TCM formula Xiaoyaosan consists of the following eight Chinese botanical drugs: Bupleurum chinense DC (Description: Apiaceae), Angelica sinensis (Oliv.) Diels (Description: Apiaceae), Paeonia lactiflora Pall (Description: Paeoniaceae), Poria cocos (Schw.) Wolf (Description: Polyporaceae), Atractylodes macrocephala Koidz (Description: Compositae), Glycyrrhiza uralensis Fisch (Description: Leguminosea), Zingiber officinale Rosc (Description: Zingiberaceae), and Mentha haplocalyx Briq (Description: Lamiaceae) at a ratio of 5:5:5:5:5:4:5:1. The prepared drug in pieces of Xiaoyaosan were purchased from Beijing Tongrentang (Bozhou) Pieces Co. Ltd., Bozhou, China, while the ultra-performance chromatography-electrospray tandem mass spectrometry (UPLC-MS/MS) was used to identify the samples of these decoction pieces (Ding et al., 2017; Yuan et al., 2020). The Xiaoyaosan dry powder (J2447) used in experimental studies was prepared by Jiuzhitang Co. Ltd, Changsha, China, in accord with the process recorded in the Chinese Pharmacopoeia 2015 Edition (Pan et al., 2019; Zhu et al., 2019). 2.36 g of crude drug yielded One Gram the finished dry powder administered experimentally.

The active ingredients and potential targets of TCM formula Xiaoyaosan for application to depression were investigated based on network pharmacology. The active ingredients in Xiaoyaosan were screened according to absorption, distribution, metabolism and enhancement (ADME) of drugs, while the bioavailability (OB) and drug likeness (DL) were used as screening parameters (Vugmeyster et al., 2012). According to ADME (OB ≥ 30% and DL ≥ 0.18), the active ingredients of Xiaoyaosan were collected from traditional Chinese medicine databases including the TCM Systems Pharmacology database and Analysis Platform (TCMSP, https://tcmsp-e.com/) (Ru et al., 2014), the Encyclopedia of Traditional Chinese Medicine (ETCM, http://www.tcmip.cn/ETCM/) (Zhang et al., 2019) and the High-throughput Experiment- and Reference-guided database of Traditional Chinese Medicine (HERB, http://herb.ac.cn/) (Fang et al., 2021), then SwissTargetPrediction database (http://www.swisstargetprediction.ch/) (Daina et al., 2019) was used to predict the targets of active ingredients in Xiaoyaosan, the reliability screening of predicted targets was also carried out (Probability ≥30%).

The related genes with depression and necroptosis pathway were collected from GeneCards database (https://www.genecards.org/) (Stelzer et al., 2016), Online Mendelian Inheritance in Man (OMIM, https://omim.org/) (Amberger et al., 2015), Comparative Toxicogenomic database (CTD, http://ctdbase.org/) (Davis et al., 2020), Therapeutic Target database (TTD, http://db.idrblab.net/ttd/) (Wang et al., 2020), and DrugBank database (http://www.drugbank.ca/) (Wishart et al., 2018), using “Depression”, “Depressive disorder” or “Necroptosis” as the search term. Then, the gene symbols of the ingredients in Xiaoyaosan, depression and necroptosis pathway were searched in the universal Protein Resource (UniProt, https://www.UniProt.org/) to get the unified UniProt gene ID.

The potential therapeutic targets of Xiaoyaosan in depression treatment via necroptosis pathway were obtained by overlapping the predicted targets of the ingredients in Xiaoyaosan and the target genes of depression and necroptosis pathway. Furthermore, the STRING database (https://string-db.org/) (Szklarczyk et al., 2019) was used to generate the protein–protein interaction (PPI) network of potential target genes of Xiaoyaosan with the species limited to human (Homo sapiens) and the interaction score ≥0.4. Then, the Cytoscape software (version 3.7.2, http://www.cytoscape.org/, Boston, MA, United States) was used to construct the “botanical drugs-active ingredients-potential targets” network, and identify the core target genes from the results of PPI network with the cytoHubba plugin (Guo et al., 2021).

The study was approved by the Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine (BUCM-4-2018120401-4053). Experimental protocols applied in our study were performed in agreement with the existing current animal welfare guidelines. Specific-pathogen free (SPF) male C57BL/6 mice aged 8-week-old (SYXK (Jing) 2016-0006, Beijing Vital River Laboratory Animal Technology Limited Company, Beijing, China) were housed separately in a standard animal feeding room (room temperature: 22 ± 2°C; relative humidity: 30–40%; light condition: a 12 h/12 h dark/light cycle) and fed standard rodent diet. Animals were adapted to their new environment for 7 days before CUMS procedure.

A total of 75 mice were randomly assigned to five groups (n = 15), including control group (no stress + physiological saline), CUMS group (CUMS + physiological saline), solvent group [CUMS + dimethyl sulphoxide (DMSO)/phosphate buffered saline (PBS)], Nec-1 group (CUMS + Nec-1), and Xiaoyaosan treatment group (CUMS + Xiaoyaosan). The CUMS modeling was performed as previously reported (Yan et al., 2019). Briefly, animals except the control group mice were subjected to the following stressors for six consecutive weeks: food deprivation for 24 h; water deprivation for 24 h; empty cages for 11 h; crowded cages for 24 h; restraint stress for 3 h; exposed to wet and soiled cages for 24 h; and 4°C cold water swimming for 5 min. The schedule of the study was showed in Figure 2.

Drug intervention lasted for 3 weeks, starting from the 4th week of modeling. The dosage of Xiaoyaosan and the duration of administration were confirmed according to the previous publications (Ding et al., 2017; Liu Y. et al., 2019). The Xiaoyaosan dry powder was dissolved in distilled water, then the mice in Xiaoyaosan treatment group received Xiaoyaosan by intragastric administration (0.254 g/kg/d), meanwhile mice in the CUMS group received 0.5 ml physiological saline (Yan et al., 2018). The concentration and volume of the gavage administration were adjusted once a week according to the changes in the body weight of the mice. Nec-1 (product number: RH82010-10mg, CAS number: 4311-88-0, BIORuler, Danbury, CT, United States) was dissolved in DMSO/PBS (1 mg in 125 μL of DMSO diluted with 1,375 μL of 0.1 M PBS). The Nec-1 group were injected intraperitoneally (i.p.) with Nec-1 (10 mg/kg) daily for 3 weeks, and the equivalent volume of DMSO/PBS (8.3%) was injected into solvent group mice (Dara et al., 2016).

The SP test was performed every week during the modeling process. The scheme lasted for successive 6 weeks, and the 3-weeks’s daily medication of Xiaoyaosan or Nec-1 was conducted. After the modeling, the FS test, and NSF test were carried out, then the mice were sacrificed for further detection.

To evaluate the depressive behaviors of model animals, the sucrose preference (SP) test, forced swimming (FS) test and novelty-suppressed feeding (NSF) test were conducted as previous described with minor modifications (Barfield et al., 2013; Liao et al., 2018).

The SP test was performed every week during the modeling process. In brief, each mouse in this study was given two bottles filled with deionized water and 1% sucrose solution after 24-h fasting and water deprivation, then the consumption of sucrose solution and deionized water in 1 h was recorded, respectively. The sucrose preference rate was presented as the percentage of the 1% sucrose solution consumed out of the total amount of liquid consumed, which represented the parameter of hedonic behavior.

The FS test and NSF test were conducted after the 6-weeks modeling to further assess the depressed behaviors in mice. In the FS test, each mouse was placed in a clear glass aquarium (24 cm height and 19 cm diameter) containing approximately 6 cm of water (24 ± 1°C) for 5 min, and the whole time was recorded using a high definition camera. The mouse was accepted and considered to be immobile when it stopped swimming and kept afloat on the water which reflected the despair of animals after they failed to cope with the attempt, the immobility time was recorded in our study. The NSF test was performed in a white plastic box (25 × 25 × 20 cm) with a single food pellet of regular chow placed in the center. After 24 h of food deprivation, the mouse was placed in a corner of the box, the latency to chew the food pellet was recorded, followed by the measurement of the food consumption in the subsequent 5 min.

After 6 weeks of modeling, mice sacrificed by decapitation after behavioral tests. The hippocampal tissues of five mice in each group were collected for western blot analysis, the RNA preservation solution (Biotech, #2714) was used to preserve the hippocampi of five mice in each group for quantitative RT-PCR assay. The whole brains of the remaining mice in each group were fixed in 4% paraformaldehyde (PFA) solution (4% PFA, 2.5% glutaraldehyde, and 0.1 M PBS) and prepared for tissue slicing.

Total RNA of hippocampus was extracted using Trizol^® reagent (Applied Biosystems, Waltham, MA, United States), then the quality and concentration of total RNA were determined by 1% agarose gel electrophoresis and Q3000 micro-volume spectrophotometer (Quawell Technology, San Jose, CA, United States). The RevertAid First Strand cDNA Synthesis Kit (Termo Fisher Scientifc, Waltham, MA, United States) was used to transfer total RNA into cDNA. Table 1 showed the sequences for primers used in our study which were designed based on published mRNA sequences in NCBI and synthesized by Sangon Biotech Co., Ltd, Shanghai, China. HPRT1 (BBI LifeScience, Amherst, MA, United States) was selected as the housekeeping gene in the experiment through reference genes selection method by geNorm, NormFinder, and BestKeeper software (Dongliang et al., 2011). The quantitative RT-PCR (qRT-PCR) reaction system was prepared by SYBR ^® Green PCR Master Mix (Applied Biosystems) in a final volume of 25 μL, then performed on Multicolor Real-time PCR Detection System (Bio-Rad, Hercules, CA, United States) with the following thermal cycling conditions: preincubation at 94 °C for 3 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extending at 72°C for 30 s. The signals were normalized to HPRT1 and the relative expression of mRNA in each sample was calculated by 2^−∆∆Ct method.

Protein levels of IBA1, IL-1β, Lipocalin-2, RIPK1, RIPK3, MLKL, and p-MLKL were measured by western blot analysis. The total protein of hippocampal tissues was extracted using RIPA Lysis buffer (Biomiga, Santiago, CA, United States). The proteins were loaded onto 12% SDS-PAGE gels at 30 µg per lane, and then transferred onto polyvinylidene fluoride (PVDF) membranes. The 5% non-fat dry milk in 0.05% tris buffered saline tween (TBST) was used to block the membranes, and the membranes were incubated with primary antibodies at 4 °C overnight (IBA1, 1:1,000 dilution, ab178847, Abcam, San Francisco, CA, United States; IL-1β, ab9722, 1:1,000 dilution, Abcam; Lipocalin-2, ab216462, 1:100 dilution, Abcam; RIPK1, 1:1,000 dilution, ab72139, Abcam; RIPK3, 1:1,000 dilution, ab56164, Abcam; phosphorylated MLKL (p-MLKL), 1:1,000 dilution, ab196436, Abcam; MLKL, 1:2000 dilution, orb32399, Biorbyt, Cambridge, UK; β-actin, 1:5,000 dilution, #5125, Cell Signal, Danvers, MA, United States). Then, the membranes were incubated with horseradish peroxidase- (HRP-) conjugated secondary antibody [Goat Anti-Rabbit IgG H&L (HRP), 1:10,000 dilution, ab205718, Abcam; Goat Anti-Mouse IgG H&L (HRP), 1:10,000 dilution, ab205719, Abcam] for 1 h at room temperature. The enhanced chemiluminescence (ECL) detection reagent (Thermo Fisher Scientific) was used to develop the membranes for 1 min, and the Tanon-5200 system (Tanon, Shanghai, China) was used to visualize the protein bands. The optical density of protein bands was quantified by Tanon Gis software (Tanon).

The brain samples fixed in 4% PFA solution were used to prepared paraffin-embedded tissue sections which included hippocampal CA1 and CA3 regions based on the stereotaxic atlases of the mouse brain (Konsman, 2003). The sections were stained with hematoxylin-eosin (H&E) to observe the cellular morphology and infiltration of inflammatory cells. A four-point severity scale (0, normal; 1, mild; 2, moderate; 3, severe) was used to score the H&E stained sections by an experienced pathologist in a blinded manner.

Immunohistochemical (IHC) staining was used to observe the levels of IBA1 and p-MLKL in hippocampus followed the previous described procedures (Yan et al., 2018). After dewaxing, dehydration and antigen retrieval, the brain sections were incubated with 3% H₂O₂ solution at room temperature for 10 min. Next, sections were incubated with primary antibodies (IBA1, 1:8,000 dilution, ab178847, Abcam; p-MLKL, 1:250 dilution, ab196436, Abcam) overnight at 4 °C. Then sections were incubated with secondary antibodies, and the diaminobezidin 3, 3 (DAB) solution (Invitrogen, Carlsbad, CA, United States) was used for coloration. For quantitative analysis, the original immunohistochemical images were collected with an image analyzer (MIAS99, Fubo-Tech Co., Beijing, China) with a color video camera (TK-C1381, JVC, Beijing, China) and BX50 microscope (Olympus Co., Tokyo, Japan), and the positive staining of hippocampal CA1 and CA3 regions at high power magnification (×400) was assessed by mean optical density (MOD) using Image-pro Plus software (version 6.0, Rockville, MD, United States).

Immunofluorescence (IF) staining was used to observe the expression of p-MLKL as previous described (Qu et al., 2017). The brain sections were incubated with 0.5% TritonX-100 solution at room temperature for 10 min. Next, sections were incubated with primary antibody (p-MLKL, 1:100 dilution, ab196436, Abcam) overnight at 4°C. Then sections were incubated with fluorescent-conjugated goat anti-rabbit IgG, follow by the Thioflavin T and DAPI staining. The fluorescence signal of sections was observed and photographed under an Olympus BX53 fluorescence microscope (Olympus Co., Tokyo, Japan), and the mean fluorescence intensity (MFI) was analyzed using Image-pro Plus software.

In this study, all data were analyzed using SPSS software (version 20.0, Chicago, IL, United States) and expressed as means ± SEM. The statistical analyses were performed by one-way analysis of variance (ANOVA) with least significant difference (LSD) post hoc multiple comparisons when equal variances were assumed, Dunnett’s T3 test was used when the data had a normal distribution but the variances are not homogeneous. A p-value of 0.05 was considered statistically significant.

The network pharmacology method was used to initial investigate whether the active components of Xiaoyaosan could improve necroptosis pathway in depression.

As showed in Figure 3, a total of 148 active ingredients in the eight botanical drugs of Xiaoyaosan were collected based on the ADME parameters (OB ≥ 30% and DL ≥ 0.18), and we obtained 23 potential treatment targets (BAX, BCL2, CAPN1, CAPN2, CASP1, CASP8, EIF2AK2, IL1B, JAK1, JAK2, JAK3, MLKL, NLRP3, PARP1, PPIA, RIPK1, RIPK3, STAT1, STAT3, STAT6, TLR4, TNF, XIAP) that might be associated with the antidepressant mechanism of Xiaoyaosan through necroptosis pathway by taking the intersection of 1708 putative targets of the active ingredients in Xiaoyaosan, 12,608 therapeutic targets for depression and 97 necroptosis related targets.

Figure 4A showed the “botanical drugs-active ingredients-potential targets” network, eight botanical drugs of TCM formula Xiaoyaosan had 96 active ingredients which could regulate the 23 key target genes. Therefore, based on the network pharmacological analysis we can speculate that traditional Chinese formula Xiaoyaosan could regulate the necroptosis pathway in depression. Figure 4B showed the PPI network of potential target genes of Xiaoyaosan and the sub-network of the top 10 core targets which was picked up using the cytoHubba plugin in Cytoscape software. As showed in Table 2, the hub nodes of sub-network were ranked by maximal clique centrality (MCC) method, and MLKL, IL1B, CASP8, CASP1, RIPK1, TLR4, NLRP3, RIPK3, TNF, STAT3 were the top 10 potential treatment targets of Xiaoyaosan.

We performed the sucrose preference (SP) test, forced swimming (FS) test and novelty-suppressed feeding (NSF) test to observe the effects of Xiaoyaosan on depressive-like behaviors. As shown in Figure 5A, the baseline sucrose preference rate of control group, CUMS group, solvent group, Nec-1 group and Xiaoyaosan group had no significant differences. However, after CUMS modeling, the mice in CUMS group had a significant drop in sucrose preference rate compared with the control group mice (p < 0.01), while the Nec-1 group and Xiaoyaosan treatment group had no differences with the stress groups. Then, the CUMS progress continued for another 3 weeks with medication procedure, the mice in CUMS group and solvent group still had a low sucrose preference rate (p < 0.01), while the sucrose preference rate of Nec-1 group and Xiaoyaosan treatment group mice was significant increased (both p < 0.01). The CUMS group and solvent group mice exhibited significantly longer immobility time in the FS test (p < 0.01), and Xiaoyaosan or Nec-1 ameliorated this depressive-like behavior in comparison with the CUMS group and solvent group (both p < 0.01) (Figure 5B). For the NSF test, it was shown that mice in CUMS group had a significant longer latency to chew the food pellet compared with the control mice (p < 0.01) (Figure 5C), and the food consumption significantly decreased (p < 0.01) (Figure 5D). There was no significant difference between the solvent group and the CUMS group. Xiaoyaosan or Nec-1 could reverse these changes in the NSF test (both p < 0.05).

In order to explore the possible mechanism of necroptosis in the mouse model of depression, the expressions of RIPK1, RIPK3, MLKL, p-MLKL were detected by western blot assay. As shown in Figure 6, the levels of RIPK1, RIPK3, MLKL and p-MLKL significant up-regulated in the hippocampi of CUMS-exposed mice compared to the control group (p < 0.01). Compared with the solvent group, these necroptosis biomarkers in Nec-1 group were significant down-regulated (both p < 0.01). Also, the effects of Xiaoyaosan were similar to Nec-1, which could mitigate the stress-induced elevation of RIPK1, RIPK3, MLKL and p-MLKL levels, as shown in the comparison between Xiaoyaosan treatment group and CUMS group (p < 0.05 or p < 0.01).

To further verified the process of necroptosis in CUMS-induced mice, the IHC and IF staining methods were used to observe the distribution of p-MLKL in hippocampus. Figure 7 exhibited the representative micrographs of IHC and IF staining for p-MLKL in the CA1 and CA3 regions of hippocampus, and the results were set out in Figure 8. The MOD of p-MLKL in both CA1 and CA3 regions of CUMS group mice was significantly increased when compared with the control group (p < 0.05, p < 0.01, respectively), and there was no significant difference between solvent group and CUMS group. Then the treatment of Nec-1 or Xiaoyaosan reversed the CUMS-induced p-MLKL changes in the CA1 and CA3 (p < 0.05 or p < 0.01, Figures 8A,B). Compared with the control group, the MFI of p-MLKL of CUMS group mice was significantly increased, and CUMS group had no significant difference with solvent group. Similarly to IHC staining results, the p-MLKL MFI was decreased in Nec-1 and Xiaoyaosan group mice (p < 0.05, p < 0.01, Figures 8C,D). The outcomes of thioflavine T staining showed that the MFI of thioflavine T labelled amyloid-like polymers was also increased in the CA1 and CA3 regions of CUMS group mice compared to the control group (both p < 0.01, Figures 8E,F). In the end, the IF staining results revealed that high levels of the p-MLKL colocalized with thioflavin T staining was detected in the CA1 and CA3 regions of CUMS group mice, in contrast with the low colocalization in control group (p < 0.01, Figures 8G,H). The low levels of colocalization were observed in the CA1 and CA3 regions of mice in Xiaoyaosan group and Nec-1 group as well (p < 0.05, p < 0.01).

The H&E staining was performed in order to evaluate the inflammatory infiltrates in mice hippocampal CA1 and CA3 regions. As exhibited in Figure 9A, a large number of inflammatory cell infiltration was observed in the hippocampus of the mice in chronically stressed groups treated with vehicle (physiological saline and solvent group). The histological scores of the hippocampus in each group were judged, it was found that the scores of CUMS group and solvent group in CA1 and CA3 regions were significantly higher than that of control group (both p < 0.01), Xiaoyaosan or Nec-1 treatment notably mitigated CUMS-induced inflammatory infiltrates (both p < 0.05) (Figures 9B,C). The expression levels of IL-1β and Lipocalin-2 were measured by qRT-PCR and western blot. The results indicated that the 6-weeks CUMS significant up-regulated the mRNA and protein levels of IL-1β in the hippocampus of CUMS-exposed mice (both p < 0.01), and the mice in the Xiaoyaosan treatment group and Nec-1 group showed a significant increase in IL-1β expression compared with the CUMS group mice (both p < 0.01) (Figures 10A,C). The CUMS modeling also increased the Lipocalin-2 level (p < 0.01), whereas the administration of Xiaoyaosan and Nec-1 noticeably inhibited the expressions of Lipocalin-2 (both p < 0.01) (Figures 10B,D).

To initial test the level of microglial activation in the hippocampi of CUMS-exposed mice, the expression of IBA1 was detected. Figure 11A presented the representative micrographs of IHC staining for IBA1 in the CA1 and CA3 regions of hippocampus. As showed in Figures 11B,C, the MOD of IBA1 in CA1 and CA3 of CUMS mice was significantly increased when compared to the control group (both p < 0.05), whereas the expression of IBA1 in CA1 and CA3 was noticeably reduced by the interference of Nec-1 and Xiaoyaosan (p < 0.05, p < 0.01). As showed in Figures 11D,E, the qRT-PCR and western blot data also demonstrated the high level of IBA1 in the hippocampus of the chronically stressed mice (both p < 0.01), and the treatment with Nec-1 or Xiaoyaosan significantly reduced the expression of IBA1 as compared to stressed groups (both p < 0.01).