Benefits of Camelina sativa Supplementation in Morphine Treatment: Enhanced Analgesia, Delayed Tolerance and Reduced Gut Side Effects Through PPAR-α Receptor Engagement

Abstract

Long-term opioid therapies are severely limited by the development of analgesic tolerance and gastrointestinal side effects. Camelina sativa, a plant of the Brassicaceae family, modulates the activity of peroxisome proliferator-activated receptor α (PPAR-α receptor), which is involved in the regulation of pain processing and gut physiology. The aim of this study was to evaluate the efficacy of Camelina sativa defatted seed meal (DSM) supplementation on the development of analgesic tolerance and side effects after repeated treatment with morphine in naïve mice. Co-administering Camelina sativa DSM (1 g kg^-1 p.o.) and morphine (10 mg kg^-1 s.c.) increased the efficacy and duration of the opioid-induced acute analgesic effect. Camelina supplementation also delayed the onset of tolerance to the morphine analgesic effect. The same result was obtained through either simultaneously administering morphine and camelina or administering camelina 24 h before morphine injection for the entire duration of the experiment. Camelina also counteracted intestinal damage and visceral hypersensitivity caused by morphine treatment. The beneficial effects of camelina on morphine-related analgesic efficacy and gut side effects were prevented via pre-treatment with the PPAR-α antagonist GW6471, though the latter did not influence the development of morphine tolerance. In conclusion, Camelina sativa DSM could be used as a supplement to improve the therapeutic profile of morphine.

Keywords: Brassicaceae; constipation; flavonoids; glucosinolate; opioid tolerance; peroxisome proliferator-activated receptor; polyunsaturated fatty acids; visceral hypersensitivity.

Figure 1

Effect of Camelina sativa DSM acute administration on mice pain threshold and morphine-induced antinociception. Camelina sativa DSM (0.5–1 g kg⁻¹) with and without myrosinase (Myr; enzyme promoting bioactivation of glucosinolates) was suspended in 1X PBS and administered orally. The acute analgesic effect was measured by Hot Plate test (50 °C, (A)) and by Paw Pressure test (B). The data shown represent the mean ± SEM of 5 mice per experimental group. ** p < 0.01 vs. pre-test (0 min). Acute analgesic effect of co-administering morphine (10 mg kg⁻¹ subcutaneously) and Camelina sativa DSM (1 g kg⁻¹ per os, bioactivated with Myr). The analgesic effect was measured by Hot Plate test (49 °C, (C)) and by Paw Pressure test (D). The data shown represent the mean ± SEM of 6 mice per experimental group. Statistical significance was assessed by one- or two-way analysis of variance (ANOVA) with a Bonferroni post-test. ** p < 0.01 vs. vehicle + vehicle group. ^ p < 0.05 and ^^ p < 0.01 vs. morphine + vehicle group.

Figure 2

Effect of repeated administration of Camelina sativa DSM on the development of tolerance to the analgesic effect of morphine. Morphine (10 mg kg⁻¹ subcutaneously) and Camelina sativa DSM (1 g kg⁻¹ + Myr per os) were administered once daily in mice for 13 consecutive days according to the experimental scheme (A). Behavioral tests were performed daily 24 h after the administration of Camelina sativa DSM, before ((B,D); mice basal threshold) and 30 min after morphine injection in mice (C,E) by Hot Plate test (50 °C, (B,C)) and by Paw Pressure test (D,E). The data obtained represent the mean ± SEM per each experimental group (vehicle + vehicle, n = 11; vehicle + Camelina sativa DSM, n = 6; morphine + vehicle, n = 16; morphine + Camelina sativa DSM, n = 13–14). Statistical significance was assessed by one- or two-way analysis of variance (ANOVA) with a Bonferroni post-test. * p < 0.05 and ** p < 0.01 vs. vehicle + vehicle. ^ p < 0.05 and ^^ p < 0.01 vs. morphine + vehicle.

Figure 3

Effect of Camelina sativa DSM and morphine co-administration on the development of opioid-related analgesic tolerance and gastrointestinal side effects. Morphine (10 mg kg⁻¹ subcutaneously) and Camelina sativa DSM (1 g kg⁻¹ + Myr per os) were concomitantly administered in mice once daily for 9 consecutive days (experimental scheme, (A)). Behavioral tests were daily performed 30 min after the co-administration of Camelina sativa DSM and morphine in mice by Hot Plate test (50 °C, (B)). The gastrointestinal side effects were evaluated at the end of the behavioral tests, through the measure of the intestinal transit rate by the Carmine Red test, expressed as the average latency of excretion of the red-labeled pellet in the three experimental groups (C), the measure of mice visceral sensitivity by scoring (0–4) AWR response to colorectal distension (50–200 µL; (D)) and the analysis of microscopic damage to the colon (E), performed on H&E-stained slices (representative images in (F); Original magnification 4×; scale bar 500 μm). The data obtained represent the mean ± SEM of 6 mice per experimental group. Statistical significance was assessed by one- or two-way analysis of variance (ANOVA) with a Bonferroni post-test. * p < 0.05 and ** p < 0.01 vs. vehicle + vehicle group. ^ p < 0.05 and ^^ p < 0.01 vs. morphine + vehicle group.

Figure 4

The involvement of PPAR-α in enhancing antinociception and reducing the gastrointestinal side effects of morphine related to Camelina sativa DSM supplementation. Morphine (10 mg kg⁻¹ subcutaneously) and Camelina sativa DSM (1 g kg⁻¹ + Myr per os) were concomitantly administered in mice once daily for 14 consecutive days. The selective antagonist of PPAR-α, GW6471 (2 mg kg⁻¹, i.p.), was administered 15 min before Camelina sativa DSM and morphine, as described in the experimental scheme (A). The effect of PPAR-α antagonism on the increase in efficacy and duration of the morphine analgesic effect due to co-administration with Camelina sativa DSM was assessed on day 1 (B) by Hot Plate test (50 °C). The same behavioral test was repeated before ((C) pre-test) and 30 min after the daily co-administration of Camelina sativa DSM and morphine in mice (D) pre-treated or not with GW6471, until analgesic tolerance developed in all experimental groups. The tolerability of treatments was evaluated by monitoring body weight through all the experiments (E). At the end of the behavioral tests, mice visceral sensitivity was measured by scoring (0–4) the AWR response to colorectal distension (50–200 µL; (F)). The data obtained represent the mean ± SEM of 8 mice (or 6 mice for visceral pain assessment) per experimental group. Statistical significance was assessed by one- or two-way analysis of variance (ANOVA) with a Bonferroni post-test. * p < 0.05 and ** p < 0.01 vs. vehicle + vehicle group. ^ p < 0.05 and ^^ p < 0.01 vs. morphine + vehicle group. ° p < 0.05 and °° p < 0.01 vs. morphine + Camelina sativa DSM group.