Modified Xiaoyao San Ameliorates Depression-Like Behaviors and Neuronal Necroptosis in a Mouse Model of Chronic Unpredictable Mild Stress

Summary

This protocol demonstrates the evaluation of Modified Xiaoyao San in a mouse model of chronic unpredictable mild stress-induced depression, including behavioral, histological, and molecular assessments of neuronal necroptosis.

Abstract

Neuronal loss and damage are important pathological features of depression, and necroptosis has been proposed as a potential mechanism of neuronal cell death. Analysis of Gene Expression Omnibus (GEO) datasets (GSE54565, GSE54566, and GSE54568) identified an association between depression and necroptosis, suggesting that inhibition of this pathway may represent a potential therapeutic strategy. Modified Xiaoyao San (MXYS), a modified formulation of the traditional Chinese medicine Xiaoyao San, was developed by incorporating heat-clearing, blood-cooling, cognitive-enhancing, and resuscitation-promoting herbs to improve therapeutic efficacy. However, its effects on neuronal necroptosis and the active constituents reaching brain tissue remain unclear. Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was used to tentatively identify MXYS constituents detected in brain tissue. Therapeutic efficacy was evaluated in a chronic unpredictable mild stress (CUMS) mouse model of depression using behavioral assessments, histological examination, immunofluorescence, western blotting, and bioinformatic analyses. MXYS administration was associated with improved depression-like behaviors, attenuation of pathological brain injury, and reduced phosphorylation of receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like protein (MLKL). These findings suggest that MXYS exerts antidepressant-like effects that are associated with reduced neuronal necroptosis markers, providing experimental support for further investigation of MXYS as a potential therapeutic strategy for depression.

Introduction

Depression, a severe mental disorder within the spectrum of mood disorders, is characterized primarily by persistent low mood and is influenced by multiple factors¹. Clinically, patients with depression commonly exhibit symptoms such as depressed mood, cognitive slowing, reduced verbal communication and physical activity, and, in severe cases, self-injurious behavior and suicidal ideation². Owing to its high prevalence, recurrence, disability, and suicide rates, depression is a leading cause of disability worldwide and imposes substantial social and economic burdens³. According to the World Health Organization, depression is projected to become the leading contributor to the global disease burden by 2030, surpassing cancer and cardiovascular and cerebrovascular diseases³. In addition, the coronavirus disease 2019 (COVID-19) pandemic was associated with a 27.6% increase in the global prevalence of major depressive disorder⁴. In China, the annual prevalence of depression is 3.6%, with a lifetime prevalence of 6.8%⁵. The pathogenesis of depression remains incompletely understood because of its complex etiology and multiple contributing factors. Consequently, treatment options remain limited, and approximately 30%–40% of patients develop treatment-resistant depression because of an inadequate response to currently available antidepressants⁶. Furthermore, the delayed onset of action, residual symptoms, neurotoxicity, and adverse effects associated with existing antidepressants continue to present significant therapeutic challenges⁷.

Programmed cell death has long been a major focus of biomedical research. Increasing evidence suggests that depression is associated with necroptosis^8,9,10,11. Necroptosis, like apoptosis, is a form of programmed cell death; however, unlike apoptosis, it elicits pronounced immune and inflammatory responses¹². In the absence of caspase-8 activity, tumor necrosis factor-α (TNF-α) binds to tumor necrosis factor receptor 1 (TNFR1), resulting in the activation of receptor-interacting protein kinase 1 (RIPK1)¹³. Activated RIPK1 interacts with receptor-interacting protein kinase 3 (RIPK3) to form the necrosome complex, which subsequently activates mixed lineage kinase domain-like protein (MLKL)¹⁴. Following oligomerization, MLKL translocates from the cytoplasm to the plasma membrane, causing organelle swelling^15,16, disruption of cell membrane integrity, and release of intracellular contents, including damage-associated molecular patterns (DAMPs) and inflammatory mediators such as TNF-α, interleukin (IL)-1, and IL-6, thereby exacerbating inflammatory responses¹⁷. Necroptosis has been implicated in numerous inflammatory and neurodegenerative diseases, including multiple sclerosis, depression, amyotrophic lateral sclerosis, Parkinson disease, and Alzheimer disease^18,19,20. Proteomic analyses have shown that chronic unpredictable mild stress (CUMS)-induced depression is associated with increased expression of RIPK3 and MLKL and activation of the necroptosis pathway². In another CUMS mouse model of depression, fluoxetine exerted antidepressant-like effects that were associated with inhibition of RIPK1-mediated necroptosis¹⁰. Furthermore, Nec-1s, an inhibitor of necroptosis, has been shown to ameliorate lipopolysaccharide (LPS)-induced depression-like behaviors²¹. Because neuronal injury is an important pathological feature of depression, targeting necroptosis may represent a promising therapeutic strategy for the treatment of depression.

Traditional Chinese medicine (TCM) has a long history of treating depression. According to TCM theory, the liver governs the free flow of qi; therefore, depression is primarily attributed to emotional distress leading to liver qi stagnation. Accordingly, the principal therapeutic strategy is to soothe the liver and relieve depression. Xiaoyao San (XYS) is a classical herbal prescription widely used for this purpose, and extensive evidence supports its application in the treatment of depression²². Furthermore, XYS has been reported to possess anti-necroptotic properties^23,24. Based on the TCM concepts of “liver depression transforming into fire” and “clear yang ascending to the brain,” XYS was modified by adding Moutan Cortex (Mudan Pi) and Gardeniae Fructus (Zhizi) to clear heat and cool the blood, together with Acori Tatarinowii Rhizoma (Shi Changpu) and Polygalae Radix (Yuan Zhi) to promote cognitive function and resuscitation. The resulting formulation was designated Modified Xiaoyao San (MXYS) and was developed to provide enhanced therapeutic effects based on TCM principles. However, direct comparison with the original XYS formulation was not performed in this study²⁵. Clinical studies have demonstrated the antidepressant efficacy of MXYS^26,27,28, and its therapeutic effects may be associated with regulation of the hypothalamic-pituitary-adrenal (HPA) axis and inhibition of neuroinflammation^29,30,31. Nevertheless, whether MXYS is associated with reduced neuronal necroptosis remains unclear.

In the present study, ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was used to tentatively identify MXYS-derived compounds detected in brain tissue. Publicly available Gene Expression Omnibus (GEO) datasets and bioinformatic analyses, including weighted gene co-expression network analysis (WGCNA), differential expression analysis, and enrichment analysis, were used to investigate the associations among MXYS, depression, and necroptosis. Based on these findings, we hypothesized that MXYS may exert antidepressant-like effects that are associated with reduced neuronal necroptosis markers, with the activating transcription factor 4 (ATF4)/cAMP response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) pathway representing a candidate correlative mechanism. To evaluate this hypothesis, compounds detected in brain tissue were tentatively identified using UHPLC-HRMS, bioinformatic analyses were performed to identify potential therapeutic targets, and in vivo experiments were conducted to examine whether MXYS treatment is associated with changes in necroptosis markers and the ATF4/CREB/BDNF pathway. These findings provide preliminary evidence supporting the potential therapeutic application of MXYS in depression while acknowledging that comparative studies with the original XYS formulation and dose-response validation remain necessary.

Protocol

The animal experiments were approved by the Animal Ethics Committee of Henan University of Chinese Medicine (approval no. DWLLGZR202200023). All procedures were conducted in accordance with institutional and national guidelines for animal welfare and complied with the ARRIVE guidelines. Male Sprague–Dawley (SD) rats were obtained from a licensed laboratory animal supplier. The animals were housed under controlled temperature and humidity with a 12 h light/dark cycle and provided standard chow and water ad libitum.

Identification of the Brain Tissue–Contained Components of MXYS
Preparation of Freeze-Dried MXYS Powder:
The 11 herbs of MXYS were steeped in 10 volumes of distilled water for 2 h and then boiled for 40 min. Peppermint was added during the final 5 min of decoction. A second decoction was prepared in the same manner using 8 volumes of distilled water. The two decoctions were combined, filtered through gauze, concentrated under reduced pressure to a concentration of 1 g/mL (crude drug equivalent), and freeze-dried under vacuum for 48 h to obtain freeze-dried MXYS powder. The extraction yield was 21.7% (w/w)^25,29,31.

Animals and Administration of MXYS:
Twelve male SD rats were randomly assigned to two groups and administered either MXYS or purified water twice daily by oral gavage. The two daily administrations were performed at 8:00 AM and 8:00 PM (12-h interval). MXYS powder was dissolved in purified water and administered at 24.4 g/kg (approximately twice the clinical dose for rats)²⁵. The control group received purified water at an equivalent volume (10 mL/kg).

Preparation of MXYS-Containing Brain Tissue:
After 14 days, all rats were anesthetized with sodium pentobarbital (40 mg/kg, intraperitoneally) 2 h after the final MXYS administration. Brain tissues were collected following transcardial perfusion with normal saline. Whole brains were immediately excised and placed on ice to obtain blank brain tissue and MXYS-treated brain tissue samples.

Chromatographic/Mass Spectrometry Conditions and Database:
Freeze-dried MXYS powder, blank brain tissue spiked with MXYS powder, blank brain tissue, and MXYS-containing brain tissue extracts were analyzed using an ultra-high-performance liquid chromatography system equipped with an HSS-T3 column (100 × 2.1 mm, 1.8 μm) maintained at 35°C. The mobile phases consisted of water containing 0.1% formic acid (mobile phase A) and acetonitrile containing 0.1% formic acid (mobile phase B). Samples were separated at a flow rate of 0.3 mL/min using a gradient of 5% B for 1 min, increasing to 98% B over 16 min, returning to 5% B in 0.5 min, and holding at 5% B for 2.5 min.

Mass spectra were acquired in both positive and negative electrospray ionization modes using data-dependent acquisition over an m/z range of 90–1,300. MS/MS spectra were generated from the 10 most intense precursor ions using stepped higher-energy collisional dissociation collision energies of 20, 40, and 60 eV. The capillary temperature was maintained at 320°C, and the probe heater temperature was set at 350°C.

Compound annotation was performed by matching accurate mass, isotopic distribution, and MS/MS spectra against an in-house traditional Chinese medicine database and the public GNPS³², ReSpect³³, and MassBank³⁴ databases. Compounds with reliable MS/MS matching and consistent chromatographic peak shapes were retained. The complete list of tentatively identified compounds is provided in Supplementary Table 1.

Bioinformatics
Data Set Selection and Preprocessing from the GEO Database:
The potential involvement of necroptosis in the pathology of depression was investigated by searching the GEO database using the keyword “depression.” Based on the selection criteria, three datasets (GSE54566, GSE54565, and GSE54568) were identified for further analysis. The datasets were derived from different brain regions: GSE54565 (anterior cingulate cortex), GSE54566 (amygdala), and GSE54568 (dorsolateral prefrontal cortex, BA9). All datasets were generated using the Affymetrix Human Genome U133 Plus 2.0 Array platform (GPL570) and consisted of postmortem brain tissue samples from patients with major depressive disorder (MDD) and non-psychiatric controls. Detailed sample characteristics for each dataset are summarized in Supplementary Table 2. These datasets comprised transcriptomic data from three brain regions implicated in depression. Differential gene expression analysis was performed between the MDD and control groups for each dataset, and consistently upregulated and downregulated genes were integrated using the Robust Rank Aggregation (RRA) R package.

All datasets were preprocessed using the robust multi-array average (RMA) algorithm for background correction and normalization. Following RMA normalization, the ComBat algorithm implemented in the sva R package was applied to adjust for batch effects across datasets, with dataset origin treated as the batch covariate. This procedure reduced technical variation among datasets and improved comparability for downstream analyses.

Bioinformatic analyses were performed using R software (version 4.2.1). The principal R packages included ClusterProfiler (version 4.8.2) for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, WGCNA (version 1.72) for weighted gene co-expression network analysis, RRA for differential gene integration, and sva (version 3.48.0) for ComBat batch correction. The analytical workflow comprised data acquisition, RMA normalization, ComBat batch correction, differential expression analysis with RRA integration, WGCNA module construction, GO and KEGG enrichment analyses with a false discovery rate (FDR) < 0.05, and target prediction with protein–protein interaction (PPI) network analysis.

Employing WGCNA to Construct Co-Expression Gene Networks and Identifying Modules Significantly Associated with Depression And Necroptosis:
WGCNA constructs weighted gene co-expression networks by applying a soft-thresholding power (β value) to approximate a scale-free topology. The β value was selected when the correlation coefficient between log(k) and log(p(k)) reached at least 0.9. After selecting the β value, gene modules were identified using the dynamic tree cut method. Additional parameters were set as follows: minModuleSize = 30, cutHeight = 0.25, and deepSplit = 2. The β value was selected when the correlation coefficient between log(k) and log(p(k)) reached at least 0.9. A soft-thresholding power of β = 7 was used for network construction. The Module Eigengene (ME) was calculated for each module to summarize gene expression patterns and evaluate associations with biological traits. Gene Significance (GS) and Module Membership (MM) were subsequently calculated, and module-trait relationships were evaluated using the correlation coefficient (cor) and corresponding p-value. Higher correlation coefficients and lower p-values indicated stronger associations between gene modules and biological traits.

Functional Enrichment Analysis and Construction of the PPI Network:
ClusterProfiler was used to perform KEGG pathway enrichment analysis. The Benjamini–Hochberg FDR method was applied for multiple-testing correction, and an adjusted p-value (FDR) < 0.05 was considered statistically significant. After identifying the target module, key genes within the module were intersected with genes associated with MXYS and necroptosis. A PPI network was subsequently constructed using the STRING database.

Target Prediction for Compounds Detected in Brain Tissue:
The 143 compounds tentatively identified in rat brain tissue following MXYS administration were used for target prediction. For each compound, potential targets were retrieved from the GeneCards database using the Related Targets function with a relevance score > 5. Duplicate targets were merged, and only human genes with official gene symbols were retained, resulting in 3,164 unique targets. These targets were subjected to GO and KEGG enrichment analyses using the ClusterProfiler R package, with an FDR < 0.05 considered statistically significant. The background gene set comprised all human protein-coding genes annotated in the KEGG database.

Experimental Verification
Cell Culture:
HT-22 cells were obtained from Procell Life Science & Technology Co., Ltd. (CL-0697, RRID: CVCL_0321) and cultured in growth medium supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin/streptomycin. Cells were maintained at 37°C in a humidified incubator with 5% CO₂.

Anti-Necroptosis Activity of MXYS:
Freeze-dried MXYS powder was reconstituted in cell growth medium to a concentration of 1.0 g/mL, sterilized through a 0.22 μm membrane filter, and stored at −20°C until use. HT-22 cells were seeded at a density of 8,000 cells per well in 96-well plates, with three wells per group. MXYS was diluted in growth medium to final concentrations of 2, 4, and 6 mg/mL. After incubation for 12 h, cells were treated with SM-164 hydrochloride (10 nM) and the caspase inhibitor z-VAD-fmk (20 μM) for 30 min, followed by stimulation with human tumor necrosis factor-α (h-TNF-α; 20 ng/mL) for an additional 12 h. Cell viability was assessed using a luminescent cell viability assay according to the manufacturer's instructions. Luminescence was measured using a microplate reader. Cell morphology was examined under an inverted microscope, and representative images were acquired for analysis.

Animals:
The animal experiments were approved by the Animal Ethics Committee of Henan University of Chinese Medicine (approval no. DWLLGZR202200023). All procedures were conducted in accordance with institutional and national guidelines for animal welfare and complied with the ARRIVE guidelines. Only male BALB/c mice aged 6–8 weeks were used for establishment of the CUMS model. Animals were housed under controlled temperature and humidity with a 12 h light/dark cycle and provided standard chow and water ad libitum.

CUMS-Induced Depression Model:
Mice were randomly assigned to a control group or a CUMS group. Following one week of acclimatization, mice in the CUMS group were subjected to intermittent and unpredictable mild stressors, whereas control mice remained undisturbed. The stress paradigm consisted of 13 different stressors, including cage tilting, cage shaking, cage exchange, wet bedding, water deprivation, stroboscopic illumination, noise exposure, continuous light, continuous darkness, intermittent lighting, cold-water swimming (4°C), warm-water swimming (40°C), restraint stress, and other mild stressors. Each stressor was applied for 2–6 h per session in a randomized sequence to maintain unpredictability. Mice were exposed to one or two different stressors each day according to a randomized schedule.

Groups and Drug Treatment:
After six weeks of CUMS exposure, mice were randomly assigned to the CUMS, MXYS, paroxetine, or Nec-1 treatment groups. The MXYS group received freeze-dried MXYS powder by oral gavage at 35.2 g/kg/day. The paroxetine group received paroxetine (20 mg/kg) by oral gavage, whereas the Nec-1 group received Nec-1 (10 mg/kg) by intraperitoneal injection for four consecutive weeks. During the same period, the control and CUMS groups received an equivalent volume of normal saline by oral gavage.

Behavioral Experiments:
Mice were randomly assigned to experimental groups using a computer-generated random number sequence before the start of drug administration. The randomization sequence was generated by an independent investigator who was not involved in subsequent experiments or data analysis.

Behavioral tests were conducted by an investigator blinded to the treatment groups. The same investigator performed video-based behavioral scoring (immobility time, distance traveled, and time spent in the center) using automated tracking software to minimize subjective bias. The blinded investigator did not have access to the group allocation codes until all behavioral data had been collected and recorded.

Immunofluorescence analyses were performed by a separate investigator who was also blinded to the experimental groups. Samples were labeled using coded identifiers, and group allocation was revealed only after densitometric or fluorescence intensity measurements had been completed. Statistical analyses were conducted by a third investigator who was blinded to both group allocation and the corresponding experimental outcomes until all quantitative data had been collated.

Sucrose Preference Test (SPT): Behavioral testing was performed after the final drug administration. Before testing, mice were acclimated to a 1% sucrose solution by providing two bottles containing 1% sucrose solution for 12 h, followed by one bottle containing 1% sucrose solution and one bottle containing drinking water for an additional 12 h. Mice were then deprived of food and water for 12 h before the formal test. During the test, each mouse was provided with one bottle containing 1% sucrose solution and one bottle containing drinking water. After 12 h, the volume consumed from each bottle was measured. Sucrose preference was calculated as:

Tail Suspension Test (TST): The tail of each mouse was secured to the top of the test apparatus using adhesive tape. Mouse behavior was recorded for 6 min using behavioral video recording and analysis software. The duration of immobility during the final 4 min of the test was quantified.

Open Field Test (OFT): Each mouse was individually placed in the center of the open-field apparatus. Behavioral activity was recorded and analyzed for 5 min using behavioral tracking software, including locomotor distance and movement activity. After each test, the apparatus was cleaned with 75% ethanol.

Sample Collection:
Mice that died during the CUMS procedure or exhibited signs of severe illness (e.g., >20% body weight loss, visible infection, or inability to move) were excluded from the study. Euthanasia was performed by certified personnel using an overdose of sodium pentobarbital (150 mg/kg, intraperitoneally). Following loss of consciousness, cervical dislocation was performed as a secondary method to ensure death. For Nissl staining and immunofluorescence analyses, three mice were randomly selected from each group. Hippocampal tissues for western blot analysis were immediately dissected on ice and snap-frozen in liquid nitrogen.

Nissl Staining:
For Nissl staining and immunofluorescence analyses, mice were deeply anesthetized with sodium pentobarbital (150 mg/kg, intraperitoneally) and decapitated without transcardial perfusion. Brains were rapidly removed and fixed in 4% paraformaldehyde for 24–48 h before paraffin embedding. Paraffin sections were cut to a thickness of 3 μm. Paraffin sections were deparaffinized by sequential incubation in dewaxing solution, absolute ethanol, and 75% ethanol before rinsing with running water. Frozen sections, when applicable, were brought to room temperature, fixed with tissue fixative for 15 min, and rinsed with running water. Tissue sections were stained with Nissl staining solution for 2–5 min, rinsed with water, differentiated with 0.1% glacial acetic acid, washed to terminate the reaction, monitored microscopically to control differentiation, dried, cleared in xylene for 10 min, and mounted with neutral mounting medium.

Immunofluorescence:
Immunofluorescence procedures were performed using standard methods. Brain tissue was fixed in 4% paraformaldehyde and embedded in paraffin for immunofluorescence staining. Tissue sections were incubated overnight at 4°C with primary antibodies against NeuN (1:200), MLKL (1:300), ATF4 (1:5000), and CREB (1:1000). Following incubation with primary antibodies, sections were washed four times with PBS and incubated for 1 h in the dark with the corresponding fluorescent secondary antibodies: CY3-conjugated goat anti-mouse IgG (1:300, Servicebio, GB21301) for mouse anti-ATF4; Alexa Fluor 488-conjugated goat anti-mouse IgG (1:400, Servicebio, GB25301) for mouse anti-MLKL; CY3-conjugated goat anti-rabbit IgG (1:300, Servicebio, GB21303) for rabbit anti-NeuN; and Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400, Servicebio, GB25303) for rabbit anti-CREB. Sections were then stained with DAPI for 10 min at room temperature, washed with PBS, mounted with an antifade mounting medium, and fluorescence images were acquired using confocal microscopy.

Western Blot:
Brain tissues were homogenized in radioimmunoprecipitation assay (RIPA) lysis buffer. Protein concentrations were determined, and 20 μg of total protein per lane was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) before transfer to polyvinylidene fluoride (PVDF) membranes. After blocking, membranes were incubated overnight at 4°C with primary antibodies (1:1000), followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h. Protein bands were visualized using a chemiluminescence imaging system, and band intensities were quantified using ImageJ software. For western blot analysis of hippocampal tissues, two mice per group were analyzed, and representative immunoblots are shown. For the HT-22 cell experiments, western blot analysis was performed once as a representative experiment. These data are presented as qualitative trend observations rather than statistically quantified findings.

Statistical Analysis:
Differences among groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Data are presented as the mean ± standard deviation (SD). A p value < 0.05 was considered statistically significant.

Data Availability:
The datasets generated and analyzed during this study are included in this published article and its supplementary materials. The UHPLC-HRMS compound identification results are provided in Supplementary Table 1, and the characteristics of the GEO datasets used for the bioinformatic analyses are summarized in Supplementary Table 2. Additional supporting data, including GFAP immunofluorescence (Supplementary Figure 1), immunofluorescence staining of p-RIPK3 and p-MLKL (Supplementary Figure 2), the experimental design and animal allocation timeline (Supplementary Figure 3), and the original uncropped western blot images (Supplementary Figure 4), are available in the supplementary materials.

Results

WGCNA Identifies a Depression-Associated Gene Module Enriched in the Necroptosis Pathway
The RRA package was used to integrate consistently upregulated and downregulated differentially expressed genes across the GSE54566, GSE54565, and GSE54568 datasets, resulting in the identification of 2,513 genes. A heat map of these genes is shown in Figure 1A. The three datasets were derived from distinct brain regions—the anterior cingulate cortex, amygdala, and dorsolateral prefr...

Discussion

Abnormal neuronal death plays a crucial role in the pathogenesis of depression⁸. Necroptosis, a form of programmed cell death, has been extensively investigated, leading to significant advances in the development of inhibitors targeting this pathway³⁹. Since the identification of the necroptosis inhibitor Nec-1 in 2005⁴⁰, considerable progress has been made in developing compounds that target key components of the necroptotic signaling pathway. Nec-1...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Acori Tatarinowii Rhizoma (Acorus tatarinowii Schott; Shi Changpu)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	231001	Herbal ingredient (12 g)
Adhesive tape	Not applicable	Not applicable	Tail suspension test
Angelicae Sinensis Radix (Angelica sinensis (Oliv.) Diels; Dang Gui)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	G01230507-16	Herbal ingredient (10 g)
Anti-ATF4 antibody (immunofluorescence)	Servicebio	GB111137	Primary antibody
Anti-ATF4 antibody (western blot)	MedChemExpress	HY-P80486	Primary antibody
Anti-BDNF antibody	MedChemExpress	HY-P80567	Primary antibody for western blot
Anti-CREB antibody	Beyotime	AF1018	Primary antibody for western blot and immunofluorescence
Anti-GAPDH antibody	Abcam	ab181602	Loading control
Anti-MLKL antibody	Proteintech	66675-1-Ig	Primary antibody for western blot and immunofluorescence
Anti-NeuN antibody	Servicebio	GB11138	Primary antibody for immunofluorescence
Anti-phospho-CREB antibody	Beyotime	AF5785	Primary antibody
Anti-phospho-MLKL antibody	Bioss	bsm-54104R	Primary antibody
Anti-phospho-RIPK1 antibody	Biolynx	BX60008	Primary antibody
Anti-phospho-RIPK3 antibody	Abcam	ab195117	Primary antibody
Anti-RIPK1 antibody	Cell Signaling Technology	3493S	Primary antibody
Anti-RIPK3 antibody	Proteintech	17563-1-AP	Primary antibody
Atractylodes macrocephala (Atractylodes macrocephala Koidz.; Bai Zhu)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	230801	Herbal ingredient (10 g)
Automated behavioral tracking software	Panlab (Harvard Apparatus)	N/A	SMART 3.0 video tracking software for open field and tail suspension tests
BCA Protein Assay Kit	CWBIO	CW0014S	Protein quantification
Behavioral recording system	Panlab (Harvard Apparatus)	SMART V3.0	Open field and tail suspension tests
Bio-Rad chemiluminescence imaging system	Bio-Rad	12003154	ChemiDoc MP Imaging System for chemiluminescence western blot detection
CellTiter-Lumi II Luminescent Cell Viability Assay Kit	Beyotime	C0056XL	Cell viability assay
Confocal microscope	Nikon Eclipse C1	N/A	Immunofluorescence imaging
DAPI	Servicebio	G1012	Nuclear stain
Environmental dewaxing solution	Servicebio	G1128	Paraffin deparaffinization
Fetal bovine serum (FBS)	BI	C04001-500	Cell culture supplement
Gardeniae Fructus (Gardenia jasminoides J. Ellis; Zhi Zi)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	22102109	Herbal ingredient (12 g)
Glycyrrhizae Radix (Glycyrrhiza uralensis Fisch.; Gan Cao)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	230301	Herbal ingredient (3 g)
CY3-conjugated goat anti-mouse IgG	Servicebio	GB21301	Fluorescent secondary antibody for mouse anti-ATF4 (IF, CY3, 1:300)
Alexa Fluor 488-conjugated goat anti-mouse IgG	Servicebio	GB25301	Fluorescent secondary antibody for mouse anti-MLKL (IF, Alexa Fluor 488, 1:400)
CY3-conjugated goat anti-rabbit IgG	Servicebio	GB21303	Fluorescent secondary antibody for rabbit anti-NeuN (IF, CY3, 1:300)
Alexa Fluor 488-conjugated goat anti-rabbit IgG	Servicebio	GB25303	Fluorescent secondary antibody for rabbit anti-CREB (IF, Alexa Fluor 488, 1:400)
HRP goat anti-rabbit IgG	Absin	abs20040	Secondary antibody for western blot
HRP-conjugated mouse anti-rabbit IgG light chain	ABclonal	AS061	Secondary antibody for western blot
Human TNF-α	Novoprotein	C008	Necroptosis inducer
HT-22 cells	Procell	CL-0697	RRID: CVCL_0321
HyClone growth medium	Cytiva (HyClone)	SH30022.01	Cell culture medium
ImageJ	NIH	Free software	Band quantification
Inverted microscope	Nikon	Eclipse Ti-SR	Cell morphology
Liquid nitrogen	Henan Jilai New Energy Technology Co., Ltd.	N/A	Tissue preservation
Molecular Devices microplate reader	Molecular Devices	SpectraMax M5	Luminescence measurement
Moutan Cortex (Paeonia suffruticosa Andr.; Mu Danpi)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	22121714	Herbal ingredient (12 g)
Necrostatin-1 (Nec-1)	Selleck	S8037	RIPK1 inhibitor
Paraformaldehyde	Servicebio	G1101-500ML	Tissue fixation
Paroxetine hydrochloride hemihydrate	Aladdin	P129711	Positive control
PBS	Servicebio	G4202-500ML	Washing buffer
Peppermint (Mentha haplocalyx Briq.; Bo He)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	230101	Herbal ingredient (6 g)
Penicillin–streptomycin	Gibco	15140-122	Cell culture antibiotic
Polygalae Radix (Polygala tenuifolia Willd.; Yuan Zhi)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	20221101	Herbal ingredient (12 g)
Polyvinylidene fluoride (PVDF) membrane	Merck Millipore	ISEQ00010	Western blot
Poria (Wolfiporia cocos; Fu Ling)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	D23070601	Herbal ingredient (15 g)
Radioimmunoprecipitation assay (RIPA) lysis buffer	CWBIO (Jiangsu Cowin Biotech Co., Ltd.)	CW2333S	Protein extraction
Radix Bupleuri (Bupleurum chinense DC.; Chai Hu)	Beijing Tongrentang Co., Ltd., Zhengzhou, China	230801	Herbal ingredient (12 g)
SDS-PAGE reagents	Solarbio; Servicebio; Shanghai Yuanye Bio-Technology Co., Ltd.	Not applicable	Acrylamide, bis-acrylamide, Tris, SDS, APS, and TEMED prepared in-house
Sodium pentobarbital	Sinopharm Chemical Reagent Co., Ltd.	69020181	Anesthetic
Ultra-high-performance liquid chromatography system	Thermo Fisher Scientific	Vanquish UHPLC System	LC-MS analysis
UHPLC HSS-T3 column (100 × 2.1 mm, 1.8 μm)	Waters	186003539	Chromatographic separation
z-VAD-fmk	TargetMol	T6013	Pan-caspase inhibitor