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Molecular mechanisms of the Xiao-chai-hu-tang on chronic stress-induced colorectal cancer growth based on an integrated network pharmacology and RNA sequencing approach with experimental validation

BMC Complementary Medicine and Therapies volume 25, Article number: 135 (2025) Cite this article

Abstract

Background

Chronic stress is a risk factor for the development of colorectal cancer (CRC). Xiao-chai-hu-tang (XCHT) is a traditional Chinese medicine prescription and has been widely used to treat chronic stress-related diseases and cancer. However, its role in chronic stress-induced CRC remains unclear.

Methods

Our study aimed to investigate the roles of XCHT in CRC development under chronic stress. A xenografted CRC mouse model subjected to chronic restraint stress (CRS) was utilized to determine the effects of XCHT on CRC growth in vitro and in vivo. XCHT was administered via oral gavage once daily at dosages of 10.27 g/kg and 20.54 g/kg. RNA-sequencing was combined with network pharmacology to investigate potential target and pathway in this study. ELISA, RT-qPCR and immunofluorescence were performed to detect the expression of inflammation related genes. Glycolysis related genes and phenotype were evaluated by western blot, RT-qPCR and seahorse.

Results

XCHT significantly alleviated depression-like behaviors in CRS mice (p < 0.05) and effectively reduced tumor size and weight in a dose-dependent manner (p < 0.01). Mechanistic studies revealed that XCHT inhibited the CRS-induced upregulation of IL-6, attenuated the IL-6/JAK2/STAT3 signaling pathway (p < 0.05), and suppressed glycolysis by downregulating glycolytic enzymes (p < 0.01). Additionally, XCHT treatment reversed the CRS-induced decrease in immune cell infiltration, including CD4+ and CD8+ T cells, and reduced F4/80+ macrophage levels.

Conclusions

XCHT could reverse the tumor energy metabolism reprogramming and improve the inflammatory microenvironment in CRC under chronic stress through the IL-6/JAK2/STAT3 pathway. Therefore, XCHT might represent a promising therapeutic strategy for suppressing psychologically associated CRC progression.

Graphical Abstract

Peer Review reports

Introduction

Colorectal cancer (CRC) is one of the most common types of malignancy worldwide that significantly threatens human health. The GLOBOCAN 2020 statistics showed that CRC ranks third in terms of incidence, but second in terms of mortality [1]. The development of CRC results from complex interactions with genetic, epigenetic and environmental factors [2]. Epidemiological studies show that, as environmental factor, high level of chronic psychological stress is associated with an increased risk of neoplasia and cancer mortality, and a worse prognosis for cancer patients [3]. Chronic stress induced depressive-like symptoms are also associated with the progression of CRC [4]. Thus, it is necessary to develop new therapeutic strategies to improve the outcome of CRC patients with chronic psychological stress.

Cancer always undergoes extensive reprogramming of cellular energy metabolism to conquer the harsh tumor microenvironment [5]. The ‘Warburg effect’ indicates that even under aerobic conditions, tumor cells prefer to glycolysis rather than oxidative phosphorylation in glucose metabolism [6]. Glucose metabolism is rewired to support aggressive tumor growth and survival in cancer cells. For example, a previous study indicated that chronic stress facilitated the stem-like properties of breast cancer by upregulating the expression level of lactate dehydrogenase A (LDHA) [7]. Glucose deprivation induced the upregulation of long non-coding RNA GLCC1 which stabilized c-Myc, leading to enhanced LDHA-mediated glycolysis and CRC progression [8]. These results imply that metabolism reprogramming plays a critical role in CRC development.

Interleukin 6 (IL-6) is a multifunctional cytokine that regulates immune responses, inflammation and hematopoiesis [9]. The Janus kinase 2 (JAK2) -signal transducer and activator of transcription 3 (STAT3) signaling cascade is a classical downstream pathway triggered by IL-6 [10]. The JAK2/STAT3 signaling pathway is activated in various types of cancers including CRC [11, 12], to promote tumor proliferation, invasion, metastasis and survival. Previous studies showed that the IL-6/JAK2/STAT3 pathway might be activated in depression [13, 14]. Hence, blockage of the IL-6/JAK2/STAT3 signaling pathway might provide a novel therapeutic strategy for the treatment of chronic stress-related tumor progression.

Traditional Chinese medicine (TCM) is considered an effective method for the prevention and treatment of major diseases. XCHT was firstly used in the Treatise on Febrile Diseases during the Eastern Han Dynasty [15]. At present, multiple studies have demonstrated that XCHT has therapeutic effects on patients with mood and anxiety disorders clinically [16], which is similar to the “Shaoyang diseases”, and XCHT could alleviate depression-like behaviors in the mouse model [17]. It could ameliorate disturbances in steroid hormone homeostasis [18], dysregulated neurotransmission [19, 20] and enhance the serotonergic system [21]. Our previous study has demonstrated that XCHT could not only exert antidepressant effects by regulating the gut microbiota, but also inhibit tumor growth and prolong mice survival [22]. This study aims to elucidate the mechanisms through which XCHT mitigates colorectal cancer progression under chronic stress conditions, thereby offering novel insights into its potential application for managing colorectal cancer within this context.

Materials and methods

Preparation of the XCHT formula

The XCHT formula was prepared according to the clinical prescription. XCHT (provided by Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, China) contains 7 species of herbal medicines. Plant names have been checked with http://www.theplantlist.org, and the detailed information were shown in Table 1. The herbs were extracted for 1 h with a tenfold volume of boiling distilled water, then extracted again for 1 h with a sixfold volume of boiling distilled water. The extracts were concentrated into 200 mL decoction and then lyophilized into powder. The major components of XCHT were analyzed by Ultra Performance Liquid Chromatography (UPLC) mass spectrum technique in our previous study, and 63 components were identified in the extracted powders, including puerarin, baicalin, ginsenoside, glycyrrhizic acid, saikoside, et al. [22]. The XCHT extract was stored in a freezer at −80 °C, and the compound contents were measured monthly to ensure the quality of the XCHT preparation.

Table 1 The details of XCHT
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The pharmacokinetic study of XCHT in rats demonstrated effective absorption of its primary components, with accurate concentration measurements obtained through timed plasma sampling and antioxidant treatment, supporting a favorable safety profile for potential therapeutic use, [23].

Animal experiments

Mice

The C57BL/6 male mice (weighed 16-to-18 g) were purchased from the Shanghai JSJ Laboratory Animal Co., Ltd. (Animal Quality Certificate: SCXK (Shanghai) 2021–0006). All mice (5 per cage) were housed in a specific pathogen-free (SPF) environment and were housed in cages for 3 days prior to measurements. Mice were subjected to 12 h light/dark cycles, constant room temperature between 22 and 25◦C with a humidity of 60%.

Chronic stress model

CRS procedure was executed in accordance with an established protocol from prior research [24]. Mice were restrained in well-ventilated 50 mL falcon tubes from 8:00 AM to 16:00 PM without water and food for 14 consecutive days. Mice in the control group were placed in cages with food and water. After 14 days of CRS, mice were no longer restrained in the 50 mL falcon tubes.

Subcutaneous tumor model and intervention

MC38 cells were subcutaneously injected into the right axillary fossa of each mouse. Mice were randomized into 5 groups, including control, CRS, CRS + XCHT-L (XCHT-L), CRS + XCHT-H (XCHT-H), and CRS + fluoxetine (FXT). Here, a conversion ratio of 9.1 was used for humans and mice [25], with a human weight set at 70 kg [26]. The daily human dosage of XCHT is 79 g. XCHT was administered daily via oral gavage at a low dosage 10.27 g/kg (79 g/70 kg × 9.1) or a high dosage 20.54 g/kg (79 g/70 kg × 9.1 × 2), with a volume of 0.4 mL per mouse. The tumor volume was measured every three days after inoculation. An open field test was performed after continuous gavage administration for 28 days. Animal experiments approval from the Ethics Committee of Shanghai University of Traditional Chinese Medicine was obtained (PZSHUTCM210507013).

Animal sacrifice procedure

The animal sacrifice procedure was in accordance with guidelines for pain management and humane animal sacrifice. Pentobarbital sodium (150 mg/kg) was administered intraperitoneally to anesthetize the mice [27]. Blood samples were collected via the retro-orbital method. After blood collection, the mice were euthanized by cervical dislocation.

Behavioral analysis

To assess the possible effect of chronic stress on locomotor and exploratory activities, mice were subjected to open field test (OFT). The OFT was performed in a box (50 × 50 cm and 40 cm high) illuminated with white light (10 lx) in a quiet room, and activity traces of each mouse were continuously recorded for 5 min and the data were analyzed with an automatic detection system (Duoyi, Shanghai, China).

Cell culture

The MC38, HT-29, HCT-116 cells and subcutaneous xenograft tumor isolated cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco) with penicillin and streptomycin (Gibco). Cells were maintained at 37◦C in an atmosphere containing 5% CO2. Cells were treated with epinephrine (10 μM), IL-6 (20 ng/mL), 5-Fu (5 μM), and XCHT (50 μg/mL, 100 ug/mL, 200 ug/mL). Tumor cells were isolated by enzymatic digestion from control and CRS xenografts, then, the non-stressed cells and stressed cells were treated with different concentrations of XCHT for 72 h.

Reagents

2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)−2-Deoxyglucose (2-NBDG; #N13195) was purchased from Invitrogen. Antibodies against HK2 (#2867S), PFKP (#8164S), PKM2 (#4053S), LDHA (#3582S), JAK2(#3230), P-JAK2(#3776), STAT3 (#30,835), P-STAT3 (#9145) were purchased from Cell Signaling Technology. Antibodies against GLUT1 (#ab115730), Ki-67 (#ab15580) were purchased from Abcam. Antibody against Tubulin (#11,224–1-AP) was purchased from Proteintech.

Proliferation assay

Cell Counting Kit-8 (CCK-8; Dojindo, Japan) was performed to detect the effect of XCHT on the viability of tumor isolated cells or MC38 cells. Briefly, cells were respectively seeded into 96-well plates at an initial density of 3000 cells per well. On the second day, XCHT, 5-Fu or IL-6 was added to the cells at different concentrations for 72 h. Cells were incubated for 1 h with 10% CCK8 (v/v) medium, the absorbance was read at 450 nm using a microplate reader (Bio-Tek).

Colony formation assay

Cells were seeded into 12‑well plates at a density of 1 × 103 cells per well. The medium was changed on day three and then cells were treated with XCHT in different concentrations for 10 days. The colonies were fixed in 4% paraformaldehyde solution for 10 min and stained with crystal violet for 10 min. Finally, the colonies were photographed and analyzed with ImageJ software.

Western blot

Cells and tumor tissues were lysed with RIPA buffer supplemented with protease and phosphatase inhibitors. The protein lysates were separated by SDS-PAGE, and the proteins were transferred to 0.45 μm PVDF membranes. Membranes were blocked with 5% BSA in TBS-T (blocking buffer) for 1 h and incubated overnight with primary antibodies (diluted in blocking buffer) at 4◦C. Then, the membranes were incubated with secondary antibodies (Proteintech, Wuhan, China) for 1 h at room temperature. Proteins were detected using an enhanced chemiluminescence (ECL) substrate and visualized using an imaging system (Tanon Science & Technology Co., Ltd.). The relative expression was determined by comparing it to the internal reference protein.

RT-qPCR

Total RNA was extracted from CRC tissues and cells using TRI reagent (#T9424, Sigma-Aldrich). Complementary DNA was generated from total RNA using the HiScript® III RT SuperMix (#R323, Vazyme) according to the manufacturer’s protocol. Quantitative PCR analyses were performed using the ChamQ SYBR qPCR Master Mix (#Q331, Vazyme) and the 7500 Real-Time PCR System (Thermo Fisher). Relative mRNA levels were calculated using the 2−ΔΔCT method.The primer sequences for RT-qPCR were listed in Table 2.

Table 2 The primer sequences for RT-qPCR
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Immunohistochemistry (IHC)

Tissue samples were fixed with 4% paraformaldehyde for 24 h. Paraffin-embedded tumor sample sections were cut at 4 μm and dried overnight at 37◦C. Then, the sections were deparaffinized in xylene twice for 10 min each and rehydrated through graded alcohol. Sections were incubated with primary antibodies overnight at 4 ◦C followed by incubation with secondary antibodies. Immunoreactivity was detected with 3,3-diaminobenzidine tetrachloride (DAB). Then, sections were counterstained with hematoxylin, dehydrated, and mounted. Images were acquired using M8 Microscope and Scanner (Precipoint).

Immunofluorescence

Sections were fixed with 4% paraformaldehyde. After washing with PBS and blocking with blocking buffer, sections were incubated with the primary antibodies. After washing with PBS, the slides were incubated with the secondary antibody at a 1:500 dilution (Invitrogen). Cell nuclei were counterstained with DAPI and then images were taken by Leica microscope.

Glucose uptake

Cells were inoculated in a 96-well plate for 24 h. Then, cells were washed in PBS and incubated with prepared 2-NBDG solution (diluted in serum-free DMEM to a final concentration of 20 µM) for 1 h at 37℃. All subsequent steps were performed in the dark. The 2-NBDG uptake was evaluated using a microplate reader (Bio-Tek), and Hoechst 33,342 was used as the cell number control.

Intracellular ATP level assay

The intracellular ATP levels were measured with a commercially available intracellular ATP assay kit (S0026, Beyotime, Shanghai, China) according to the manufacturer’s instructions. Cells were inoculated in a 6-well plate. Then, cells were lysed on ice and centrifuged at 4◦C, 12,000 g for 10 min at 4◦C to obtain supernatant. 100 μL ATP detection reagent was added to each well of a 96-well plate and incubated for 5 min at room temperature. 20 μL supernatant was then added to each well. The amounts of ATP were calculated from a log–log plot of a standard curve and normalized to protein concentration.

Lactate production assay

Cells were seeded into six-well plates in 2 mL DMEM medium per well. lactate production was detected using LA assay kit (BC2230, Solarbio, Beijing, China) according to the instruction of the manufacturers. the lactate concentrations were normalized by the cell numbers.

ELISA measurement

Norepinephrine (NE) (MEN012, Bogoo, Shanghai, China), cortisol (Cort) (MEC065, Bogoo, Shanghai, China), epinephrine (Epi) (MEE013, Bogoo, Shanghai, China), interleukin 6 (IL-6) (MEI021, Bogoo, Shanghai, China), interleukin 12 (IL-12) (MEI026, Bogoo, Shanghai, China) and tumor necrosis factor alpha (TNF-α) (HET019, Bogoo, Shanghai, China) were measured by ELISA Kit according to the manufacturer’s instructions.

RNA sequencing of colorectal cancer tissue

RNA isolated from colorectal cancer tumors in different groups (n = 3 per group) was utilized for RNA-seq. RNA samples were converted to cDNA fragments using Illumina adapters, and 150 bp single end read sequences were obtained with the Illumina HiSeq system.

Network pharmacology analysis

The main active compounds of XCHT were searched in TCMSP (https://tcmsp-e.com/tcmsp.php) database. The screening criteria were oral bioavailability (OB) ≥ 30% and drug similarity (DL) ≥ 0.18, and total of 124 components were obtained. Swiss Target Prediction (https://www.swisstargetprediction.ch/) databases were used to search the gene targets for active ingredients. Concurrent with component analysis, disease-specific targets for “Colorectal Cancer" and "Depression" were identified through comprehensive searches in the GeneCards (https://www.genecards.org/) and DisGeNET (https://www.disgenet.org/) databases using disease-related keywords. Common targets were subjected to further analysis through the construction of a Protein–Protein Interaction (PPI) network via the STRING database (https://string-db.org/cgi/input.pl). The GO and KEGG analyses were performed using the the “clusterProfiler” package and “ggplot2” of R. Cytoscape software was used to construct the network.

Statistical analysis

Statistical analysis was conducted using GraphPad v10.1.2. The data are presented as the means ± SEM. One-way analysis of variance (ANOVA) was utilized to conduct comparisons across multiple groups, and comparisons between the two groups were analyzed using Student’s t test. P-value < 0.05 was considered statistically significant.

Results

XCHT ameliorates depression-like behavior in mice

Stress hormones, such as epinephrine and norepinephrine, are major mediators of the stress response [28]. In our previous studies, MC38 cells were stimulated by epinephrine and we discovered that epinephrine can enhance the proliferation of CRC cells [29]. In a separate study, we found that XCHT could inhibit CRC growth under chronic stress in vivo [30]. We established an in vitro chronic stress model by epinephrine stimulation (Fig. 1A), and we found that chronic stress promoted he proliferation of CRC cells and this phenomenon was reversed by XCHT (Fig. 1B). To investigate the role of XCHT in CRC growth under chronic stress, the CRS mice model bearing CRC xenograft was established as the flowchart in Fig. 1C. The male C57BL/6 mice were restrained daily for 8 h for two weeks, then, MC38 cells were injected subcutaneously. Three days after injection, mice were treated daily with XCHT or fluoxetine (10 mg/kg) by oral gavage, and 0.9% saline was used as a control. An OFT was performed before mice were sacrificed. The CRS model is commonly used to induce depression-like behavior in mice [31]. Compared to the control group, chronic stress obviously led to a reduction in total distance traveled, number of entries into the center, time spent in the center, and an increase in rest time. Interestingly, XCHT administration apparently improved the depression-like state in the mice caused by CRS (Fig. 1D).

Fig. 1
figure 1

XCHT ameliorates depression-like behavior in mice induced by CRS. A Schematic flowchart of cell experiments. B MC38 cells were treated with epinephrine and different concentrations of XCHT for 72 h. Cell viability was measured by CCK8 assay. C Schematic flowchart of animal experiments. D Behavioral analysis of OFT. Distance moved, center entries, time spent in the center, rest time are tested

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XCHT inhibits colorectal cancer growth under chronic stress

Next, we accessed the effect of XCHT on CRC growth. Although CRS significantly accelerated tumor growth rate, leading to a larger tumor volume and heavier tumor weight than that of the control group, XCHT administration could significantly reduce tumor growth and tumor weight induced by CRS in a dose-dependent manner (Fig. 2A,B). Moreover, the expression level of Ki-67 was accessed by immunohistochemistry. Compared to the stress group, XCHT blocked the promoting effect of chronic stress on Ki-67 index in the high dose group (Fig. 2C). The effect of XCHT on cell viability was accessed in colorectal cancer cell lines HT-29 and HCT-116. 5-fluorouracil (5-Fu) was used as the positive control. Our results demonstrated that XCHT had no effect on cell viability in HT-29 and HCT-116 cells compared with 5-Fu treatment (Fig. 2D). Consistent with the in vivo results, cells isolated from the CRS group xenografts grew faster than that from the control group. XCHT inhibited the stressed cells proliferation in a dose dependent manner while XCHT showed slightly effect on cell viability in non-stressed cells (Fig. 2E). Moreover, the colony formation assay results also showed that exposure to gradually increased concentrations of XCHT markedly inhibited colony formation in stressed cells (Fig. 2F). Taken together, these findings demonstrated that XCHT could inhibit colorectal cancer growth under chronic stress.

Fig. 2
figure 2

XCHT inhibited colorectal cancer growth induced by CRS. A Tumor size and tumor weight in different groups. Intragastric XCHT was given at a low dose (XCHT-L, 10.27 g/kg/day) or a high dose (XCHT-H, 20.54 g/kg/day). Fluoxetine was given at a dosage of 10 mg/kg/day via gavage. B Tumor growth curves in each group. C The proliferation marker Ki-67 was evaluated in tumor tissues by immunohistochemistry. Scale bar, 50 μm. D and (E) HT-29, HCT-116 and tumor isolated cells were treated with 5-Fu and different concentrations of XCHT for 72 h. Cell viability was measured by CCK8 assay. Tumor cells were isolated by enzymatic digestion from control and CRS xenografts. Cells were cultured in vitro For 48 h. Then, the non-stressed cells and stressed cells were treated with different concentrations of 5-Fu or XCHT for 72 h. F Cells were seeded in a 12-well plate and treated with different concentrations of XCHT (50, 100 and 200 μg/mL) (Left) and the colonies area were quantified (right). These cell experiments were repeated three times

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XCHT modulates gene expression in CRC under chronic stress

In order to explore the underlying mechanism of XCHT in chronic stress-induced CRC progression, an RNA-sequencing analysis of tumor tissues was performed. Heatmap showed differential gene expression among the control, stress and XCHT-treated groups. In comparison with the control group, chronic stress resulted in gene expression change, and XCHT treatment remarkably led to different gene expression profile when compared with the stress group (Fig. 3A). Next, the differentially expressed genes (DEGs) were identified by DESeq2 analysis. The DEGs between the control and the stress group were visualized using a volcano plot. Among these DEGs, CRS resulted in 154 upregulated genes and 134 downregulated genes compared with the control group (Fig. 3B). The DEGs between the stress group and the XCHT-H group were also visualized. Among these DEGs, XCHT treatment led to 275 upregulated genes and 192 downregulated genes compared to the CRS group (Fig. 3C). Moreover, KEGG analysis further revealed the top 20 significantly different pathways between the stress and the XCHT-H group (Fig. 3D). Considering that stress promoted tumor growth is accompanied by dysregulated tumor microenvironment with changes in cytokines and chemokines mediated inflammatory response [32, 33], we focused on cytokine-cytokine receptor interaction and chemokine signaling pathways within the enriched pathways for further study.

Fig. 3
figure 3

XCHT regulated CRS mediated gene expression. A The heat map revealed changes in the gene-expression profile of the control group (n = 3), the stress group (n = 3), the XCHT-L group (n = 3), and the XCHT-H group (n = 3). B Volcano plots showed differential gene expression analysis of tumor tissues between the control group and the stress group. C Volcano plots showed differential gene expression analysis of tumor tissues between the stress group and the XCHT-H group. D KEGG pathway enrichment analyses were performed on differential genes

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Network pharmacology analysis

Trough the TCMSP databases, a total of 124 active components in XCHT were collected. A total of 905 potential drug target proteins were identified from the active components of XCHT. After matching the 2230 depression-related targets with 1479 colorectal cancer-related target, 576 shared targets were identifed as potential targets. After all, 131 shared targets were identifed as potential targets for XCHT to treat CRC associated with depression (Fig. 4A). To screen out the hub genes and crucial compounds of XCHT against CRC associated with depression, we performed ingredients-genes disease and PPI networks through Cytoscape 3.8 (Fig. 4B). Figure 4C uncovered the hub genes based on the degree value, indicating these genes were core targets. We found that TNF, EGFR, STAT3, AKT1, TP53, and IL6 are six core targets (Fig. 4C). To decipher the molecular mechanism of the identifed potential targets, ClusterProfile and ggplot2 were used to perform GO enrichment and KEGG pathway analyses for overlapping genes. A total of 4902 terms in GO function enrichment analysis were acquired, including 276 in Cellular Component (CC), 474 terms in Molecular Function (MF), and 4152 in Biological Process (BP) (Fig. 4D). KEGG pathway enrichment analysis revealed several significantly enriched pathways, such as the PI3K-Akt pathway, lipid pathway and HIF-1 signaling pathway (Fig. 4E).

Fig. 4
figure 4

The potential molecular mechanism of XCHT to treat CRC-depression related diseases based on network pharmacology analysis. A Venn diagram showed targets among XCHT, CRC and depression. B Ingredients—genes—diseases network diagram. C PPI network diagram. D GO enrichment analysis. E KEGG pathway enrichment

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XCHT inhibits CRC growth under CRS via IL-6

To reveal the potential roles of cytokines and chemokines in XCHT mediated retraction of CRC under chronic stress, cytokines and chemokines expression levels in RNA-sequencing data were accessed (Fig S3). We found that the expression levels of IL-6, IL-12, IL-16 and IL-18 were significantly upregulated following CRS, and XCHT treatment alleviated the increase in IL-6 and IL-12 induced by CRS (Fig. 5A). TNF acts as an endogenous tumor promoter, facilitating the progression of malignant diseases by creating an inflammatory ecological environment [34], but we did not observe an increase in the expression of TNF-related factors in RNA-sequencing data. Next, we measured the plasma levels of IL-6 and IL-12 with ELISA assay, the plasma levels of IL-6 and IL-12 were upregulated by chronic stress when compared with the control group, but these increments were downregulated by XCHT treatment (Fig. 5B). Similar results were found that the expression level of IL-6 was upregulated in response to chronic stress and downregulated by XCHT treatment in both mRNA and protein levels, but not IL-12 (Fig. 5C,D). Previous reports indicated that tumor cells could produce IL-6 autocrine to sustain its aggressiveness [35, 36]. Hence, we evaluated the role of IL-6 on MC38 cell proliferation. We found that IL-6 indeed promoted MC38 proliferation, but XCHT administration could dose-dependently inhibit IL-6 induced cell proliferation (Fig. 5E,F). Thus, these data indicated that XCHT could suppress stress-induced CRC growth by abrogating IL-6 expression.

Fig. 5
figure 5

XCHT suppressed chronic stress-induced IL-6 expression and reversed the immune cells infiltration. A The expression levels of cytokines and chemokines at the transcriptomic level from RNA-sequencing data in different groups were shown. B The plasma levels of IL-6 and IL-12 were detected by ELISA. C The mRNA expression levels of IL-6 and IL-12 were measured in tumor isolated cells and tumor tissue by RT-qPCR. D The expression level of IL-6 in tumor isolated cells was measured by western blot. E MC38 cells were treated with IL-6 (20 ng/mL) and XCHT (50, 100 and 200 μg/mL) for 72 h as indicated, and cell viability was measured by CCK8 assay. F Cells were seeded in a 12-well plate and treated with IL-6 and XCHT (50, 100 and 200 μg/mL) (Left) and the colonies area were quantified (right). G The CD4 and CD8 positive cells were detected by immunohistochemistry. Scale bar, 50 μm. H The F4/80 positive cells were detected by immunohistochemistry. Scale bar, 50 μm. Data were presented as the mean ± SEM. These cell experiments were repeated three times

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XCHT modulates immune cells infiltration in CRC under CRS

IL-6 plays a critical role in immune responses and tumorigenesis [37]. Previous studies showed that tumors with IL-6 overexpression had fewer CD8+ T cells and CD4+ T cells [38], and F4/80+ macrophages were the major source of IL-6 [39]. Considering that chronic stress could upregulate the expression of IL-6, thus, we used immunohistochemistry to evaluate infiltrated CD4+ T cells, CD8+ T cells and F4/80+ macrophages in tumor tissues. Our results showed that CRS suppressed infiltration of CD4+ and CD8+ cells and XCHT treatment obviously reversed this effect of CRS (Fig. 5G). Moreover, flow cytometric analysis revealed that the percentages of CD4+ and CD8+ T cells in the XCHT group were higher than those of the stress group (Figure S1). Furthermore, we found that F4/80+ macrophage infiltration in the stress group was significantly increased when compared to the control group, which was reduced after XCHT treatment (Fig. 5H). Hence, these data showed that XCHT could inhibit CRC growth under chronic stress by improving immune function in mice.

XCHT inhibits CRS-mediated activation of IL-6/JAK2/STAT3 pathway

The JAK2/STAT3 pathway is a major signal pathway downstream of IL-6 in cancer [40, 41]. To evaluate whether STAT3 phosphorylation levels could be stimulated by IL-6, MC38 cells were treated with IL-6. We found that IL-6 induced phosphorylation of STAT3 in a time-dependent manner (Fig. 6A). Since XCHT could suppress IL-6 induced CRC cell proliferation, we found that XCHT also gradually suppressed the effect of IL-6 on STAT3 phosphorylation (Fig. 6B). Moreover, the stress-elevated level of phosphorylated STAT3 was attenuated by XCHT in CRC tissues (Fig. 6C). Previous reports had indicated that IL-6 promoted STAT3 phosphorylation mainly via JAK2 [42]. XCHT indeed attenuated the phosphorylation of JAK2 and STAT3 induced by IL-6 in tumor isolated cells and tumor tissues, respectively (Fig. 6D and E). To confirm whether XCHT blocked the IL-6/JAK2/STAT3 signaling activated by CRS, we treated stressed cells with AG490 and tocilizumab. AG490 is a potent inhibitor that has suppressive activity toward the JAK2/STAT3 signaling [43], and tocilizumab (TCZ) is an anti-IL-6 monoclonal antibody used to block IL-6 signaling [44]. Although CRS induced the activation of the JAK2/STAT3 pathway, AG490 and tocilizumab blocked this effect of CRS on the phosphorylation of JAK2 and STAT3. XCHT could barely attenuate JAK2 and STAT3 phosphorylation when combined with AG490 and tocilizumab treatment simultaneously (Fig. 6F). Taken together, these results suggested that XCHT abrogated chronic stress activated the IL-6/JAK2/STAT3 signaling in CRC.

Fig. 6
figure 6

XCHT inhibited the IL-6/JAK2/STAT3 pathway in CRC under CRS. A MC38 cells were stimulated with IL-6 (20 ng/mL) at the indicated time points, the expression levels of STAT3, p-STAT3 were measured by western blot. B MC38 cells were treated with IL-6 and XCHT in different concentrations for 48 h, the expression levels of JAK2, p-JAK2, STAT3, p-STAT3 were measured by western blot. C The expression levels of p-STAT3 in tumor tissues were measured by immunofluorescence. Scale bar, 20 μm. D,E Cells were treated with XCHT in different concentrations for 48 h, the expression levels of JAK2, p-JAK2, STAT3, p-STAT3 were measured by western blot in tumor isolated cells and tumor tissues. F Cells were treated with AG490, tocilizumab and XCHT in different concentrations for 48 h, the expression levels of JAK2, p-JAK2, STAT3, p-STAT3 were measured by western blot in tumor isolated cells. These experiments were repeated three times

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Chronic stress enhances glycolysis through JAK2/STAT3 pathway

The IL-6/JAK2/STAT3 signaling pathway is closely correlated with glucose metabolism which is deregulated in cancer [45]. We evaluated the effects of XCHT on glucose metabolism. Firstly, we observed that XCHT treatment abolished the promoting effect of CRS on glucose uptake and lactate production in stressed cells (Fig. 7A,B), and the level of ATP was significantly decreased by CRS stimulation and XCHT could increase ATP level in a dose-dependent manner (Fig. 7C). We then analyzed the extracellular acidification rate (ECAR) using a Seahorse XF instrument. Results showed that XCHT-treated groups exhibited lower levels of glycolysis and glycolytic capacity compared to the stress group (Fig. 7D). Our previous report has indicated that chronic stress could induce the upregulation of glycolytic related enzymes [29]. Consistently, we found that CRS induced the expression levels of glycolytic enzymes, including GLUT1, HK2 and PFKP, but XCHT administration significantly reversed this effect of CRS on the expression levels of glycolytic enzymes in both protein and transcriptional levels (Fig. 7E-G). Then, cells were treated with AG450 and tocilizumab with or without XCHT to investigate whether XCHT could inhibit glycolysis via the JAK2/STAT3 pathway. We found that co-treated stressed cells with AG490 and tocilizumab weakened the expression levels of glycolytic-associated enzymes, and XCHT treatment slightly enhanced this inhibitory effect of AG490 and tocilizumab on the expression levels of glycolytic enzymes (Fig. 7H). Moreover, STAT3 is positively correlated with GLUT1(SLC2A1), HK2, PFKP (Figure S2). Hence, these results showed that XCHT could mitigate the promoting effect of CRS on glycolysis via the JAK2/STAT3 pathway in CRC cells.

Fig. 7
figure 7

XCHT blunted CRS-promoted glycolysis via the JAK2/STAT3 pathway. A Glucose uptake was measured in tumor isolated cells. B Lactate production was measured in tumor isolated cells. C The ATP level was measured in tumor isolated cells. D Seahorse analysis was conducted to detect the ECAR in indicated cells, and the glycolysis and glycolytic capacity were also shown. E,F Cells were treated with XCHT in different concentrations for 48 h, the expression levels of GLUT1, HK2, and PFKP were detected by RT-qPCR and western blot. G The expression levels of HK2, PFKP, and GLUT1 in tumor tissues were detected by RT-qPCR and western blot. H Tumor isolated cells were treated with AG490, tocilizumab and XCHT in different concentrations for 48 h, the expression levels of GLUT1, HK2, and PFKP were detected by western blot. These experiments were repeated three times

Full size image

The schematic diagram of the molecular mechanisms underlying XCHT in CRS-induced CRC

In Fig. 8, we detail how XCHT inhibits the progression of CRS-induced CRC through the IL-6/JAK2/STAT3 signaling pathway.

Fig. 8
figure 8

Schematic diagram of the mechanisms. XCHT inhibited CRS-induced CRC growth via modulating the IL-6/JAK2/STAT3 pathway

Full size image

Discussion

Emerging clinical and epidemiological studies have indicated that chronic psychological stress is a risk factor for cancer development, including CRC [28, 46]. TCM has shown immense potential and has been widely used in the treatment of psychiatric disorders for thousand years [47]. The application of TCM formulas in the treatment of cancer patients with psychological stress has attracted increasing attention. XCHT is a classic formula widely used in relieving depression-like symptoms. Previous studies have shown that XCHT could alleviate perimenopausal depression-like behaviors in different animal models [18, 48]. Besides, XCHT has shown an anti-tumor effect in a subcutaneous transplanted tumor model with CRS [22]. Here, we found that chronic stress could lead to depression-like behaviors and facilitate CRC tumor growth, while XCHT could significantly inhibit CRC progression under chronic stress in vivo and in vitro (Fig. 2A-B, Fig. 2E-F).

Chronic stress triggers a series of inflammatory responses and the systemic release of inflammatory cytokines, resulting in long-term pathological changes. However, there is limited information on how chronic stress affects CRC progression by modulating inflammation. It has been reported that XCHT exerted anti-inflammatory effects in the CRS model by decreasing levels of IL-6, IL-1β, and TNF-α [22]. PPI network diagram showed that the core targets were IL-6, STAT3, etc. (Fig. 4C). As a proinflammatory cytokine, IL-6 is usually elevated and correlated with advanced tumor progression and poor prognosis in CRC patients [49], suggesting that IL-6 might facilitate the progression of CRC. Recent research has reported that the expression level of IL-6 was upregulated by chronic stress [50], and we also found that IL-6 was elevated under CRS (Fig. 5A,B). IL-6 could be produced by both tumor cells and immune cells [42, 51]. We found that chronic stress upregulated IL-6 expression in both in mRNA and protein levels, while XCHT could reverse chronic stress-induced elevation of IL-6 (Fig. 5C-D). Moreover, IL-6 treatment promoted CRC cell proliferation and enhanced colony formation ability, whereas XCHT inhibited the proliferation-promoting effect of IL-6 in MC38 cells (Fig. 5E,F). The IL-6-activated JAK2/STAT3 signaling pathway plays a role in the regulation of inflammation and cell proliferation [40]. We observed that the phosphorylation of JAK2 and STAT3 induced by IL-6 was abrogated by XCHT administration (Fig. 6B). Thus, XCHT could mitigate chronic stress-activated IL-6/JAK2/STAT3 signaling pathway to suppress CRC progression. IL-6 signaling could be driven in both autocrine and paracrine manners to facilitate tumor progression [52]. Here, we revealed that XCHT deactivated the JAK2/STAT3 signaling by downregulating the expression of IL-6 (Fig. 6F).

Increasing evidence has shown that TCM exert favorable effects on the immune regulation [53]. Compound kushen injection could relieve tumor-associated macrophage-mediated immunosuppression by increasing CD8+ T cells and M1-TAMs in the tumor microenvironment [54]. As one of the cytokines produced by M1-TAMs, IL-6 plays an important role in immune function [13]. Blocking IL-6 resulted in better tumor suppression and higher CD4+/CD8+ effector T cell numbers with a reduction in Th17, macrophages, and myeloid cells [55]. Our results indicated that XCHT treatment reversed chronic stress-mediated diminishment of CD4+, CD8+, and increasement of F4/80+ immune cells infiltrated in tumor tissues (Fig. 5G,H). Thus, we speculated that chronic stress exerted immunosuppressive function in tumor progression, and IL-6 might promote this effect under chronic stress. Although the detailed mechanism still needs to be further investigated, it is reasonable to speculate that targeting IL-6 might be an effective strategy for chronic stress-associated CRC, and XCHT could be used to improve the inflammatory microenvironment in cancer.

Recent studies have illustrated that microenvironment-mediated adaptions in cellular metabolism and inflammation responses played an important role in various tumors [56, 57]. More importantly, chronic stress could promote metabolic reprogramming, thereby facilitating the rapid growth of cancer [7, 29, 58]. Our results indicated that CRS led to significant alterations in glucose metabolism by enhancing glycolysis, while XCHT was able to reverse the increased glycolysis induced by CRS in CRC (Fig. 7A-D). Moreover, XCHT inhibited the expression levels of glycolytic enzymes, including GLUT1, HK2, and PFKP, which were upregulated under chronic stress (Fig. 7E-G). Glycolysis could be modulated by targeting the IL-6/JAK2/STAT3 pathway in cancer [45]. When we treated tumor isolated cells with IL-6 antagonist tocilizumab and JAK/STAT3 inhibitor AG490, the expression levels of glycolytic-enzymes were obviously attenuated. Co-treatment with XCHT could barely enhance the inhibitory effect of AG490 and tocilizumab (Fig. 7H). These findings implied that blocking the IL-6/JAK2/STAT3 signaling pathway represented a promising therapeutic strategy for modulating aberrant glucose metabolism in chronic stress-associated cancer. These findings highlight its potential utility as a complementary therapy in clinical settings, particularly for patients whose cancer progression may be exacerbated by psychological stress. We propose that these results warrant further exploration through clinical trials to evaluate the efficacy and safety of XCHT in CRC patients with chronic stress. We acknowledge potential limitations of this study, including species differences affecting, effective dosing and safety profiles. In the future, particularly human trials, should be conducted.

Conclusions

In conclusion, our study revealed that chronic stress could promote CRC progression by activating the elevated IL-6-mediated JAK2/STAT3 pathway to enhance glycolysis and modulate immune response. XCHT could suppress chronic stress-induced CRC progression by regulating glycolysis and inflammatory responses via the IL-6/JAK2/STAT3 pathway. Our findings not only uncovered the mechanism of chronic psychological stress on CRC but also highlighted XCHT as a promising treatment for alleviating chronic psychological stress in colorectal cancer patients.

Data availability

The data sets that were utilized and/or examined throughout the course of this study can be obtained from the corresponding authors if requested with justifiable reasons. The clean reads from transcriptomes were submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi). The accession number is GSE276601.

Abbreviations

CRC:

Colorectal cancer

CRS:

Chronic restraint stress

ECAR:

Extracellular acidification rate

LDHA:

Lactate dehydrogenase A

IL-6:

Interleukin 6

OFT:

Open field test

STAT3:

Signal transducer and activator of transcription 3

TCM:

Traditional Chinese medicines

TCZ:

Tocilizumab

JAK2:

The Janus kinase 2

XCHT:

Xiao-Chai-Hu-Tang

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Funding

This work was funded by the National Natural Science Foundation of China (82122075, 82104607, 82074232, 82030118, 81830120, 82004131, 82405498), China Postdoctoral Science Foundation (2021M700090), Shanghai Frontier Research Base of Disease and Syndrome Bi-ology of Inflammatory Cancer Transformation (2021KJ03-12), “Shu Guang” project supported by Shanghai Municipal Education Commission and Shanghai Education Development Foundation (21SG43), Three-year Plan Project of Shanghai Traditional Chinese Medicine (ZY(2021–2023)-0208), Clinical Research Plan of SHDC and Shanghai Youth Talent Support Program (SHDC2020CR4043).

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  1. Wang Yao and Dong-Ming Hua contributed equally to this work.

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  1. Department of Medical Oncology, Pudong New Area, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, No. 528, Zhangheng Road, Zhangjiang Hi-Tech Park, Shanghai, 201203, China

    Dong-Ming Hua, Ying-Ru Zhang, Yi-Yang Zhao, Zhong-Ya Ni, Yun-Feng Guan & Yan Wang

  2. Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai, 201203, China

    Wang Yao, Ying Feng, Zhao-Zhou Zhang, Hai-dong Guo & Yan Wang

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Contributions

Wang Yao, Dong-Ming Hua (First Author): Investigation, Formal Analysis, Writing- Original Draft, Methodology, Software; Ying-Ru Zhang and Yi-Yang Zhao: Data Curation, Visualization, Investigation; Ying Feng, Zhao-Zhou Zhang, Zhong-Ya Ni and Hai-dong Guo: Supervision, Software, Validation; Yun-Feng Guan and Yan Wang (Corresponding Authors): Conceptualization, Funding Acquisition, Resources, Supervision, Writing—Review & Editing.

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Correspondence to Yun-Feng Guan or Yan Wang.

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Yao, W., Hua, DM., Zhang, YR. et al. Molecular mechanisms of the Xiao-chai-hu-tang on chronic stress-induced colorectal cancer growth based on an integrated network pharmacology and RNA sequencing approach with experimental validation. BMC Complement Med Ther 25, 135 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04860-8

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12906-025-04860-8

Keywords

BMC Complementary Medicine and Therapies

ISSN: 2662-7671

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