Shugan granule contributes to the improvement of depression‐like behaviors in chronic restraint stress‐stimulated rats by altering gut microbiota

Abstract Aim The investigation aims to evaluate the potential effect of Shugan Granule (SGKL) on the gut, brain, and behaviors in rats exposed to chronic restraint stress (CRS). Methods The fecal microbiota and metabolite changes were studied in rats exposed to CRS and treated with SGKL (0.1 mg/kg/day). Depressive behaviors of these rats were determined through an open‐field experiment, forced swimming test, sucrose preference, and weighing. Moreover, LPS‐stimulated microglia and CRS‐stimulated rats were treated with SGKL to investigate the regulation between SGKL and the PI3K/Akt/pathway, which is inhibited by LY294002, a PI3K inhibitor. Results (i) SGKL improved the altered behaviors in CRS‐stimulated rats; (ii) SGKL ameliorated the CRS‐induced neuronal degeneration and tangled nerve fiber and also contributed to the recovery of intestinal barrier injury in these rats; (iii) SGKL inhibited the hippocampus elevations of TNF‐α, IL‐1β, and IL‐6 in response to CRS modeling; (iv) based on the principal coordinates analysis (PCoA), SGKL altered α‐diversity indices and shifted β‐diversity in CRS‐stimulated rats; (v) at the genus level, SGKL decreased the CRS‐enhanced abundance of Bacteroides; (vi) Butyricimonas and Candidatus Arthromitus were enriched in SGKL‐treated rats; (vii) altered gut microbiota and metabolites were correlated with behaviors, inflammation, and PI3K/Akt/mTOR pathway; (viii) SGKL increased the LPS‐decreased phosphorylation of the PI3K/Akt/mTOR pathway in microglia and inhibited the LPS‐induced microglial activation; (ix) PI3K/Akt/mTOR pathway inactivation reversed the SGKL effects in CRS rats. Conclusion SGKL targets the PI3K/Akt/mTOR pathway by altering gut microbiota and metabolites, which ameliorates altered behavior and inflammation in the hippocampus.


| INTRODUC TI ON
Depression, a common illness dependent on genomic and other biological factors, is defined as a mental disorder that evokes functional impairment and represses quality of life. 1 Depression has been considered a global public health event independent of age and socioeconomic factors. 2 Among depression symptoms, major depressive disorder (MDD) is the most serious symptom characterized by cognitive dysfunction, affective disorder, and psychosocial impairment. 3 According to a cross-sectional study, 4 patients with MDD have a high risk of developing suicidal ideation. The 12-month prevalence of MDD is 2.3% in China. 5 Treatment and management of MDD are challenging. For example, the pathogenesis of MDD is a complex biological network involved in neuronal remodeling, epigenetics, immune inflammation, gliocyte function, and gut microbiota, 6,7 indicating the definite mechanism of MDD has been unclear. Microglial activation in the central nervous system results from neuroinflammation, which develops into depression and associated impairments of neuroplasticity and neurogenesis. 8,9 Increasing numbers of investigations have determined that altered gut microbiota was explicitly associated with the development of depression. In the process of the gut to brain, extrinsic nociceptors may respond to altered gut microbiota during intestinal impairment, thereby resulting in the transmission of the extrinsic signal to the central nervous system (CNS). 10 Zhang et al. 11 found that there is a significant alteration in gut microbiota composition during CNS disorder. Interestingly, the altered gut microbiota is correlated to spontaneous brain activity and cognitive function. 12 A previous investigation 13 reported that gut microbiotacorrelated amino acid metabolism developed depression-like behaviors in vivo. Han et al. 14 suggested that treatments with Lactobacillus reuteri NK33 and Bifidobacterium adolescentis NK98 contributed to the alleviation of depression by improving the altered populations of gut microbiota and regulating NF-κB activation. A report 15 based on an animal model with unpredictable chronic mild stress revealed that the gut microbiota-regulated endocannabinoid system significantly alleviated fatty acid metabolism and subsequently depression-like behaviors. Valles-Colomer 16 concluded that Dialister and Coprococcus are positively related to the quality of life and depleted in treatment-free depression, indicating the potential role of gut microbiota in depression progress. Alterations in gut microbiota are involved in pro-inflammatory cytokine production and cell death in neurons. 17 Thus, targeting gut microbiota will be instrumental in the improvement of neuroinflammation-associated CNS disorder. 18 In neuroinflammation conditions, exposure to microbiota induces inflammasome complex to be enriched in cells in CNS when cells activate and then trigger the release of pro-inflammatory cytokines. 19 The special mechanism suggests a possible relation between gut microbiota and microglia, one of the CNS cells, during depression progression. However, the concrete mechanism of microbiota to microglia has been not yet fully understood in MDD.
The lack of an effective therapy for this physiological and psychological disease makes MDD treatment challenging. It is estimated that a half of the patients with depression receive inadequate treatment, partly because of standard treatment resistance. 20 Thus, a novel and effective drug is required for depression treatment.
Shugan granule (SGKL) is a Chinese patent medicine that comprises angelica sinensis, radix bupleuri, rhizoma cyperi, radix paeoniae alba, rhizoma atractylodis macrocephalae, mint, poria cocos, gardenia jasminoides, cortex moutan, and licorice. SGKL functions as the inhibitor of hyperplastic disease of the breast by repressing cell proliferation and p53 expression. 21 SGKL suppresses the population of gut mucosal serotonin-positive cells to improve irritable bowel syndrome. 22 Importantly, SGKL significantly evoked the reductions in CRF and ACC in the hypothalamus and alleviated depression-like behaviors in an animal model, 23 which suggest its potential role in depression treatment. Nevertheless, the molecular therapeutic mechanism of SGKL remains unknown for the depression process.
Clinically, SGKL administration is processed orally, possibly indicating the potential role in the gut microenvironment, particularly in microbiota composition. Given the gut-brain axis effecting depression progression, we guess SGKL may play the anti-depression role via gut microbiota-mediated pathogenesis of MDD, which requires experimental evidence.
Our previous investigation based on network pharmacology revealed that SGKL targets the PI3K/Akt/mTOR pathway, indicating that SGKL might contribute to alleviating depression symptoms through the PI3K/Akt/mTOR pathway. The PI3K/Akt/mTOR pathway is not only a critical cellular signaling pathway involved in malignant tumor such as breast cancer, 24 prostate cancer, 25 acute myeloid leukemia, 26 osteoarthritis, 27 and ovarian cancer, 28 but also contributes to is depression-like behaviors and autophagy of hippocampal neurons in animal models. 29 Tao et al. 30 showed that PI3K/ Akt/mTOR pathway activation could improve BDNF-TrkB pathwaymediated depression-like behaviors in rats. Notably, the mTOR pathway mediates autophagy, growth, and lipid metabolism stimulated by gut metabolism in cells and monitors gut microbiota composition through the gut barrier. 31 A study 32 revealed that microbiotaderived short-chain fatty acids elevated the production of IL-22 in CD4+ T cells through mTOR mediation. These reports suggest that the PI3K/Akt/mTOR pathway might regulate gut metabolites in depression progress.
Thus, this investigation is based on a novel hypothesis that SGKL targets the PI3K/Akt/mTOR pathway by altering gut microbiota and metabolites, thereby contributing to the amelioration of altered behavior and neuroinflammation. Ltd (Beijing, China). The rats were randomly divided into three groups: control group (n = 8) containing rats not exposed to any stress; CRS group (n = 8) exposed to CRS; and SGKL group (n = 8) exposed to CRS on SGKL treatment. The rats were subjected to 6 h of restraint stress in a mineral water bottle every day at random times after 1-week habituation to the environment.
CRS modeling was performed continually for 4 weeks. After CRS modeling each day, SGKL treatment was immediately administered at the dose of 0.63 g/kg/day. The treatment was continued for 4 weeks. Behavioral tests and weighing were performed after modeling. All animal works were conducted with the approval of the Ethics Committee of China-Japan Friendship Hospital (zryhyy21-20-09-9).

| Experiment 2
The rats were randomly divided into four groups: control group (n = 8) without any stress; CRS group (n = 8) exposed to CRS; SGKL group (n = 8) exposed to CRS with SGKL treatment; and SGKL+LY294002 group (n = 8) based on the administration of SGKL group and infused with LY294002 (25 μg in 0.5μl DMSO, Solarbio, Beijing, China), a PI3K inhibitor, in the hippocampus. The method of intrahippocampal infusion was performed, as described in a previous study. 33

| Behavioral test
We performed the sucrose preference test, open-field test, and forced swimming test to assess the behaviors of rats. The rats were subjected to behavioral tests after modeling, and they were acclimated to text condition for 30 min before the tests. Sucrose preference test: The rats in cages were cultured without food and water for 24 h, after which two bottles were simultaneously placed into each cage. The rat could freely access the two bottles. We weighed the two bottles after the 10-h test. Sucrose preference was calculated as follows: sucrose preference = [intake of sucrose/(intake of sucrose + intake of water)] × 100%. The reduction of sucrose preference mean animals was in depression. Open-field test (OFT): Openfield test was performed in a plastic chamber (72 × 72 × 40 cm 3 ) without a ceiling. The chamber was cleaned with 70% ethanol and covered with corn kernels on the base before each test. Each rat was placed at the center of the chamber and allowed to move for 4 min freely. The latency time to enter the central part, crossing and rearing behaviors of rats, was recorded by a video camera system

| Histological staining
After the last trial, we assessed pathological changes in the hippocampus or colonic tissue isolated from rats through H&E staining or Nissl staining. Each rat provided one sample of the hippocampus and one of colonic tissue. We investigated the activation of microglia in the CA1 hippocampus through IBA-1. Before histological staining, the tissues were incubated with 4% paraformaldehyde solution for fixation and embedded in paraffin after dehydration. These samples were sectioned into 4μm slices for histological staining.

| Enzyme-linked immunosorbent assay
The colon or hippocampus tissue was homogenized with pre-cooled PBS, respectively, and then the supernatant was obtained from homogenate centrifugated with 5000 × g for 5-10 min at 4 °C. Also, the serum samples were obtained from caudal venous blood centrifugated with 5000 × g for 5-10 min at 4 °C. ELISA was used to determine the levels of cytokines, namely TNFα, IL-1β, and IL-6, in the colon, serum, and hippocampus. TNFα (ab108913), IL-1β (ab255730), and IL-6 (ab234570) kits were purchased from Abcam (UK). The unit of concentration was recorded in a picogram per milliliter.

| Fecal sample collection
Fresh fecal samples were collected from rats after they were starved for 12 h, and the collected samples were transferred to Eppendorf tubes supplemented with a drop of sodium azide (1:100, v/v) per tube, followed by flash-freezing in liquid nitrogen for 15 min. The frozen samples were stored at −80°C for further tests.

| Bioinformatic analysis for gut microbiota
We investigated the community alteration of gut microbiota among the three groups based on operational taxonomic units (OTUs), community structure, α-diversity, and β-diversity. Indices of α-diversity included Chao1, Shannon, and Simpson. We discuss β-diversity according to the simple distance obtained from the Bray-Curtis method and PCoA. We used linear discriminant analysis coupled with effect size measurements (LEfSe) to assess the altered microbiota among the groups. We predicted the functional categories by using the phylogenetic investigation of communities through the reconstruction of unobserved states (PICRUSt).
Additionally, Spearman's analysis demonstrated the relationship between metabolites/microbiota and depression-like behaviors/ inflammation/pathways.

| GC-MS
GC-MS analysis was performed using Oebiotech (Shanghai, China). Prior to GC-MS analysis, the fecal samples stored at −80°C were transferred to Eppendorf tubes containing 500 μl of water and centrifuged at 10,000 × g for 15 min. We used the Agilent 7890B gas chromatography system coupled with the Agilent 5977A MSD system (Agilent, USA) to determine metabolites in gut microbiota. The DB-5MS-fused silica capillary column (30 m × 0.25 mm × 0.25 μm, Agilent, USA) was used to separate the samples. Helium (>99.999%) was used as the carrier gas at a constant flow rate of 1 ml/min through the column. The oven temperature for GC-MS was initially set to 60°C for 30 s, which was ramped to 125°C at a rate of 8°C per min, 210 °C at a rate of 5°C per min, 270°C at a rate of 10°C per min, 305°C at a rate of 20°C per min, and finally held at 305°C for 5 min. The temperature of MS quadrupole and ion source (electron impact) was set to 150°C and 230°C, respectively. The collision energy was 70 eV. Mass spectrometric data were acquired in a full-scan mode from 50 to 500 m/z.

| Metabolome analysis
Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to distinguish the altered metabolites among the groups. We determined the altered metabolites according to the variable importance of projection (VIP) > 1 and p value <0.05 and visualized the result by using volcano plots based on ggplot2. The fold change value of >1 indicated upregulation, and that of <1 indicated downregulation.

| Statistical analysis
Experiment data are expressed as mean ± SEM, followed by visualization using GraphPad Prism 8.0 and using SPSS 22.0 (IBM, USA). Experiment data were subject to test for normality by Shapiro-Wilk test and then statistical analysis was performed with one-way analysis of variance, followed by Dunnett's multiple comparison test. p value <0.05 with a 95% confidence interval indicated a significant difference among the groups.

| Effect of SGKL on depression-like behaviors of CRS-stimulated rats
After the rats were adapted to a new environment for 1 week, they were stimulated with CRS to evoke depression-like behaviors for 4 weeks, during which they were treated with SGKL. Their body weight and depression-like behavior were recorded weekly after modeling ( Figure 1A

| Effect of SGKL on inflammation in CRSstimulated rats through microglial inactivation
We investigated the role of SGKL in histopathological alterations in the colon and hippocampus CA3 area through HE staining. IBA-1 immunofluorescence was used to determine microglial cell activation and Franna Nissl assay to assess neuron alteration. Levels of inflammatory cytokines, TNFα, IL-1β, and IL-6, in the colon, serum, and hippocampus were measured through ELISA. Compared with the control group, the colon tissue in the CRS group displayed a disordered arrangement of colon gland cells and intestinal mucosa with impaired integrity (Figure 2A). CRS-stimulated rats exhibited triangular or polygonal cells, shrinkage in the nuclear membrane, and unclear nucleolus in the hippocampus CA3 area compared with the control group rats ( Figure 2B). Compared with the CRS group, rats in the SGKL group had intestinal mucosa with better integrity and well-arranged cells in the colon tissue and hippocampus CA3 area (Figure 2A & 2B). As shown in Figure 2C  Collectively, altered microbiota was associated with the behavior, inflammation in the hippocampus, and PI3K/Akt/mTOR pathway.

| Effect of SGKL in metabolites of CRSstimulated rats
Interestingly, 34 of the altered metabolites were upregulated, whereas 40 of the altered metabolites were downregulated in the SGKL group compared with those in the CRS-stimulated rats ( Figure 5A and Table S1). The CRS group had 114 of the altered metabolites (89 upregulated and 25 downregulated) compared with the control group ( Figure 5B and Table S2). Taking the intersection of

| Effect of SGKL in depression through the PI3K/Akt/mTOR pathway in vitro/vivo
The We investigated the role of SGKL in the PI3K/Akt/mTOR pathway.
SGKL-improved histological alterations in the colon tissue and hippocampus CA3 of CRS rats were significantly reversed by LY294002 ( Figure 7A&7B). LY294002 repressed SGKL-evoked elevations of F I G U R E 1 Effect of SGKL on behaviors of CRS-evoked mice. After the adaptation to a new environment for 1 week, rats (n = 8) were stimulated with CRS to evoke depression-like behaviors for 4 weeks, during which these were rats treated with SGKL.  A mechanism that links microbiota/metabolites in the gut to microglia might exist. PI3K/Akt/mTOR pathway is the target of SGKL based on the network pharmacology (data not shown), providing a hypothesis that it is the molecular approach linking SGKL treatment to metabolite variation in MDD. Meanwhile, we discovered that the PI3K/Akt/mTOR pathway was significantly associated with altered gut microbiota, metabolites, behaviors, and inflammation in the hippocampus. These findings suggest that, during SGKL treatment, the PI3K/Akt/mTOR pathway may be involved in mediating the pathological mechanism of altered gut microbiota for the depression process. In the cellular process, mTOR is activated by PI3K/Akt pathway to play a regulatory role in survival, growth, proliferation, and metabolism. 45  induced the alteration of gut microbiota that led to changes in metabolite production, which caused the inactivation of the PI3K/Akt/ mTOR pathway in the hippocampus and then inhibited the development of behaviors and inflammation. The regulation of gut microbiota in the PI3K/Akt/mTOR pathway may be the potential mechanism of SGKL between microglia and intestinal barrier. SGKL treatment improves the depression-induced alteration of gut microbiota that stimulates the phosphorylation of the PI3K/Akt/mTOR pathway, which represses microglial activation through PI3K/Akt/mTOR pathway-regulated inflammation in the hippocampus.
We demonstrated a novel mechanism of gut microbiota in causing depression; gut microbiota cause changes in phosphorylation of the PI3K/Akt/mTOR pathway, which mediates microglial activation and subsequent inflammation, eventually causing the development of depression. Importantly, SGKL treatment targets the PI3K/Akt/ mTOR pathway in improving depression-like behavior, microglial activation, and inflammation. Our investigation revealed the molecular mechanism of SGKL in gut microbiota-associated depression and may be useful in promoting its clinical value in depression treatment.

| LI M ITATI O N
Gender plays a significant role in brain vessels, cerebral blood flow, brain metabolism, and animal behavioral deficits in CNS F I G U R E 6 Effect of SGKL on LPS-stimulated microglia through the PI3K/Akt/mTOR pathway in vitro. Microglial cells were stimulated with LPS and then treated with SGKL, in which LY294002 inhibited the activity of PI3K. (A) Protein levels of PI3K, Akt, p-Akt, mTOR, and p-mTOR determined through western blotting. (B) Protein levels of TNFα, IL-1β, and IL-6 determined through western blotting. (C) Activities of microglia determined through immunofluorescence (scale = 50 μm). NS, p > 0.05. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 disorders, 51-54 which possibly affect the therapeutic effect of SGKL in depression. In the present study, we only explored the therapeutic mechanism of SGKL based on the gut-brain axis in male rats.
In the present study, we only explored the therapeutic mechanism of SGKL based on the gut-brain axis in male rats. Actually, gender has been determined as a significant role affecting brain vessels, cerebral blood flow, brain metabolism, and animal behavioral deficits in CNS disorders, which possibly affect the therapeutic effect of SGKL in depression. We will design two experiments to investigate the role of gender during SGKL treatment. One experiment will contain the male rat group (n = 8) and female rat group (n = 8), both of which are exposed to chronic restraint stress and treated with SGKL. Another will include three groups: control without any stress, CRS exposed to chronic restraint stress, and SGKL treated with SGKL undergoing CRS modeling; in this experiment, each group contains eight mice in half genders. Also, it remains unclear how Candidatus Arthromitus is related to depression-involved metabolites, which warrants further investigations based on fecal microbiota transplantation.

ACK N OWLED G M ENTS
This work was supported by the National Natural Science Foundation of China (No. 81874422, No. 81573843).

CO N FLI C T O F I NTE R E S T
None.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used or analyzed during the current study are available from the corresponding author on reasonable request.

E TH I C A L A PPROVA L
All animal works were conducted with the approval of the Ethics Committee of China-Japan Friendship Hospital.