Therapeutic Effect of Brucea Javanica Oil Emulsion in Mice with Irinotecan-Induced Delayed Diarrhea

Introduction

Chemotherapy can improve patient survival, and is currently essential for cancer treatment. However, its adverse effects, particularly chemotherapy-induced diarrhea (CID), can severely impact quality of life, often leading to premature discontinuation and reduced survival.¹ Clinically, irinotecan is an important chemotherapy drug commonly used for solid tumors such as lung, gastric, cervical, and ovarian cancers.² However, irinotecan severely damages the proliferative mucosal lining of the digestive tract, leading to intestinal inflammation, ulceration, impaired absorption, mucosal barrier dysfunction, and heightened immune suppression.³ Delayed diarrhea is a significant adverse reaction associated with irinotecan, with an incidence ranging from 60% to 90% and severe diarrhea (grade 3–4) occurring in 28% to 40% of cases.⁴ The primary medications used clinically to treat irinotecan-induced diarrhea are loperamide and octreotide.⁵ However, available therapies often come with various adverse effects. Consequently, developing drugs that do not compromise the anticancer efficacy of irinotecan while alleviating its major adverse effect delayed diarrhea, is a pressing concern.

Brucea javanica oil emulsion (BJOE), an approved and widely marketed formulation, represents the primary application of Brucea javanica as an anti-inflammatory and anti-dysentery^6,7 agent. Various studies have indicated that BJOE has low toxicity in clinical application and exhibits numerous bioactive activities, including anti-inflammatory, antimalarial, and antitumor effects.⁸ In clinical practice, BJOE is primarily used in conjunction with chemotherapeutic agents such as irinotecan and docetaxel⁹ to enhance their anticancer effects, improve safety and efficacy, and increase treatment compliance among oncology patients.¹⁰ Notably, it has been shown to significantly increase the disease remission rate, protect immune function, and improve the quality of survival for patients with brain metastases, while also significantly reduce the incidence of gastrointestinal reactions.¹¹ Our preceding studies have shown that Brucea javanica and its active ingredient exhibit anti-inflammatory and anti-inflammatory bowel disease (IBD) effects,¹² supporting its traditional use in treating dysentery. Our previous work has also reported that Brucea javanica oil effectively ameliorated symptoms of weight loss and diarrhea, increased food intake, and alleviated intestinal mucosal damage in mice with 5-fluorouracil-induced intestinal mucosal injury.¹³ Therefore, we hypothesized that BJOE might exert therapeutic effect on irinotecan-induced delayed diarrhea.

It has been shown that damaged double-stranded DNA (dsDNA) induced by irinotecan accumulates and releases in the cytoplasm, leading to abnormal activation of the cGAS-STING pathway. This subsequently triggers the activation and production of a series of downstream immune cells and inflammatory factors, thereby promoting the inflammatory response in the organism.¹⁴ Irinotecan, derived from the plant alkaloid camptothecin, is a topoisomerase I (Topo1) inhibitor that forms a Topo1-drug-DNA complex, preventing DNA reattachment and resulting in double-strand breaks. Cyclic GMP-AMP synthase (cGAS) acts as both a biosynthetic enzyme and an intracellular DNA receptor, recognizing aberrant double-stranded DNA (dsDNA) and catalyzing the generation of second messengers to activate the adaptor molecule stimulator of interferon genes (STING). Activated STING subsequently triggers downstream signalling pathways, including TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), leading to the activation and production of various immune cells and inflammatory factors, thereby promoting the inflammatory response in the body.

The properly functioning cGAS-STING signalling pathway is crucial for maintaining gut homeostasis. However, its overactivation is associated with irinotecan-induced delayed diarrhea.¹⁵ This indicated that abnormal activation of the cGAS-STING signalling might constitute an important pathological basis for delayed diarrhea in the context of irinotecan treatment. Given its critical role in innate immunity, targeting this pathway presents a promising strategy to alleviate inflammation and maintain intestinal barrier integrity. To date, the therapeutic efficacy of BJOE in mitigating irinotecan-associated delayed diarrhea has not been systematically examined, nor has its mechanistic inhibition of the cGAS–STING pathway been characterized in this context. Therefore, in this study, we investigated the therapeutic effect and underlying mechanism of BJOE in irinotecan-induced delayed diarrhea mice model. Results indicated that BJOE demonstrated partial efficacy in alleviating irinotecan-induced delayed diarrhea by protecting the intestinal barrier and reducing inflammation via inhibition of aberrant cGAS-STING pathway activation.

This study was the pioneering endeavor to explore the therapeutic potential of BJOE in irinotecan-induced delayed diarrhea and also the first time to indicate the inhibitory effect of BJOE on the cGAS-STING pathway. These findings are expected to provide further support for the traditional use of Brucea javanica in the treatment of diarrhea, and add new therapeutic dimension to its current clinical application as an anti-cancer adjunct, simultaneously endowed with beneficial effect in alleviating irinotecan-associated delayed diarrhea.

Materials and Methods

Chemicals and Reagents

BJOE was obtained from Tonghua Renmin Pharmaceutical Co., Ltd. (Jilin, China), which has been approved by National Medical Products Administration for marketing (230601036). Irinotecan (purity ≥ 98%) was purchased from Dalian Meilun Biotechnology Co., Ltd. (Liaoning, China), and loperamide was sourced from Xi’an Janssen Pharmaceutical Co., Ltd. (Shanxi, China). Vadimezan (DMXAA, purity ≥ 98%) was acquired from KKL MED (Ashland, USA). Rabbit anti-cGAS pAb (A8335) and rabbit anti-STING/TMEM173 mAb (A21051) were obtained from ABclonal (Wuhan, China). Rabbit anti-NAK/TBK1 (phospho-Ser172) mAb (14259) and rabbit anti-TBK1 mAb (52074) were obtained from Signalway Antibody (Maryland, USA). IRF3 recombinant rabbit monoclonal antibody (ET1612-14) was acquired from Huabio (Zhejiang, China). Rabbit anti-phospho-IRF3 (Ser396) (AF2436) was obtained from Affinity Biosciences (Jiangsu, China).

Gas Chromatography-Mass Spectrometry Analysis

The total composition of BJOE was analyzed by gas chromatography-mass spectrometry (GC-MS) after derivatization. Using helium as the carrier gas, the test solution was injected into a DB-WAX strong polarity column (30 cm × 250 μm, 0.25 μm, Agilent) at a flow rate of 1 mL/min with a split ratio of 1:10. The ion source temperature was set to 220°C, interface temperature to 250°C, and injection temperature to 250°C, with an injection volume of 1 μL. The column temperature was maintained at 100°C for 10 min, then ramped to 180°C at 10°C/min and held for 5 min, followed by an increase to 250°C at 20°C/min, where it was held for 10 min.

Animals and Treatments

BALB/c mice (weighed 22–25 g, male) were provided by Guangzhou Rigel Biotechnology Co., Ltd. (SCXK (Guangdong) 2023–0059). Mice were maintained under standard laboratory conditions (temperature: 22°C ± 2°C; humidity: 50% ± 10%; 12 h dark cycle). They were fed a standard diet and provided free access to water for one week to acclimatize to the environment before the experiment. All experimental procedures were approved by the Animal Ethics Committee of Guangzhou University of Chinese Medicine (Approval No. 20231211006). All procedures complied with international animal welfare guidelines, and all personnel received mandatory training to minimize animal suffering.

After one week of acclimatization, 96 BALB/c mice were randomly divided into eight groups (n = 12 for each group): normal group, model group (irinotecan, 45 mg/kg/day, days 1–4, administered intraperitoneally), positive control group treated with loperamide (LO, 4 mg/kg/day, days 1–7, orally), and three groups orally receiving low, medium, and high doses of BJOE (0.25, 0.5, and 1.0 g/kg/day, days 1–7). The dose was administered as previously described with minor modifications.^13,15 Additionally, there was a STING agonist group (DMXAA, 10 mg/kg/day, administered intraperitoneally every other day) and a DMXAA + BJOE combination group. Mice in the normal group received saline injection and blank emulsion orally. The remaining groups were treated with irinotecan to establish the delayed diarrhea model. Mice were allowed free access to water and food, while the corresponding doses of the positive control, BJOE, and agonist were administered via gavage for seven consecutive days. During this process, water and food intake, body weight, faecal output, and general behaviour of mice were recorded. After the final gavage, mice were fasted but watered for 12 h, followed by cervical dislocation for euthanasia and tissue and blood collection.

Disease Activity Index

Body weight, faecal pattern, and faecal occult blood were measured daily. The scoring criteria followed the previous report,¹⁶ where the disease activity index (DAI) was calculated as follows: DAI = (weight change score + blood stool score + dilute stool score) / 3. The DAI score was classified according to the criteria outlined in Table 1.

Table 1 Disease Activity Index (DAI)

Hematoxylin and Eosin (H&E) Staining

Immediately following euthanasia, colonic tissues were excised and placed in 4% paraformaldehyde fixative for 24 h. Subsequently, the tissues were embedded in paraffin, and sections (5 μm thick) were deparaffinized and stained according to standard operating protocols. Hematoxylin and eosin (H&E) staining of the colonic sections was then observed under a microscope and scored by two independent and blinded researchers.¹⁷ The scoring was conducted according to the criteria specified in Table 2.

Table 2 Histological Colitis Score

Alcian Blue Staining

The colon tissue was taken, fixed, dehydrated, and embedded in paraffin according to standard protocols. The resulting sections were sequentially placed in wax solution and ethanol before being rinsed with tap water. Subsequently, the sections were stained with alcian blue staining solution for 10–15 min, followed by another wash with tap water to facilitate nuclear staining. The slices were then dehydrated in anhydrous ethanol and xylene, sealed with neutral gum, and examined microscopically under a light microscope for imaging.

Immunohistochemical Staining

Paraffin-embedded sections of colon tissue were dewaxed and rehydrated, then incubated in citrate antigen retrieval buffer (pH 6.0) for antigen retrieval. Following this, endogenous peroxidase activity was quenched, and the sections were blocked with 3% BSA. Primary and secondary antibodies were then applied sequentially according to the experimental protocol. Subsequently, the sections were stained using the DAB (diaminobenzidine) method, followed by hematoxylin staining of the cell nuclei for 3 min. Immunohistochemical staining was performed to assess the PCNA expression, and images were captured under light microscopy by a blinded technician, with positive cells identified by the presence of brown granules.

RNA Extraction and Quantitative Real-Time PCR

Total RNA was extracted from mouse colon tissues using TRIzol reagent (Vazyme, R401-01). Complementary DNA (cDNA) was synthesized via reverse transcription of RNA using a reverse transcription kit (Vazyme, R223-01). The amplification procedure was subsequently performed according to the manufacturer’s protocol. Primer design was conducted using Oligo 7 software (Molecular Biology Insights, Inc., Colorado Springs, USA) based on sequences from GenBank, with the synthesized primers provided by Sangon Bioengineering (Shanghai, China) Co., Ltd. as detailed in Table 3.

Table 3 Primer Sequences for Real-Time PCR

Western Blotting

Colon tissues were sheared and rinsed with PBS, followed by lysis using RIPA lysis buffer. The samples were centrifuged at 14,000 rpm for 20 min at 4°C, and the supernatant was collected for protein quantification using the BCA method. Each sample was prepared with 40 μg of protein and subjected to separation on a 10% SDS-PAGE gel, after which the proteins were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk powder at room temperature for 1 h. Following washing with TBST, the diluted primary antibody was added and incubated overnight at 4°C, using β-actin (1:5000) as the internal reference. The next day, the membrane was washed with TBST, and an HRP-conjugated secondary antibody (1:10,000) was applied, followed by 1 h incubation at room temperature. After further washing with TBST, ECL chemiluminescent solution was added for detection. Images were captured using a gel imaging system (Tanon 4200SF, China), and protein bands were analyzed with ImageJ (National Institutes of Health, Maryland, USA). The expression levels of cGAS, STING, p-TBK1, TBK1, p-IRF3, IRF3 were evaluated in the colonic tissues of each experimental group.

Statistical Analysis

All data were presented as mean ± standard deviation (SD) and statistically analyzed using SPSS software version 26.0 (Chicago, USA). To ensure reliability, each independent experiment was replicated a minimum of three times. The experimental data were tested for normal distribution. If they met the criteria, one-way ANOVA was employed to compare differences between groups. If the data did not conform to normal distribution, the Kruskal–Wallis test was utilized. For homogeneous variance (P > 0.05), post hoc comparisons were conducted using the Bonferroni method; conversely, if the variance was heterogeneous (P < 0.05), the Dunn’s T3 test was applied. A P-value < 0.05 was considered statistically significant, while a P-value < 0.01 was considered very significant.

Results

The Components of BJOE

The total components of BJOE were analyzed using GC-MS. As shown in Figure 1, nine main components were identified as: hexadecanoic acid (C₁₇H₃₄O₂, 5.13%), methyl stearate (C₁₉H₃₈O₂, 2.27%), 11-octadecenoic acid (C₁₉H₃₆O₂, 19.16%), linoleic acid (C₁₉H₃₄O₂, 20.67%), 9,12,15-octadecatrienoic acid (C₁₉H₃₂O₂, 1.88%), n-hexadecanoic acid (C₁₆H₃₂O₂, 8.35%), octadecanoic acid (C₁₈H₃₆O₂, 19.16%), oleic acid (C₁₈H₃₄O₂, 8.35%), and 9,12-octadecadienoic acid (C₁₈H₃₂O₂, 18.79%) (Table 4). Among the nine components detected, linoleic acid was identified as the major component, which was in concert with previous investigation.¹⁸

Table 4 The Composition of BJOE

Figure 1 BJOE was subjected to GC-MS analysis to identify its major chemical constituents. The percentages of the nine ingredients in BJOE were hexadecanoic acid (C17H34O2, 5.13%), methyl stearate (C19H38O2, 2.27%), 11-octadecenoic acid (C19H36O2, 19.16%), linoleic acid (C19H34O2, 20.67%), 9,12,15-octadecatrienoic acid (C19H32O2, 1.88%), n-hexadecanoic acid (C16H32O2, 8.35%), octadecanoic acid (C18H36O2, 19.16%), oleic acid (C18H34O2, 8.35%), and 9,12-octadecadienoic acid (C18H32O2, 18.79%), respectively.

BJOE Mitigated Enterotoxicity in Mice Experiencing Delayed Diarrhea

Irinotecan was administered to BALB/c mice via intraperitoneal injection for 4 consecutive days, while BJOE and loperamide were administered in each dose group from days 1 to 7 (Figure 2a). As shown in Figure 2b, the rate of body weight change in the model group declined progressively, with a noticeable deceleration in this decline observed following the administration of BJOE and loperamide. The DAI scores for each group are depicted in Figure 2c Mice in the model group exhibited symptoms of perianal soiling and bloody stools. However, those treated with BJOE had significantly lower DAI scores compared to the model group (P < 0.01).

Figure 2 Effect of BJOE on symptoms of mice with delayed diarrhea. (a) Schematic of animal experimental protocol. (b) Rate of change in body weight of mice. (c) DAI score. (d–e) Length of mouse colon. (f–g) HE staining and scoring of mouse colon tissue (200× and 400×). Data are expressed as mean ± SD (n = 6). #P < 0.05, ##P < 0.01, compared with the normal group. *P < 0.05, **P < 0.01, compared with the model group.

Furthermore, Figure 2d–e showed that a marked reduction was observed in colon length following modelling. While the high-dose BJOE group exhibited a notable recovery in colon length relative to the model group (P < 0.01). In the normal group, the colon maintained structural integrity, with intact mucosa, closely arranged and orderly crypts (glands) with no atrophy, and minimal inflammatory cell infiltration in the lamina propria. In contrast, the model group displayed pronounced necrosis in the intestinal tissues of both the mucosal and epithelial layers, along with significant cellular detachment, disrupted intestinal villi, disordered crypt arrangements, and extensive inflammatory cell infiltration within the lamina propria, indicative of successful establishment of delayed diarrhea mouse model (P < 0.01). Nevertheless, administration of BJOE and loperamide resulted in varying degrees of recovery, characterized by a reduction in inflammatory infiltration and a restoration of glandular architecture (P < 0.01). Notably, the high-dose BJOE group showed recovery levels comparable to those in the positive control group (Figure 2f–g). These findings collectively suggested that BJOE exerted beneficial effect in ameliorating irinotecan-induced delayed diarrhea.

BJOE Mitigated Inflammation and Preserved Intestinal Barrier Function in Mice with Delayed Diarrhea

To investigate whether BJOE could reduce intestinal inflammation and protect the intestinal barrier, we measured the mRNA expression levels of TNF-α, IL-1β, IL-6, and iNOS in mouse colon tissues. As shown in Figure 3a, the mRNA expression levels of TNF-α, IL-1β, IL-6, and iNOS were significantly elevated in the model group (P < 0.01). In contrast, administration of varying doses of BJOE and loperamide resulted in a significant reduction in these inflammatory markers (P < 0.01).

Figure 3 Effect of BJOE on inflammation level and intestinal barrier in mice with delayed diarrhea. (a) mRNA expression levels of TNF-α, IL-1β, IL-6, and iNOS by quantitative real-time PCR (n = 6). (b) Representative graphs of alcian blue staining in mice of each group (200×). (c) mRNA expression levels of occludin and ZO-1 detected by quantitative real-time PCR (n = 6). (d) Immunohistochemical staining to detect PCNA expression (200× and 400×). (e) Disruption of the intestinal barrier and inflammatory response induced by irinotecan. Data are expressed as mean ± SD. #P < 0.05, ##P < 0.01, compared with the normal group. *P < 0.05, **P < 0.01, compared with the model group.

Intestinal mucin expression in mice with delayed diarrhea was assessed using alcian blue staining. Mucins were almost absent in the colons of the model group, while varying degrees of restoration were observed in both the BJOE-treated and positive control groups (Figure 3b). The expression of PCNA in colonic tissues, indicative of the level of intestinal proliferation, was also restored following drug administration (Figure 3c).

Additionally, the key tight junction genes, occludin and ZO-1, were evaluated using quantitative PCR to further assess the ameliorative effect of BJOE on irinotecan-induced delayed diarrhea. As depicted in Figure 3d, the mRNA expression of occludin and ZO-1 was significantly downregulated in irinotecan-treated mice compared to the normal group. Notably, the barrier protein expression was upregulated following administration of three doses of BJOE and loperamide. The high-dose BJOE group displayed significant increases in both occludin and ZO-1 levels (P < 0.01). These results suggested that BJOE effectively downregulated the intestinal inflammation and protected the intestinal barrier in mice experiencing delayed diarrhea (Figure 3e).

Effect of BJOE on cGAS-STING Pathway in Mice with Delayed Diarrhea

RNA and protein expression levels of cGAS-STING pathway-related factors were assessed in the colonic tissues of mice across different groups using PCR and Western blotting. (Figure 4a). The mRNA expression levels of cGAS-STING pathway-related genes including cGAS, STING, CXCL10, CCL5, and IFN-β in the colon tissues across the experimental groups are depicted in Figure 4b. Compared to the model group, treatment with BJOE significantly downregulated the mRNA expression of cGAS, STING, CXCL10, CCL5, and IFN-β in colonic tissues (P < 0.01). Furthermore, while the levels of cGAS-STING pathway-related proteins were significantly elevated after modelling (P < 0.05). However, these proteins were significantly downregulated following BJOE administration (P < 0.05). These findings suggested that BJOE effectively inhibited the activation of the cGAS-STING signalling pathway (Figure 4c and d).

Figure 4 Effect of BJOE on cGAS-STING pathway-related factors in delayed diarrhea mice. (a) PCR and Western blotting workflow diagram. (b) mRNA expression levels of cGAS, STING, CXCL10, CCL5, and IFN-β measured by quantitative real-time PCR (n = 6). (c) Representative protein imprinted bands of cGAS, STING, p-TBK1, TBK1, p-IRF3, and IRF3. (d) Protein expression levels of cGAS, STING, p-TBK1, TBK1, p-IRF3, and IRF3 were measured by Western blot (n = 5). Data are expressed as mean ± SD. #P < 0.05, ##P < 0.01, compared with the normal group. *P < 0.05, **P < 0.01, compared with the model group.

cGAS-STING Pathway Activation Aggravated Intestinal Toxicity in Mice with Delayed Diarrhea

Based on these experiments, we employed DMXAA, a STING agonist, to verify whether BJOE alleviated irinotecan-induced delayed diarrhea by modulating the cGAS-STING signalling pathway. The experimental design for each group of animals was depicted in Figure 5a. As illustrated in Figure 5b–g, both the DMXAA and model groups exhibited a continuous decline in body weight, significantly elevated DAI scores, and markedly shortening of colon length (P < 0.01). Histopathological analysis revealed the loss of crypts in the model group, accompanied by extensive inflammatory cell infiltration in the lamina propria. Notably, colonic tissue damage was exacerbated in the DMXAA group. However, the BJOE treatment group exhibited substantial recovery of colonic architecture (P < 0.01). In contrast, the therapeutic effect of BJOE was markedly diminished in the DMXAA+BJOE group (P < 0.05).

Figure 5 Enhancing STING secretion diminished the effect of BJOE on symptoms in mice with delayed diarrhea. STING agonist DMXAA was used to activate the cGAS-STING pathway. (a) Animal experimental design diagram. (b) Rate of change in body weight of mice. (c) DAI score. (d–e) Length of mouse colon. (f–g) HE staining and scoring of mouse colon tissue (200× and 400×). Data are expressed as mean ± SD (n = 6). #P < 0.05, ##P < 0.01, compared with the normal group. *P < 0.05, **P < 0.01, compared with the model group. &P < 0.05, &&P < 0.01, compared with the BJOE group.

cGAS-STING Pathway Activation Influenced Inflammation and Intestinal Barrier in Mice with Delayed Diarrhea

We further assessed the levels of inflammatory factors, namely TNF-α, IL-1β, IL-6, and iNOS, in the colonic tissues of mice across different groups. The model and agonist groups exhibited a significant elevation in inflammation. However, the BJOE-treated group displayed a marked reduction in these inflammatory markers compared to the model group. Notably, this improvement was attenuated in the DMXAA+BJOE group (Figure 6a). The expression of intestinal mucins in mice was evaluated using alcian blue staining. Mucin levels were nearly absent in the model and DMXAA groups, while significant improvement was observed in the BJOE group. Improvement in mucin expression in the DMXAA + BJOE group was curtailed (Figure 6b).

Figure 6 Promoting STING secretion mitigated BJOE’s effects on inflammation and intestinal barrier in delayed diarrhea mice. STING agonist DMXAA was used to activate the cGAS-STING pathway. (a) mRNA expression levels of TNF-α, IL-1β, IL-6, and iNOS measured by quantitative real-time PCR (n = 6). (b) Representative graphs of mice stained with alcian blue in each group (200×). (c) Immunohistochemical staining to detect PCNA expression (200× and 400×). (d) mRNA expression levels of occludin and ZO-1 detected by quantitative real-time PCR (n = 6). Data are expressed as mean ± SD. #P < 0.05, ##P < 0.01, compared with the normal group. *P < 0.05, **P < 0.01, compared with the model group. &P < 0.05, &&P < 0.01, compared with the BJOE group.

The number of proliferating crypt cells in the colons of mice was significantly reduced in both the model and agonist groups. However, PCNA expression level was markedly restored in the BJOE group. Conversely, the DMXAA + BJOE group exhibited reduced PCNA level compared to the BJOE group (Figure 6c). Furthermore, mRNA levels of barrier genes occludin and ZO-1 were significantly decreased in the model and agonist groups, while their expression was up-regulated following treatment with BJOE (Figure 6d). However, the mRNA levels of occludin (P < 0.01) and ZO-1 were down-regulated in the DMXAA + BJOE group compared to the BJOE group. Collectively, these findings indicated that BJOE enhanced the gene and protein expression of intestinal barrier in mice with delayed diarrhea, and this effect was compromised by DMXAA.

BJOE Suppressed Aberrant Activation of the cGAS-STING Pathway

In comparison to the normal group, the mRNA expression levels of cGAS-STING pathway-related factors were significantly elevated in both the model and agonist groups (P < 0.05). Following the administration of BJOE, the mRNA expression levels of cGAS, STING, CXCL10, CCL5, and IFN-β were notably down-regulated (P < 0.05). Additionally, the expression levels of cGAS-STING pathway-related factors were evidently increased in the DMXAA+BJOE group compared to the BJOE group (P < 0.05) (Figure 7a).

Figure 7 Promoting STING secretion attenuated BJOE’s effects on cGAS-STING pathway-related factors in delayed diarrhea mice. STING agonist DMXAA was used to activate the cGAS-STING pathway. (a) mRNA expression levels of cGAS, STING, CXCL10, CCL5, and IFN-β measured by quantitative real-time PCR (n = 6). (b) Representative protein imprinted bands of cGAS, STING, p-TBK1, TBK1, p-IRF3, and IRF3. (c) Protein expression of cGAS, STING, p-TBK1, TBK1, p-IRF3, and IRF3 were measured by Western blot (n = 5). (d) BJOE suppressed aberrant activation of the cGAS-STING pathway. Data are expressed as mean ± SD. #P < 0.05, ##P < 0.01, compared with the normal group. *P < 0.05, **P < 0.01, compared with the model group. &P < 0.05, &&P < 0.01, compared with the BJOE group.

Western blot analysis (Figure 7b and 7c) indicated that BJOE treatment significantly downregulated the protein expression levels of cGAS, STING, p-TBK1, and p-IRF3 when compared to the model group (P < 0.05). While the expression levels of these cGAS-STING pathway-related proteins were remarkably elevated in the DMXAA+BJOE group (P < 0.05). These results suggested that BJOE might alleviate irinotecan-induced delayed diarrhea in mice by inhibiting the aberrant activation of the cGAS-STING pathway (Figure 7d).

Discussion

Recent advancements in cancer treatment, especially in immunotherapy and targeted therapy, have achieved significant breakthroughs, yet adverse effects continue to limit their efficacy.¹⁹ Irinotecan, a widely used chemotherapeutic agent for solid tumors like colorectal, pancreatic, and lung cancers,^20,21 is limited by the adverse effect of delayed diarrhea, which restricted its dosage and completion of chemotherapy regimens.²² This side effect profoundly impacts patients’ performance status and their ability to engage in daily activities, leading to decreased work capacity, strained interpersonal relationships, and psychological distress.

Currently, several Western medications are available to address irinotecan-induced diarrhea, including the opioid receptor agonist loperamide and the long-acting growth hormone analogue octreotide.²³ However, the incidence of adverse effects, such as paralytic intestinal obstruction, rises with escalating doses of loperamide.²⁴ A Phase III randomized clinical trial demonstrated that long-acting octreotide neither prevented nor alleviated the severity of diarrhea in colorectal cancer patients undergoing chemotherapy-induced diarrhea.²⁵ Currently, there is no standardized and effective clinical treatment for delayed diarrhea induced by irinotecan. Modern pharmacology has confirmed that traditional Chinese medicine effectively alleviates various adverse effects associated with chemotherapy, demonstrating a favorable safety profile.^26,27 Furthermore, several clinical trials^28–32 have demonstrated that adjuvant therapy with Chinese herbal medicine can significantly prevent and treat chemotherapy-associated diarrhea without compromising the anti-tumor activity of chemotherapy. This approach offers a promising and effective strategy for managing delayed diarrhea induced by irinotecan.

Brucea javanica is recognized as a pivotal treatment for dysentery. It is primarily used in clinical settings as a traditional Chinese medicine preparation, specifically BJOE, as a monotherapy or an adjunct to anti-tumor therapies in modern practice. Research has indicated that this emulsion possessed the capability to inhibit cell proliferation, induce apoptosis in tumor cells, and mitigate resistance to chemotherapy agents.³³ For example, the combination of BJOE with conventional chemotherapeutic drugs has been shown to enhance the quality of life for patients suffering from malignant pleural effusion.³⁴ Furthermore, the administration of BJOE has been shown to mitigate the myelotoxicity and gastrointestinal reactions associated with conventional chemotherapeutic agents. Existing research has reported that BJOE possessed significant anti-ulcer activity, exhibiting protective effects against gastric ulcers in both mice and rats.³⁵

GC-MS analysis showed that linoleic acid was the main component of BJOE, which was consistent with previous report.³⁶ Linoleic acid reduced the production of pro-inflammatory mediators via the nuclear hormone receptor PPARγ in macrophages.³⁷ Thus, the anti-inflammatory effect of BJOE may be linked to its component linoleic acid. Our previous studies have confirmed that BJOE effectively alleviated ulcerative colitis (UC) induced by dextran sulfate sodium salt in mice. This effect was attributed to its ability to improve intestinal inflammation and promote the repair of intestinal mucosal damage, thereby exerting notable anti-UC effects.^36,38 The aforementioned studies indicated that BJOE enhanced the efficacy of chemotherapeutic agents and exhibited significant anti-inflammatory properties. Given that delayed diarrhea induced by the chemotherapeutic agent irinotecan was closely associated with inflammation, we hypothesized that BJOE could alleviate the intestinal adverse effects associated with irinotecan. Our results indicated that BJOE significantly improved the physical condition of mice, including reductions in weight loss, colon shortening, and DAI scores.

Chemotherapy-induced intestinal adverse effects frequently lead to mucositis, where inflammatory factors play a critical role in inflammation and tissue damage resulting from cytotoxic treatments. Subsequent studies have demonstrated that chemotherapeutic agents stimulated the activation of NF-κB, leading to the production of pro-inflammatory cytokines, including TNF-α, IL-6, and IL-1β, which contributed to mucosal injury.³⁹ In addition to enhancing the physical condition of mice with delayed diarrhea, BJOE significantly reduced the elevation of inflammatory factors associated with delayed diarrhea, as well as the mRNA expression levels of TNF-α, IL-1β, IL-6, and iNOS.

The dynamic regulation of tight junction proteins is fundamental to various physiological processes. Disruption of this regulation significantly alters mucosal barrier function and intestinal permeability, establishing tight junction proteins as critical markers of numerous pathological states. Irinotecan has been shown to induce defects in tight junction proteins, resulting in mucosal barrier dysfunction and diarrhea.⁴⁰ Consequently, comparison of mRNA levels of the tight junction proteins occludin and ZO-1 across groups revealed a significant increase in the colon of mice treated with BJOE compared to the model group. The mucus barrier served as the primary defense layer separating the microbiota from the epithelial cells, comprising mucus secreted by goblet cells that coated the intestinal epithelium.⁴¹

Irinotecan has been reported to decrease the number of mucin-producing goblet cells in the colon.⁴² Our study corroborated with the existing literature, indicating that goblet cells were significantly reduced in the model group. The mucin expression in the BJOE treatment group was comparable to that in the positive control group, suggesting that BJOE enhanced mucin secretion and played a protective role in maintaining the intestinal barrier. PCNA is essential for DNA replication, DNA repair, cell cycle regulation, and apoptosis.⁴³ PCNA is involved in DNA damage repair and promotes the proliferation of intestinal epithelial cells in the mouse intestine. Maintaining a balance between cell proliferation and apoptosis is essential for preserving the integrity of the intestinal barrier.⁴⁴ Our results indicated that irinotecan administration led to a significant loss of proliferating cells in the colons of mice. In contrast, treatment with BJOE restored cell proliferation across all dosage groups.

The innate immune system is crucial for maintaining mucosal homeostasis. Dysregulated innate immune responses are linked to gastrointestinal inflammation and cancer. Dysfunction in intestinal immune defense and imbalances in tolerance regulatory mechanisms can result in mucosal injury and gastrointestinal disorders. The cGAS-STING signaling pathway plays a crucial role in maintaining intestinal homeostasis by detecting both self and non-self DNA. However, excessive activation of this pathway can lead to gastrointestinal inflammation. Therefore, the cGAS-STING signaling pathway may provide enlightening insights for developing new therapeutic strategies.⁴⁵

Irinotecan, a selective DNA topoisomerase I (ToPoI) inhibitor, covalently binds to the DNA-topoisomerase complex,⁴⁶ leading to the accumulation of damaged dsDNA in the cytoplasm that activates the cGAS-STING pathway in intestinal epithelial cells. Upon activation, STING initiates downstream signaling cascades involving TBK1 and IRF3.⁴⁷ Phosphorylated IRF3, upon dimerization and translocation to the nucleus, activates gene transcription of factors such as IFN-β, CXCL10, and CCL5.^48,49 In our work, the mRNA levels of cGAS, STING, CXCL10, CCL5, and IFN-β, as well as the protein expression levels of cGAS, STING, p-TBK1, and p-IRF3 in the cGAS-STING pathway were significantly upregulated in the model group. In contrast, the expression of cGAS-STING pathway-related factors in the BJOE group was significantly ameliorated in parallel to the irinotecan model group.

STING knockout mice demonstrated a reduced inflammatory response and decreased intestinal permeability. Conversely, mice treated with the STING agonist DMXAA exhibited increased intestinal apoptosis and exacerbated systemic inflammation, indicating that the activation of the STING pathway contributed to intestinal inflammatory responses.⁵⁰ Therefore, we employed the agonist DMXAA to investigate whether BJOE alleviated irinotecan-induced delayed diarrhea by inhibiting aberrant activation of the cGAS-STING pathway. As anticipated, our experimental results indicated a significant increase in the mRNA levels of cGAS, STING, CXCL10, CCL5 and IFN-β, as well as in the protein expression levels of cGAS, STING, p-TBK1, and p-IRF3 in the DMXAA+BJOE group compared to those of the BJOE group. Irinotecan induced delayed diarrhea by disrupting the balance of the cGAS-STING pathway, resulting in hyperactivation, while BJOE inhibited this activation, thereby exerting a therapeutic effect.

Nevertheless, there were also some limitations in this study. Firstly, the absence of clinical validation challenged translational relevance. While species differences may affect the pharmacokinetics and pharmacodynamics of BJOE, highlighting the need for further evaluation in human trials. Secondly, potential off-target or dose-related adverse effects also warrant dedicated safety assessments, particularly when combined with irinotecan. Thirdly, a relatively small sample size may reduce power to detect subtle effects.

Further in-depth investigation was warranted. For example, future research should focus on identifying the active compounds in BJOE responsible for its therapeutic effects, particularly in modulating the cGAS-STING pathway. Advanced preclinical platforms, such as human intestinal organoids and STING-humanized mouse models, should be used to better understand BJOE’s therapeutic mechanism. Furthermore, long-term safety and pharmacokinetics studies should be performed before clinical applications. These efforts are envisaged to further define BJOE’s clinical potential.

Conclusion

In conclusion, BJOE significantly alleviated irinotecan-induced delayed diarrhea in mice, at least in part, via inhibiting the aberrant activation of the cGAS-STING signaling pathway and subsequent intestinal homeostasis (Figure 8). BJOE may serve as a promising adjuvant strategy to mitigate chemotherapy-associated gastrointestinal toxicity. Future studies should aim to isolate its active constituents and evaluate their safety and efficacy in clinically relevant models to advance translational application.

Figure 8 Schematic illustration of the potential protective mechanism of BJOE in irinotecan-induced delayed diarrhea mice. BJOE attenuated intestinal inflammation, as indicated by decreased levels of TNF-α, IL-1β, IL-6, and iNOS. It also preserved intestinal barrier integrity by upregulating the expression of mucins and tight junction proteins, including ZO-1 and Occludin. These protective effects may be associated with the inhibition of aberrant activation of the cGAS-STING signaling pathway.