TRPV1 channel antagonist capsazepine alleviates morphine tolerance and morphine-induced neurotoxicity by preventing mitochondrial damage and apoptosis: an in vivo and in vitro study

Abstract

Morphine is one of the drugs frequently used for severe pain in chronic diseases such as cancer, but long-term use leads to morphine tolerance. The mechanism of morphine tolerance is not yet fully understood. This study aimed to investigate the effects of the TRPV1 channel antagonist capsazepine (CPZ) on morphine tolerance and morphine-induced neurotoxicity, mitochondrial damage, and apoptosis by in vivo and in vitro methods. Thirty-six male Wistar Albino rats, aged 12-14 weeks (weight 230-250 g), were included in the study. To evaluate the effect of morphine on mitochondrial damage and apoptosis, cytochrome c, apoptosis-inducing factor (AIF), caspase-9, and caspase-3, Bax, and Bcl-2 levels were determined from tissue samples by ELISA and immunohistochemical (IHC) methods. For in vitro analysis, CPZ C6 glioma cells were treated for 1 h, and then neurotoxic morphine (4 mM) was added to the cell medium. Cell viability was measured by the XTT method. Biochemical methods and immunofluorescence staining were used to evaluate mitochondrial damage and apoptosis. The findings indicated that co-administration of CPZ with morphine significantly reduced morphine tolerance (p < 0.05). Furthermore, in vivo and in vitro tests showed that CPZ administration decreased the levels of mitochondrial markers cytochrome c and AIF and proapoptotic markers caspase-3, caspase-9, and Bax and significantly increased the expression of antiapoptotic Bcl-2 (p < 0.01). In conclusion, both in vivo and in vitro test findings demonstrated that CPZ ameliorated morphine-induced mitochondrial dysfunction and attenuated apoptosis, reducing morphine-induced toxicity and tolerance.

Keywords: Apoptosis; Capsazepine; Mitochondrial damage; Morphine tolerance; Neurotoxicity.

Fig. 1

Timeline diagram representing in vivo and in vitro experimental design. CPZ, capsazepine; MORP, morphine; IHC, immunohistochemistry; OFT, open field test; TF, tail-flick test; HT, hot-plate test

Fig. 2

Effects of CPZ on morphine analgesia and tolerance. The analgesic effect (% MPE) of CPZ + MT group was significantly higher in both tail-flick (A) and hot-plate test (B) compared to the MT group (p < 0.05). Each point represents the mean ± SEM of % MPE for 6 rats. *p < 0.05, **p < 0.01 vs. control and ^#p < 0.05 vs. MT group (one-way ANOVA followed by Tukey HSD post hoc test). %MPE, Percent maximum possible effect; CPZ, capsazepine; MT, morphine tolerant

Fig. 3

Effects of CPZ and morphine on locomotor activity. The values are presented as means ± SEM (n = 6)

Fig. 4

Effect of CPZ on AIF (A), cytochrome c (B), caspase-3 (C), caspase-9 (D), Bax (E), and Bcl-2 (F) expressions in DRG. Values are presented as mean ± SEM for 6 rats. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control and ^#p < 0.05, ^##p < 0.01 vs. MT group (one-way ANOVA followed by Tukey HSD post hoc test). CPZ, capsazepine; MT, morphine toleran

Fig. 5

Effect of capsazepine on neuronal necrosis, satellitosis, Cyt-c, AIF, caspase-9, and cleaved caspase-3 levels in DRG. A H&E and IHC stainings in DRG slides, and B, C, D, and E quantitative IHC analysis of Cyt-c, AIF, caspase-9, and caspase-3, respectively. (Magnification × 200). Values are presented as mean ± SEM for 6 rats. *p < 0.05 and **p < 0.01, vs. control and ^#p < 0.05, vs. MT group (one-way ANOVA followed by Tukey HSD post hoc test). Black arrows indicate necrotic and satellite cells. Scale bar, 20 μm

Fig. 6

The data illustrate the impact of CPZ at varying concentrations on cell viability following morphine-induced cytotoxicity in C6 cells. A The C6 cells were exposed to morphine at various concentrations (1.5, 2, 3, 4, 6, 8, 12, and 16 mM) for a duration of 24 h. C6 cells were treated with CPZ at various concentrations (20, 10, 5, 2.5, and 1.25 µM) in combination with 4 mM morphine. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 vs. control group and ^##p < 0.01, ^###p < 0.001 vs. MORP group. MORP, morphine; CPZ, capsazepine

Fig. 7

Effect of CPZ on AIF (A), cytochrome c (B), caspase-3 (C), caspase-9 (D), Bax (E), and Bcl-2 (F) expressions in C6 cells. Values are presented as mean ± SEM (n = 3). ***p < 0.001 vs. control and ^#p < 0.05, ^##p < 0.01 vs. MORP group (one way ANOVA followed by Tukey HSD post hoc test)

Fig. 8

Effects of CPZ on cytochrome c, AIF, caspase-9, and caspase-3 protein expressions in C6 cells. A Immunofluorescence staining of cytochrome c, AIF, caspase-9, and caspase-3 protein expressions in C6 cells and B, C, and D quantitative analysis of Bcl-2, Cyt-c, and AIF proteins, respectively. (Magnification × 200). Values are presented as mean ± SEM (n = 6). **p < 0.01, ***p < 0.001 vs. control and ^#p < 0.05, ^##p < 0.01 vs. MORP group. Scale bar, 25 μm

Fig. 9

Effects of CPZ on Bax, caspase-9, and caspase-3 protein expressions in C6 cells. A Immunofluorescence staining of Bax, caspase-9, and caspase-3 protein expressions in C6 cells and B, C, and D quantitative analysis of Bcl-2, Cyt-c, and AIF proteins, respectively. (Magnification × 200). Values are presented as mean ± SEM (n = 6). ***p < 0.001 vs. control and ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 vs. MORP group. Scale bar, 25 μm