Anxiolytic Effects of Acanthopanax senticosus-Schisandra chinensis Combination: Mechanistic Insights via HPA and Neuroinflammatory Modulation

Introduction

Mental disorders account for a substantial proportion of the global disease burden. Evidence currently available suggests that mental disorders are responsible for roughly 14.3% of deaths worldwide.¹ Among the ten leading contributors to the global disease burden, there is no evidence that this burden has decreased since 1990.² Anxiety disorders are common, highly prevalent, and typically chronic psychiatric conditions. A nationwide epidemiological study in China reported that among all mental disorders, anxiety disorders have the highest lifetime rate, estimated at 7.57%.³ The onset of anxiety disorders is often closely linked to prolonged exposure to high levels of state anxiety. In recent years, findings from ecological momentary assessment (EMA) and longitudinal studies have consistently shown that individuals on the anxiety spectrum not only maintain elevated state anxiety in daily life, but also exhibit greater emotional reactivity to everyday stressors.^4,5 Such exaggerated stress responses may contribute to impairments in cognition, emotion, and behavior, thereby substantially compromising quality of life and social functioning.⁶

Stress-induced HPA axis dysfunction elevates circulating corticosterone, which downregulates glucocorticoid receptor (GR) expression and blunts feedback sensitivity within the hippocampus and prefrontal cortex, ultimately weakening negative feedback control and facilitating anxiety-like behaviors.⁷ This is accompanied by structural alterations in the amygdala, hippocampus, and prefrontal cortex-core nodes of the fear-anxiety and extinction circuits-including dendritic atrophy, cortical thinning, and amygdala hyperactivity.⁸ Chronic HPA overactivation also promotes neuroinflammation in these regions, with elevated pro-inflammatory cytokines and microglial activation further compromising neuroplasticity and perpetuating circuit dysfunction.⁹ In addition, HPA axis activity is also tightly coupled to circadian rhythmicity, as the 24-hour oscillation of glucocorticoids serves as a major internal synchronizer of the circadian system. Disruption of this rhythm, whether through chronic stress or intrinsic clock gene alterations, has been implicated in the pathophysiology of mood disorders.¹⁰

At present, pharmacotherapy remains the mainstay of treatment for anxiety disorders, commonly prescribed agents include benzodiazepine-class drugs and selective serotonin reuptake inhibitors (SSRIs). Nevertheless, such medications carry risks including addiction potential, tolerance, and dependence, and relapse is frequently observed after discontinuation.¹¹ These limitations not only restrict their long-term use but also pose substantial risks to patients’ health and quality of life. Therefore, identifying anxiolytic, food-based interventions derived from natural dietary resources that offer both safety and therapeutic efficacy is essential.

The root and rhizome of Acanthopanax senticosus (Rupr. et Maxim). Harms. (Araliaceae) serve as its medicinal part, while the ripe fruit of Schisandra chinensis (Turcz). Baill. (Magnoliaceae) is used. Both are rich in bioactive constituents such as lignans and polysaccharides and have been reported to exert multiple pharmacological effects, including anxiolytic, anti-stress, neuroprotective, and sedative-hypnotic activities.^12,13 According to traditional Chinese medicine theory, these two herbs are frequently combined for mutual reinforcement. Notably, a standardized fixed combination containing Acanthopanax senticosus and Schisandra chinensis (together with Rhodiola rosea) has demonstrated clinical efficacy in patients with acute non-specific pneumonia, including improved quality of life, enhanced cognitive performance under stress, and shortened recovery time.^14,15 At the cellular level, this combination has been reported to upregulate neuropeptide Y and heat shock protein 72 in neuroglial cells, which serve as upstream mediators of the HPA axis stress response.¹⁶ Existing studies indicate that bioactive constituents from adaptogenic herbs-including Acanthopanax senticosus, Schisandra chinensis, Rhodiola rosea, Withania somnifera, Panax ginseng, and Bacopa monnieri-produce multi-target anxiolytic effects through normalizing HPA axis activity, attenuating stress-induced neuroendocrine dysregulation, modulating neurotransmitter systems, and suppressing neuroinflammatory pathways.^17,18 Although botanical hybrid preparations containing Acanthopanax senticosus and Schisandra chinensis have shown evidence of synergistic interaction and superior efficacy over individual components, the specific anxiolytic mechanisms and therapeutic potential of their combined use (Acanthopanax senticosus + Schisandra chinensis, AS) in stress-related anxiety models remain to be fully elucidated.¹⁹

Liquid chromatography-mass spectrometry (LC-MS/MS) has substantially advanced the ability to identify and profile bioactive constituents in traditional Chinese medicine.²⁰ Leveraging such techniques enables precise profiling of herbal constituents and supports mechanistic studies of complex botanical interventions.²¹

In this study, LC-MS/MS was first employed to characterize the phytochemical constituents of the AS herb pair, and a systematic investigation was then conducted on its dose-dependent effects in a state-anxiety model established by conditioned fear stimulation. These findings are expected to provide robust experimental evidence for AS as an anxiolytic intervention and offer insights for further mechanistic studies, including potential applications of multi-omics approaches in elucidating herbal mechanisms in anxiety disorders.

Materials and Methods

Preparation of the AS Aqueous Decoction

Acanthopanax senticosus (No. 20230523) and Schisandra chinensis (No. 20230219) used in this study were produced in Heilongjiang, China, and purchased from Heilongjiang Beicaotang Chinese Medicine Co., Ltd. The plant materials were formally identified by Professor Huifeng Sun, Department of Traditional Chinese Medicine Identification. 100 g of Acanthopanax senticosus and 25 g of crushed Schisandra chinensis were mixed and placed in a decoction pot with an appropriate amount of water, and then decocted for 30 min. The first decoction was filtered and collected. The residue was subsequently decocted again for 30 min after adding water equivalent to two-thirds of the volume of the crude materials, and the second decoction was collected by filtration. The pooled filtrates were brought to a concentration of 2.5 g/mL and stored at 4°C until further use.

Qualitative Analysis by UPLC-QTOF-MS

Following centrifugation (10,000 r/min, 10 min), the decoction was passed through a 0.22 μm filter. Chromatographic and mass spectrometric analyses were carried out on a TripleTOF 6600+ UPLC-QTOF-MS platform (SCIEX, USA) controlled by SCIEX OS software. The source settings were as follows: spray voltage, 5500 V; collision energy, 35 ± 15 V; source temperature, 550 °C. Separation was achieved on a Phenomenex Kinetex XB-C18 column (2.6 μm, 100 × 2.1 mm) with a mobile phase consisting of (A) water containing 0.1% formic acid and (B) methanol. The flow rate was maintained at 0.4 mL/min, and the gradient was programmed as: 0–3 min, 5% B; 3–28 min, 5–95% B; 28–40 min, 95% B; 40–41 min, 5% B; 41–45 min, 5% B.

Experimental Animals

Male SPF-grade SD rats weighing 240–260 g were supplied by Liaoning Changsheng Biotechnology Co., Ltd. (license No. SCXK (Liao) 2020–0001). They were kept at 23 ± 1 °C in a facility with adequate ventilation and shielding from light, sound, and electromagnetic interference. A 12-h illumination cycle (illuminated from 07:00 to 19:00) was maintained automatically. All procedures received approval from the Animal Ethics Committee of Heilongjiang University of Chinese Medicine (permit No. 20231115003) and were performed in compliance with its guidelines.

Animal Grouping and Drug Administration

Male SD rats were allocated to five groups (n = 6 each) by a computer-generated list: Control, Model, L-AS, H-AS, and Diazepam. The sample size was determined via G*Power 3.1 using preliminary Elevated T-Maze data (effect size = 0.95, α = 0.05, 1−β = 0.80), which indicated a minimum of six animals per group. Intragastric administration began at 08:00 one day after the state-anxiety model was established. The L-AS and H-AS groups received AS aqueous decoction at 12.5 g/kg (1.25 g/mL) and 25 g/kg (2.5 g/mL), respectively; the Diazepam group was given 4.5 mg/kg diazepam (0.45 mg/mL). Control and Model groups received an equivalent volume of distilled water. All treatments were delivered by gavage at 1 mL/100 g daily for 7 consecutive days.

Establishment of the State Anxiety Rat Model

Rats in the Model, L-AS, H-AS, and Diazepam groups were individually placed in a conditioned fear monitoring system. After a 3-min habituation period, an auditory cue was presented for 5 s (75 dB, 100 Hz). At the fifth second of the tone, a footshock was delivered simultaneously (0.5 mA, 1 s). After stimulation, the rats stayed in the chamber for a further 3 min and were then removed. Rats in the Control group received the auditory cue only, with all other parameters kept identical. After each session, the waste tray was replaced, and the apparatus was rinsed with water, disinfected with 75% ethanol, and rinsed again with water to minimize odor-related interference. This training procedure was conducted once daily for 4 consecutive days.

Body Weight Measurement

During the experiment, body weight was recorded on day 1 and day 7 after model induction. Net body weight was used to evaluate changes in body weight over the experimental period.

Open Field Test

For the open-field test, animals were placed individually in the central zone of the apparatus (55 × 55 cm) and allowed to move freely for 5 min. Locomotor trajectories were captured with the TM-Vision behavioral analysis system. Over the 5-min session, the analysis quantified total distance traveled, grid-crossing frequency, and central-zone indices, including both distance traveled and time spent in the central area.

Elevated T-Maze Test

For the avoidance trials, rats were placed at the distal end of a closed arm facing the center, and the time taken to leave the arm completely was scored as the baseline avoidance latency (Test 1); the procedure was repeated twice more (Tests 2–3) with a 30-s pause between trials. Escape latency was then assessed by placing each animal at the far edge of an open arm facing toward the central area, with three successive trials (Tests 1–3) also separated by 30-s intervals.

Forced Swimming Test

For the forced swimming test, each animal was placed singly in a cylindrical container (inner diameter, 30 cm; water depth, 45 cm; water temperature, 26–30 °C), and immobility was scored over a 5-min session.

Pentobarbital Sodium Potentiation Test

Animals in each group received an intraperitoneal dose of 0.5% pentobarbital sodium (55 mg/kg; Meirida Biotechnology Co., Ltd., Shanghai, China) and were positioned supine on a warming pad set to 37 °C. The time from injection to disappearance of the righting reflex was recorded as sleep latency, and the interval between loss and return of this reflex was taken as total sleep time. To confirm complete recovery, each rat was returned to a supine posture immediately after it first righted itself; if it could right itself a second time within 1 min, the initial response was regarded as the recovery point, otherwise the later response was used.

Determination of Corticosteroids, Inflammatory Cytokines, and Neurotransmitters in Serum and Hippocampus

After being anesthetized with 0.5% pentobarbital sodium (55 mg/kg), the rats were subjected to blood collection, followed by decapitation for the harvest of hippocampal tissues. Blood samples were centrifuged to obtain serum (3000 r/min, 20 min) and stored at −80°C until analysis. Commercial ELISA kits (Jiangsu Enzyme Immunoassay Industry Co., Ltd., Yancheng, China) were used to quantify the levels of ACTH (MM-0565R2), CORT (MM-0559R2), IL-1β (MM-0047R1), IL-6 (MM-0190R1), TNF-α (MM-0180R1), ACh (MM-0517R1), DA (MM-0355R2), GABA (MM-0441R1), Glu (MM-060R2), NE (MM-0556R2), and 5-HT (MM-7117R2) in serum and hippocampal samples.

H&E Staining

Rats were anesthetized using the same protocol as described above, followed by transcardial perfusion. After the perfusion, the brains were removed rapidly and post-fixed in 4% paraformaldehyde at 4 °C for 48 h. Using bregma as the reference landmark, anatomical coordinates were determined based on a standard rat brain stereotaxic atlas, the hippocampal region (AP: −2.80 to −4.52 mm) was dissected using a brain matrix. The pre-fixed brains were cut into 2-mm-thick slices, immersed in 4% paraformaldehyde for an additional 24 h, and subsequently placed in 75% ethanol for overnight storage prior to paraffin embedding. Following deparaffinization, the sections underwent hematoxylin-eosin staining and were subsequently examined and photographed using a light microscope (Nikon Eclipse Ci-L, 400× magnification; Nikon, Tokyo, Japan).

Immunohistochemistry

According to the rat brain stereotaxic atlas, with bregma as the reference point, the hypothalamic paraventricular nucleus (PVN) region (AP: −0.80 to −1.80 mm) and the hippocampus were dissected using a brain matrix and processed for paraffin embedding. Localization and semi-quantitative assessment of CRH protein in the hypothalamus, as well as MR and GR proteins in the hippocampus, were performed by immunohistochemistry.

The primary antibodies were CRH (1:400, 26,848-1-AP), MR (1:500, 18,704-1-AP), and GR (1:200, 24,050-1-AP), all purchased from Sanying Biotechnology (Wuhan, China). A goat anti-rabbit IgG polymer was used as the secondary antibody (2413D0825; Zhongshan Golden Bridge Biotechnology, Beijing, China). The PVN and hippocampal regions were observed with a light microscope. Image-Pro Plus 6.0 was applied for image analysis and for quantification of mean optical density (MOD). In the same sections, the suprachiasmatic nucleus (SCN) and lateral hypothalamus (LH) were also evaluated as adjacent control regions to verify the specificity and reliability of the findings.

Statistical Analysis

All behavioral tests, ELISA measurements, H&E staining and immunohistochemical evaluations were performed by experimenters blinded to group allocation. Statistical analyses were performed using IBM SPSS Statistics 26.0, and graphs were generated using GraphPad Prism 8.0. All data were tested for normality and homogeneity of variances. Normally distributed variables with equal variances are expressed as mean ± standard deviation (SD), and comparisons among multiple groups were conducted using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for pairwise comparisons. When Levene’s test indicated unequal variances, Welch’s ANOVA was applied, followed by the Tamhane’s T2 post hoc test. Avoidance and escape latencies in the elevated T-maze were analyzed using repeated-measures ANOVA; when Mauchly’s test indicated violation of sphericity, the Greenhouse-Geisser correction was applied, and post hoc pairwise comparisons were performed with Sidak correction. When the assumptions of data normality were not met, the Kruskal–Wallis non-parametric test was used, and results are expressed as median (first quartile, third quartile) [M (P25, P75)], followed by Dunn’s post hoc test for pairwise comparisons. Correlations between serum CORT levels and behavioral, neurochemical, and neuroinflammatory outcomes were assessed using Spearman’s rank correlation. A two-sided P < 0.05 was considered statistically significant. For all parametric tests, F-values, degrees of freedom, and exact P-values are reported; for non-parametric tests, the test statistic and exact P-values are reported.

Results

Chemical Constituents of AS Identified by UPLC-QTOF-MS

A total of 39 monomeric compounds were identified in AS by UPLC-QTOF-MS (two of which were detected in both positive and negative ion modes). The corresponding mass spectra acquired in positive ion mode are shown in Figures S1–S34), and those in negative ion mode are presented in Figures S35–S41). The overall results are displayed in Figure 1, Tables 1 and 2. These compounds comprised 11 terpenoids, 5 phenolic acids, 5 flavonoids, 5 glycosides, 3 phenols, 2 fatty acids, 2 triterpenoids, and one each of amides, coumarins, lignans, organic acids, steroids, and xanthones. Notably, caffeic acid, benzoic acid, chlorogenic acid, 3-dicaffeoylquinic acid, ferulic acid, isoquercitrin, schisandrin B, isofraxidin, secoisolariciresinol, rutin, gomisin H, magnolin, and stearic acid were present at relatively high levels in AS, suggesting that they may constitute the principal bioactive components responsible for the anxiolytic effects of AS.

Table 1 Positive-Ion Information of AS Determined by UPLC-QTOF-MS (ESI+)

Table 2 Negative-Ion Information of AS Determined by UPLC-QTOF-MS (ESI−)

Figure 1 Qualitative characterization of AS constituents by UPLC-QTOF-MS. (A) Base peak ion chromatogram (BPC) of AS in positive ion mode. (B) BPC of AS in negative ion mode. (C) Chemical structures of all compounds identified.

Effects of AS on Body Weight in Rats

In the present study, Welch’s ANOVA indicated a significant main effect of group on net body weight [F(4, 11.004) = 62.422, P < 0.001]. The Model group exhibited significantly lower net body weight than the Control group (P < 0.001). Relative to the Model group, net body weight increased significantly in both the H-AS and Diazepam groups (P < 0.001) (Figure 2A).

Figure 2 Effects of Acanthopanax senticosus + Schisandra chinensis on anxiety-related outcomes in a conditioned fear-induced anxiety rat model. (A) Body weight change. Welch’s ANOVA: F(4, 11.004) = 62.422, P < 0.001. (B) Representative open-field trajectories. (C) Open-field parameters. ANOVA: total distance, F(4, 25) = 11.902, P < 0.001; grid crossings, F(4, 25) = 17.504, P < 0.001; central time, F(4, 25) = 11.458, P < 0.001; central distance, F(4, 25) = 4.541, P = 0.007. (D) Immobility time in the forced swimming test. ANOVA: F(4, 25) = 7.960, P < 0.001. (E) Elevated T-maze. Repeated-measures ANOVA (Greenhouse-Geisser): avoidance, trial F(1.607, 40.173) = 98.238, P < 0.001, trial × group F(6.428, 40.173) = 7.420, P < 0.001; escape, trial F(2, 50) = 19.136, P < 0.001, trial × group F(8, 50) = 0.331, P > 0.05. (F) Pentobarbital sodium potentiation test. Sleep latency, Welch’s ANOVA: F(4, 12.000) = 6.524, P = 0.005; total sleep time, ANOVA: F(4, 25) = 6.502, P < 0.001. Groups: Control, Model, L-AS, H-AS, Diazepam; n = 6 per group. Data are mean ± SD. P < 0.05, *P < 0.01, **P < 0.001, ***P < 0.001. Abbreviations: L-AS, low-dose AS; H-AS, high-dose AS.

Effects of AS on Anxiety-Like Behaviors in Rats

The open field test (OFT), forced swimming test (FST), and elevated T-maze (ETM) are widely used behavioral paradigms for assessing anxiety-like phenotypes. In this study, these assays were applied to validate the establishment of the state anxiety model and to evaluate the therapeutic effects of AS. Representative locomotor trajectories in the OFT are shown in Figure 2B. One-way ANOVA revealed significant differences among groups in total distance [F(4, 25) = 11.902, P < 0.001], number of grid crossings [F(4, 25) = 17.504, P < 0.001], central-zone time [F(4, 25) = 11.458, P < 0.001], and central-zone distance [F(4, 25) = 4.541, P = 0.007]. Locomotor activity was markedly lower in the Model group than in the Control group, as indicated by a shorter total distance (P < 0.001) traveled and fewer grid crossings (P < 0.001), together with reduced central-zone time (P < 0.001) and distance (P = 0.002). Both the H-AS and Diazepam treatments significantly improved locomotor performance. Specifically, the H-AS treatment led to significant increases in total distance (P < 0.001), grid crossings (P = 0.006), and central-zone time (P = 0.004). Similarly, the Diazepam treatment also significantly increased total distance (P < 0.001), grid crossings (P < 0.001), and central-zone time (P < 0.001) (Figure 2C).

In the FST, one-way ANOVA indicated a significant main effect of group on immobility time [F(4, 25) = 7.960, P < 0.001]. The Model group exhibited a significantly longer immobility time than the Control group (P < 0.001). The L-AS, H-AS, and Diazepam treatments all significantly reduced immobility time compared with the Model group (P = 0.012, P = 0.002, P < 0.001, respectively) (Figure 2D). In the ETM, a significant effect across the three avoidance trials emerged only in Test 3. Repeated measures ANOVA (Greenhouse-Geisser corrected) revealed a significant main effect of trial [F(1.607, 40.173) = 98.238, P < 0.001], along with a significant Trial × Group interaction [F(6.428, 40.173) = 7.420, P < 0.001]. In Test 3, avoidance latency was significantly higher in the Model group than in the Control group (P < 0.001). Relative to the Model group, the L-AS, H-AS, and Diazepam groups all exhibited significantly reduced avoidance latency in Test 3 (all P < 0.001). For the escape trials, repeated measures ANOVA revealed a significant main effect of trial [F(2, 50) = 19.136, P < 0.001], the Trial × Group interaction was not significant [F(8, 50) = 0.331, P > 0.05]. Only in Escape 1 did the Diazepam group show a significant increase in escape latency compared with the Model group (P = 0.016); no other statistically significant differences were observed among groups (Figure 2E).

Effects of AS on Sleep Latency and Duration in Rats

Accumulating pharmacological evidence indicates a close bidirectional relationship between anxiety and sleep. Anxiety can impair sleep initiation and maintenance and reduce overall sleep quality, while insufficient sleep further compromises emotional regulation and exacerbates anxiety symptoms, thereby forming a vicious cycle.^22,23 Based on this rationale, we performed a pentobarbital sodium potentiation test. One-way ANOVA with Welch’s correction revealed significant differences among groups in sleep latency [Welch’s F(4, 12.00) = 6.524, P = 0.005] and total sleep time [F(4, 25) = 6.502, P < 0.001]. Relative to the Control group, the Model group exhibited longer sleep latency (P = 0.007) and shorter total sleep time (P = 0.007). Relative to the Model group, the H-AS group significantly reduced sleep latency (P = 0.018) and increased total sleep time (P = 0.004); the Diazepam group also significantly reduced sleep latency (P = 0.037) and increased total sleep time (P = 0.006). However, no significant changes were observed in the L-AS group (P > 0.05) (Figure 2F).

Effects of AS on Corticosteroids, Inflammatory Cytokines, and Neurotransmitter Levels in Rats

At the end of the experiment, multiple biochemical indices were assessed in rat serum and hippocampal tissue (Figure 3).

Figure 3 Effects of Acanthopanax senticosus + Schisandra chinensis on corticosteroid, inflammatory cytokine, and neurotransmitter levels in a conditioned fear-induced anxiety rat model. (A) Serum ACTH and CORT. ANOVA: ACTH, F(3, 20) = 11.157, P < 0.001; CORT, F(3, 20) = 8.173, P < 0.001. (B) Serum cytokines. IL-1β: KW, P = 0.004; IL-6: ANOVA, F(3, 20) = 5.246, P = 0.008; TNF-α: KW, P = 0.048. (C) Hippocampal cytokines. ANOVA: IL-1β, F(3, 20) = 8.598, P < 0.001; IL-6, F(3, 20) = 6.529, P = 0.003; TNF-α, F(3, 20) = 2.760, P > 0.05. (D) Hippocampal neurotransmitters. DA: Welch’s ANOVA, F(3, 9.539) = 54.899, P < 0.001; ACh: ANOVA, F(3, 20) = 5.911, P = 0.005; Glu: ANOVA, F(3, 20) = 11.537, P < 0.001; NE: ANOVA, F(3, 20) = 7.567, P = 0.001; 5-HT: KW, P < 0.001; GABA: KW, P = 0.003. Groups: Control, Model, H-AS, Diazepam; n = 6 per group. Data are mean ± SD. P < 0.05, *P < 0.01, **P < 0.001, ***P < 0.001. Abbreviation: H-AS, high-dose AS.

Serum stress hormone assays showed that one-way ANOVA revealed significant group effects for both ACTH [F(3, 20) = 11.157, P < 0.001] and CORT [F(3, 20) = 8.173, P < 0.001]. Serum ACTH and CORT levels were significantly higher in the Model group than in the Control group (both P < 0.001). Compared with the Model group, the H-AS group showed significant decreases in ACTH (P = 0.011) and CORT (P = 0.012), and the Diazepam group also exhibited significant decreases in ACTH (P < 0.001) and CORT (P = 0.011) (Figure 3A).

With respect to serum cytokines, Kruskal–Wallis tests revealed significant group effects for IL-1β [M (21.773, 24.803), P = 0.004] and TNF-α [M (55.495, 63.465), P = 0.048], while one-way ANOVA revealed a significant group effect for IL-6 [F(3, 20) = 5.246, P = 0.008]. Serum IL-1β (P = 0.013), IL-16 (P = 0.005) and TNF-α (P = 0.043) levels were significantly higher in the Model group than in the Control group. Relative to the Model group, the H-AS group significantly reduced IL-1β (P = 0.011), and the Diazepam group significantly reduced IL-1β (P < 0.001) and TNF-α (P = 0.007)] (Figure 3B).

In the hippocampus, one-way ANOVA revealed significant group effects for IL-1β [F(3, 20) = 8.598, P = < 0.001], IL-6 [F(3, 20) = 6.529, P = 0.003] and TNF-α[F(3, 20) = 2.760, P > 0.05]. Hippocampal IL-1β (P < 0.001) and IL-6 (P = 0.008) levels were elevated in the Model group relative to the Control group. Compared with the Model group, the H-AS group showed reduced levels of IL-1β (P = 0.012) and IL-6 (P = 0.014), and the Diazepam group also exhibited reduced levels of IL-1β (P = 0.005) and IL-6 (P = 0.006) (Figure 3C).

Regarding hippocampal neurotransmitters, one-way ANOVA with Welch’s correction revealed a significant group effect for DA [Welch’s F(3, 9.539) = 54.899, P < 0.001]. One-way ANOVA revealed significant group effects for ACh [F(3, 20) = 5.911, P = 0.005], Glu [F(3, 20) = 11.537, P < 0.001], and NE [F(3, 20) = 7.567, P = 0.001]. Kruskal–Wallis tests revealed significant group effects for 5-HT [M (353.213, 446.515), P < 0.001] and GABA [M (17.165, 24.275), P = 0.003]. The Model group displayed higher levels of ACh (P = 0.002), Glu (P < 0.001), NE (P = 0.003), and 5-HT (P = 0.002) than the Control group, together with lower DA (P < 0.001) and GABA (P < 0.001) levels. Compared with the Model group, the H-AS group showed a significant increase in DA (P < 0.001) and a marked decrease in 5-HT (P < 0.001). The Diazepam group also showed a significant increase in DA (P < 0.001) and GABA (P = 0.023), and a marked decrease in Glu (P = 0.004), NE (P = 0.003), and 5-HT (P < 0.001) (Figure 3D).

Correlations Between Serum CORT and Behavioral, Neurochemical, and Neuroinflammatory Outcomes

Spearman correlation analysis across all four groups (n = 24) revealed that serum CORT levels were significantly associated with multiple behavioral, neurochemical, and inflammatory parameters (Figure 4). Serum CORT was negatively correlated with body weight (r = −0.646, P < 0.001), open-field total travel distance (r = −0.526, P = 0.008), time spent in central zone (r = −0.616, P = 0.001), distance traveled in central zone (r = −0.572, P = 0.003), number of grid crossings (r = −0.517, P = 0.009), total sleep time (r = −0.549, P = 0.005), hippocampal DA (r = −0.681, P < 0.001), and hippocampal GABA (r = −0.430, P = 0.036). Positive correlations were observed between serum CORT and FST immobility time (r = 0.510, P = 0.011), hippocampal TNF-α (r = 0.534, P = 0.007), and hippocampal ACh (r = 0.458, P = 0.024).

Figure 4 Spearman correlations between serum CORT and behavioral, neurochemical, and neuroinflammatory outcomes. Experimental groups: Control, Model, H-AS, Diazepam; n = 6 per group. *P < 0.05, **P < 0.01, ***P < 0.001.

Effects of AS on Hippocampal Morphology in Rats

To investigate the effects of elevated CORT-associated anxiety on hippocampal structure and neuronal integrity, H&E staining was performed. As shown in Figure 5, the Control group displayed intact hippocampal architecture in the CA1, CA3, and DG regions, with neatly arranged neurons, plump cell bodies, and well-defined layers. In the Model group, marked damage was observed in all three regions. In CA1, neurons were disorganized and some cell bodies appeared shrunken. In CA3, cells were loosely arranged, with chromatin condensation and degeneration, focal structural disruption, and inflammatory cell infiltration. In DG, granule cells showed irregular alignment, blurred lamination, reduced cell density, and darker staining, accompanied by varying degrees of inflammatory responses. Relative to the Model group, the H-AS group showed a clear attenuation of pathological alterations. In CA1, neuronal arrangement was relatively regular and cellular morphology was largely preserved. In CA3, cellular organization and structural integrity were improved, with a marked reduction in inflammatory cells. In DG, granule cells tended to be more orderly, with increased density and reduced inflammatory changes. The Diazepam group showed recovery closer to that of the Control group, with well-organized neurons, clear structure, mild inflammation, and an overall morphology approaching normal.

Figure 5 Effects of Acanthopanax senticosus + Schisandra chinensis on hippocampal morphology in a conditioned fear-induced anxiety rat model. Scale bars, 50 μm. High-dose Acanthopanax senticosus + Schisandra chinensis: H-AS. Experimental groups: Control, Model, H-AS, Diazepam; n = 6 per group.

Effects of AS on Hypothalamic CRH Protein Expression in Rats

Immunohistochemical results are shown in Figure 6A and B. One-way ANOVA revealed a significant group effect for CRH MOD in the PVN [F(3, 20) = 5.449, P = 0.007], whereas no significant group differences were observed in the SCN (Kruskal–Wallis test, M (0.090, 0.110), P = 0.442) and LH [F(3, 20) = 0.419, P = 0.741]. Compared with the Control group, the Model group exhibited significantly higher CRH MOD in the PVN (P = 0.010). Relative to the Model group, CRH MOD in the PVN was significantly decreased in both the H-AS (P = 0.036) and Diazepam groups (P = 0.018). No significant changes were observed in the SCN or LH across groups (P > 0.05).

Figure 6 Effects of AS (Acanthopanax senticosus + Schisandra chinensis) on hypothalamic CRH expression in a conditioned fear anxiety model. (A) Representative CRH immunohistochemistry in the PVN, SCN, and LH. (B) Quantitative analysis of CRH expression (mean optical density, MOD). (C–E) Percentage change of CRH expression: (C) Model vs. Control, (D) H-AS vs. Model, (E) Diazepam vs. Model. High-dose Acanthopanax senticosus + Schisandra chinensis: H-AS. Experimental groups: Control, Model, H-AS, Diazepam; n = 6 per group. Data are mean ± SD. *P < 0.05, ***P < 0.001.

For within-group comparisons among brain regions, a repeated measures ANOVA with Region (PVN, SCN, LH) as the within-subject factor and Group as the between-subject factor was conducted. Mauchly’s test indicated a violation of sphericity (P < 0.001), and Greenhouse-Geisser correction was applied. A significant main effect of Region was found [F(1.266, 25.328) = 46.937, P < 0.001], along with a significant Region × Group interaction [F(3.799, 25.328) = 3.405, P = 0.025]. Post hoc pairwise comparisons with Sidak correction showed that in the Control group, CRH MOD did not differ significantly among the three brain regions (all P > 0.05). In the Model group, the PVN exhibited significantly higher CRH MOD than both the SCN and LH (both P < 0.001). In the H-AS group, the PVN was significantly higher than the SCN (P = 0.016) and LH (P = 0.030). In the Diazepam group, the PVN was significantly higher than the SCN (P = 0.047), but the difference between the PVN and LH was not statistically significant (P > 0.05). Although the regional expression pattern varied slightly among groups, the PVN consistently displayed the highest CRH MOD, confirming its role as the primary site of CRH protein expression (Figure 6B–E). Together, these data suggest that elevated CRH is a component of HPA-axis dysfunction, and that AS may improve HPA-axis abnormalities by suppressing CRH signaling dynamics and regulatory plasticity.

Effects of AS on Hippocampal MR and GR Protein Expression in Rats

Immunohistochemistry indicated that MR protein expression in the hippocampal CA1, CA3, and DG regions showed a downward trend in the Model group relative to the Control group. One-way ANOVA revealed no significant group differences in CA1 MR [F(3, 20) = 1.413, P > 0.05], CA3 MR [F(3, 20) = 0.201, P > 0.05] and DG MR [F(3, 20) = 2.981, P > 0.05]. Relative to the Model group, MR expression in these hippocampal subregions tended to increase in the H-AS and Diazepam groups, but the changes did not reach statistical significance (P > 0.05) (Figure 7A and C). In contrast, GR protein expression was significantly altered. One-way ANOVA revealed significant group effects for GR MOD in CA1 [F(3, 20) = 10.565, P < 0.001] and DG [F(3, 20) = 6.316, P = 0.003], whereas no significant group difference was observed in CA3 [F(3, 20) = 1.831, P > 0.05]. Relative to the Control group, GR MOD decreased in the hippocampal CA1 (P < 0.001) and DG (P = 0.003) regions in the Model group. Treatment with H-AS increased GR expression in both CA1 (P = 0.018) and DG (P = 0.044) relative to the Model group. Similarly, treatment with Diazepam also increased GR expression in both CA1 (P < 0.001) and DG (P = 0.020) relative to the Model group, whereas GR MOD in CA3 remained statistically unchanged (P > 0.05) (Figure 7B and D).

Figure 7 Effects of Acanthopanax senticosus + Schisandra chinensis on hippocampal MR and GR protein expression in a conditioned fear-induced anxiety rat model. (A) Representative MR immunoreactivity in CA1, CA3, and DG. Scale bars, 50 μm. (B) Representative GR immunoreactivity in CA1, CA3, and DG. Scale bars, 50 μm. (C) MR expression. ANOVA: CA1, F(3, 20) = 1.413, P > 0.05; CA3, F(3, 20) = 0.201, P > 0.05; DG, F(3, 20) = 2.981, P > 0.05. (D) GR expression. ANOVA: CA1, F(3, 20) = 10.565, P < 0.001; DG, F(3, 20) = 6.316, P = 0.003; CA3, F(3, 20) = 1.831, P > 0.05. Groups: Control, Model, H-AS, Diazepam; n = 6 per group. Data are mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. Abbreviation: H-AS, high-dose AS.

Discussion

The results suggest that AS may dose-dependently ameliorate anxiety- and depression-like behaviors and partially normalize sleep parameters in rats with state anxiety. These effects were accompanied by decreased serum CORT and ACTH, lowered levels of the inflammatory mediators IL-1β, IL-6, and TNF-α, and altered hippocampal neurotransmitters DA and 5-HT. AS was also correlated with decreased hypothalamic CRH and partial restoration of hippocampal MR and GR balance, which may contribute to improved HPA axis regulation. Overall, these findings indicate that AS potentially exerts anxiolytic effects.

Using UPLC-QTOF-MS analysis, several representative constituents of the formulation were identified, including caffeic acid, chlorogenic acid, ferulic acid, isoquercitrin, and schisandrin B. These compounds may constitute the material basis underlying the anxiolytic effects of AS. Previous studies have reported that caffeic acid exerts neuroprotective effects in chronic stress-induced anxiety rats.²⁴ Building on evidence that Acanthopanax senticosus has anxiolytic activity, Miyazaki²⁵ et al further identified chlorogenic acid as an active anxiolytic constituent of Acanthopanax senticosus. Ferulic acid has also been proposed to have anxiolytic potential, potentially involving the serotonergic (5-HT) system.²⁶ In addition, numerous studies have documented anxiolytic activity for flavonoids and terpenoids, which further supports our findings.^27,28

The model rats displayed anxiety- and depression-like phenotypes-body weight loss, decreased motor activity, prolonged immobility in the forced swim test, and sleep disruption-consistent with our previous work.²⁹ Diazepam alleviated these abnormalities, while H-AS also improved behavior and sleep, indicating potential dose-dependent anxiolytic and antidepressant effects. In the present study, the L-AS group showed partial restoration of body weight without full normalization of anxiety-like behaviors, indicating a dissociation between physiological and behavioral recovery. This pattern is consistent with prior reports in chronic stress models, where physiological measures (eg, body weight, HPA-axis indices) can recover earlier or independently of behavioral measures.³⁰ Therefore, body weight change should be considered a complementary marker of systemic recovery rather than a specific indicator of anxiolytic efficacy in this model. Behaviorally, L-AS improved some active-coping measures, but did not significantly affect inhibition-related measures. Such domain-specific responsiveness is frequently observed in anti-psychopharmacology,³¹ suggesting that L-AS produces partial, measure-dependent anxiolytic effects-potentially enhancing active stress-coping before fully reversing anxiety-related behavioral inhibition. Consistent with our results, Jung³² reported that standardized Acanthopanax senticosus extract attenuated stress-induced behavioral abnormalities, and Chen³³ et al showed that Schisandra lignans mitigated stress-induced anxiety-like behaviors. These observations support the view that AS can help improve motor activity and reduce avoidance in anxiety-like rats.

In this study, rats in the anxiety model group exhibited prolonged sleep latency and reduced total sleep time, consistent with anxiety-related insomnia. These sleep disturbances were alleviated following AS intervention. This sleep-promoting effect may involve both HPA axis normalization and GABAergic modulation. The HPA axis is closely linked to circadian rhythmicity, and disruption of glucocorticoid oscillations can impair sleep architecture.^10,34 Previous studies indicate that Acanthopanax senticosus extract attenuates HPA axis responses to stress, reducing serum corticosterone and hippocampal/hypothalamic c-Fos activation,³⁵ while Schisandra chinensis suppresses stress-induced hypothalamic CRH and peripheral corticosterone elevations.³⁶ In parallel, schisandrin B, a major lignan of Schisandra chinensis, has been shown to promote sleep by upregulating GABAA receptor expression and increasing the GABA/Glu ratio.³⁷ These findings suggest that the sleep-promoting effects of AS likely involve both HPA axis recovery and GABAergic enhancement, consistent with the relationship between circadian cortisol rhythmicity and sleep architecture.¹⁰

The present study showed that serum CORT and ACTH levels were elevated in anxious rats, accompanied by increased IL-1β, IL-6, and TNF-α in serum and hippocampus, decreased hippocampal DA, and increased 5-HT. These findings are consistent with HPA axis hyperactivation and a pro-inflammatory state, which were partially attenuated following AS treatment. ACTH, secreted by the anterior pituitary, stimulates glucocorticoid synthesis in the adrenal cortex,³⁸ and elevated CORT is closely linked to anxiety states.^39,40 Glucocorticoids can also modulate inflammatory responses.⁴¹ Pro-inflammatory cytokines, normally low, increase under pathological conditions and can cross the blood-brain barrier to act on circumventricular organs.^42–44

Prior evidence indicates that bioactive constituents in Acanthopanax senticosus, including flavonoids and lignans, can modulate the neuroendocrine-immune axis,⁴⁵ and schisandrin B from Schisandra chinensis suppresses pro-inflammatory mediators and alleviates neuroinflammation.⁴⁶ Multiple neurotransmitters contribute to anxiety-related behaviors, and aberrant neurotransmitter profiles often accompany inflammatory changes.^47,48 DA and 5-HT are key modulators of anxiety,^49,50 and our findings suggest that AS may alleviate anxiety, at least in part, through modulation of these systems, potentially linked to improved neuroendocrine function. Central serotonergic neurotransmission regulates emotional behavior in rodents.⁵¹ Liu⁵² et al reported that the Ziziphi Spinosae Semen-Schisandra chinensis herbal pair alleviated anxiety by regulating 5-HT and DA levels, which is consistent with our findings. Using MALDI-MSI, Zheng⁵³ et al demonstrated that Acanthopanax senticosus increased DA levels in the mouse brain. Together, these previous reports provide support for the present findings.

The correlation analysis showed that elevated serum CORT was associated with reduced central-zone exploration and increased immobility in the forced swimming test, consistent with evidence that glucocorticoid elevation promotes anxiety-like behaviors.⁵⁴ Serum CORT was negatively correlated with hippocampal DA, the strongest association observed, which is consistent with findings that acute stress decreases hippocampal DA levels in rats.⁵⁵ Serum CORT was also negatively correlated with hippocampal GABA and positively correlated with hippocampal ACh, consistent with evidence that stress reduces hippocampal GABAergic tone and activates septo-hippocampal cholinergic pathways.^56,57 Serum CORT was also positively correlated with hippocampal TNF-α, in line with reports that glucocorticoids can enhance hippocampal pro-inflammatory responses.⁵⁸ These correlations are consistent with a role of CORT in linking HPA axis hyperactivation to the behavioral and neurochemical changes observed in this anxiety model.

The model rats exhibited disorganized neuronal arrangement in the hippocampal CA1, CA3, and DG regions, with shrunken cell bodies, intensified staining, degenerative necrosis, and inflammatory cell infiltration, indicating marked hippocampal injury. AS intervention largely restored structural integrity, reduced inflammatory cells, and alleviated pathological damage, suggesting protective effects against stress-related hippocampal injury. Mechanistically, transient CORT elevation facilitates stress adaptation, but prolonged elevation may be neurotoxic, inducing neuronal degeneration, apoptosis, suppressed neurogenesis, and impaired GR function, which weakens HPA-axis negative feedback and creates a “GC elevation-hippocampal injury-feedback dysregulation” cycle.^59–61 Inflammatory cytokines (IL-1β and IL-6) were also elevated in the model group, consistent with H&E findings, indicating a persistent inflammatory state that may exacerbate neural injury.^62,63 Previous studies show that Acanthopanax senticosus extracts and Gomisin J from Schisandra chinensis can alleviate neural damage, suppress neuroinflammation, and restore neuronal numbers,^64–66 aligning with our observations. The coexistence of hippocampal neuroinflammation, sleep disturbances, and HPA axis hyperactivation is consistent with emerging evidence implicating the glymphatic system in the sleep-neuroinflammation-hippocampal integrity axis in psychiatric disorders.^67–69 Although glymphatic function was not directly assessed, the observed impairments suggest a potential integrative pathway linking these pathophysiological features, providing a direction for future investigation.

The results showed that CRH protein expression was increased in the hypothalamic PVN of model rats, while hippocampal GR expression was downregulated. This pattern may reflect impaired HPA axis negative-feedback regulation. Following AS intervention, CRH protein expression was reduced and hippocampal GR protein expression was restored, suggesting that AS may influence HPA axis negative-feedback function. The hippocampus contributes to negative-feedback control of the HPA axis, and hippocampal GR expression is thought to affect the sensitivity of this feedback.⁷⁰ Reduced hippocampal GR has been observed in anxiety model animals, and this reduction has been associated with impaired CORT-mediated feedback inhibition and persistent CRH elevation.^71,72 In the present study, GR-positive cell density was lower in the CA1 and DG regions of model rats, which may be related to the sustained HPA-axis activation observed in this group. MR expression did not differ significantly among groups. One possible explanation is that MR is largely occupied under basal conditions due to its high affinity for low CORT concentrations and is therefore less sensitive to hormonal fluctuations.⁷³ GR, by contrast, is more responsive to elevated CORT levels and may better reflect the functional status of HPA-axis feedback in this anxiety model.⁷⁴ AS intervention reduced CRH protein expression in the PVN and restored hippocampal GR expression, raising the possibility that AS may enhance feedback inhibition of CRH through GR upregulation. It should be noted that the restoration of hippocampal GR protein observed after 7-day AS treatment likely reflects an acute-to-subacute regulatory response. GR expression in the hippocampus is rapidly modulated by stress and circulating glucocorticoids; acute stress decreases GR mRNA within hours, and chronic stress effects on GR mRNA are first detectable at 7 days.⁷⁵ A week-long treatment with escitalopram has also been shown to increase GR protein and decrease CRH expression and circulating corticosterone in stress-exposed rats,⁷⁶ indicating that GR function can respond to pharmacological intervention over short timescales.

Despite the integrative design, this study has several limitations. First, the modest sample size (n=6 per group) reduce the statistical power to detect smaller but potentially meaningful effects and may increase the risk of Type II error. Future studies with larger sample sizes are needed to confirm and extend these findings. Second, while the protein and serum data are consistent with the involvement of hippocampal HPA axis and neuroinflammatory pathways, they do not establish mechanistic causality. Integrating longitudinal behavioral assessment with multi-omics analyses may help establish links between molecular alterations and anxiety behaviors. Further molecular validation using targeted approaches would help clarify the sequence of events. Third, the study used only male rats. Given well-documented sex differences in HPA-axis reactivity, GR expression, and anxiety-like behavior, the generalizability of the findings to females remains uncertain. Future experiments should include both sexes to determine whether the observed anxiolytic effects are sex-dependent. Fourth, although UPLC-QTOF-MS identified several bioactive constituents in the AS decoction, including caffeic acid, chlorogenic acid, ferulic acid, isoquercitrin, and schisandrin B, whether these constituents reach the CNS at pharmacologically relevant concentrations following oral gavage remains unclear. Future pharmacokinetic studies are needed to address this gap.

Conclusion

Taken together, the findings demonstrate that Acanthopanax senticosus plus Schisandra chinensis produces dose-dependent improvements in state-anxiety rats, including the alleviation of anxiety- and depression-like behaviors, shortened sleep latency, and extended sleep duration. These behavioral benefits were accompanied by reduced serum CORT and ACTH, alterations in inflammatory cytokine profiles (IL-1β, IL-6, and TNF-α), regulation of neurotransmitter levels (DA and 5-HT), attenuation of hippocampal pathological damage, downregulation of hypothalamic CRH expression, and restoration of hippocampal GR protein levels. Collectively, these observations suggest that AS may exert anxiolytic effects, at least in part, through restoration of hippocampus-HPA axis negative feedback regulation. This study provides new insights into the anxiolytic potential of the traditional Chinese medicine herb pair Acanthopanax senticosus and Schisandra chinensis, and offers a theoretical foundation for the further development of this combination as an anti-anxiety preparation.