Levo-corydalmine Attenuates Vincristine-Induced Neuropathic Pain in Mice by Upregulating the Nrf2/HO-1/CO Pathway to Inhibit Connexin 43 Expression

Abstract

Antimicrotubulin chemotherapeutic agents, including plant-derived vincaalkaloids such as vincristine, can cause peripheral neuropathic pain. Exogenously activated heme oxygenase 1 (HO-1) is a potential therapy for chemotherapy-induced neuroinflammation. In this study, we investigated a role for Nrf2/HO-1/CO in mediating vincristine-induced neuroinflammation by inhibiting connexin 43 (Cx43) production in the spinal cord following the intrathecal application of the HO-1 inducer protoporphyrin IX cobalt chloride (CoPP) or inhibitor protoporphyrin IX zinc (ZnPP), and we analyzed the underlying mechanisms by which levo-corydalmine (l-CDL, a tetrahydroprotoberberine) attenuates vincristine-induced pain. Treatment with levo-corydalmine or oxycodone hydrochloride (a semisynthetic opioid analgesic, used as a positive control) attenuated vincristine-induced persistent pain hypersensitivity and degeneration of the sciatic nerve. In addition, the increased prevalence of atypical mitochondria induced by vincristine was ameliorated by l-CDL in both A-fibers and C-fibers. Next, we evaluated whether nuclear factor E2-related factor 2 (Nrf2), an upstream activator of HO-1, directly bound to the HO-1 promoter sequence and degraded heme to produce carbon monoxide (CO) following stimulation with vincristine. Notably, l-CDL dose-dependently increased HO-1/CO expression by activating Nrf2 to inhibit Cx43 expression in both the spinal cord and in cultured astrocytes stimulated with TNF-α, corresponding to decreased Cx43-mediated hemichannel. Furthermore, l-CDL had no effect on Cx43 following the silencing of the HO-1 gene. Taken together, our findings reveal a novel mechanism by which Nrf2/HO-1/CO mediates Cx43 expression in vincristine-induced neuropathic pain. In addition, the present findings suggest that l-CDL likely protects against nerve damage and attenuates vincristine-induced neuroinflammation by upregulating Nrf2/HO-1/CO to inhibit Cx43 expression.

Keywords: Vincristine; connexin-43; heme oxygenase 1; neuropathic pain; nuclear factor E2-related factor 2.

Fig. 1

l-CDL attenuates vincristine-induced pain hypersensitivity in mice. (A) Timeline of vincristine, l-CDL, CoPP, and oxycodone treatments, behavioral tests, histological analysis, and the detection of various factors. (B, C) The PWT and the TFL were both significantly decreased at day 6 and maintained at day 14 in the vincristine-treated group. l-CDL and the CoPP and oxycodone positive control groups exhibited a reversal of vincristine-induced mechanical allodynia and thermal hyperalgesia after the first injection, and the effect was maintained at day 14. ^###P < 0.001 compared with the sham control; **P < 0.01 and ***P < 0.001 compared with the vincristine-treated group, two-way ANOVA followed by Bonferroni’s post hoc tests, n = 6 mice/group. All data are presented as means±S.D.

Fig. 2

l-CDL attenuates vincristine-induced degeneration of the sciatic nerve and the prevalence of atypical mitochondria. (A) All six micrographs are shown at the same magnification (× 5000, bar = 2 μm). Normal myelinated (m) and unmyelinated fibers (n) were observed in the sham group. Severe degeneration of myelin and axons in large myelinated fibers was observed in the vincristine-treated group (day 14), whereas unmyelinated fibers appeared in looser bundles and were swollen. Shrinkage of the axons of myelinated fibers was observed on day 14 (p). l-CDL, particularly the high dose of l-CDL, and oxycodone reduced the degeneration of myelinated and unmyelinated fibers compared with the vincristine-treated group. n = 5 mice/group. (B–E) Representative ultrastructural images show the prevalence of atypical mitochondria in A-fibers (B) and C-fibers (C) in the six different groups (× 30,000, bar = 0.5 μm). The application of all three doses of l-CDL, especially the high dose of l-CDL, significantly decreased the prevalence of atypical mitochondria (indicated by arrows) compared with the vincristine-treated group. ^###P < 0.001 compared with the sham control; **P < 0.01 and ***P < 0.001, compared with the vincristine-treated group, ANOVA followed by Tukey’s post hoc test, n = 5 mice/group. All data are presented as means±S.D.

Fig. 3

l-CDL induces Nrf2/HO-1/CO upregulation in the spinal cord after the vincristine treatment. (A–C) l-CDL and oxycodone induced HO-1-overexpression in the same region of the superficial dorsal horn (laminae I–III) compared with the vincristine group, as assessed using immunostaining. Scale bar = 100 μm. (D) Western blots also showed that l-CDL increased HO-1 expression. (E–F) The l-CDL and oxycodone groups exhibited lower cytoplasmic Nrf2 levels and higher nuclear Nrf2 levels. (G) The l-CDL and oxycodone groups also exhibited increased CO release in the spinal cord, and the high dose of l-CDL (20 mg/kg) was the most effective at increasing CO levels. ^###P < 0.001 compared with the sham control; *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the vincristine-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 mice/group. All data are presented as means±S.D.

Fig. 4

l-CDL decreases Cx43 expression by the regulating HO-1/CO pathway in the spinal cord after the vincristine treatment. (A, B) CoPP (8 μg), l-CDL (20 mg/kg), and ZnPP (8 μg) + l-CDL (20 mg/kg) treatments attenuated vincristine-induced mechanical allodynia and heat hyperalgesia. **P < 0.01 and ***P < 0.001 compared with the vehicle-treated group, two-way ANOVA followed by Bonferroni’s post hoc tests, n = 6 mice/group. (C–E) The CoPP group, the l-CDL group and the ZnPP + l-CDL group exhibited increased HO-1 and CO expression at 7 h after the intrathecal injection, but significantly attenuated Cx43 expression compared with the vehicle group. **P < 0.01 and ***P < 0.001 compared with the vehicle-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 mice/group. (F–G) l-CDL inhibited the vincristine-induced increase in Cx43 immunoreactivity in the spinal cord 14 days after the vincristine injection. Confocal images of spinal sections showed the co-localization of Cx43 with the astrocytic marker GFAP but not with neuronal marker NeuN or microglial marker Iba-1. Scale bar = 50 μm. ^###P < 0.001 compared with the sham control; **P < 0.01 and ***P < 0.001 compared with the vincristine-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 mice/group. (H, I) l-CDL inhibited vincristine-induced increases in the levels of the Cx43 protein and mRNA at 14 days. ^###P < 0.001, compared with the sham control; *P < 0.05 and ***P < 0.001, compared with the vincristine-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 mice/group. All data are presented as the means±S.D.

Fig. 5

l-CDL increases Nrf2-activated HO-1 expression in cultured astrocytes. (A) The Nrf2-HO-1-wt group exhibited significantly reduced HO-1 promoter activity, whereas the Nrf2-HO-1-mut promoter group, HO-1-wt promoter group, and HO-1-mut promoter group did not exhibit changes in basic activity. The promoter activity is represented by the level of luciferase activity indicated by relative light units (RLU). Wild-type Renilla luciferase plasmids were also transfected into cells and used as an internal control. ***P < 0.001 compared with the Nrf2-HO-1-wt promoter; N.S., not significant, ANOVA followed by Tukey’s post hoc test. n = 3 cultures/group. (B, C) l-CDL and CoPP evoked lower cytoplasmic Nrf2 levels and higher nuclear Nrf2 levels in astrocyte cultures at 1 h after TNF-α incubation. ^#P < 0.05 compared with the sham control; **P < 0.01 and ***P < 0.001 compared with the TNF-α group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. (D) Real-time quantitative PCR results showed that l-CDL and CoPP increased the expression of the HO-1 mRNA. **P < 0.01 and ***P < 0.001 compared with the TNF-α group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. All data are presented as means±S.D.

Fig. 6

l-CDL increased HO-1/CO levels in cultured astrocytes after the TNF-α treatment. (A, B) l-CDL and CoPP significantly increased the HO-1 fluorescence intensity at 24 h after the TNF-α incubation (10 ng/ml, 60 min at 37 °C). Scale bar = 100 μm. *P < 0.05 and ***P < 0.001 compared with the TNF-α group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. (C) Double staining for HO-1 and GFAP showed the expression of HO-1 in astrocytes. Scale bar = 100 μm. (D) ELISA results showed that both l-CDL and CoPP substantially increased CO levels in cultured astrocytes. ^#P < 0.05 compared with the sham control; **P < 0.01 and ***P < 0.001 compared with TNF-α group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. All data are presented as means±S.D.

Fig. 7

l-CDL reduced TNF-α-induced increases in Cx43 levels and hemichannel activity in cultured astrocytes. (A, B) Cx43 was expressed at very low levels in sham control astrocytes and increased 1 h after TNF-α incubation. Double staining for Cx43 and GFAP revealed the expression of Cx43 in astrocytes. Scale bar = 100 μm. ^##P < 0.01 and ^###P < 0.001 compared with the sham control, Student’s t test, n = 3 cultures/group. (C) Cx43 expression in cultured astrocytes stimulated with TNF-α (10 ng/ml, 60 min) was suppressed by treatment with l-CDL, CoPP, and Gap 27. ^###P < 0.001, compared with the sham control and ***P < 0.001 compared with the TNF-α group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. (D, E) The TNF-α treatment (10 ng/ml, 60 min) increased hemichannel function, as revealed by ethidium bromide uptake in astrocytes. This increase was suppressed by l-CDL, Gap 27, and CoPP. Scale bar = 50 μm. ^###P < 0.001 compared with the sham control and ***P < 0.001 compared with the TNF-α group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. All data are presented as means±S.D.

Fig. 8

l-CDL has no effect on Cx43 expression following HO-1 gene silencing. (A, B) Low levels of the HO-1 mRNA and protein were detected expression after a TNF-α incubation in the presence of l-CDL with an HO-1 small interfering RNA pretreatment, indicating that HO-1 gene silencing was stable. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the vehicle-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. (C) l-CDL induced lower CO production in response to HO-1 silencing. ***P < 0.001 compared with the vehicle-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. (D, E) l-CDL had no effect on Cx43 expression following HO-1 silencing. ***P < 0.001 compared with the vehicle-treated group; N.S., not significant compared with the vehicle-treated group, ANOVA followed by Tukey’s post hoc test, n = 3 cultures/group. All data are presented as means±S.D.