Soyasaponin Bb Attenuates Morphine Withdrawal-Induced Anxiety-Like Behaviors in Male Mice by Rescuing GSK-3β/Nrf2 Signaling

Jie Wu; Chenchen Zhu; Yin Shu; Yongli Lu; Lu Liu; Zhigang Xiao; Zicheng Li

doi:10.30773/pi.2025.0091

Psychiatry Investig > Volume 22(7); 2025 > Article

Wu, Zhu, Shu, Lu, Liu, Xiao, and Li: Soyasaponin Bb Attenuates Morphine Withdrawal-Induced Anxiety-Like Behaviors in Male Mice by Rescuing GSK-3β/Nrf2 Signaling

Original Article

Psychiatry Investigation 2025;22(7):821-831.

Published online: July 16, 2025

DOI: https://doi.org/10.30773/pi.2025.0091

Soyasaponin Bb Attenuates Morphine Withdrawal-Induced Anxiety-Like Behaviors in Male Mice by Rescuing GSK-3β/Nrf2 Signaling

Jie Wu^1,^2,^*

, Chenchen Zhu^1,^2,^*

, Yin Shu^1,^2,^*

, Yongli Lu^1,²

, Lu Liu^1,²

, Zhigang Xiao³

, Zicheng Li^1,²

¹College of Basic Medical Science, China Three Gorges University, Yichang, China

²Third-grade Pharmacological Laboratory on Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang, China

³Yichang Mental Health Center, Yichang, China

Correspondence: Zicheng Li, PhD Third-grade Pharmacological Laboratory on Traditional Chinese Medicine, State Administration of Traditional Chinese Medicine, China Three Gorges University, Yichang 443002, China Tel: +86-7176484962, E-mail: zichengli@ctgu.edu.cn

^* These authors contributed equally to this work.

Received March 15, 2025 Revised April 23, 2025 Accepted May 18, 2025

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Objective

To examine the effect of Soyasaponin Bb on morphine withdrawal-induced anxiety-like behaviors in male mice and the corresponding mechanism.

Methods

The chronic intermittent escalating-dose morphine administration paradigm and spontaneous withdrawal were used to induce anxiety-like behaviors in male mice. Open field test (OFT) and elevated plus maze test (EPM) were used to evaluate anxiety-like behaviors. Soyasaponin Bb was intragastrically administered. Western blot was used to monitor the level of GSK-3β, Nrf2, and superoxidase dismutase in the basolateral amygdala (BLA). Virus-mediated gene overexpression and knockdown were used to manipulate the expression of GSK-3β and Nrf2 in the BLA. Two-way analysis of variance followed by Tukey’s test were used to make comparisons for the experiments between four groups. Student’s t-test was used to make comparisons for the experiments between two groups.

Results

Intragastric administration of Soyasaponin Bb attenuated morphine withdrawal-induced anxiety-like behaviors in male mice. Soyasaponin Bb rescued GSK-3β/Nrf2 signaling in the BLA in morphine withdrawn mice. Overexpression of GSK-3β in the BLA was sufficient to induce anxiety-like behaviors in male mice. Knockdown of GSK-3β in the BLA attenuated anxiety-like behaviors in male mice. Knockdown of Nrf2 in the BLA was sufficient to induce anxiety-like behaviors in male mice.

Conclusion

Soyasaponin Bb could play anxiolytic effect by regulating GSK-3β/Nrf2 signaling and inhibition of GSK-3β in the BLA may represent a novel therapeutic approach to morphine addiction and anxiety disorders.

Keywords: Morphine withdrawal, GSK-3β, Basolateral amygdala, Anxiety

INTRODUCTION

Opioid use disorder (OUD) is a complex relapsing disorder following repeated opioid exposure. Despite our increasing knowledge about the pathophysiology and existing medical treatments of OUD, it has remained a relapsing disorder for decades, with rising deaths rather than declining [1]. According to the World Drug Report 2023, there were about 60 million people living with OUD worldwide in 2021. Therefore, it is crucial to develop new targets to treat OUD.

It has been proposed that opioid withdrawal-induced anxiety is apt to play motivational roles in opioid relapse [2]. It also has been reported that oxidative stress is involved in the pathophysiology of anxiety [3] and addiction [4] and antioxidant supplementation plays protective roles in anxiety disorders [5]. However, it remains unclear whether antioxidant treatment has protective effect on morphine withdrawal-induced anxiety. Furthermore, it has been reported that the glycogen synthase kinase 3β (GSK-3β) is involved in anxiety [6] and oxidative stress [7]. Nevertheless, the role of GSK-3β-mediated oxidative signaling pathways in anxiety induced by morphine withdrawal remains to be elucidated.

Soyasaponin Bb is a triterpenoid saponin that has been found in soy and has been reported to play antioxidative effect [8]. However, it is elusive whether and how Soyasaponin Bb rescues morphine withdrawal-induced anxiety by regulating antioxidative signaling.

In the present study, the effect of Soyasaponin Bb on morphine withdrawal-induced anxiety was first explored. Then the possible roles of GSK-3β/nuclear factor-erythroid 2-related factor 2 (Nrf2)/superoxidase dismutase (SOD) pathway involved in the protective effect of Soyasaponin Bb in morphine withdrawal-induced anxiety was tested. Finally, the role of virus-mediated manipulation of GSK-3β/Nrf2 pathway in anxiety was studied.

METHODS

Animals

Male C57BL mice, aged 7 to 8 weeks upon arrival, were utilized for the study. The mice were housed in a temperature, noise and humidity well-controlled room and maintained on a standard 12-hour light/dark cycle (lights on 7:00 AM-7:00 PM) with food and water ad libitum. All efforts were made to minimize animal suffering and to reduce the number of animals used. All animal experiments complied strictly with the Guidelines for the Care and Use of Laboratory Animals of CTGU under approval by the Ethics Committee (number of approval: No. 42010200008724).

Chronic intermittent escalating-dose morphine administration paradigm and withdrawal

The mice were randomly assigned to two groups: one group received chronic saline treatment and the other group received chronic morphine treatment. They were given intraperitoneal injections (i.p.) of either saline or morphine (Northeast Pharmaceutical Group Shenyang NO.1 Pharmaceutical Co., LTD), following a chronic, intermittent, escalating-dose regimen (starting with 20 mg/kg on the first day, increasing to 40 mg/kg on the second and third day, 80 mg/kg on the fourth and fifth day, and culminating at 100 mg/kg on the sixth and seventh day). The injections were administered twice daily at 8:00 AM and 4:00 PM, following a protocol previously published with slight modifications [9]. The animals were allowed to withdraw spontaneously in their home cages without any further injections.

Behavioral tests

The room temperature, illumination, humidity and noise in the testing room were well-controlled. The behaviors of mice were tracked with the automated video-tracking system and recorded for 5 minutes.

Open field test

The open field is a square area, made of 40×40×40 cm transparent plexiglass without a top box. Each animal was gently placed in the upper right corner of the open field, allowing the mice to move freely. The entries across the center, the time spent in the center, and the total distance traveled were measured by a software (TopScan Lite, CleverSys Inc). The intensity of light was 10 lux.

Elevated plus maze test

The maze consists of a plus-shaped apparatus with two open arms (30 cm length×5 cm width) that have small walls (0.5 cm in height), two closed arms (30 cm length×5 cm width) that have high walls (30 cm in height) and a center platform (5×5 cm) kept on a stand whose height is around 40 cm. The mice were placed in the central area with their heads facing the open arm (all animals were placed in the same direction), and the mice were allowed to move freely. The software (TopScan Lite, CleverSys Inc) was used to record the number of entries into the center, the duration spent in the center, and the total distance covered by the mouse. The lighting intensity was set to 10 lux.

Virus-mediated manipulation of GSK-3β and Nrf2 in the basolateral amygdala

To investigate the impact of GSK-3β and Nrf2 suppression in the basolateral amygdala (BLA) on anxiety-like behaviors in mice, mice were treated with intra-BLA microinjections of pAAV-U6-shRNA (Gsk3b)-CMV-EGFP-WPRE, pAAV-U6-ShRNA (Nrf2)-CMV-EGFP-WPRE, or control virus (OBiO Technology). Briefly, the AAV vectors (0.3 μL per side) were delivered bilaterally into the BLA region (AP=-1.45 mm, ML=±3.45 mm, and DV=-4.9 mm) at 0.1 μL/min by micro syringe in a stereotactic apparatus, followed by 5-10 minutes of rest to allow diffusion. Behavioral tests were done 4 weeks following viral injection.

To examine the effect of the overexpression of GSK-3β in the BLA on the anxiety-like behaviors in normal mice, intra-BLA microinjection of pAAV-CMV-GSK-3β-3xFlag-EF1a-EGFP or control virus (OBiO Technology) was applied. Briefly, the AAV vectors (0.3 μL per side) were delivered bilaterally into the BLA region (anterior-posterior [AP]=-1.45 mm, middle-lateral [ML]=±3.45 mm, dorsal-ventral [DV]=-4.9 mm) at 0.1 μL/min by micro syringe in a stereotactic apparatus, followed by 5-10 minutes of rest to allow diffusion. Behavioral tests were done 4 weeks following viral injection.

After the behavioral tests, mice were subjected to intracardiac perfusion, and their brains were sectioned for analysis. Fluorescence microscopy and western blotting were employed to evaluate the efficiency of in vivo transfection.

Western blot

Following elevated plus maze test (EPM) test, the mice were euthanized and sacrificed. The brain tissue from the BLA was dissected and cryopreserved at -80°C for subsequent analysis.

In order to detect the change of GSK-3β, p-GSK-3β (S9), Nrf2, and SOD in the BLA, the total protein fraction was extracted by using the RIPA lysis and extraction buffer (89901, Thermo Fisher) and the nuclear protein fraction was extracted by Subcellular Protein Fractionation Kit for Tissues (87790, Thermo Fisher).

Proteins were quantified using Bradford assay (Bio-Rad Laboratories) and 30 μg per sample were loaded on 10% SDS-PAGE gel and transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Bio-Rad Laboratories) to perform western blot analysis. The blots were blocked with 5% skimmed milk for 1 hour at room temperature and then incubated with GSK-3β (1:1,000, 701102, Thermo Fisher), p-GSK-3β (S9) (1:1,000, PA5-104555, Thermo Fisher), Nrf2 (1:1,000, 16396-1-AP, Protein-tech), SOD (1:1,000, 10269-1-AP, Proteintech), and GAPDH (1:1,000, A19056, ABclonal) polyclonal antibody for 12 hours at 4°C. Following extensive washing, membranes were then incubated with goat anti-rabbit horseradish peroxydase-conjugated secondary antibody (1:3,000, AS014, ABclonal) for 1 hour at room temperature. The washing was repeated. Protein blots were detected using a chemiluminescence detection kit (G2014, Servicebio) and analyzed quantitatively by densitometry with Image J (National Institutes of Health).

Chemical and preparation

Soyasaponin Bb (PCS1429, Chengdu Herb Substance Co., Ltd) was dissolved in 2% Tween-80 and the concentration of working solution was 1 nM. For intragastric administration, the dosage was 10 mg/kg.

Data analysis and statistics

Data were statistically analyzed by Image J and GraphPad Prism 8.0.2. Two-way analysis of variance (ANOVA) followed by Tukey’s test was used to make comparisons for the behavioral experiments between four groups. Student’s t-test was used to make comparisons for other experiments. Data were presented as mean±standard error of the mean (SEM). p<0.05 was considered statistically significant. In our lab, the male mice showed stable and obvious anxiety-like behaviors on day 3 and day 4 after last injection of morphine. Therefore, the open field test (OFT) and EPM were conducted on day 3 and day 4, respectively after last injection of morphine in this study.

RESULTS

Intragastric administration of Soyasaponin Bb attenuated morphine withdrawal-induced anxiety-like behaviors in male mice

To test whether Soyasaponin Bb has protective effect on morphine abstinence-induced anxiety-like behaviors, the effect of intragastric administration of Soyasaponin Bb was studied. Soyasaponin Bb was dissolved in 2% Tween-80. Mice were divided into 4 groups: saline withdrawal+vehicle (N+T) (n=15), saline withdrawal+Soyasaponin Bb (N+S) (n=15), morphine withdrawal+vehicle (M+T) (n=15), and morphine withdrawal+Soyasaponin Bb (M+S) (n=15). Soyasaponin Bb was administered at 10 mg/kg. The procedure of the experiment and data are shown in Figure 1.

In the OFT, the two-way ANOVA revealed significant differences for the morphine treatment (total distance: F_{(1, 56)}=8.055, p=0.006; duration in the centre: F_{(1, 56)}=2.939, p=0.092; faeces: F_{(1, 56)}=5.667, p=0.021), the Soyasaponin Bb treatment (total distance: F_{(1, 56)}=3.174, p=0.080; duration in the centre: F_{(1, 56)}=3.636, p=0.062; faeces: F_{(1, 56)}=15.24, p<0.001) and morphine treatment×Soyasaponin Bb treatment interaction (total distance: F_{(1, 56)}=6.442, p=0.014; duration in the centre: F_{(1, 56)}=6.309, p=0.015; faeces: F_{(1, 56)}=12.41, p<0.001).

Post-hoc analysis confirmed that Soyasaponin Bb significantly increased the center duration (p=0.021) and total distance (p=0.002) in M+S mice compared with M+T mice. Post-hoc analysis also confirmed that Soyasaponin Bb was effective in decreasing the faeces (p<0.001) in M+S mice compared with M+T mice.

In the EPM, the two-way ANOVA revealed significant differences for the morphine treatment (total distance: F_{(1, 56)}=1.966; p=0.166; duration in open arms: F_{(1, 56)}=3.891, p=0.054; open arm entries: F_{(1, 56)}=1.750, p=0.191), the Soyasaponin Bb treatment (total distance: F_{(1, 56)}=4.453, p=0.039; duration in open arms: F_{(1, 56)}=3.435, p=0.069; open arm entries: F_{(1, 56)}=2.968, p=0.090), and morphine treatment×Soyasaponin Bb treatment interaction (total distance: F_{(1, 56)}=5.518, p=0.022; duration in open arms: F_{(1, 56)}=3.767, p=0.057; open arm entries: F_{(1, 56)}=6.209, p=0.016).

Post-hoc analysis confirmed that Soyasaponin Bb was effective in increasing total distance (p=0.049), the duration in open arms (p=0.037), and open arm entries (p=0.044) in M+S mice compared with M+T mice.

Soyasaponin Bb rescued GSK-3β/Nrf2 signal pathway in the BLA in morphine withdrawn mice

To test whether oxidative stress plays important roles in morphine withdrawal-induced anxiety-like behaviors and whether Soyasaponin Bb has an antioxidative effect, the role of Soyasaponin Bb on GSK-3β/Nrf2 signal pathway was studied. The experimental procedure and data were shown in Figure 2.

The two-way ANOVA revealed significant differences for the morphine treatment (GSK-3β: F_{(1, 12)}=2.296, p=0.156; p-GSK-3β: F_{(1, 12)}=3.208, p=0.099; Nrf2: F_{(1, 16)}= 6.794, p=0.019; nuclear Nrf2: F_{(1, 12)}=8.400, p=0.013; SOD: F_{(1, 12)}= 5.186, p=0.042), the Soyasaponin Bb treatment (GSK-3β: F_{(1, 12)}=0.852, p=0.117; p-GSK-3β: F_{(1, 12)}=21.40, p<0.001; Nrf2: F_{(1, 16)}=0.1117, p=0.743; nuclear Nrf2: F_{(1, 12)}=9.148, p=0.010; SOD: F_{(1, 12)}=6.350, p=0.027), and morphine treatment×Soyasaponin Bb treatment interaction (GSK-3β: F_{(1, 12)}=9.723, p=0.009; p-GSK-3β: F_{(1, 12)}=6.517, p=0.025; Nrf2: F_{(1, 16)}=14.82, p=0.001; nuclear Nrf2: F_{(1, 12)}=3.133, p=0.102; SOD: F_{(1, 12)}=4.282, p=0.061).

Post-hoc analysis confirmed that Soyasaponin Bb significantly decreased total GSK-3β (p=0.029), and increasing p-GSK-3β (p=0.042), Nrf2 (p=0.002), nuclear Nrf2 (p=0.028), and SOD (p=0.042) in M+S mice compared with M+T mice.

At the same time, there was no significant effect of morphine and Soyasaponin Bb treatment on Heme oxygenase (HO-1) and NAD(P)H dehydrogenase, quinone 1 (NQO1) (the data were not shown).

Overexpression of GSK-3β in the BLA induced anxiety-like behaviors in male mice

To test whether upregulated GSK-3β in the BLA is sufficient to induce anxiety-like behaviors, GSK-3β was overexpressed in the BLA of male mice by intra-BLA microinjection of pAAV-CMV-Gsk3b-3xFLAG-EF1a-EGFP-tWPA. The mice were divided into 2 groups: control group (n=14) and GSK-3β overexpression group (OE-GSK-3β) (n=14). The experimental procedure and data are shown in Figure 3.

The t-test analysis revealed that overexpression of GSK-3β in the BLA induced obvious anxiety-like behaviors in mice (in OFT: total distance: t₍₂₆₎=0.5797, p=0.567; duration in the centre: t₍₂₆₎=2.572, p=0.016; faeces: t₍₂₆₎=2.620, p=0.015 and in EPM: total distance: t₍₁₈₎=1.354, p=0.193; duration in open arms: t₍₁₈₎=2.227, p=0.039; open arm entries: t₍₁₈₎=2.710, p=0.014).

At the same time, the western blot test revealed that overexpression of GSK-3β in the BLA induced downregulation of Nrf2 (t-test, t₍₆₎=7.067, p<0.001) and SOD (t-test, t₍₆₎=2.596, p<0.001).

The AAV injection site was verified under fluorescence microscope (Supplementary Figure 1).

Knockdown of GSK-3β in the BLA attenuated anxiety-like behaviors in male mice

To confirm whether downregulated GSK-3β in the BLA could reduce anxiety-like behaviors, shRNA-mediated knockdown of GSK-3β in the BLA was done. Mice were divided into 2 groups: shRNA scramble (n=15) and shRNA GSK-3β (pAAV-U6-shRNA [Gsk3b]-CMV-EGFP-WPRE) (n=15). The experimental procedure and data are shown in Figure 4.

The t-test analysis revealed that knockdown of GSK-3β in the BLA played anxiolytic effect in mice (in OFT: total distance: t₍₂₈₎=3.158, p=0.004; duration in the centre: t₍₂₈₎=2.873, p=0.008; faeces: t₍₂₈₎=2.357, p=0.026 and in EPM: total distance: t₍₂₈₎=2.638, p=0.014; duration in open arms: t₍₂₈₎=4.065, p<0.001; open arm entries: t₍₂₈₎=2.528, p=0.017).

At the same time, the Western blot test revealed that knockdown of GSK-3β in the BLA induced upregulation of Nrf2 (t-test, t₍₁₂₎=2.645, p=0.021) and SOD (t-test, t₍₆₎=3.734, p=0.010).

The AAV injection site was verified under fluorescence microscope (Supplementary Figure 2).

Knockdown of Nrf2 in the BLA induced anxiety-like behaviors in male mice

To confirm whether downregulated Nrf2 in the BLA is sufficient to induce anxiety-like behaviors, shRNA-mediated knockdown of Nrf2 in the BLA was done. Mice were divided into 2 groups: shRNA scramble (n=14) and shRNA Nrf2 (pAAV-U6-shRNA (NRF2)-CMV-EGFP-WPRE) (n=15). The experimental procedure and data are shown in Figure 5.

The t-test analysis revealed that knockdown of Nrf2 in the BLA had an anxiogenic effect in mice (in OFT: total distance: t₍₂₇₎=2.066, p=0.049; duration in the centre: t₍₂₇₎=3.364, p=0.002; faeces: t₍₂₇₎=0.7359, p=0.468 and in EPM: total distance: t₍₂₇₎=2.737, p=0.011; duration in open arms: t₍₂₇₎=3.061, p=0.005; open arm entries: t₍₂₇₎=3.335, p=0.003).

At the same time, the western blot test revealed that knockdown of Nrf2 in the BLA induced downregulation of SOD (t-test, t₍₆₎=5.112, p=0.002).

The AAV injection site was verified under fluorescence microscope (Supplementary Figure 3).

DISCUSSION

Here, we presented that intragastric administration of Soyasaponin Bb attenuated morphine withdrawal-induced anxiety-like behaviors in male mice. Moreover, Soyasaponin Bb-mediated attenuation of morphine withdrawal-induced anxiety-like behaviors in male mice is dependent on GSK-3β/Nrf2 signaling, which is supported by the following results: 1) Soyasaponin Bb rescued morphine withdrawal-induced upregulation of GSK-3β and downregulation of Nrf2 in the BLA in male mice. 2) Overexpression of GSK-3β in the BLA was sufficient to induce anxiety-like behaviors and downregulation of NRF2 and SOD in male mice. 3) Downregulation of Nrf2 in the BLA was sufficient to induce anxiety-like behaviors in male mice and downregulation of SOD in male mice. 4) Downregulation of GSK-3β in the BLA attenuated anxiety-like behaviors in male mice and induced upregulation of Nrf2 and SOD.

One important finding in this study is that downregulation of antioxidation system may play critical roles in morphine withdrawal-induced anxiety, which strongly supports the idea that substance use disorder (SUD) could be partially associated with an imbalance in reactive oxygen species and antioxidant defenses [10]. Therefore, antioxidant therapy may be a promising strategy for treating morphine withdrawal-induced anxiety disorders. Importantly, GSK-3β could serve as a significant target for antioxidation therapy.

Another important finding is that Soyasaponin Bb could be a promising candidate in antioxidant therapy. Soyasaponins, natural chemical compounds in legume plants, are composed of soyasapogenol (aglycone) and oligosaccharide moieties. They are generally classified into four subgroups depending on their aglycone (sapogenin) structures, with soyasaponin group A-E and group DDMP [11,12]. Soyasaponin Bb is a representative group B soyasaponin. Soyasaponin Bb has been reported to promote health due to its antioxidative effects [13,14]. Considering wide involvement of oxidant/antioxidant imbalance in pathophysiology in many kinds of disorders such as SUD [10,15], Alzheimer’s disease [16], anxiety [3,17], depression [18] and so on, Soyasaponin Bb could have considerable potential for translational applications in treating neuropsychiatric diseases.

The third important finding is that BLA could be deeply involved in the pathophysiology of morphine withdrawal-induced anxiety mediated by upregulated GSK-3β. These results align with prior research showing that GSK-3β in the BLA is critical for the reconsolidation of cocaine reward memory [19] and inhibition of GSK-3β in the BLA attenuates heroin relapse [20]. Together, GSK-3β in the BLA could be the critical target for anti-addiction treatment.

One limitation is that it is unclear whether the anxiolytic effect of Soyasaponin Bb depends on itself or its metabolites. It has been reported that 11 metabolites of Soyasaponin Bb were first identified from urine, plasma, bile, and feces of rats after intragastric administration of soyasaponin Bb [21]. Hydroxylation and hydrolysis were the major metabolic pathways of soyasaponin Bb in rat. Radiolabeling methods could be further used to trace the distribution of Soyasaponin Bb and its metabolites in the mice brain.

The second limitation is that it is not clear whether other downstream of GSK-3β is responsible for the anxiolytic effect of Soyasaponin Bb except for Nrf2/SOD signaling. The GSK-3 is a highly specific serine/threonine kinase for glycogen synthase. It was isolated in 1980, from rabbit skeletal muscle. There are two isozymes of GSK-3, α and β, and both are expressed at similar levels in the mouse brain [22]. In the human brain, the β isozyme predominates, which suggests that GSK-3β could play crucial roles in brain function [23]. Researches regarding GSK-3β not only demonstrate GSK-3β plays its regulatory effect on oxidative pathway [7,24], but also plays critical roles in physiological synaptic plasticity and aberrant GSK-3β activity contributes to the development of dysfunctional synaptic plasticity in neuropsychiatric and neurodegenerative disorders [23,25]. Therefore, it should be identified whether GSK-3β-mediated synaptic plasticity mechanism is engaged in the anxiolytic effect of Soyasaponin Bb.

The third limitation is that further studies should be done to identify whether other upstream of Nrf2/SOD signaling is responsible for the anxiolytic effect of Soyasaponin Bb except for GSK-3β. It has been reported that p38, PI3K, PKC, ERK, and JNK are critical regulators for Nrf2 [26]. Therefore, it should be identified whether other upstream is regulated by Soyasaponin Bb except for GSK-3β.

Whatever, the present study suggests Soyasaponin Bb exerts anxiolytic effect via its antioxidative activity. GSK-3β/Nrf2/SOD signaling could be the important targets mediating the anxiolytic effect of Soyasaponin Bb.

In conclusion, Soyasaponin Bb is a powerful antioxidant and could be used to treat anxiety disorders induced by morphine withdrawal. The GSK-3β signaling and downstream components of this pathway are disrupted during morphine abstinence and thus are promising targets for anxiolytic therapies for morphine addicts.

Supplementary Materials

The Supplement is available with this article at https://doi.org/10.30773/pi.2025.0091.

Supplementary Figure 1.

The high-fidelity image for fluorescence figure in Figure 3A showing AAV-mediated EGFP expression in the basolateral amygdala.

pi-2025-0091-Supplementary-Fig-1.pdf

Supplementary Figure 2.

The high-fidelity image for fluorescence figure in Figure 4A showing AAV-mediated EGFP expression in the basolateral amygdala.

pi-2025-0091-Supplementary-Fig-2.pdf

Supplementary Figure 3.

The high-fidelity image for fluorescence figure in Figure 5A showing AAV-mediated EGFP expression in the basolateral amygdala.

pi-2025-0091-Supplementary-Fig-3.pdf

Notes

Availability of Data and Material

The datasets generated or analyzed during the study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors have no potential conflicts of interest to disclose.

Author Contributions

Conceptualization: Chenchen Zhu, Zicheng Li. Data curation: Jie Wu, Yin Shu. Formal analysis: Jie Wu, Yin Shu. Funding acquisition: Zicheng Li. Investigation: Jie Wu, Yin Shu. Methodology: Jie Wu, Yin Shu, Chenchen Zhu, Zicheng Li. Project administration: Chenchen Zhu, Zicheng Li. Resources: Zicheng Li, Zhigang Xiao. Supervision: Chenchen Zhu, Zicheng Li. Validation: Yongli Lu, Lu Liu, Zhigang Xiao. Visualization: Jie Wu, Yin Shu. Writing—original draft: Jie Wu, Yin Shu, Chenchen Zhu, Zicheng Li. Writing—review & editing: Yongli Lu, Lu Liu, Zhigang Xiao.

Funding Statement

This work was supported by the National Natural Science Foundation of China (NSFC) (grant numbers 81971248, 31200814) and Open Fund of Yichang City Clinical Research Center for Mental Disorders (YCXL-23-13).

Acknowledgments

None

Figure 1.

Intragastric injection of Soyasaponin Bb alleviated morphine abstinence-induced anxiety-like behaviors. A: Experimental procedure. B: The histogram illustrates total distance, duration in center, faeces, and representative traces of movement in the OFT. C: The histogram illustrates total distance, duration in open arms, entries in open arms, and representative traces of movement in the EPM. All data were presented as the mean±standard error of the mean. N, saline withdrawal; M, morphine withdrawal; T, vehicle; S, Soyasaponin Bb; B4 and C4, N+T group; B5 and C5, N+S group; B6 and C6, M+T group; B7 and C7, M+S group; OFT, open field test; EPM, elevated plus maze test. *p<0.05, ****p<0.001 versus saline group; ^#p<0.05, ^##p<0.01, ^###p<0.001 versus morphine group using two-way analysis of variance followed by Tukey’s test.

Figure 2.

The expression of GSK-3β signaling in the BLA. Representative Western blot and quantification of GSK-3β (A), p-GSK-3β (B), Nrf2 (C), nuclear Nrf2 (D), and SOD (E) in the BLA. All data were presented as the mean±standard error of the mean. N, saline withdrawal; M, morphine withdrawal; T, vehicle; S, Soyasaponin Bb; BLA, basolateral amygdala; SOD, superoxidase dismutase. *p<0.05, **p<0.01 versus saline group; ^#p<0.05, ^##p<0.01 versus morphine group using two-way analysis of variance followed by Tukey’s test.

Figure 3.

Overexpression of GSK-3β in the BLA was sufficient to produce anxiety-like behaviors. A: Experimental procedure. B: The histogram illustrates total distance, duration in center, faeces, and representative traces of movement in the OFT. C: The histogram illustrates total distance, duration in open arms, entries in open arms, and representative traces of movement in the EPM. D: Representative immunoblots and histogram illustrates total protein fraction of Nrf2 and SOD in the BLA after behavior test. Data represent mean±standard error of the mean. OFT, open field test; EPM, elevated plus maze test; SOD, superoxidase dismutase; ns, no significance; BLA, basolateral amygdala. *p<0.05, ***p<0.001 versus control group using Student’s t test.

Figure 4.

shRNA-mediated knockdown of GSK-3β in the BLA reduced anxiety-like behaviors. A: Experimental procedure. B: The histogram illustrates total distance, duration in center, faeces, and representative traces of movement in the OFT. C: The histogram illustrates total distance, duration in open arms, entries in open arms, and representative traces of movement in the EPM. D: Representative immunoblots and histogram illustrates total protein fraction of Nrf2 and SOD in the BLA after behavior test. Data represent mean±standard error of the mean. OFT, open field test; EPM, elevated plus maze test; SOD, superoxidase dismutase; BLA, basolateral amygdala. *p<0.05, **p<0.01, ***p<0.001 versus control group using Student’s t test.

Figure 5.

shRNA-mediated knockdown of Nrf2 in the BLA was sufficient to produce anxiety-like behaviors. A: Experimental procedure. B: The histogram illustrates total distance, duration in center, faeces, and representative traces of movement in the OFT. C: The histogram illustrates total distance, duration in open arms, entries in open arms, and representative traces of movement in the EPM. D: Representative immunoblots and histogram illustrates total protein fraction of SOD in the BLA after behavior test. Data represent mean±standard error of the mean. OFT, open field test; EPM, elevated plus maze test; SOD, superoxidase dismutase; BLA, basolateral amygdala; ns, no significance. *p<0.05, **p<0.01 versus control group using Student’s t test.

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