Preparation of the YNJY Prescription

YNJY prescription was purchased from Kangrentang Pharmaceutical Co. (Beijing, China). It comprises five different Chinese medical herbs: 20 g of Acanthopanax senticosus, 13 g of Curcuma longa, 10 g of Fructus Gardeniae sp., 10 g of Schisandra chinensis, and 10 g of Bupleurum. Before using YNJY, the granules were dissolved in 100 mL of distilled water and then stored at 4 °C.

Animals

Sixty male Sprague Dawley (SD) rats (weighing 250–280 g) were supplied by Si Pei Fu Biotechnology Co. (Beijing, China). Before the experiments, the rats were subjected to adaptive feeding for 1 week in a 22 °C ± 2 °C room with a 12-h day/night cycle. All of these rats were given ad libitum access to food and water. The Animal Committee of the MDKN Biotech Co. (Beijing, China) approved all animal studies (Approval No.: MDKN-2023–010). Furthermore, all animal experiments were conducted according to the “Guiding Opinions on the Good Treatment of Laboratory Animals” issued by the Ministry of Science and Technology of the People’s Republic of China.

Construction of the PSD Rat Model

Stroke following CUMS stimulation is the common method for the construction of PSD (Li et al. 2021; Zhang et al. 2021a, b, c). Thus, in this work, the PSD rat model was established via two steps: developing the intracerebral hemorrhage (ICH) model and then the PSD model. The procedure was implemented as previously reported (Li et al. 2021; Zhang et al. 2021a, b, c; Liu et al. 2022).

To develop the ICH model, collagenase was injected into the rats via the caudate nucleus. Specifically, the rats were subjected to deep anesthesia via 2.5% isoflurane (Reward Life Technology Co., Shenzhen, China) inhalation. Thereafter, they were fixed on a brain stereotaxic apparatus (Yuyan Scientific Instrument Co., Shanghai, China). Head hair was shaved, and the skin of the exposed head was sterilized and incised. At the site of the caudate nucleus ( the injection coordinates were 3 mm to the right of the breggma, 1 mm posterior to the breggma, and 5 mm in depth, with breggma as the zero point), a 5-mm deep hole was drilled into the skull. Then, a microsyringe containing 0.6 µL of collagenase type VII (Sigma, St Louis, MO, USA) was directly inserted into the caudate nucleus; the microsyringe was withdrawn 5 min after the injection. At last, the head skin was sutured and then sterilized. After the surgery, rats were intraperitoneally injected with penicillin (800,000 IU/kg/d; North China Pharmaceutical Group Co., Shijiazhuang, China) to prevent infection. Based on the Berderson scoring criteria (Desland et al. 2014), rats without stroke signs or those still not awake 2 h after the surgery were excluded from this study.

Next, rats with ICH were subjected to CUMS stimulation and solitary rearing to develop the PSD model. After the ICH surgery, rats were routinely housed for 1 week for recovery. In the subsequent 4 weeks, they were subjected to CUMS stimulation every day. Seven stimulation methods were included to stimulate CUMS, with one method conducted daily per week. The CUMS stimulation methods were as follows: (1) rats were subjected to fasting from food and water from 7:00 to 7:00 the next day; (2) 250 mL of water was poured into the rat bedding and the rats were left in the damp bedding for 24 h; (3) rats were treated with circadian inversion; (4) rats were placed in cold water (4 °C) for swimming for 5 min; (5) rats were placed in warm water (40 °C) for swimming for 5 min; (6) rats were kept in a bottle with its tail exposed; the tail was clamped with a clip for 1 min at 1 cm from the tail root; and (7) rats were placed in a fixator (20 cm long) for 2 h.

Animal Treatment

Sixty SD rats were randomly divided into six groups: Sham, ICH, PSD, low-dose YNJY (YNJY-L), high-dose YNJY (YNJY-H), and YNJY-H + ML385 groups. There were 10 rats in each group. ML385 is an inhibitor of Nrf2, which was used to block the Nrf2 pathway in rats in this study. The dose of ML385 used in rats was referred to the study of Chen et al. (Chen et al. 2023a, b).

The rats in the Sham group were subjected to ICH surgery; notably, 0.6 µL of saline (rather than collagenase type VII) was injected via the caudate nucleus. After the surgery, rats were routinely housed and not subjected to any other interventions or treatments.

The rats in the ICH group were only subjected to ICH surgery but not any other interventions or treatments.

The rats in the PSD group underwent ICH surgery and PSD model construction. However, no other interventions or treatments were performed.

The rats in the YNJY-L and YNJY-H groups underwent ICH surgery and PSD model construction. During CUMS stimulation, low-dose (4.19 g/kg/day, equivalent to normal clinical minimum dosage) and high-dose (8.38 g/kg/day, equivalent to normal clinical minimum dosage) YNJY were administered to the rats in the YNJY-L and YNJY-H groups, respectively, via gavage.

The rats in the YNJY-H + ML385 group were subjected to ICH surgery and construction of PSD. During CUMS stimulation, high-dose (8.38 g/kg/day) YNJY administration combined with intraperitoneal injection of 30 mg/kg/day ML385 (Chen et al. 2023a, b) (Nrf2 inhibitor) were given to rats.

After surgery, mice were injected subcutaneously with meloxicam (dose: 2 mg/kg/d) for 3 days for analgesia. During the last week of the experiment, the rats in each group were subjected to behavioral tests to evaluate depression-like behaviors. At the end of the entire experiment, the rats were weighed and then euthanized to collect whole brains. The hippocampus was isolated and preserved in a refrigerator at − 80 °C.

Sucrose Preference Test

Two identical water bottles containing 1% sucrose solution were placed in the cages one day before the sucrose preference test for habituation. The next day, rats were deprived of water for 4 h. Subsequently, one bottle was filled with 1% sucrose solution, whereas the other one was filled with pure water. To avoid the rats from developing positional preference, the two bottles were displaced after 12 h. After 24 h, sucrose preference was evaluated using the following formula: Sucrose preference = (sucrose water intake/the total of pure water and sucrose water intake) × 100%.

Tail Suspension Test (TST)

The tail of each rat was secured with tape and then hung upside down on a stand. The distance of the tape from the tail tip was 1 cm, and the head of the rat was 15 cm from the ground. After 6 min, the immobility time of the rats was recorded. The entire experiment was recorded using a digital camera (Nikon, Tokyo, Japan).

Forced Swimming Test (FST)

A transparent glass cylinder (diameter of 15 cm and height of 30 cm) was prepared. The cylinder was filled with water at 23 °C to a depth of 15 cm. Then, the rats were placed into this cylinder and kept for 6 min. The immobility time was recorded. The entire experiment was recorded using a digital camera (Nikon).

Open-Field Test (OFT)

Each rat was placed in a box (100 cm long, 100 cm wide, and 50 cm height) for 5 min. The center of the box was a square that was 50 cm long and 50 cm wide. After the test, the counts of rearing and crossing the center were recorded. The entire experiment was recorded using a digital camera (Nikon).

Hematoxylin–eosin (HE) and Nissl Staining

The hippocampus of each rat was fixed with 4% paraformaldehyde (Beyotime Biotechnology Co., Shanghai, China) for 48 h at 4 °C. After dehydrating the hippocampal samples and embedding them in paraffin, 5-µm thick slices were prepared. Then, the slices were treated with xylene and gradient alcohol to dewax and rehydrate them, respectively.

For HE staining, hematoxylin and eosin solutions (both from Lianmai Biological Engineering Co., Shanghai, China) were sequentially added to the sections for staining for 5 and 2 min, respectively. For Nissl staining, the sections were stained with Nissl solution (Yiyan Biotechnology Co., Shanghai, China) for 5 min. After dehydration using gradient alcohol and transparency using xylene, the dried sections were sealed with neutral resin for histopathological and neuronal observations under an optical microscope (CX41, Olympus, Tokyo, Japan).

Immunofluorescence Staining

The dewaxed and rehydrated 5-µm thick hippocampal tissue sections were permeabilized with 0.5% Triton X-100 (Beyotime Biotechnology Co.) for 30 min at room temperature. Then, the sections were blocked with 5% normal goat serum (Beyotime Biotechnology Co.) for 30 min at room temperature. To measure neuronal nuclear antigen (NeuN) expression, the sections were treated with mouse anti-NeuN (1:200, ab104224, Abcam, Shanghai, China) primary antibodies overnight at 4 °C. Then, the sections were treated with Alexa Fluor 488-conjugated goat anti-mouse (1:200, SY0683, Biolab Technology Co., Beijing, China) secondary antibodies for 2 h at room temperature. Further, to detect the co-localization of NeuN and nuclear factor erythroid 2-related factor-2 (Nrf2), the sections were incubated with both mouse anti-NeuN (1:200, ab104224, Abcam) and rabbit anti-Nrf2 (1:200, ab62352, Abcam) primary antibodies overnight at 4 °C. Thereafter, the sections were treated with both Alexa Fluor 594-conjugated goat anti-mouse secondary antibody (1:200, SY0688, Biolab Technology Co.) and Alexa Fluor 488-conjugated goat anti-rabbit secondary antibody (1:200, SY0669, Biolab Technology Co.) for 2 h at room temperature. Then, 4’, 6-diamidino-2-phenylindole (DAPI) (Beyotime Biotechnology Co.) was used to stain the sections for 5 min in the dark. After drying and treating the sections with neutral resin, they were observed and imaged under a fluorescence microscope (IX70, Olympus).

Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from rat hippocampus using TRIzol reagent (Lianmai Biological Engineering Co.). Total RNA was reverse-transcribed into cDNA through adopting the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) based on the manufacturer’s instructions. Then, quantitative PCR was performed using the cDNA template with the SYBR Green Master Mix (Applied Biosystems) on the 7500 Real-Time PCR System (Applied Biosystems). The cycling conditions were as follows: 94 °C for 3 min; 39 cycles at 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 35 s; and 72 °C for 5 min. The primers used were as follows: Nrf2: sense, 5′-GCCTTCCTCTGCTGCCATTAGTC-3′, and antisense, 5′-TGCCTTCAGTGTGCTTCTGGTTG-3′; glutathione peroxidase (GPX4): sense, 5′-CCAGCAACAGCCACGAGTTCC-3′, and antisense, 5′-CACACGCAACCCCTGTACTTATCC-3′; solute carrier family 7 member 11 (SLC7A11): sense, 5′-CCATCATCATCGGCACCGTCATC-3′, and antisense, 5′-TACTCCACAGGCAGACCAGAACAC-3′; and glyceraldehyde-3-phosphate dehydrogenase (GAPDH): sense, 5′-AGTGCCAGCCTCGTCTCATA-3′, and antisense, 5′-TGAACTTGCCGTGGGTAGAG-3′. The relative mRNA expression of Nrf2, GPX4, and SLC7A11 was calculated using the 2−ΔΔCt method and normalized using GAPDH.

Isolation and Identification of Primary Rat Hippocampal Neurons

Primary hippocampal neurons were isolated from newborn SD rats (Kaixue Biotechnology, Shanghai, China) using a previously described method (Combs et al. 2021). Briefly, the hippocampal samples isolated from 10 newborn SD rats were sliced into small pieces. To dissociate the hippocampal neurons, these small hippocampal pieces were incubated with 0.125% trypsin (Lianmai Biological Engineering Co.) for 15 min at 37 °C. The dissociated hippocampal neurons were cultured in neurobasal medium (Yanhui Biotechnology Co., Shanghai, China) supplemented with 2% B-27 (Yanhui Biotechnology Co.) and 1% GlutaMAX (Kemin Biotechnology Co., Shanghai, China) at 37 °C and 5% CO2 in 6-well plates precoated with poly-D-Lysine (Beyotime Biotechnology Co.).

After culturing for 5 days, the isolated primary hippocampal neurons were identified via immunofluorescence staining for NeuN and microtubule-associated protein2 (MAP2). Hippocampal neurons were fixed in 4% paraformaldehyde for 15 min, permeabilized in 0.5% Triton X-100 for 10 min, and then blocked in 5% normal goat serum for 30 min at room temperature. After probing with both mouse anti-NeuN (1:200, ab104224, Abcam) and rabbit anti-matrix metalloproteinase 2 (MMP2) (1:200, ab235167, Abcam) primary antibodies overnight at 4 °C, these hippocampal neurons were treated with both Alexa Fluor 488-conjugated goat anti-mouse (1:200, SY0683, Biolab Technology Co.) and Alexa Fluor 594-conjugated goat anti-rabbit (1:200, SY0673, Biolab Technology Co.) secondary antibodies for 2 h at room temperature. Then, DAPI solution was added dropwise, and the hippocampal neurons were incubated for 5 min in the dark. Finally, hippocampal neurons were observed and imaged under a fluorescence microscope (IX70, Olympus).

Transfection of Hippocampal Neurons

Hippocampal neurons were plated into the 6-well plates with 1 × 105 cells in 1 mL serum-free neurobasal medium. Short interfering RNA (siRNA) against Nrf2 and the corresponding siRNA negative control (NC) were both commercially supplied by GeneChem (Shanghai, China). The two kinds of siRNA were separately transfected into hippocampal neurons by using Lipofectamine 3000 (Thermo Fisher Scientific, San Jose, CA USA). After transfection, hippocampal neurons were cultivated in normal neurobasal medium at 37 °C and 5% CO2.

Treatment of Primary Hippocampal Neurons

To determine the safe and effective concentrations of the YNJY prescription, hippocampal neurons were treated with neurobasal medium supplemented with different concentrations of YNJY prescription (1 µg/mL, 10 µg/mL, 100 µg/mL, 500 µg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, and 100 mg/mL) for 48 h at 37 °C and 5% CO2.

To induce ferroptosis, erastin (Beyotime Biotechnology Co.) was dispersed to the neurobasal medium at a final concentration of 10 µM (Dahlmanns et al. 2017) and hippocampal neurons were pretreated for 8 h. Then, these hippocampal neurons were incubated with the neurobasal medium in the presence or absence of YNJY prescription for 48 h at 37 °C and 5% CO2.

To inactivate Nrf2, hippocampal neurons were pretreated with ML385 (Nrf2 inhibitor) at a final concentration of 1 µM (Chen et al. 2023a, b) for 1 h. Furthermore, to cotreat erastin and ML385, hippocampal neurons were pretreated with erastin for 8 h and then with ML385 for 1 h. All subsequent experiments were performed by culturing these hippocampal neurons in the presence or absence of YNJY prescription for 48 h at 37 °C and 5% CO2. Hippocampal neurons cultured under normal conditions were used as a control.

Cell Counting Kit-8 (CCK-8) Assay

The hippocampal neurons were inoculated in 96-well plates at a density of 5 × 103 cells/mL. Five replicates were prepared in each group. After the relevant treatment, the CCK-8 solution (Beyotime Biotechnology Co.) was dispersed into the wells and incubated for 2 h at 37 °C. Finally, the optical density (OD) was measured using a microplate reader (BioTek, Winooski, VT, USA).

Lactate Dehydrogenase (LDH) Assay

The culture medium of the hippocampal neurons isolated from each group was collected and centrifuged at 4 °C and 120,000 rpm for 10 min. The supernatant was collected. The LDH activity in each supernatant sample was measured using the LDH activity assay kit (Sangon Biological Engineering Co., Shanghai, China) according to the manufacturer’s instructions.

Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick end Labeling (TUNEL) Staining

After the relevant treatments, hippocampal neurons in 6-well plates were fixed with 4% paraformaldehyde for 10 min and then permeated with 0.1% Triton X-100 for 2 min. Then, the neurons were reacted with the TUNEL reaction solution (Beyotime Biotechnology Co.) for 40 min in the dark, followed by treatment with DAPI for 5 min at room temperature. The residual staining solution was removed using phosphate buffer saline (Beyotime Biotechnology Co.). Finally, hippocampal neurons were observed under a fluorescence microscope (IX70, Olympus) with green fluorescence indicating apoptotic neurons.

Detection of Ferroptosis-Related Factors

The levels of ferroptosis-related factors in the hippocampus and hippocampal neurons of rats were measured, including iron, malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and reactive oxygen species (ROS). The hippocampus and hippocampal neurons were collected from each group and lysed with lysis buffer (Kanglang Biotechnology Co., Shanghai, China) for 30 min on ice. The composition of the lysis buffer included 20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton X-100, and 100 mM phenylmethanesulfonyl fluoride. The supernatant was obtained by centrifuging the samples at 4 °C and 12,000 rpm for 10 min. The levels of these ferroptosis-related factors were measured by using the iron assay kit, MDA assay kit, SOD assay kit, GSH assay kit, and ROS assay kit according to the manufacturers’ instructions. All these kits were commercially supplied by Yubo Biotechnology Co. (Shanghai, China).

Transmission Electron Microscopy (TEM)

After fixed the hippocampal tissues with 4% paraformaldehyde for 48 h, they were routinely dehydrated, paraffin-embedded, and sliced into 60-nm thick sections. Furthermore, after relevant treatments, the hippocampal neurons were collected from each group, fixed with 4% paraformaldehyde for 2 h, paraffin-embedded, and then sliced into 60-nm-thick sections. These hippocampal tissue and neuronal sections were routinely dewaxed and dehydrated and then transferred to copper grids. After staining with uranyl acetate (Kanglang Biotechnology Co.) for 10 min and with lead citrate (Head Biotechnology Co., Beijing, China) for 2 min, the sections were imaged under a transmission electron microscope (HT7700, Hitachi, Tokyo, Japan) at an accelerating voltage of 80 kV.

JC-1 Staining

To measure mitochondrial membrane potential, hippocampal neurons were cultured under specific conditions in 6-well plates and then fixed with 4% paraformaldehyde for 2 h. Next, the neurons were stained with JC-1 solution (Beyotime Biotechnology Co.) for 30 min in the dark. After staining the nucleus with DAPI, the hippocampal neurons from each group were imaged under a fluorescence microscope (IX70, Olympus). The mitochondrial membrane potential was indicated as the ratio of JC-1 aggregate/JC-1 monomer.

C11-BODIPY Staining

Hippocampal neurons were cultured in 6-well plates under relevant conditions and then fixed with 4% paraformaldehyde for 2 h. To measure lipid peroxidation, the C11-BODIPY 581/591 probe (27086–1, AmyJet Scientific, Wuhan, China) was added into the wells and incubated for 30 min in the dark. After DAPI staining, the hippocampal neurons were observed and imaged under a fluorescence microscope (IX70, Olympus). In the presence of lipid peroxidation, the color of C11-BODIPY 5 shifted from red fluorescence to green fluorescence.

Mito-Tracker Red Staining

Hippocampal neurons were cultured under the relevant conditions in 6-well plates and then fixed with 4% paraformaldehyde for 2 h. Thereafter, Mito-Tracker Red solution (Beyotime Biotechnology Co.) was added to stain the neurons and incubated in the dark for 30 min at 37 °C. DAPI solution was added for routine nuclear staining. The mitochondria length were observed and imaged under a fluorescence microscope (IX70, Olympus) and analyzed by Mitochondria Analyzer software.

Western Blotting

Total proteins were extracted from the hippocampus and hippocampal neurons using the radioimmunoprecipitation assay lysis buffer (Kanglang Biotechnology Co.). The lysed samples were centrifuged at 4 °C and 120,000 rpm for 15 min. The supernatant of each sample was collected to measure the total protein concentration using a BCA kit (Biolab Technology Co.). Total proteins were then separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transferring the gels onto polyvinylidene fluoride (PVDF) membranes (Lianshuo Biotechnology Co., Shanghai, China), the proteins were subjected to blocking with 5% skimmed milk for 2 h at room temperature. Then, the following primary antibodies were added onto the PVDF membranes to treat the proteins overnight at 4 °C: rabbit anti-Nrf2 (1:1000, ab92946, Abcam), rabbit anti-GPX4 (1:1000, AF7020, Beyotime Biotechnology Co.), rabbit anti-SLC7A11 (1:1000, PAB14937, Kemin Biotechnology Co., Wuhan, China), and rabbit anti-GAPDH (1:1000, ab37168, Abcam). The remaining primary antibodies were removed using Tris-buffered saline with 0.1% Tween 20 (TBST). Horseradish peroxidase-labeled goat anti-rabbit secondary antibody (1:3000, ab97051, Abcam) was added onto the PVDF membranes and allowed to react for 2 h at room temperature. After washing with TBST, the PVDF membranes were treated with the enhanced chemiluminescent reagent (Absin Biotechnology Co., Shanghai, China) to visualize protein blots. Protein blots were quantified using Image J software (NIH, Bethesda, Maryland, USA). The relative protein expression of Nrf2, GPX4, and SLC7A11 was normalized to that of GAPDH.

Statistical Analysis

Data were represented as mean ± standard deviation and analyzed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). In this work, data were shown to be normally distributed by the Kolmogorov–Smirnov and the Shapiro–Wilk tests. One-way analysis of variance combined with Tukey’s post-hoc test was conducted to analyze statistical differences in multiple groups (at least three groups). A P-value of < 0.05 was considered statistically significant.