Sprague-Dawley rats (200–220 g) were purchased from the Animal Center of Peking University Health Science Center (Beijing, China; certificate No. SCXK, 2006-0001). All animals were individually housed in plastic cages under a standard 12 h/12 h light/dark cycle (lights on at 9:00 p.m.) in a temperature-controlled room (temperature of 23°C ± 1 °C and humidity of 50% ± 10%). Food and water were available ad libitum unless otherwise specified. Animals were given 1 week to acclimatize to the new environment before surgery. All animals were handled daily. All experimental animal protocols were conducted in accordance with “Reporting animal research: Explanation and elaboration for the ARRIVE guidelines 2.0” (Percie du Sert et al., 2020) and approved by the Peking University Committee on Animal Care and Use (permission no. LA 2020279).

For habituation, the animals were connected to the recording apparatus at least 1 day before the experiments. Rats were anesthetized with isoflurane (5% induction and 2% maintenance) and the heads were secured in a stereotaxic apparatus. The guide cannula (O.D. 0.64 mm × I.D. 0.25 mm, C.C. 1.2 mm, RWD Life Science Co. Ltd., Shenzhen, China) was directed toward the lateral cerebral ventricle at the following coordinates relative to the bregma from the stereotaxic atlas of Paxinos and Watson (Paxinos and Watson, 1986): AP = −0.8 mm, ML = −1.50 mm and DV = −3.50 mm. Dental acrylic cement and another three stainless steel screws were fixed to the skull in order to affix the cannula. A dummy stylet was inserted into the cannula to prevent obstruction. Subsequently, rats were kept warm until full recovery from anesthesia. Topical analgesic (lidocaine/prilocaine cream) and penicillin was used to prevent signs of pain and/or infection. All rats were handled once time daily after surgery. The rats were allowed to recover for at least 7 days before exposure to STZ.

STZ (Sigma-Aldrich, S0130, Saint Louis, Missouri, United States) was resolved in 4 μL artificial cerebrospinal fluid (aCSF; Tocris Bioscience, 3,525/25 mL, Bristol, UK) at a dose of 3 mg/kg prior to administration. STZ was slowly and continually injected in the ventricle using a Hamilton micro syringe attached to the injection cannula over 5 min. The needle was left in the same spot for an additional 3 min to allow for diffusion. STZ were microinjected at a rate of 0.8 μL/min.

RGLS was provided by Zhejiang ShouXianGu Pharmaceutical Co. Ltd. (batch No. 20181101, Zhejiang, China). As described in our previous report, this product was approved by the State Food and Drug Administration of China in 2016 (China health food approval No. G20160280) and the representative chromatograms of RGLS obtained by LC-QTOF-MS in negative ion mode were presented in our previous reports (Li et al., 2020; Zhao et al., 2021). The results showed that RGLS contained 99 triterpenes, 1 linoleic acid and 9 potential new compounds. The chemical composition and content of RGLS were much higher than that of G. lucidum spore powder extracts without broken wall. The voucher specimen (SXG-GLS-20181006016) was preserved in our laboratory and the company. The samples were kept in the Department of Pharmacology, School of Basic Medical Science, Peking University. RGLS was dissolved in distilled water to a final concentration of 45, 90 and 180 mg/mL. The dose of RGLS (180, 360, 720 mg/kg) used in this study.

The experimental design is presented in Figure 1. Rats were respectively assigned in a random manner to five groups: Vehicle; STZ; STZ + RGLS (180 mg/kg); STZ + RGLS (360 mg/kg) and STZ + RGLS (720 mg/kg). After 7 days of recovery from surgery, the STZ and STZ + RGLS groups received twice intracerebroventricularly administration of STZ at an interval of 48 h. The STZ + RGLS group was administered orally with 180 or 360 or 720 mg/kg RGLS by gavage daily from day 1 to day 7. Vehicle group was treated with aCSF intracerebroventricularly and distilled water intragastrically. Subsequently, all the behavioral tests were conducted on day 7. On day 8, the animals were decapitated, the mPFC region was rapidly dissected using a brain blade and frozen at −80 °C for subsequent biochemical determinations.

All operations were conducted during the light phase, and each animal was used only once time in each behavioral test. Before the tests, animal was introduced to the experiment room to acclimate to the environment for at least 2 h. Depression-like behaviors of the animals were evaluated by the sucrose preference test, novelty-suppressed feeding test and forced swim test. Anxiety-like behaviors of the animals were evaluated by open field test.

The rats were decapitated and mPFC tissue was homogenized in RIPA lysis buffer and complete protease and phosphatase inhibitors. Then the homogenate was followed by centrifugation at 12,000 g for 15 min at 4 °C. The 5 X loading buffer was added to the supernatant and then used to detect protein levels. The subsequent procedure was essentially the same to Zhao et al. (Zhao et al., 2021). Proteins of 40–50 μg isolated from tissues were resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferring to polyvinylidene difluoride membranes (PVDF; Bio-Rad, United States). The membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated at 4 °C overnight with primary antibodies against β-actin, (1:4,000, ABclonal, AC038, Wuhan, China), ionized calcium-binding adaptor molecule-1 (Iba1, 1:1,000, Cell Signaling Technology, 17,198, MA, United States), NF-κB (1:1,000, ABclonal, A2547, Wuhan, China), NLRP3 (1:1,000, Abcam, ab263899, Cambridge, UK), caspase-1 (1:1,000, Abcam, ab179515, Cambridge, UK), apoptosis-associated speck-like protein (ASC, 1:1,000, Abcam, ab180799, Cambridge, UK), and brain-derived growth factor (BDNF, 1:500, Abcam, ab108319, Cambridge, UK). After washing three times with Tris-buffered saline with Tween-20 (TBST), blots were incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. Lastly, protein expression was detected with the ECL Enhanced Kit (ABclonal, RM00021, Wuhan, China), and the intensity of each blot was analyzed with ImageJ software.

Rats were euthanized and transcardially perfused with 0.01 M phosphate buffered saline (PBS), followed by 4% paraformaldehyde (PFA) in PBS. The brains were harvested, post fixed in 4% PFA overnight at 4 °C, and then consecutively dehydrated with 20% and 30% sucrose solution in sequence until brain sank. Subsequently, the brains were cut to a thickness of 20 µm on a cryostat microtome (Leica Microsystems UK, Leica CM 1950, Milton Keynes, UK). The slides were washed with PBS twice for 5 min each and were then placed in metal bath at 96 °C containing 0.01 M citrate buffer (pH 6.0) for 5 min. After return to room temperature, slides were washed three times and permeabilized with 0.01 M PBS containing 0.1% Triton X-100 for 20 min. After blocking with 10% donkey serum for 40 min at room temperature, the slides were incubated with primary antibodies rabbit anti-Iba1 (1:500, Cell Signaling Technology, 17,198, MA, United States) at 4 °C overnight. Alexa Fluor 448-conjugated donkey anti-rabbit antibody (1:2000, Abcam, ab150073, Cambridge, UK) was used as the secondary antibody. Nuclei were stained with DAPI before slides were viewed using a Nikon DS-Ri2 microscope camera (Nikon, Tokyo, Japan). For quantitative analysis, Fiji software was used to measure integrated density. All images of each section were acquired at 200× magnification. The number of branches and end-points can reflect microglia complexity and distinguish the state of microglia. In brief, bidimensional images were converted to 8-bit images and adjusted for brightness-contrast before thresholding to minimize background noise. Following these steps, the images were processed with the despeckle function and skeletozine. Then, we run the analysis of the skeleton program.

The generations of tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and interleukin-1beta (IL-1β) in the mPFC were measured using ELISA kits (ABclonal, RK00029, RK00020, RK00009, Wuhan, China) according to manufacturer’s instructions. The optical density (OD) of each well was quantified at 450 nm with an ELISA plate reader (Thermo Fisher Scientific Multiskan Mk3, MA, United States). Results are shown as pg/mg of protein.

Golgi-Cox staining was used to visualize dendrites and dendritic spines. Brain was stained using the FD Rapid Golgi Stain Kit (FD Neurotechnologies, PK401A, MD, United States). Brains were immersed in a mixture of Solution A (potassium dichromate and mercuric chloride) and Solution B (potassium chromate) in the dark for 2 weeks, and the tissue was then immersed in Solution C for 1 week, according to the manufacturer’s instructions. 150 μm slices were then mounted on gelatin-coated slides using additional Solution C, then left to dry in the dark overnight. After dehydration and decoloration, the slides were sealed with neutral balsam (Solarbio, G8590, China). Slides were stored in the dark. 3 dendrites per rat were traced, and a total of 5 rats per group were counted. The images were captured under Nikon Eclipse Ci-L microscope (Nikon, Tokyo, Japan) using DP controller software with 100×A/1.25 oil immersion lens. The density of dendritic spine and the dendritic spine morphologies in mPFC were analyzed using the ImageJ software.

Data analysis was performed by using GraphPad Prism (Version 8.0.1). The sample size was the number of independent values, and statistical analysis was performed using these independent values. The data from the western blot were normalized to control group values and determined as ‘Fold change’ in figures. Data were analyzed using one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test. The results were considered significant at p < 0.05. All data were presented as the mean ± SEM.

ICV-STZ significantly prolonged immobility time in the FST (F_{(4, 25)} = 13.73, p < 0.001, Figure 2A), decreased sucrose intake in the SPT (F_{(4, 25)} = 44.16, p < 0.001, Figure 2B), and increased the latency time to feed in the NSFT (F_{(4, 25)} = 11.54, p < 0.001, Figure 2C), indicating depression-like behaviors in ICV-STZ rats. There was no significant difference in food consumption in the NSFT among all groups when excluding ingestion bias (F_{(4, 25)} = 0.0313, p > 0.99, Figure 2D). The administration of RGLS (360 and 720 mg/kg) prevented despair- and anhedonia-like behaviors that were induced by ICV-STZ (Figures 2A–D). For anxiety-like behavior, ICV-STZ markedly decreased the time spent in the center in the OFT, which was reversed by 360 and 720 mg/kg RGLS (F_{(4, 25)} = 9.41, p < 0.001, Figure 2E). There was no significant difference in the total distance travelled among groups, indicating that exposure to ICV-STZ and RGLS did not impair general motor ability in rats (F_{(4, 25)} = 0.2759, p = 0.8908, Figures 2F,G). These results indicated that 360 and 720 mg/kg RGLS alleviated depression- and anxiety-like behaviors that were induced by ICV-STZ.

Depression-like behavior in rodents highly correlates with inflammatory responses in the central nervous system, and NF-κB/NLRP3 pathway activation is generally considered the initial inflammatory response. We examined proinflammatory factor and protein expression levels of several components of the NF-κB/NLRP3 pathway. Compared with rats in the vehicle group, TNF-α in the mPFC increased in ICV-STZ rats, but this increase was not statistically significant. RGLS treatment (360 mg/kg) reversed the ICV-STZ-induced increase in TNF-α expression (F_{(4, 25)} = 3.231, p < 0.05, Figure 3A). Similarly, ICV-STZ significantly increased IL-6 and IL-1β in the mPFC. The administration of RGLS (180, 360, and 720 mg/kg) reversed the ICV-STZ-induced increase in IL-6 (F_{(4, 25)} = 6.325, p < 0.05 or p < 0.01), and 360 and 720 mg/kg RGLS reversed the ICV-STZ-induced increase in IL-1β (F_{(4, 24)} = 4.362, p < 0.05, Figures 3B,C).

The NLRP3 inflammasome contains NLRP3 protein, adapter protein ASC, and caspase-1 (Shao BZ et al., 2015). NF-κB activates pattern recognition receptors and cytokine receptors license NLRP3 inflammasome activation by regulating NLRP3 expression (Bauernfeind et al., 2009). NF-κB, NLRP3, caspase-1, and ASC expression in the mPFC significantly increased in ICV-STZ rats compared with the vehicle group (p < 0.05 or p < 0.01, Figures 3D–H). These alterations were reversed by RGLS treatment (360 and 720 mg/kg) in ICV-STZ rats, and inflammation-related protein expression returned to levels of the vehicle group (NF-κB, F_{(4, 25)} = 6.286; NLRP3, F_{(4, 25)} = 34.22; caspase-1, F_{(4, 25)} = 35.67; ASC, F_{(4, 25)} = 15.43, p < 0.05 or p < 0.01, Figures 3D–H). These results indicate that RGLS exerts antiinflammatory effects by suppressing the NF-κB/NLRP3 pathway.

Microglia play an important role in neuroinflammation. We examined the activation of microglia in the mPFC to determine the potential antidepressant mechanism of action of RGLS. Functional and morphological features of microglia were evaluated by Western blot and the immunofluorescence staining of Iba1, a microglia-specific calcium-binding protein. Iba1 expression (F_{(4, 25)} = 8.558, p < 0.05, Figures 4A,B) and Iba1-positive cells (F_{(4, 25)} = 9.093, p < 0.01, Figures 4C,D) in the mPFC significantly increased in ICV-STZ rats, which were completely reversed by RGLS treatment (360 and 720 mg/kg). The morphology of microglia in the mPFC showed the significant attenuation of branch (F_{(4, 25)} = 7.122, p < 0.01) and end-point numbers (F_{(4, 25)} = 6.219, p < 0.01) in ICV-STZ rats, which were reversed by RGLS treatment (360 and 720 mg/kg, Figures 4E–G). These data indicated that RGLS treatment reversed ICV-STZ-induced pathological activation and morphological alterations of microglia in the mPFC.

The central inflammatory response is associated with impairments in neuronal plasticity. BDNF levels were measured, and Golgi staining was performed to evaluate synaptic plasticity in the mPFC. BDNF expression in the mPFC significantly decreased in the ICV-STZ group, which was reversed by 360 and 720 mg/kg RGLS (F_{(4, 25)} = 7.545, p < 0.01, Figures 5A,B). Golgi staining of the mPFC was used to detect the damage of dendritic spines in ICV-STZ rats (Figures 5C,D). Neuronal dendrites with significant spine loss (F_{(4, 70)} = 150.9, p < 0.01, Figure 5E) and less of a mushroom type (F_{(4, 70)} = 14.93, p < 0.01, Figure 5F) were detected in the mPFC in the ICV-STZ group. RGLS (360 and 720 mg/kg) prevented these neuronal impairments in the mPFC in ICV-STZ rats. Altogether, these results suggest that ICV-STZ damages synaptic plasticity in the mPFC, which is significantly reversed by RGLS.