Ojeok-san ameliorates visceral and somatic nociception in a mouse model of colitis induced colorectal cancer

Abstract

Cancer patients can develop visceral, somatic, and neuropathic pain, largely due to the malignancy itself and its treatments. Often cancer patients and survivors turn to the use of complementary and alternative medicine (CAM) to alleviate pain and fatigue. Thus, it is necessary to investigate how CAM therapies work as novel analgesics to treat cancer pain. Ojeok-san (OJS) is an herbal formula consisting of seventeen herbs. This herbal formula has been shown to possess anti-inflammatory, immunoregulatory, and analgesic properties. In this study, we examined the potential beneficial effects and mechanism of action of OJS in a preclinical model of colitis-associated colorectal cancer. Male and female C57BL/6J mice were exposed to the carcinogen, azoxymethane (AOM, 10 mg/kg) and a chemical inflammatory driver, dextran sulfate sodium (DSS1-2%), to promote tumorigenesis in the colorectum. OJS was given orally (500, 1000, and 2000 mg/kg) to determine its influence on disease activity, tumor burden, nociception, sedation, Erk signaling, and behavioral and metabolic outcomes. In addition, in vitro studies were performed to assess CT-26 cell viability, dorsal root ganglia (DRG) activation, and bone-marrow-derived macrophage (BMDM) inflammatory response to lipopolysaccharide stimulation after OJS treatment. We found that administration of 2000 mg/kg of OJS was able to mitigate mechanical somatic and visceral nociception via Erk signaling without affecting symptom score and polyp number. Moreover, we discovered that OJS has sedative properties and elicits prolonged total sleeping time in AOM/DSS mice. Our in vitro experiments showed that OJS has the capacity to reduce TNFα gene expression in LPS-stimulated BMDM, but no changes were observed in DRG spike number and CT-26 cell proliferation. Taken together, these data suggest that OJS ameliorates nociception in mice and warrants further examination as a potential CAM therapy to promote analgesia.

Fig 1. Graphical depiction of the experimental designs.

(A) Experiment 1, (B) Experiment 2, and (C) Experiment 3. Created with Biorender.com.

Fig 2. OJS (2000 mg/kg) mitigates referred somatic hyperalgesia in colitis-induced colorectal cancer without impacting tumor burden.

(A) Mechanical nociceptive threshold to von Frey filament (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, and 0.60 gr). (B) Disease activity was calculated using score-based symptoms including body weight loss, fecal consistency, and blood in the stool. (C) Polyp count. Number of mice per group as indicated: AOM/DSS-Vehicle, n = 8; AOM/DSS-OJS 500 mg/kg, n = 7; AOM/DSS-OJS 1000 mg/kg, n = 8; and AOM/DSS-OJS 2000 mg/kg, n = 6. * Indicates statistical significance (p<0.05) for AOM/DSS-Vehicle versus AOM/DSS OJS 2000 mg/kg; # indicates statistical significance (p<0.05) for AOM/DSS-OJS 500 mg/kg versus AOM/DSS-OJS 2000 mg/kg from a one-way ANOVA Tukey’s multiple comparisons test. (D) CT-26 cell impedance (proliferation) was evaluated pre- (6 and 24 hrs) and post- (30, 50, and 72 hrs) OJS and Tergazyme (1%, cell lysis) administration. The assay was run in 96-well plate in the presence of OJS at doses ranging from 10–500 ug/mL. Each point represents the mean + SE obtained in three biological replicates; each group consist of twelve technical replicates. ^ Indicates statistical significance (p<0.05) for all groups (CT26) versus media only; $ indicates statistical significance (p<0.05) for CT26-NTC and CT26-OJS (all doses) versus media only and CT26-tergazyme.

Fig 3. OJS reduces visceral pain-related intracolonic pressure during ascending phasic distensions in AOM/DSS mice.

Each colorectal distension (CRD, 10, 25, 40, 65, and 80 mmHg) included a 20 sec duration followed by a 5 min interval between distensions and each distension was repeated 3 times. (A) Control non-cancer mice and (B) AOM/DSS mice were orally gavaged with Vehicle (water) or OJS (2000 mg/kg dissolved in water) 10 min prior to the initiation of CRD. * Indicates statistical significance (p<0.05) from a two-way RM ANOVA Fisher LSD, n = 10 in non-cancer (control) groups, and n = 7–8 in AOM/DSS groups. (C) Symptom score, (D) colon length, and (E) polyp number were assessed in AOM/DSS-Vehicle and AOM/DSS-OJS mice. Spinal cord protein expression of (F) phosphorylated and (G) total Erk and (H) blot images. * Indicates statistical significance (p<0.05) from two-way ANOVA Fisher LSD, n = 4–5 in non-cancer (control) groups, and n = 3–4 in AOM/DSS groups.

Fig 4. OJS administration did not elicit changes in cell blood counts.

(A) White blood cells and (B) Red blood cells were measured using a VetScan HMT. Two-way ANOVA, n = 8–9 in Control non-cancer group and n = 4–7 in AOM/DSS group.

Fig 5. OJS exhibits sedative properties in the AOM/DSS model.

Exploratory behavior was assessed using a custom acrylic box with two distinct compartments. Percent time that the mice spent in (A) center, (B) black box, and (C) elevated platform were evaluated at baseline (pre-OJS) and after OJS (post-OJS) administration. n = 17 Control non-cancer mice and n = 13 AOM/DSS mice. * Indicates statistical significance between groups (p<0.05) from Mixed-effects model (REML) Sidak post hoc. (D) Neuromuscular performance in rotarod test from pre-OJS to post-OJS treatment. n = 8 Control non-cancer mice and n = 8 AOM/DSS mice. * Indicates statistical significance between groups (p<0.05) from two-way RM ANOVA Fisher LSD post hoc.

Fig 6. OJS decreases locomotor activity and increases sleep time during the night cycle in the AOM/DSS model.

Behavioral phenotyping was achieved by use of the automated Promethion System. Percent time of the cycle that (A) Control non-cancer and (B) AOM/DSS mice spent walking. Total hours that (C) Control non-cancer mice and (D) AOM/DSS mice spent sleeping during the light and night cycle. * Indicates statistical significance between groups (p<0.05) from two-way RM ANOVA Fisher LSD, n = 8/group.

Fig 7. Behavioral time budget pre- and post-OJS administration in the drinking water.

Percentage time Control non-cancer mice and AOM/DSS mice spent eating (A, B), drinking water (C, D), inside the habitat (E, F) and long roaming (G, H). * Indicates statistical significance between groups (p<0.05) from paired t-test, n = 8/group.

Fig 8. Mice decreased water intake when OJS was added to the water bottle.

Food (A, B) and water (C, D) intake in grams during the light and night cycle for control (non-cancer) and AOM/DSS mice. * Indicates statistical significance between groups (p<0.05) from two-way RM ANOVA Fisher LSD, n = 8, control, and n = 8 AOM/DSS.

Fig 9. Pre-treatment of OJS did not mitigate DRG activation but decreased inflammatory responses in BMDM.

(A) Rat DRG sensory afferents were given inflammatory mediators (IS, inflammatory soup) and U0126 (Erk inhibitor) to determine DRG excitability. (B) The impact of an OJS dose response (100, 200, and 500 μg/mL) on spontaneous DRG firing. (C) Pre-treatment of OJS (100, 200, and 500 μg/mL) 15 min prior to activating DRG afferents with IS. (D) TNFα mRNA expression resulting from LPS-stimulated BMDM. Each bar represents the mean + SE obtained from at least three biological replicates. * Indicates statistical significance between groups (p<0.05) from one-way ANOVA Tukey’s post hoc, n = 3/4 per group.