Abstract
Introduction: This study aimed to explore the impact and mechanism of Scutellariae radix (SR), dried root of Scutellaria baicalensis Georgi of Labiatae, on prenatal stress (PS)-induced anxiety-like and depression-like behavior in the offspring in a mouse prenatal stress model. Methods: The open field test (OFT), tail suspension test (TST), and forced swimming test (FST) were utilized to assess the behavior of the offspring. Histological changes were evaluated using HE staining and Nissl staining. ELISA was employed to detect the levels of related factors in the serum and fetal brains of offspring mice. Immunohistochemistry was used to determine the expressions of doublecortin and neurotrophic factors in the hippocampus, and RT-PCR reflected the expression of factors in the hippocampus and placenta of offspring mice. These various techniques collectively provided insight into the neurodevelopmental status by detecting indicators related to neurodevelopmental status. LC-MS/MS and molecular docking were used to clarify the chemical constituents and the pharmacodynamic components in S. radix. Results:S. radix ameliorated prenatal stress-induced anxiety-like and depression-like behavior in the offspring. It also alleviated hippocampal neurogenesis impairment caused by prenatal stress and restored abnormal expression of hippocampal glutamate (Glu) and brain-derived neurotrophic factor in the offspring. Additionally, S. radix maintained normal 11β-HSD1 expression in the placenta of prenatal stress mice, ensuring a normal level of glucocorticoids (GCs) and glucocorticoid receptors (GRs) in the fetus. Furthermore, S. radix increased the mRNA expression of GR and 11β-HSD2 while decreasing the mRNA expression of 11β-HSD1, thereby normalizing levels of serum CRH, ACTH, and GC in the offspring. Finally, docking results indicated that baicalein, wogonin, wogonoside, and baicalin exhibited stronger binding ability with the target. Conclusion: The results of our study indicate that S. radix may have the potential to alleviate prenatal stress-induced anxiety-like and depression-like behaviors in offspring, at least partially through protecting placental barrier function, reversing HPA axis hyperfunction, and ameliorating neurodevelopmental dysfunction.
Introduction
Prenatal stress (PS) is a systemic nonspecific response produced by the stimulation of internal and external environment during pregnancy [1‒3]. PS is a relatively common occurrence during maternal pregnancy. The occurrence of PS, whether psychological or physical, has been linked to adverse outcomes in fetal development [4], with long-term implications for the offspring [5]. Epidemiologic studies reveal that exposure of pregnant women to a stressful environment is associated with neurodevelopmental disturbances that increase the susceptibility to emotional problems and abnormal motor behaviors in their children [6‒9]. Findings from animal behavior studies confirm that PS can induce anxiety-like and depression-like behavior in offspring [10‒12]. However, the impact of PS on anxiety-like and depression-like behaviors in offspring exhibits sex-specific effects. For instance, it induces anxiety-like and depression-like behavior in male offspring but not in females [13]. Nevertheless, PS might heighten the vulnerability of female offspring to depression-like behavior [14]. Further research is required to elucidate these nuances. Despite these sex-specific variations, it is clear that PS results in adverse outcomes in offspring. Therefore, the alleviation of PS represents a significant challenge in the prevention of anxiety-like and depression-like behaviors in offspring, underscoring the urgent need for effective coping strategies.
Adverse conditions during pregnancy can elevate maternal glucocorticoids (GCs) and increase fetal circulating GCs through placental transport. The placental enzyme 11β-hydroxysteroid dehydrogenase (11β-HSD) functions as the rate-limiting enzyme in GC metabolism, thereby playing a pivotal role in regulating the quantity of GCs transported between the mother and fetus through the placenta. Two subtypes have been identified: 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2), which inactivates GCs, and 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1), an enzyme with both oxidation and reduction effects. The latter is primarily a reductase in vivo and has the effect of increasing GCs. In addition, the hypothalamic-pituitary-adrenal (HPA) axis, consisting of the paraventricular nucleus of the hypothalamus, anterior pituitary, and adrenal cortex, is an important part of the neuroendocrine system, which is involved in controlling stress response [15, 16]. The HPA axis is subject to negative feedback regulation. GCs, synthesized and secreted by the adrenal cortex, can exert a negative regulatory effect on the hypothalamus and pituitary gland, leading to a reduction in the secretion of corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). This, in turn, enables the joint regulation of the body’s stress response [17, 18]. PS also disrupts the negative feedback function of the offspring’s HPA axis, resulting in increased exposure to GCs [19]. This heightened GC level serves as the foundation for abnormal neural development in the fetus. The hippocampus is composed of several subregions, including CA1, CA2, CA3, and dentate gyrus (DG) [20, 21]. The hippocampus is a crucial region of the brain for investigating learning and cognitive processes, and it is also implicated in the modulation of emotional responses, including fear, anxiety, and stress [22]. The hippocampus, rich in glucocorticoid receptors (GRs), is highly susceptible to various stressors [23]. Neural stem cells in the DG of the hippocampus exhibit continuous neurogenesis, generating new neurons that integrate into existing neural circuits throughout various stages of development [24]. This neurogenesis is associated with emotions such as anxiety and depression [25]. Several studies emphasize the necessity of enhanced hippocampal neurogenesis in treating depression [25]. Brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) are pivotal in neurogenesis [26]. Notably, research indicates that PS reduces the expression of BDNF and DCX in the brains of offspring, impairing neural development and increasing the possibility of mental illness [27].
Scutellariae radix (SR), the dried root of Scutellaria baicalensis Georgi, has the effect of clearing away heat, which is interpreted as antibacterial, antiviral, and anti-inflammatory by modern pharmacology [28‒30]. In addition, SR is traditionally believed to have properties that support a healthy pregnancy and prevent miscarriage. In Traditional Chinese Medicine, PS is attributed to the mother’s negative emotions, leading to fetal heat anxiety. The root extract of SR contains a variety of chemical components, including flavonoids [31, 32], polysaccharides [33, 34], volatile oils [35], and other compounds like β-sitosterol and benzoic acid [33]. Flavonoids, particularly baicalin, baicalein, wogonoside, and wogonin, are considered characteristic components of SR. Modern pharmacology recognizes SR for its neuroprotective effects [36, 37], with the main components – baicalin, baicalein, and wogonin – reported to have neuroprotective properties [38‒41]. Considering both traditional and modern perspectives, SR is believed to have the potential to improve PS. In this study, we explored the effects of SR on PS-induced anxiety-like and depression-like behavior in the offspring and elucidated its underlying pathways in a mouse PS model.
Methods
Preparation of Scutellariae radix Extract
SR was purchased from Beijing Tongrentang (lot number: 270226301). The preparation began by soaking 500 g of SR decoction pieces in cold water for 30 min, using a water volume approximately 6 times that of the medicinal materials. Following 1 h of heating and decoction, the mixture was filtered. Subsequently, water was added, and the decoction continued for an additional 45 min. Following another filtration process, the two decoctions were combined and concentrated under reduced pressure. The resulting solution was then vacuum-dried to obtain a powdered form.
Liquid Chromatography-Tandem Mass Spectrometry Analysis of SR
Data were collected using an ultra-high-performance liquid phase (Thermo Vanquish UHPLC, Thermo Fisher Scientific) coupled with a high-resolution mass spectrometer (Q-Exactive HF, Thermo Fisher Scientific). A C18 chromatographic column (Zorbax Eclipse C18 [1.8 μm × 2.1 mm × 100 mm]) was employed for the separation. Chromatographic separation conditions: The column temperature was maintained at 30°C and the flow rate was set at 0.3 mL/min. The mobile phase composition A was 0.1% fomic acid solution, while composition B was pure acetonitrile, with an injection volume of 2 μL. The autosampler temperature was maintained at 4°C. Gradient elution was employed (shown in Table 1). The total ion current diagram of the sample was obtained in both positive and negative modes. Positive/negative mode: heater temperature 325°C; sheath gas flow: 45 arb (arbitrary units); aux gas flow: 15 arb; sweep gas flow: 1 arb; electrospray voltage: 3.5 kV; capillary temperature: 330°C; and S-lens RF level: 55%. Scanning mode: full scan (full scan, m/z 100–1,500) and data-dependent mass spectrometry (dd-MS2, TopN = 10); resolution: 120,000 (MS1) and 60,000 (MS2). Collision mode: High-energy collision dissociation.
LC mobile phase conditions
Time, min . | Flow rate, μL/min . | Gradient . | B% acetonitrile . | A% fomic acid . |
---|---|---|---|---|
0–2 | 300 | - | 5 | 95 |
2–6 | 300 | Linear gradient | 30 | 70 |
6–7 | 300 | - | 30 | 70 |
7–12 | 300 | Linear gradient | 78 | 22 |
12–14 | 300 | - | 78 | 22 |
14–17 | 300 | Linear gradient | 95 | 5 |
17–20 | 300 | - | 95 | 5 |
20–21 | 300 | Linear gradient | 5 | 95 |
21–25 | 300 | - | 5 | 95 |
Time, min . | Flow rate, μL/min . | Gradient . | B% acetonitrile . | A% fomic acid . |
---|---|---|---|---|
0–2 | 300 | - | 5 | 95 |
2–6 | 300 | Linear gradient | 30 | 70 |
6–7 | 300 | - | 30 | 70 |
7–12 | 300 | Linear gradient | 78 | 22 |
12–14 | 300 | - | 78 | 22 |
14–17 | 300 | Linear gradient | 95 | 5 |
17–20 | 300 | - | 95 | 5 |
20–21 | 300 | Linear gradient | 5 | 95 |
21–25 | 300 | - | 5 | 95 |
Animal Models and Drug Administration
Eight-week-old male and female ICR mice were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Approval No. SCXK [Beijing] 2021-0006). The mice were provided with unrestricted access to food and water and were housed in a controlled environment with a 12-h light/dark cycle, consistent temperature (22 ± 2°C), and humidity (55 ± 5%) throughout the experiment. All animal experiments strictly adhered to the guidelines outlined in the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine, with approval reference TCM-LAEC2022224. We were committed to making every possible effort to alleviate the suffering of animals.
The experiment was conducted according to the experimental scheme (shown in Fig. 1a). After 1 week of adaptive feeding, male and female mice (1:1) were housed in single cages at 18:00 and separated at 8:00 the next morning. Breeder pairs were only put together once, and mice that failed to mate once were excluded from the experiment. A total of 45 breeding pairs were included in the experiment, resulting in 45 pregnant mice. Stress exposure for pregnant mice commenced on gestational day 12 (G12). This entailed restraining pregnant mice in a ventilated glass tube measuring 12 cm in length and 3.5 cm in diameter for 45 min, with the timing of PS fixed at 9:00, 13:00, and 17:00 three times a day for 45 min. The rationale behind the timing of PS is that G12 corresponds to the late stage of pregnancy in mice. The processes of neuronal differentiation, dendritic arborization, axonal elongation, synapse formation, and collateralization, as well as myelination, commence at the late stage of pregnancy [42]. The late stage of gestation represents a critical period for neural development. It can be reasonably deduced that stress experienced during the late stage of pregnancy is more likely to result in hippocampal dysfunction, which may subsequently lead to the development of emotional disorders such as depression and anxiety. This provides an important foundation for the prenatal restraint stress intervention conducted during the late gestational phase of this study. The pregnant mice were divided into five experimental groups using the random number method in Microsoft Excel 2016, with the grouping starting from G12. The control and stress groups were administered pure water daily; however, the control group was not subjected to stress. By the 2020 edition of the China Pharmacopoeia, the recommended clinical dose of SR decoction pieces is 10 g for adults, with a dose converted to mice of 1.5 g/kg, based on the conversion coefficient of 9.1 between humans and mice. The low dose of SR is 0.5 g/kg and the high dose is 4.5 g/kg, according to the ratio of 1:3:9, which is based on the middle dose. The low-, medium-, and high-dose groups of SR underwent daily gavage at doses of 0.5 g/kg, 1.5 g/kg, and 4.5 g/kg, respectively, while also being subjected to stress. It is worth noting that the administration of SR was conducted once daily at 8:00 a.m. during the PS period of pregnant mice, with the administration completed before the onset of PS. Thirty pregnant mice waited to give birth naturally. After which all pups within the same litter were kept together with their mother mouse for co-feeding for 21 days before being weaned and separated by gender into cages housing 5 weaned mice each. Litters were raised until 1 month old for behavioral experiments with only two female and two male offspring randomly selected from each litter for subsequent experiments. Additionally, 15 pregnant mice had placenta and fetal brain tissue collected on G18.
SR ameliorated anxiety-like and depression-like behaviors in the offspring induced by PS. a Timeline of the experimental protocol. b Heat maps of path tracing in the OFT. c Trajectory of path tracing in OFT. d The average speed of offspring mice in OFT. e Immobility time of offspring mice in OFT. f Immobility time of offspring mice in FST. g Immobility time of offspring mice in TST. Values were expressed as the mean ± SEM. n = 9–12 in each group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PS.
SR ameliorated anxiety-like and depression-like behaviors in the offspring induced by PS. a Timeline of the experimental protocol. b Heat maps of path tracing in the OFT. c Trajectory of path tracing in OFT. d The average speed of offspring mice in OFT. e Immobility time of offspring mice in OFT. f Immobility time of offspring mice in FST. g Immobility time of offspring mice in TST. Values were expressed as the mean ± SEM. n = 9–12 in each group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PS.
Behavioral Tests
Behavioral tests were conducted at P30-P32 from 8:00 to 12:00 and 14:00 to 18:00. Adolescence is a crucial stage of development. We believe that adolescent mice are better suited to conduct behavioral experiments related to neuropsychiatric disorders than adult mice because patients often show their first symptoms during adolescence [21]. Therefore, we conducted behavioral experiments on adolescent mice, which are necessary to monitor the PS offspring at an adverse developmental stage. To facilitate acclimatization to the unfamiliar environment and minimize the influence of nonexperimental factors, the experimental mice were placed in the experimental room the night before the formal behavioral experiment.
Open Field Test
The open field test (OFT) experimental apparatus comprises four boxes of identical dimensions, each with a square bottom of 50 cm × 50 cm and a height of 40 cm. At the beginning of the experiment, the experimenter gently lifted the mouse’s tail, and the mouse was gently placed in the center of the square bottom of each box with its back facing the experimenter, and then promptly withdrew. Four mice can be tested simultaneously. Mice behavior in the open field was recorded for 5 min using Supermaze software, capturing metrics such as movement distance, speed, and immobility time. Subsequently, the mice were returned to their original cages, and the box was cleaned to remove any residual urine and feces, eliminating any potential odor. The illuminance of OFT was 200 lux.
Tail Suspension Test
The suspended tail experimental apparatus comprises a crossbar and a black background. The mice, positioned approximately 2 cm from their tail, were suspended on the bar using a medical tape to prevent them from climbing. Black partitions were utilized to segregate individual mice, and their behavior during a 5-min suspension was recorded by the software. The immobility time within these 5 min served as the criterion for assessing depression-like behavior. The illuminance of tail suspension test (TST) was 50 lux.
Forced Swimming Test
The apparatus used for the forced swimming experiment consists of a transparent plastic bucket with a diameter of 13 cm and a height of 18.5 cm. The water level was maintained at 13 cm, and the temperature was set at 23 ± 1°C. At the start of the experiment, the mice were gently placed into the bucket, and the software recorded their immobility time over 4 min. This recorded immobility time served as the criterion for assessing depression-like behavior. The illuminance of forced swimming test (FST) was 50 lux.
Blood Collection and Cytokine Detection
After completing all behavioral studies, the mice were anesthetized with isoflurane, and blood was subsequently collected. The obtained blood was allowed to stand at room temperature for 1 h and then centrifuged at 3,000 rpm at 4°C for 15 min. The serum was stored on ice and subjected to assays for ACTH, CRH, GC, and Glu using standard ELISA procedures. The ELISA kits used in the experiment were all purchased from Sino Best Biological Technology Co., Ltd. The sensitivities of CRH, ACTH, GC, and Glu ELISA kits were 1.0 pg/mL, 1.0 pg/mL, 0.1 ng/mL, and 1.0 mg/L, respectively, and none of them cross-react with other soluble structural analogs.
Tissue Collection
The mice were fixed and perfused with normal saline until the blood was washed out of each tissue. The brain and placenta for subsequent pathological sections were carefully removed and placed in 4% paraformaldehyde tissue fixative. The hippocampus was then isolated from the remaining brain tissue, and the placenta and fetal brain were temporarily stored on ice and subsequently placed in a −80°C refrigerator for later testing.
Fetal Brain Protein Detection
The fetal brain was homogenized with saline at a ratio of 1:10 (w/v) at 4°C. The supernatant of the homogenate was obtained by centrifugation at 3,000 rpm for 10 min. The ELISA procedure was used to detect GC, GR, BDNF, and Glu. The ELISA kits used in the experiment were all purchased from Sino Best Biological Technology Co., Ltd. The sensitivities of GC, GR, BDNF, and Glu ELISA kits were 0.1 ng/mL, 10 pg/mL, 1.0 pg/mL, and 1.0 mg/L, respectively, and none of them cross-react with other soluble structural analogs.
Histology
The placentas and the brains of offspring mice were fixed in 4% paraformaldehyde for more than 24 h. The fixed tissues were dehydrated and waxed. The following solutions were utilized: 50% ethanol, 75% ethanol, 95% ethanol (I) (II), 100% ethanol (I) (II), xylene (I) (II), and paraffin (I) (II). After paraffin embedding, the paraffin blocks were placed in a refrigerator at 4°C for cooling and then fixed on a rotary slicer to adjust the thickness of the slices to 4 μm. The slices were spread at 42°C on a tissue spreading machine for 20 s and baked for 2 h. The placental sections were stained with hematoxylin-eosin (HE), and brain sections were stained with HE, Nissl staining, and immunohistochemical staining.
HE Staining
The sections were baked at 60°C for 1 h and subsequently washed with xylene (I) (II) (III) (IV), 100% ethanol (I) (II) (III), 90% ethanol, 70% ethanol, and hematoxylin dyeing. Subsequently, the sections were washed with water, stained with eosin, washed again, then treated with 100% ethanol (I, II, III, IV) and xylene (I, II, III, IV), and sealed with neutral resin. Images were obtained using a microscope (Invitrogen EVOS M7000) at ×40 magnification and ×200 magnification.
Nissl Staining
The sections were baked at 60°C for 1 h and washed with xylene (I) (II) (III) (IV), 100% ethanol (I) (II) (III), 90% ethanol, and 70% ethanol. Subsequently, the tissue sections were stained using Nissl dye, followed by sequential steps of water washing, differentiation with 0.1% glacial acetic acid, additional water washing, air-drying, xylene treatment for transparency, and sealing with neutral resin. Images were obtained using a microscope (Invitrogen EVOS M7000) at ×40 magnification and ×200 magnification.
Immunohistochemistry
The sections were dewaxed and dehydrated, then blocked with 3% hydrogen peroxide (H2O2) for 25 min and 3% bovine serum albumin for 30 min. The sections were incubated at 4°C overnight with the DCX primary antibody (Abcam ab18723, 0.1 μg/mL) and the BDNF primary antibody (Abcam ab108319, 1:500). The sections were incubated with biotinylated goat anti-rabbit IgG at room temperature for 30 min. Subsequently, diaminobenzidine was applied as the substrate, followed by hematoxylin staining to visualize the nucleus. The sections were then dehydrated with a series of ethanol concentrations, followed by treatment with xylene for transparency. Lastly, the sections were sealed with an appropriate sealing agent. Subsequently, the samples were observed under a microscope. Images were obtained using a microscope (Invitrogen EVOS M7000) at ×40 magnification and ×200 magnification. Immunohistochemical results for DCX and BDNF were quantified using Image J software, as described in the online supplementary material 4 and 5 (for all online suppl. material, see https://doi.org/10.1159/000543152).
Real-Time PCR
Total RNA was extracted from the hippocampus of offspring mice and placenta using a Trizol kit (TransGen Biotech, China) according to the manufacturer’s instructions. The concentration of RNA was determined by a Microvolume-UV-Vis Spectrophotometer (Thermo Fischer Scientific). First-strand cDNA was synthesized to a final volume of 20 μL using an All-in-One First-Strand cDNA Synthesis Kit (TransGen Biotech, China). Reverse transcription to cDNA was conducted using a BIO RAD T100™ Thermal Cycler. After reverse transcription, the sample system was prepared according to the operating instructions of PerfectStart® Green qPCR Kit (TransGen Biotech, China), and real-time PCR was performed on QuantStudio 6 Flex real-time fluorescence quantitative PCR system (Thermo Fischer Scientific). The primer information is valid (shown in Table 2). The relative expression levels of the candidate genes were analyzed using the 2−ΔΔCt method.
Primer information
Gene name . | Forward primer . | Reverse primer . |
---|---|---|
GAPDH | AGGTCGGTGTGAACGGATTTG | GGGGTCGTTGATGGCAACA |
GR | GACTCCAAAGAATCCTTAGCTCC | CTCCACCCCTCAGGGTTTTAT |
11β-HSD1 | GGAGCCCATGTGGTATTGACT | CCGCAAATGTCATGTCTTCCAT |
11β-HSD2 | GAGGGGACGTATTGTGACCG | TGTGTCCATAAGCAGTGCTATTG |
BDNF | AGGTCTGACGACGACATCACT | CTTCGTTGGGCCGAACCTT |
Gene name . | Forward primer . | Reverse primer . |
---|---|---|
GAPDH | AGGTCGGTGTGAACGGATTTG | GGGGTCGTTGATGGCAACA |
GR | GACTCCAAAGAATCCTTAGCTCC | CTCCACCCCTCAGGGTTTTAT |
11β-HSD1 | GGAGCCCATGTGGTATTGACT | CCGCAAATGTCATGTCTTCCAT |
11β-HSD2 | GAGGGGACGTATTGTGACCG | TGTGTCCATAAGCAGTGCTATTG |
BDNF | AGGTCTGACGACGACATCACT | CTTCGTTGGGCCGAACCTT |
Molecular Docking
The target structures of the candidate proteins of SR were obtained from the RCSB protein database (https://www.rcsb.org/). The target information was BDNF (PDB ID: 1b8m), DCX (PDB ID: 2bqq), GR (PDB ID: 1nhz), and 11β-HSD1 (PDB ID: 1xu7). The file was downloaded in a PDB format, the protein was processed with Pymol and AutoDockTools1.5.7, and the file was saved in a PDBQT format. The 2D structures of the four monomer compounds were obtained from the TCMSP database (https://old.tcmsp-e.com/tcmsp.php), converted into 3D structures by Chem3D software with minimum energy, and saved in a mol2 format. These compounds were converted into the pdbqt format by AutoDockTools1.5.7, and docking was performed with AutoDock Vina [43]. Pymol was employed to visualize the docking results.
Statistical Analysis
The data analysis was conducted using SPSS 21 (version 21.0.0.0). The significance of variables among groups was determined using either nonparametric tests or one-way ANOVA, followed by the LSD test or Dunnett’s T3 multiple comparison test as appropriate. Data plotting was performed using GraphPad Prism 9 (version 9.0.0 [121]). Results were presented as mean ± SEM. A p value of <0.05 was considered statistically significant.
Results
Chemical Composition Analysis of SR
The samples were analyzed by liquid chromatography-tandem mass spectrometry to identify the chemical components in SR. Positive and negative ionization modes were used to characterize the corresponding signals. The total ion current diagram of the sample was generated (shown in Fig. 2a, b). Compound Discoverer 3.3 was used for retention time correction, peak identification, peak extraction, etc. According to the secondary mass spectrum information, the Thermo mzCloud online database was used for compound identification. The identified compounds were shown in the online supplementary material 3.
Analysis of the chemical composition of SR. a TIC diagram of SR (negative mode). b TIC diagram of SR (positive mode). c Chemical structures of the top 10 compounds in SR.
Analysis of the chemical composition of SR. a TIC diagram of SR (negative mode). b TIC diagram of SR (positive mode). c Chemical structures of the top 10 compounds in SR.
The results showed that SR contained the highest content of flavonoids. In addition, SR also contained many types of compounds, such as prenol lipids, coumarins and derivatives, carboxylic acids, and derivatives, phenols, indoles, and derivatives, etc., and we listed the structural formulas of 10 compounds in SR (shown in Fig. 2c).
SR Ameliorated Anxiety-Like and Depression-Like Behavior in the Offspring Induced by PS
The OFT was utilized to assess anxiety-like behavior in offspring mice. The offspring of PS mice demonstrated pronounced anxiety-like behavior compared to the control group. Analysis of the trajectory and heat map revealed that the offspring of PS mice spent less time in the central zone (shown in Fig. 1b, c). In addition, the offspring of PS mice exhibited decreased movement speed and prolonged immobility time (shown in Fig. 1d, e), supporting the presence of anxiety-like behavior in the offspring of PS mice. Remarkably, the impaired performance of the offspring of PS mice was significantly reversed upon administration of SR. These results suggest that SR has the potential to alleviate anxiety-like behaviors in offspring mice exposed to PS.
The FST is a classical experiment used to assess depression-like behavior in mice, with immobility time as a key index. The results of the FST showed that PS significantly increased immobility time in the offspring mice compared to the control group, indicating that PS induced depression-like behavior in the offspring mice. Fortunately, SR significantly reduced immobility time in male and female offspring of PS mice. Moreover, only the medium dose of SR showed a significant reduction in immobility time in male offspring mice compared to the PS group (shown in Fig. 1f). In conclusion, the FST results indicated that administration of SR at a medium dose significantly ameliorated depression-like behavior in the offspring of PS mice.
The TST is another behavioral test used to assess depression-like behavior in mice. In female offspring of PS mice, SR significantly reduced the immobility time in the TST (shown in Fig. 1g). However, PS had no significant effect on the immobility time of male offspring mice in the TST (shown in Fig. 1g). In conclusion, the TST results indicated that PS induced depression-like behavior in female offspring mice, a phenomenon that was significantly ameliorated by SR treatment. However, male offspring did not show significant changes in immobility time due to PS.
SR Ameliorated Hippocampal Neurogenesis Defects in the Offspring Induced by PS
PS caused structural changes in the hippocampus of the offspring mice. The results of the HE and Nissl staining demonstrated that the hippocampal structure of the PS group offspring mice exhibited alterations when compared to the control group. These alterations were primarily evidenced by the presence of more nuclei with condensed chromatin (pyknotic nuclei) in the hippocampus, which is a hallmark of hippocampal pathology. Conversely, the administration of SR effectively prevented the occurrence of these histopathological changes (shown in Fig. 3a, b).
SR ameliorated hippocampal neurogenesis defects in the offspring induced by PS. a HE staining in the hippocampus and CA1 region of the brain. b Nissl staining in the hippocampus and DG region of the brain. c Immunohistochemistry of DCX in the DG region of the hippocampus. d Average optical density of DCX-positive area in the DG region of the hippocampus of offspring female mice. e The average optical density of DCX-positive area in the DG region of the hippocampus of offspring male mice. Values were expressed as the mean ± SEM. n = 6–9 in each group. #p < 0.05, ##p < 0.01 vs. control. **p < 0.01 and ***p < 0.001 vs. PS.
SR ameliorated hippocampal neurogenesis defects in the offspring induced by PS. a HE staining in the hippocampus and CA1 region of the brain. b Nissl staining in the hippocampus and DG region of the brain. c Immunohistochemistry of DCX in the DG region of the hippocampus. d Average optical density of DCX-positive area in the DG region of the hippocampus of offspring female mice. e The average optical density of DCX-positive area in the DG region of the hippocampus of offspring male mice. Values were expressed as the mean ± SEM. n = 6–9 in each group. #p < 0.05, ##p < 0.01 vs. control. **p < 0.01 and ***p < 0.001 vs. PS.
Neurogenesis was measured by detecting the expression of DCX (a marker of neurogenesis) in the hippocampus. The positive expression of DCX in the hippocampal DG area in the offspring of PS mice was markedly lower than that in the control group, indicating that PS induced hippocampal neurogenesis defects in the offspring. SR administration significantly increased the positive expression of DCX in the hippocampal DG area in the offspring of PS mice (shown in Fig. 3c–e). Overall, the results indicated that SR administration alleviated PS-induced hippocampal neurogenesis defects in the offspring.
SR Restored Abnormal Expression of Glu and BDNF in the Hippocampus Induced by PS
Glu is the major excitatory neurotransmitter in the central nervous system and plays a critical role in synaptic transmission. Excessive Glu and insufficient Glu are detrimental. PS resulted in a significant reduction in Glu expression in the fetal brain and offspring serum. However, SR administration normalized Glu levels in fetal brain and offspring serum in PS mice (shown in Fig. 4a, b).
SR restored abnormal expression of glutamate (Glu) and BDNF in the hippocampus induced by PS. a Expression of Glu in fetal brain. b Expression of Glu in offspring serum. c Expression of BDNF in fetal brain. d Expression of BDNF in hippocampus CA1 and CA3 of the female offspring of PS mice. e The average optical density of BDNF-positive area in the CA1 region of the hippocampus of offspring female mice. f The average optical density of BDNF-positive area in the CA3 region of the hippocampus of offspring female mice. g Expression of BDNF mRNA in the hippocampus of female offspring. h Expression of BDNF in hippocampus CA1 and CA3 of the male offspring of PS mice. i Average optical density of BDNF-positive area in the CA1 region of the hippocampus of offspring male mice. j Average optical density of BDNF-positive area in the CA3 region of the hippocampus of offspring male mice. k Expression of BDNF mRNA in the hippocampus of male offspring. Values were expressed as the mean±SEM. n = 8–9 in each group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PS.
SR restored abnormal expression of glutamate (Glu) and BDNF in the hippocampus induced by PS. a Expression of Glu in fetal brain. b Expression of Glu in offspring serum. c Expression of BDNF in fetal brain. d Expression of BDNF in hippocampus CA1 and CA3 of the female offspring of PS mice. e The average optical density of BDNF-positive area in the CA1 region of the hippocampus of offspring female mice. f The average optical density of BDNF-positive area in the CA3 region of the hippocampus of offspring female mice. g Expression of BDNF mRNA in the hippocampus of female offspring. h Expression of BDNF in hippocampus CA1 and CA3 of the male offspring of PS mice. i Average optical density of BDNF-positive area in the CA1 region of the hippocampus of offspring male mice. j Average optical density of BDNF-positive area in the CA3 region of the hippocampus of offspring male mice. k Expression of BDNF mRNA in the hippocampus of male offspring. Values were expressed as the mean±SEM. n = 8–9 in each group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PS.
BDNF is a basic neurotrophic factor that is critical during brain development, playing a protective role in neurons and maintaining their normal physiological functions. PS resulted in a significant reduction of BDNF expression in the fetal brain, and administration of SR normalized BDNF levels in the fetal brain (shown in Fig. 4c). Immunohistochemistry revealed a significant decrease in the positive expression of BDNF in the hippocampal CA1 and CA3 regions in the offspring of PS mice, while SR administration significantly increased the positive expression of BDNF in the hippocampal CA1 and CA3 regions in the offspring of PS mice (shown in Fig. 4d–f, h–j). Additionally, PS induced a significant decrease in hippocampal BDNF mRNA expression in the offspring compared to the control group (shown in Fig. 4g, k). Notably, SR administration significantly increased the mRNA expression of hippocampal BDNF in the offspring of PS mice (shown in Fig. 4g, k). In conclusion, these results suggested that SR may preserve the normal physiological function of neurons by increasing the expression of BDNF and Glu.
SR Restores PS-Induced Structural and Functional Changes in the Placenta
The placenta serves as a vital organ for the exchange of nutrients between the mother and the fetus. GCs elevated due to PS are primarily transported to the fetus through the placenta. Therefore, the normalization of the placental structure and function of the placenta is an important link in the influence of PS on the fetus. HE staining of the placenta revealed structural changes induced by PS. Notably, abnormal placental structure was observed (shown in Fig. 5a), characterized by disordered arrangement of blood vessels in the labyrinthine region, looseness, hemocytopenia, and thinning of the blood vessel wall. All these changes were the basis of the changes in placental function. The SR groups showed increased blood cells in the placental labyrinth area and improved organization of vascular arrangement. In addition, PS induced a significant increase in placental 11β-HSD1 mRNA expression compared with the control group. The SR groups showed a significant decrease in the expression level of 11β-HSD1 mRNA compared with the PS group (shown in Fig. 5b), indicating that SR could maintain normal 11β-HSD1 expression in the placenta of PS mice, thereby ensuring normal GC levels in the fetus. Moreover, compared with the control group, PS induced a significant increase in the level of GC and a significant decrease in the expression of GR in the fetal brain. However, administration of SR significantly reduced the level of GC and increased the expression of GR in the fetal brain in PS mice offspring (shown in Fig. 5c, d). The results suggested that SR protected the fetus from excessive GC by protecting the placental structure and function.
SR restores PS-induced structural and functional changes in the placenta. a HE staining of placental labyrinthine region. b The mRNA expression of 11β-HSD1 in placenta. c Expression of GC in fetal brain. d Expression of GR in fetal brain. Values were expressed as the mean ± SEM. n = 6–8 in each group. #p < 0.05 and ##p < 0.01 vs. control. *p < 0.05 and **p < 0.01 vs. PS.
SR restores PS-induced structural and functional changes in the placenta. a HE staining of placental labyrinthine region. b The mRNA expression of 11β-HSD1 in placenta. c Expression of GC in fetal brain. d Expression of GR in fetal brain. Values were expressed as the mean ± SEM. n = 6–8 in each group. #p < 0.05 and ##p < 0.01 vs. control. *p < 0.05 and **p < 0.01 vs. PS.
SR Reversed HPA Axis Hyperfunction in the Offspring Induced by PS
PS decreased the mRNA expression of GR and 11β-HSD2, while significantly increasing the mRNA expression of 11β-HSD1 (shown in Fig. 6), thereby elevating serum levels of CRH, ACTH, and GC in the offspring. These findings indicate that PS-induced dysfunction in the HPA axis may be attributed to its impact on the function of GR and 11β-hydroxysteroid dehydrogenase in the offspring. It is noteworthy that the administration of SR led to a notable increase in the mRNA expression of GR and 11β-HSD2, accompanied by a significant reduction in the mRNA expression of 11β-HSD1. This resulted in the normalization of serum CRH, ACTH, and GC levels in the offspring. These findings indicated that SR restored the function of GR and 11β-hydroxysteroid dehydrogenase, ensuring the normal operation of the HPA axis in the offspring.
SR reversed HPA axis hyperfunction in the offspring induced by PS. a Expression of GR mRNA in the hippocampus of offspring. b Expression of 11β-HSD1 mRNA in the hippocampus of offspring. c Expression of 11β-HSD2 mRNA in the hippocampus of offspring. d Serum levels of CRH in the offspring. e Serum levels of ACTH in the offspring. f Serum levels of GC in the offspring. Values were expressed as the mean±SEM. n = 8 in each group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PS.
SR reversed HPA axis hyperfunction in the offspring induced by PS. a Expression of GR mRNA in the hippocampus of offspring. b Expression of 11β-HSD1 mRNA in the hippocampus of offspring. c Expression of 11β-HSD2 mRNA in the hippocampus of offspring. d Serum levels of CRH in the offspring. e Serum levels of ACTH in the offspring. f Serum levels of GC in the offspring. Values were expressed as the mean±SEM. n = 8 in each group. #p < 0.05, ##p < 0.01, and ###p < 0.001 vs. control. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. PS.
Prediction of Active Ingredients in SR
Our study identified BDNF, DCX, GR, and 11β-HSD1 as key proteins for SR to ameliorate PS-induced anxiety-like and depression-like behaviors in offspring. To identify the major components that played a protective role in SR, we employed molecular docking. The results of the molecular docking analysis demonstrated that these 10 compounds in SR exhibited a robust affinity (binding energy <−5 kcal/mol) for BDNF, DCX, GR, and 11β-HSD1 (shown in online suppl. material 2). Hydrogen bonds can be formed between the ligand and the amino acid residues of the receptor protein, which serves as the foundation for the compound’s effective binding to the target. The docking results revealed that baicalein, wogonin, wogonoside, and baicalin exhibited a markedly stronger binding affinity with the target. The docking sites of these four compounds with the target were subsequently visualized (shown in online suppl. material 1). We believe that these 10 compounds, especially baicalein, wogonin, wogonoside, and baicalin, play a crucial role in mediating the neuroprotective effect of SR.
Discussion
The present study aimed to investigate the impact of SR on anxiety-like and depression-like behaviors as well as neurological development in the offspring of PS mice. It has been demonstrated that PS affects the behavior of the offspring mice. The results of this study confirmed that the offspring of PS mice exhibited anxiety-like behavior. Furthermore, our results demonstrate a gender-specific aspect to depression-like behavior in the offspring of PS mice, with female offspring exhibiting more pronounced symptoms compared to male mice, which is consistent with prior research [44]. However, there are opposing views on this topic, with some studies suggesting that male offspring exposed to PS exhibit more pronounced depressive-like behavior [45]. The species of the animal and the experimental detection method are crucial factors influencing these disparate results. The administration of SR was found to ameliorate anxiety and depression behaviors in the offspring of PS mice.
Furthermore, we employed HE staining and Nissl staining to investigate neuropathological changes in the offspring’s brain. The results demonstrated that SR effectively attenuated aberrant neuronal alterations, including Nissl dissolution and disorganized neuronal configuration. Neurogenesis in the hippocampus highly modulates neurodevelopment processes by regulating information processing in the hippocampal DG [46]. Neurogenesis in the hippocampus is susceptible to internal and external environmental changes during the fetal period [47]. Consistent with previous studies, PS inhibits neurogenesis in the hippocampus, resulting in anxiety-like and depression-like behavior in the offspring [48]. The results demonstrated that SR mitigated the neurogenesis defects observed in the hippocampal DG of offspring mice. Glu serves as the primary excitatory neurotransmitter in the mammalian central nervous system [49]. Previous investigations have demonstrated that stress can induce damage to the central nervous system, leading to an increased concentration of Glu in the synaptic space. This elevation may contribute to excitotoxicity and subsequent neuron death, which may ultimately lead to the development of depression and other mental disorders [50, 51]. Recent evidence indicates that depressed patients and stressed offspring exhibit lower levels of Glu in their brains [52‒54]. This observation is consistent with our findings. Nevertheless, the precise mechanism by which this occurs remains unknown. The discrepancies in conclusions may be attributed to the species studied and the methods of intervention. Therefore, it is crucial for researchers to pay close attention to these factors. The present study revealed that SR was effective in restoring normal Glu levels in the fetal brain and offspring serum in PS mice. BDNF is a widely expressed neurotrophic factor in the central nervous system, playing an important role in the process of neuron maturation and survival, synaptic formation, and synaptic plasticity [55, 56], thereby exerting a crucial influence on the development of the nervous system. PS has been shown to influence the brain development of offspring, resulting in anxiety-like and depression-like behaviors. These behavioral changes may be associated with alterations in BDNF levels. Numerous studies have indicated that maternal stress can reduce BDNF levels in the brains of offspring [57], which is closely related to impairment of HPA axis function. HPA axis disorders influence the function of BDNF, and elevated GC levels can alter BDNF signal transduction in PS mouse offspring [58]. This aberration can impact neuronal plasticity and potentially harm brain development in the offspring. Thus, we conducted an analysis of the expression of BDNF in the hippocampal region of the offspring, which revealed a significant downregulation of BDNF in the CA1 and CA3 regions of PS mice offspring. This observation explains the compromised brain development in the offspring of PS mice. Importantly, our results indicated that SR effectively mitigated this downregulation. Overall, these findings suggest that SR ameliorates PS-induced neurodevelopmental dysfunction in the offspring by improving the aberrant structure and neurogenesis defects of the hippocampus in the offspring and increasing the expression of BDNF and Glu of the hippocampus in the offspring.
GC is extracted from the maternal system via the placenta during fetal development and is essential for maturation, development, and survival. Normally, only 10–20% of maternal GC is transmitted to the fetus due to the function of the placenta as a barrier [4]. Proper development of the placental labyrinth is crucial for the exchange of nutrients and waste between the mother and the fetus [59]. The present study demonstrated that PS induced abnormal placental structure, characterized by disordered arrangement of blood vessels in the labyrinthine region, looseness, hemocytopenia, and thinning of the blood vessel wall. These adverse conditions can affect fetal neurodevelopment. Notably, these changes constituted the basis for alterations in placental function. Structure determines function, and the change in placental structure will inevitably lead to a change in its function. Normally, maternal GCs are converted into inactive forms by the 11β-HSD2 enzyme in the placenta, to ensure proper function of the placental barrier [60, 61]. In contrast, another enzyme, 11β-HSD1, exhibits an opposing function, converting inactive GCs into their active form [62, 63]. The enzyme 11β-HSD1 can elevate the risk of exposing the fetus to an excessive GC environment, which can have negative effects on fetal development. The present study demonstrated that PS significantly increased the gene expression of 11β-HSD1 in the placenta, while reduced by the administration of SR. Therefore, it can be concluded that structural and functional changes in the placenta result in an increased flow of GCs into the fetus, further reducing the expression of GR, which is the basis of fetal neurodevelopmental dysfunction. In conclusion, SR possessed the potential to safeguard the structure and function of the placenta, thereby maintaining a normal intrauterine environment for the fetus.
Fetal long-term exposure to an excessive GC environment is also associated with HPA axis hyperfunction, which persists into childhood [64]. PS can result in the excessive release of GCs [65], a reduction in the expression of GR in the offspring’s hippocampus, the destruction of the offspring’s HPA axis negative feedback function [66‒68], and an increase in the secretion of CRH and ACTH, as confirmed by our results. GR plays a crucial role in regulating GC levels, as it can bind with excessive GC to restore normal HPA axis function [69]. Therefore, GR is a key factor in regulating HPA axis hyperfunction. Additionally, the balance between the enzymes 11β-HSD1 and 11β-HSD2 in the hippocampus contributes to the regulation of GC concentration in hippocampal cells. The results demonstrated that PS diminished the expression of 11β-HSD2 and GR while elevating the expression of 11β-HSD1 in the offspring. This led to the secretion and accumulation of GC, which further exacerbated the hyperfunction of the HPA axis. Fortunately, SR can restore the normal expression of GR, 11β-HSD1, and 11β-HSD2, thereby reducing the accumulation of high concentrations of GC in the hippocampus and alleviating HPA axis hyperfunction. Furthermore, the chemical composition of SR was analyzed, and molecular docking simulations were conducted for 10 components of SR to predict the binding pattern and affinity between the compounds and the target. The findings indicated that these 10 compounds exhibited robust binding affinity with BDNF, DCX, GR, and 11β-HSD1, especially baicalein, wogonin, baicalin, and wogonoside. Therefore, this may be the material basis for SR to play a neuroprotective role.
The present study demonstrated that SR could ameliorate PS-induced anxiety-like and depression-like behavior in the offspring, at least partially through protecting placental barrier function, reversing HPA axis hyperfunction, and ameliorating neurodevelopmental dysfunction. Additionally, we propose that baicalein, wogonin, baicalin, and wogonin may play a significant role in this process. However, our study has some limitations. First, our focus was limited to mice and we did not validate our findings in other species. This may account for the discrepancies observed in comparison to previous studies. Second, while we have elucidated the mechanism through which SR improves PS, our understanding is not exhaustive, and we plan to conduct further research to gain a deeper understanding of the mechanism in the future. Finally, we will aim to isolate the active monomer components present in the extract of SR and investigate their mechanism of action.
Statement of Ethics
This study protocol was reviewed and approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (Approval No. TCM-LAEC2022224).
Conflict of Interest Statement
The authors have no conflicts of interest to declare.
Funding Sources
This research was funded by the National Natural Science Foundation of China, Grant No. 82204775 (Q.Z.), Tianjin Municipal Education Commission research project, Grant No. 2022KJ142 (Q.Z.), and the new teachers’ research program of Tianjin University of Traditional Chinese Medicine, Grant No. XJS2022110 (Q.Z.).
Author Contributions
Conceptualization: Lixia Li, Qian Zhou, and Xiaoying Wang; methodology: Lixia Li, Wenying Zhang, and Congying Sun; software: Lixia Li and Wenying Zhang; validation: Wenying Zhang, Congying Sun, Zhiqiang Chai, and Kaiyue Wang; formal analysis: Lixia Li and Kaiyue Wang; investigation: Lixia Li, Wenying Zhang, Congying Sun, and Zhiqiang Chai; writing – original draft preparation: Lixia Li; writing – review and editing: Qian Zhou; visualization: Lixia Li and Wenying Zhang; project administration: Qian Zhou and Xiaoying Wang; and funding acquisition: Qian Zhou.
Data Availability Statement
All data generated or analyzed during this study are included in this article and its supplementary material files. Further inquiries can be directed to the corresponding author.