Effect of Rhizoma Paridis saponin on the pain behavior in a mouse model of cancer pain

Abstract

Rhizoma Paridis saponins (RPS) as active parts of P. polyphylla Smith var. yunnanensis has been used as an anti-cancer drug in traditional Chinese medicine. In this study, RPS was first found to demonstrate a potent effect on markedly reducing the pain induced by cancer. Therefore, the aim of this study was to further explore the analgesic effect of RPS and its possible reaction pathway on H22 hepatocarcinoma cells inoculated in the hind right paw of mice. Cancer-induced pain model mice were randomly divided into 5 groups (n = 10) and orally administered with RPS (50-200 mg kg^-1) for 2 weeks. On the last day of treatment, the pain behavior of mice was measured using hot-plate test and open field test, and brain tissues were sampled for detection of biochemical indices, malondialdehyde (MDA), superoxide dismutase (SOD), prostaglandin E2 (PGE2), serotonin (5-HT) and β-endorphin (β-EP). Moreover, the concentrations of NF-κB and IL-1β in the blood serum were measured by ELISA reagent kits. In addition, naloxone, the non-selective antagonist of opioid receptors, was used to identify the opioid receptors involved in RPS's action. It has been found that RPS alleviates cancer pain mainly via the suppression of inflammatory pain induced by oxidative damage, such as decreasing MDA and PGE2 levels, renewing activity of SOD, as well as increasing 5-HT and β-EP in the brain and suppressing the expression of NF-κB and IL-1β in the serum in a concentration-dependent manner. Overall, the current study highlights that RPS has widespread potential antinociceptive effects on a mouse model of chronic cancer pain, which may be associated with the peripheral nervous system and the central nervous system.

Fig. 1. Time-course of tumor growth in H22 hepatocarcinoma cells inoculated and normal saline. Each value represents the mean ± SE. Compared with the model group using one-way ANOVA with Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.01.

Fig. 2. The picture of H22 tumor after subcutaneous inoculation with H22 hepatoma cells into the plantar region of the hind right paw. (A) Control group, (B) model group, (C) INN group, (D) RPS-H group, (E) RPS-M group, and (F) RPS-L group.

Fig. 3. Analgesic effect of three dosages of RPS on established tumor-bearing paw model in hot-plate test. Each value is represented as the mean ± SE. Values within treatment groups having different letters are significantly different, as indicated by one-way ANOVA and Student's t-test. Letters a–d, means with the same letter is not significantly different (p < 0.05).

Fig. 4. Effect of RPS on the open field test of the H22 cell inoculated cancer pain model. (A) Static time of mice. (B) Movement distance of mice. Each value represents the mean ± SE. Compared with the model group using one-way ANOVA with Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.01.

Fig. 5. Movement track in cancer pain model H22 hind paw of mice, as recorded in an open field test. (A) Control group, (B) model group, (C) INN group, (D) RPS-H group, (E) RPS-M group, (F) RPS-L group.

Fig. 6. Histomorphological micrograph of the hematoxylin and eosin (HE) stained paws of the control and carcinoma-bearing rats (scale bar = 10 μm). (A) Control group, (B) model group, (C) INN group, (D) RPS-H group, (E) RPS-M group, and (F) RPS-L group.

Fig. 7. Effects of three dosages of RPS and positive drug (INN) on the expression of MDA in brain tissues. Each value represents the mean ± SE from three independent experiments (n = 6 per group). Values within treatment groups having different letters (a–d) are significantly different and vice versa, as indicated by one-way ANOVA and Student's t-test (p < 0.05).

Fig. 8. Effects of three dosages of RPS and positive drug (INN) on the expression of SOD in brain tissues. Each value represents the mean ± SE from three independent experiments (n = 6 per group). Values within treatment groups having different letters (a–c) are significantly different and vice versa, as indicated by one-way ANOVA and Student's t-test (p < 0.05).

Fig. 9. Effects of three dosages of RPS and positive drug (INN) on the expression of PGE2 in brain tissues. Each value represents the mean ± SE from three independent experiments (n = 6 per group). Values within treatment groups having different letters (a–e) are significantly different and vice versa, as indicated by one-way ANOVA and Student's t-test (p < 0.05).

Fig. 10. Effects of three dosages of RPS and positive drug (INN) on the expression of β-EP in brain tissues. Each value represents the mean ± SE from three independent experiments (n = 6 per group). Values within treatment groups having different letters (a–e) are significantly different and vice versa, as indicated by one-way ANOVA and Student's t-test (p < 0.05).

Fig. 11. Effects of three dosages of RPS and positive drug (INN) on the expression of 5-HT in brain tissues. Each value represents the mean ± SE from three independent experiments (n = 6 per group). Values within treatment groups having different letters (a–e) are significantly different and vice versa, as indicated by one-way ANOVA and Student's t-test (p < 0.05).

Fig. 12. The relationship between the paw licking latency of the hot-plate test and (A) MDA level, (B) PGE2 level, (C) SOD activity, (D) 5-HT level and (E) β-EP level are shown. The values of r and p (Pearson's test), and linear fit (black line) were also obtained.

Fig. 13. The mechanism of analgesic effect of RPS on cancer-induced pain model mice.

Fig. 14. Effects of three dosages of RPS and positive drug (INN) on the serum levels of NF-κB and IL-1β in established tumor-bearing paw model. (A) ELISA-based assay with plate-adhered oligonucleotides containing an NF-kB consensus binding sequence. (B) ELISA-based assay with plate-adhered oligonucleotides containing an IL-1β consensus binding sequence. Data represent mean ± SE from three independent experiments (n = 6 per group). Values within treatment groups having different letters (a–e) are significantly different and vice versa, as indicated by one-way ANOVA and Student's t-test (p < 0.05).

Fig. 15. Opioid receptors mediate RPS-induced antinociception. The systemic preadministration of naloxone (1 mg kg⁻¹, i.p.), an opioid receptor antagonist, 30 min before pretreatment with morphine ((A) 2.5 mg kg⁻¹, s.c.) and RPS ((B) 200 mg kg⁻¹, i.g.) significantly reverses the antinociceptive effects of these treatments, when compared to the control group, after intraplantar injection of formalin (2.5%, i.pl.) in mice. Each column represents the mean of 6 mice, and the vertical lines indicate the SE. ***P < 0.001 as compared with control group or ^###P < 0.001 when comparing treatment with naloxone + treatment (one-way ANOVA followed by and Student's t-test).