF-κB, which could regulate the production of pro-inflammatory factors, including TNF-α, IL-1β and IL-6.

On the other hand, large evidence has suggested that TKP dysfunction is regulated by microglial activation in depression. Microglia regulate KP balance by preferentially producing oxidative metabolites, including quinolinic acid known as the primary neurotoxic metabolite (Murata et al., 2019). Disruption of neuroinflammatory balance leads to the redistribution and activation of microglia within the brain. This can alter the KP metabolic balance toward oxidative metabolism of excitatory and potentially cytotoxic metabolites, which induce the dysregulation of tryptophan-KP (Wang et al., 2023). Meanwhile, clinical data indicate the correlation of depressive symptoms with plasma and increased CSF concentrations of KYN, and depressed patients showed decreased levels of KYNA, QA, and KYN, consistent with the present study. Moreover, it is generally accepted that, dependent on the cell type, kynurenine is metabolised into neurotoxic QA in microglia and macrophages or is transformed into neuroprotective KYNA in astrocytes. Studies have also indicated that mice exposed to low level inflammatory exhibit increased expression of IDO-1 and TDO, and acute stress increases plasma and brain kynurenine. Under physiological conditions, IDO-1 remains in a semi-dormant state. However, the increase of pro-inflammatory cytokines and the activation of microglia mutually promote each other, induces neuroinflammation, Importantly, this activation also boosts the activity of IDO-1, leading to increased TRP and reduced 5-HT. In this paper, the effect of ISO on the protein of KP in mice was delved into.. KYN/TRP always considered as indicator of enzyme activity for IDO-1. In this paper, the activity of IDO-1 is reduced after ISO treatment, which could play a crucial role in the administration of ISO antidepressants.

In this study, ISO was administered orally, and introduced effects on the gut microbiota. The impact of gut microbiota on brain diseases has been widely accepted (Singh et al., 2024). Gut microbiota alterations can play a role in promoting gut microbiota-derived molecules, such as LPS, within the brain. This alteration in homeostasis towards pro-inflammatory conditions sets the stage for the pathogenesis of neurogenic diseases and depressive-like behavior. Oral antibiotic treatment could effectively reduce alcohol-induced microglial activation, strongly supporting the influence of gut microbiota on the microglia activation (Buchenauer et al., 2023). In this study, it was observed that the disruption of gut microbiota caused by CUMS could improve by treatment with ISO. However, BV2 in vitro studies indicated that the improvement effect of ISO on inflammation do not depend on the regulation of gut microbiota. Besides its close relationship with inflammation, the crosstalk between gut microbial and KP metabolism also emerges as an important driving force in modulating pathological progression of depression. 5-HT, a monoamine molecule synthesized from tryptophan, which acts as a key neurotransmitter involved in the modulation of emotional control, food intake, etc. However, central serotonin only accounts for a small proportion of the body’s total serotonin. Over 90% of 5-HT is present in the GI tract. Study has demonstrated that the levels of both upstream or downstream factors in the KP in the intestine interact with the mouse gut microbiota of mice (Wiedlocha et al., 2021). For example, mice with increased levels of 5-HT in the intestine have significant alterations in microbial composition, particularly a significant increase in Firmicutes abundance where the CUMS group showed a significant decrease in Firmicutes abundance, a trend reversed by administration of ISO. However, The direct interaction between ISO and gut microbiota needs further validation.

In summary, this study presents a comprehensive investigation into the moderating effects of ISO, a flavonoid compound, on depression. A combination of behavioral, biochemical, molecular approaches and bio-informatics analysis were employed to provide a thorough understanding of its mechanisms of action. The study indicated that ISO reduced chronic stress-evoked depression-like behaviour in association with its normalization of inflammation-induced dysregulation of the KP regulating the PI3K/AKT/NF-κB pathway. Additionally, ISO was found to repair gut microbiota disorder induced by depression (Fig. 9).

Figure 9. Working model of ISO in protecting against depression. ISO mediates activation of inflammation signaling through suppression of PI3K/AKT and repairs disrupted kynurenine pathway and gut microbiota.

Future attempts will be made to integrate multisource targeted metabolomics through in-depth analysis of gut microbiota and immunity to verify whether the regulation of gut microbiota is also a key pathway in antidepressant mechanism of ISO. Besides, a multi-time-point longitudinal study design should be useful to draw a comprehensive picture of the changes in microbial composition and microglial phenotype caused by ISO. Fecal transplantation, specific gut microbiota transplantation and spatially resolved metabolomics based on Air flow-assisted desorption electrospray ionization-mass spectrometry imaging could be used in our further studies to deeply research the relationship among gut microbiota, immunity and kynurenine metabolism in depression after ISO treatment.

This work was partially supported by funding from the National Natural Science Foundation of China (No. 82304710) and Shanxi Basic Research Project of Shanxi Province Science and Technology Department (202103021223369). We express our sincere appreciation to the study participants whose participation was integral to the acquisition of our findings.

The authors declare that there is no conflict of interest.

Mengjie Xu: Conceptualization, Methodology, Investigation, Writing - Original Draft, Funding acquisition. Jiao Xiao: Conceptualization, Formal analysis, Writing - Review & Editing, Project administration. Wei He: Methodology, Investigation. Ke Yan: Methodology, Investigation. Xinru Gao: Methodology. Jun Li: Investigation. Dongyue Xu: Investigation. Tingxu Yan: Resources, Supervision.