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....

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 MXY....

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