C57BL/6 pregnant mice, purchased from Southern Medical University (Guangzhou, China) were maintained on a 12-h light/dark cycle with a constant temperature (22°C–24°C). The experiment was conducted in compliance with the Guide for the Care and Use of Laboratory Animals and was approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine, China (approval No. 2021W0050).

To assess the impact of ELS on adolescent depression, we utilized an animal model characterized by maternal separation and early weaning at postnatal day 17 (PND17), as established in previous research (Tchenio et al., 2017). Male C57/BL6 mouse pups were arbitrarily divided into two groups: those in the control group and those subjected to early life stress. The early life stress group was subjected to maternal separation from postnatal days 7–15 (PND7-15) and isolated in a new cage equipped with adequate bedding, water, and food for a duration of 6 h per day, with weaning initiated at PND17. Male pups from this group were randomly selected to constitute the ELS group on postnatal day 21 (PND21). On the other hand, the control group pups were left undisturbed until their regular weaning on PND21, after which male pups were randomly assigned to the control group.

The preparation method and doses calculating are based on our previous (Cao et al., 2019; Deng et al., 2022). SNS, derived from the Shang Han Lun, consists of Chai Hu, Shao Yao, Zhi Shi, and roasted Gan Cao in a 1:1:1:1 ratio. Each herb contributes 6 g to the formula. Both the herbal ingredients and fluoxetine procured from the First Affiliated Hospital of Guangzhou University of Chinese Medicine. The preparation of SNS followed traditional modern clinical practices. The herbs were weighed in equal proportions (1:1:1:1 ratio) and were then coarsely ground. The ground herbs were soaked in distilled water at ten times their weight for 60 min. After bringing the solution to a boil, it was simmered for 40 min. The solution was then allowed to cool and was subsequently filtered through an 8-layer cheesecloth to obtain the filtrate. The process was repeated: the herbs were boiled in 8 times the amount of water, simmered, cooled, and filtered. The filtrates from both rounds were combined. This combined solution was concentrated using a rotary evaporator to achieve a concentration of 1.96 g/mL of the original herb. The solution was then aliquoted and stored at 4°C for further use. Considering an average adult body weight of 60 kg/day for dose conversion, the clinically equivalent dose for mice was determined to be 4.9 g/kg. Using a 1:2:4 scaling, the low, medium, and high doses were set at 4.9 g/kg, 9.8 g/kg, and 19.6 g/kg, respectively. The quality and consistency of the SNS preparation were validated using high performance liquid chromatography (HPLC) according to our previous study (Deng et al., 2022). Fluoxetine, SNS and saline (as a vehicle control) were administered via intragastric route from PND22 to PND42. Following the completion of this treatment, behavioral tests were conducted.

For liquid chromatographic analysis, 1 mL of SNS solution (1.96 g/mL) was mixed with 23 mL of methanol. The mixture was subjected to ultrasonic radiation for 30 min to ensure uniform dissolution. Subsequently, it was filtered using a 0.45 µm microporous membrane to obtain a clear supernatant. Reference standards were prepared for the following compounds: Hesperidin (B20182, Shanghai Yuanye Bio-Technology Co., Ltd., China), Liquiritin (B20414, Shanghai Yuanye Bio-Technology Co., Ltd., China), Glycyrrhizic acid ammonium salt (IG0740, Beijing Solarbio Science and Technology Co., Ltd., China), Gallic acid (B20851, Shanghai Yuanye Bio-Technology Co., Ltd., China), Paeoniflorin (B21148, Shanghai Yuanye Bio-Technology Co., Ltd., China), Neohesperidin (B21390, Shanghai Yuanye Bio-Technology Co., Ltd., China). Each of these compounds was accurately weighed, dissolved in methanol, and then transferred to individual 1 mL volumetric flasks.

The analysis was performed on an Agilent HPLC-1200 System, utilizing a Diamonsil C18 (2) column (150 × 4.6 mm, 5 μm) maintained at 25°C. The mobile phase comprised of Solution A (acetonitrile) and Solution B (0.01 mol/L aqueous phosphoric acid). The solvent gradients were as follows: 0–10 min: 2%–10% A; 10–20 min: 10%–22% A; 20–28 min: 22%–29% A; 28–40 min: 29%–40% A; 40–50 min: 40%–55% A. Throughout the analysis, the flow rate was consistently maintained at 1 mL/min, and each injection had a volume of 10 μL.

Anxiety-like behavior were assessed by open field test (OFT) and the elevated plus maze (EPM), while depression-like behaviors were measured using the sucrose preference test (SPT), the tail suspension test (TST), and the forced swim test (FST) according to our previous study (Zhao et al., 2023). OFT: Each mouse is placed at the center of an open field in a dimly lit room, and their movements are recorded for 10 min using a video camera. This test primarily evaluates the reluctance of the rodent to explore open spaces, which can be indicative of anxiety-like behavior. EPM: This test involves a cross-shaped elevated platform with two open arms and two closed arms. Animals are placed at the center, facing the open-arm direction. Their movement is recorded for 6 min in a dimly lit setting. SPT: To assess anhedonia, a hallmark symptom of depression, mice are initially habituated with two bottles containing a 1% sucrose solution for 2 days. On the third day (test day), they are presented with two bottles-one with 1% sucrose and the other with water-for 24 h. The sucrose preference is calculated as the ratio of the volume of sucrose solution consumed to the total liquid intake (sucrose + water) during the test day, expressed as a percentage. TST: Mice are suspended by the tail using tape, approximately 1 cm from the tail’s tip and 25 cm off the ground. The amount of time the mouse remains immobile over a 6-min period is recorded. FST: Mice are placed in a cylindrical container filled with 25°C water up to a depth of 25 cm. After an initial 6-min period, the duration of immobility is recorded for the subsequent 4 min.

Lentiviruses expressing constitutively active Rac1 driven by the CMV promoter with a double-floxed inverted open reading frame combined with eGFP (DIO-Rac1-CA) and dominant negative Rac1 (DIO-Rac1-DN), or control lentivirus-eGFP (DIO-eGFP) were constructed by Obio Technology Corp., Ltd. Recombinant adeno-associated virus serotype 2/9 (AAV 2/9) expressing mCherry in combination with the Cre enzyme driven by the CMV promoter (CMV-Cre), lentivirus-eGFP (LV-eGFP), and rAAV-hSyn-eGFP-WPRE were constructed by Obio Technology Corp., Ltd. CMV-Cre and DIO-Rac1-CA, DIO-Rac1-DN or DIO-eGFP were bilaterally infused into the NAc over 5 min (coordinates AP, +1.54 mm; ML, ±0.80 mm; and DV,−4.20 mm) according to our previous studies (Tu et al., 2019; Zhao et al., 2019; Ying et al., 2022). Upon Cre-mediated recombination of the DIO, eGFP, Rac1-CA or Rac1-DN are expressed. Rac1-CA simulates the activated state of the wild-type G-protein by mutating glutamine 61 of Rac1 to leucine (Zhang et al., 2006). Rac1-DN inhibits the activity of Rac1 by mutating threonine 17 of Rac1 to asparagine (Tong et al., 2013). In our earlier research, we’ve shown that Rac1-CA can effectively activate Rac1-GTP and its downstream effector, p-PAK, while Rac1-DN inhibits their activity (Zhao et al., 2019).

To observe the impact of ELS on the NAc dendritic spines, the virus rAAV-hSyn-eGFP-WPRE was randomly infused into the NAc of both control and ELS mice at PND22. Three mice were used for each group. To investigate the effect of Rac1 on the NAc dendritic spines, the CMV-Cre and either DIO-Rac1-CA, DIO-Rac1-DN, or DIO-eGFP were randomly infused into the NAc of control and ELS mice. Subsequently, based on the type of viral injection, mice were grouped into control + eGFP, ELS + eGFP, control + Rac1-CA, ELS + Rac1-CA, control + Rac1-DN, and ELS + Rac1-DN. After ensuring full viral expression, all groups underwent behavioral tests. After the behavioral test, three mice were randomly selected from each group for dendritic spine analysis. Furthermore, to assess the influence of Si-Ni-San on dendritic spines, three mice were randomly chosen from the control, ELS, positive, and SNS medium dose groups at PND22. The virus rAAV-hSyn-eGFP-WPRE was randomly infused into the NAc of selected mice. Following the behavioral experiments, dendritic spine analysis was conducted. Dendritic spine analysis were performed according to our previous studies (Tu et al., 2019; Zhao et al., 2019; Zhao et al., 2022). The primary antibody was anti-GFP antibody (1:500, Abcam). The second antibody was Alexa Fluor 488-conjugated anti-rabbit antibody (1:200, Invitrogen).

The pull-down assay and Western blotting were performed were performed as described before (Zhao et al., 2019). The primary antibodies included the following: anti-Rac1 (1:1000, BD Transduction Laboratories); p-Pak and Pak (1:1000, Cell Signaling); Peroxidase-conjugated goat anti-rabbit or anti-mouse IgG second antibodies (1:5000, Santa Cruz Biotechnology Inc.).

Statistical analysis was conducted using SPSS 20.0 software. One-way or two-way ANOVA followed by Bonferroni’s post hoc test were employed to evaluate differences among multiple groups, while two groups were performed using Student’s t-tests. Significance was set at p < 0.05. Details were described in Supplementary Material.

We first evaluated whether early life stress induced depression-like or anxiety-like behavior in adolescent mice. The early-life stress protocol is illustrated in Figure 1A. The OFT and EPM are used to evaluate anxiety-like behavior in adolescent mice. When compared with the control group, the ELS group did not display a significant difference in terms of total distance traversed (Figure 1B, n = 14, p = 0.427) or time spent in the center during the OFT (Figure 1C, n = 14, p = 0.591). Similarly, there was no significant difference in the percentage of open arm entries in the ELS group in EPM (Figure 1D, n = 14, p = 0.761). The FST and TST was used to assess behavioral despair and SPT was used to measure anhedonia-like phenotypes in depression-related behavior. As shown in Figure 1E, the ELS group showed a considerable reduction in the percentage of sucrose intake (Figure 1E, n = 14, p = 0.001). Additionally, an increase in immobility time was observed in the tail suspension test (Figure 1F, n = 14, p = 0.007) and the forced swimming test (Figure 1G, n = 14, p = 0.011) for the ELS group. Taken together, these findings suggest that early life stress primarily induced depression-like, not anxiety-like behaviors, in adolescent mice.

Chronic stress is known to elicit structural remodeling of dendritic spines. Such alterations can influence the strength and number of synaptic connections between neurons, thereby affecting neural circuits related to mood and emotion regulation (Gebara et al., 2021). To further explore the impact of ELS on the NAc dendritic spines, the virus rAAV-hSyn-eGFP-WPRE was randomly infused into the NAc of both control and ELS mice at PND22. Figures 2A, B schematically represents the area of rAAV-hSyn-eGFP-WPRE injection and the image of eGFP-labelled spines in the NAc. Our findings revealed notable differences in spine morphology between the ELS group and controls. Specifically, compared to the control group, the total spine density was significantly increased in the NAc of ELS group (Figure 2C, 8–12 dendrite sections per animal with 3 animals per group, p = 0.033). While thin spine density did not show any significant difference between groups (Figure 2D, p = 0.997), we observed a marked decrease in mushroom spine density (Figure 2E, p = 0.039) and a substantial increase in stubby spine density (Figure 2F, p < 0.001) for the ELS group. These findings suggest that early life stress triggers a restructuring of dendritic spines within the NAc of adolescent mice.

Rac1 plays a significant role in several psychiatric disorders, such as addiction and depressive disorder (Zhao et al., 2019; Ru et al., 2022). We further evaluated the role of Rac1 in ELS induced-depression-like behaviors. AS shown in Figures 3A–C, both Rac1-GTPase (Figure 3B, n = 5, p = 0.033) and its downstream p-PAK (Figure 3C, n = 5, p = 0.011) activities were decreased in the NAc of the ELS group compared to the control group. Then, to investigate whether the decrease of Rac1 mediated early life stress induced-depression-like behaviors, CMV-Cre and DIO-Rac1-CA, DIO-Rac1-DN or DIO -eGFP were bilaterally infused into the NAc. Upon Cre-mediated recombination of the DIO, eGFP, Rac1-CA or Rac1-DN are expressed (Figures 3D, E). As shown in Figures 3F, G the constructed Rac1 mutant viruses were able to regulate the activity of Rac1 (Figure 3F, n = 4, Rac1-CA vs. eGFP: p = 0.043; Rac1-DN vs. eGFP: p = 0.01) and its downstream p-Pak activity (Figure 3G, n = 4, Rac1-CA vs. eGFP: p = 0.044; Rac1-DN vs. eGFP: p = 0.01) in the NAc. Moreover, Rac1-CA reversed the low percentage of sucrose consumption observed in the ELS group (Figure 3H, n = 8, p = 0.001), while Rac1-DN induced a decrease in the percentage of sucrose consumed in the control group (Figure 3H, p = 0.03). Similarly, Rac1-CA reversed the increase in immobility time in the ELS group in the tail suspension (Figure 3I, n = 8, p = 0.038) and forced swimming tests (Figure 3J, n = 8, p < 0.001), while Rac1-DN induced an increase in immobility time in the control group (Figure 3I, n = 8, p < 0.001; Figure 3J, n = 8, p = 0.002). These findings suggest that Rac1 plays a significant role in regulating behaviors associated with depression that result from early life stress.

Rac1 plays a significant role in spine plasticity (Zhao et al., 2019). Therefore, we further examined its role in the spine abnormalities in the NAc induced by ELS. Figure 4A depicted a representative image of the dendrites of neurons in the NAc. We discovered that the overexpression of Rac1-CA significantly decreased the total spine density in the NAc that was induced by early life stress (Figure 4B, 8–12 dendrite sections per animal with 3 animals per group, p = 0.0.021), while the overexpression of Rac1-DN on its own resulted in a significant increase in total spine density, mirroring the effect of early life stress on spine remodeling in the NAc (Figure 4B, p = 0.009). As shown in Figure 4C, Rac1 did not affect the density of thin spines. Moreover, the reduction in mushroom spine density in the early life stress group was reversed by the Rac1-CA virus (Figure 4D, p = 0.001), and Rac1-DN was able to reduce mushroom spine density in the control group (Figure 4D, p < 0.001). Additionally, overexpression of Rac1-CA significantly decreased stubby spine density in the NAc induced by early life stress (Figure 4E, p < 0.001), whereas the overexpression of Rac1-DN led to a significant increase in stubby spine density in the NAc compared to the control group (Figure 4E, p < 0.001). All these findings suggest that a decrease in Rac1 activity in the NAc plays an important role in spine remodeling in the NAc induced by ELS.

A substantial body of research on animals suggests that SNS and its modified prescriptions have an antidepressant effect, which may be attributed to the overall regulation of the hypothalamus-pituitary-adrenal system, monoamine neurotransmitters, brain-derived neurotrophic factor, and synaptic plasticity (Shen et al., 2020; Deng et al., 2022). However, the antidepressant mechanism of SNS is rarely designed for the spine remodeling in the NAc. Therefore, we further investigated whether SNS could alleviate ELS-induced depression-like behaviors and spine abnormalities in the NAc. As shown in Figure 5A, SNS was administered intragastrically at the dose of at 4.9 g/kg (low dose group), 9.8 g/kg (medium dose group), and 19.6 g/kg (high dose group). We found that SNS exerted a significant antidepressant effect. All three doses of SNS significantly increased the percentage of consumed sucrose (Figure 5B, n = 14, ELS vs. Control: p < 0.001; Positive vs. ELS: p < 0.001; SNS-L vs. ELS: p = 0.002; SNS-M vs. ELS: p = 0.001, SNS-H vs. ELS: p = 0.001). Furthermore, all three doses of SNS decreased immobility time in both the tail suspension (Figure 5C, n = 14, ELS vs. Control: p = 0.001; Positive vs. ELS: p = 0.002; SNS-L vs. ELS: p < 0.001; SNS-M vs. ELS: p = 0.008; SNS-H vs. ELS: p = 0.001) and forced swimming experiments (Figure 5D, n = 14, ELS vs. Control: p < 0.001; Positive vs. ELS: p = 0.007; SNS-L vs. ELS: p = 0.025; SNS-M vs. ELS: p = 0.009; SNS-H vs. ELS: p = 0.002). These results suggest that SNS could improve adolescent depression-like behavior induced by ELS. We also evaluated whether SNS could ameliorate spine deficits in the NAc induced by early life stress. Figure 5E presents representative confocal images of dendritic spines in the NAc. We found that SNS reversed the increase in total spine density in the NAc induced by early life stress (Figure 5F, n = 4, ELS vs. Control: p = 0.028; Positive vs. ELS: p = 0.007; SNS vs. ELS: p = 0.035). Moreover, SNS had no effect on the density of thin spines in the NAc (Figure 5G, n = 4, ELS vs. Control: p = 0.356; Positive vs. ELS: p = 0.131; SNS vs. ELS:p = 0.092). Additionally, SNS administration significantly increased mushroom spine density (Figure 5H, n = 4, ELS vs. Control: p = 0.006; Positive vs. ELS: p = 0.048; SNS vs. ELS: p = 0.014) and decreased stubby spine density (Figure 5I, n = 4, ELS vs. Control: p = 0.005; Positive vs. ELS: p < 0.001; SNS vs. ELS: p < 0.001) in the NAc induced by ELS. These results indicate that SNS may alleviate depression-like behaviors in adolescence by modulating spine plasticity in the NAc.

Given the established association between Rac1 activity in the NAc and depression-like behaviors, as well as spine remodeling due to early life stress, we further investigated Rac1’s involvement in the antidepressant effects of SNS. We initially measured the impact of SNS on Rac1 activity in the NAc. As demonstrated in Figure 6A, SNS significantly increased Rac1 (Figure 6B, n = 4, ELS vs. Control: p = 0.004; Positive vs. ELS: p = 0.023; SNS vs. ELS: p = 0.016) and its downstream p-PAK activity (Figure 6C, n = 4, ELS vs. Control: p = 0.022; Positive vs. ELS: p = 0.035; SNS vs. ELS: p = 0.015). Furthermore, we found that overexpression of Rac1-DN significantly weakened the antidepressant effect of SNS, as shown by the decrease in the percentage of consumed sucrose (Figure 6D, n = 8, ELS vs. Control: p = 0.005; SNS vs. ELS:p = 0.007; SNS vs. SNS-Rac1-DN: p = 0.016), and the increase in immobility time in both the tail suspension (Figure 6E, n = 8, ELS vs. Control: p = 0.004; SNS vs. ELS: p < 0.001; SNS vs. SNS-Rac1-DN: p = 0.018) and forced swimming tests (Figure 6F, n = 8, ELS vs. Control: p = 0.005; SNS vs. ELS: p = 0.011; SNS vs. SNS-Rac1-DN: p = 0.038). These results suggest that the antidepressant action of SNS is mediated, at least in part, by modulating Rac1 activity within the NAc.