Ginsenoside Re protects methamphetamine-induced dopaminergic neurotoxicity in mice via upregulation of dynorphin-mediated κ-opioid receptor and downregulation of substance P-mediated neurokinin 1 receptor

Abstract

Background: We previously reported that ginsenoside Re (GRe) attenuated against methamphetamine (MA)-induced neurotoxicity via anti-inflammatory and antioxidant potentials. We also demonstrated that dynorphin possesses anti-inflammatory and antioxidant potentials against dopaminergic loss, and that balance between dynorphin and substance P is important for dopaminergic neuroprotection. Thus, we examined whether GRe positively affects interactive modulation between dynorphin and substance P against MA neurotoxicity in mice.

Methods: We examined changes in dynorphin peptide level, prodynorphin mRNA, and substance P mRNA, substance P-immunoreactivity, homeostasis in enzymatic antioxidant system, oxidative parameter, microglial activation, and pro-apoptotic parameter after a neurotoxic dose of MA to clarify the effects of GRe, prodynorphin knockout, pharmacological inhibition of κ-opioid receptor (i.e., nor-binaltorphimine), or neurokinin 1 (NK1) receptor (i.e., L-733,060) against MA insult in mice.

Results: GRe attenuated MA-induced decreases in dynorphin level, prodynorphin mRNA expression in the striatum of wild-type (WT) mice. Prodynorphin knockout potentiated MA-induced dopaminergic toxicity in mice. The imbalance of enzymatic antioxidant system, oxidative burdens, microgliosis, and pro-apoptotic changes led to the dopaminergic neurotoxicity. Neuroprotective effects of GRe were more pronounced in prodynorphin knockout than in WT mice. Nor-binaltorphimine, a κ-opioid receptor antagonist, counteracted against protective effects of GRe. In addition, we found that GRe significantly attenuated MA-induced increases in substance P-immunoreactivity and substance P mRNA expression in the substantia nigra. These increases were more evident in prodynorphin knockout than in WT mice. Although, we observed that substance P-immunoreactivity was co-localized in NeuN-immunreactive neurons, GFAP-immunoreactive astrocytes, and Iba-1-immunoreactive microglia. NK1 receptor antagonist L-733,060 or GRe selectively inhibited microgliosis induced by MA. Furthermore, L-733,060 did not show any additive effects against GRe-mediated protective activity (i.e., antioxidant, antimicroglial, and antiapoptotic effects), indicating that NK1 receptor is one of the molecular targets of GRe.

Conclusions: Our results suggest that GRe protects MA-induced dopaminergic neurotoxicity via upregulatgion of dynorphin-mediated κ-opioid receptor and downregulation of substance P-mediated NK1 R.

Keywords: Dynorphin; Methamphetamine; Microglia; Neurokinin 1 receptor; κ-opioid receptor.

Fig. 1

MA-induced changes in dynorphin level, prodynorphin mRNA, and substance P mRNA expression. Changes in dynorphin level (a), prodynorphin (b), and substance P (c) mRNA expression in the dorsal striatum of wild-type mice. Sal = saline, MA = methamphetamine 35 mg/kg, i.p. Each value is the mean ± SEM of six animals (one-way ANOVA followed by Fisher’s LSD pairwise comparisons)

Fig. 2

MA-induced changes oxidative burdens in the dorsal striatum of wild-type (WT) mice. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., ROS = reactive oxygen species, HNE = 4-hydroxynonenal. Each value is the mean ± SEM of six animals (one-way ANOVA followed by Fisher’s LSD pairwise comparisons)

Fig. 3

Role of κ-opioid receptor in GRe-mediated modulation of prodynorphin and substance P. Experimental design for determining whether κ-opioid receptor is involved in GRe-mediated effects against dopaminergic toxicity induced by MA; mice received Nor-B by two times (3 and 1.5 h) before MA treatment. Mice received ginsenoside Re twice a day for seven consecutive days (i.e., 5 days before and 1 day after MA) (a). Effect of GRe on MA-induced decrease in prodynorphin mRNA expression in the striatum of mice (b). Effects of κ-opioid receptor antagonist Nor-B on GRe-mediated pharmacological activity against MA-induced increases in substance P (SP)-immunoreactivity (c) and substance P mRNA expression (d) in the substantia nigra (SN) of DYN KO mice. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., GRe = ginsenoside Re 20 mg/kg, i.p., Nor-B = nor-binaltorphimine 3 or 6 mg/kg, i.p., WT = wild-type mice, DYN KO = prodynorphin knockout. Each value is the mean ± SEM of six animals [two-way ANOVA (B) or one-way ANOVA (C and D) followed by Fisher’s LSD pairwise comparisons]. Scale bar = 100 μm

Fig. 4

Role of κ-opioid receptor and neurokinin 1 receptor in GRe-mediated antioxidant potentials. Effects of neurokinin 1 receptor antagonist L-733,060 on Nor-B-mediated pharmacological activity in response to antioxidant effect of GRe against ROS formation (a), HNE (b), and protein carbonyl (c) levels induced by MA. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., GRe = ginsenoside Re 20 mg/kg, i.p., Nor-B = Nor-binaltorphimine 3 or 6 mg/kg, i.p., L733 = L-733,060 5 or 10 mg/kg, i.p., ROS = reactive oxygen species, HNE = 4-hydroxynonenal, WT = wild type, DYN KO = prodynorphin knockout. Each value is the mean ± SEM of six animals (one-way ANOVA followed by Fisher’s LSD pairwise comparisons)

Fig. 5

MA-induced changes in substance P-immunodistribution. Substance P-immunoreactivity was localized in the NeuN-labeled neurons (a), GFAP-labeled astrocytes (b), and Iba-1-labeled microglial cells (c). Arrow indicates co-localization. Scale bar = 100 μm

Fig. 6

Role of κ-opioid and neurokinin 1 receptors in GRe-mediated neuronal, astrocytic, and microglial modulation. Effects of neurokinin 1 receptor antagonist L-733,060 on κ-opioid receptor antagonist Nor-B-mediated pharmacological activity in response to effects of GRe against the changes in NeuN (a), GFAP (b), Iba-1 expression (c), and Iba-1-immunoreactivity (d) induced by MA in the striatum of DYN KO mice. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., GRe = ginsenoside Re 20 mg/kg, i.p., Nor-B = Nor-binaltorphimine 3 or 6 mg/kg, i.p., L733 = L-733,060 5 or 10 mg/kg, i.p., GFAP = glial fibrillary acidic protein, Iba-1 = ionized calcium-binding adapter molecule 1, WT = wild type, DYN KO = prodynorphin knockout. Each value is the mean ± SEM of six animals (one-way ANOVA followed by Fisher’s LSD pairwise comparisons). Scale bar = 100 μm

Fig. 7

Role of κ-opioid and neurokinin 1 receptors in GRe-mediated anti-apoptotic potentials. Effects of neurokinin 1 receptor antagonist L-733,060 on κ-opioid receptor antagonist Nor-B-mediated pharmacological activity in response to effects of GRe against the changes in Bax (a), cleaved caspase-3 (b), and Bcl-2 expression (c) induced by MA in the striatum of DYN KO mice. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., GRe = ginsenoside Re 20 mg/kg, i.p., Nor-B = Nor-binaltorphimine 3 or 6 mg/kg, i.p., L733 = L-733,060 5 or 10 mg/kg, i.p., WT = wild type, DYN KO = prodynorphin knockout. Each value is the mean ± SEM of six animals (one-way ANOVA followed by Fisher’s LSD pairwise comparisons)

Fig. 8

Effects of genetic depletion of DYN on MA-induced dopaminergic change. a Effects of dorsal striatum. b Effects of nucleus accumbens. c Effects of ventral midbrain. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., GRe = ginsenoside Re 20 mg/kg, i.p., Nor-B = Nor-binaltorphimine 3 or 6 mg/kg, i.p., L733 = L-733,060 5 or 10 mg/kg, i.p., WT = wild type, DYN KO = prodynorphin knockout. Each value is the mean ± SEM of six animals (two-way ANOVA followed by Fisher’s LSD pairwise comparisons)

Fig. 9

Role of κ-opioid and neurokinin 1 receptors in GRe-mediated dopaminergic neuroprotection. Effects of neurokinin 1 receptor antagonist L-733,060 on κ-opioid receptor antagonist Nor-B-mediated pharmacological activity in response to effects of GRe against changes in tyrosine hydroxylase-immunoreactivity (TH-IR; a), TH expression (b), dopamine level (c), and dopamine turnover rate (d) induced by MA in the striatum of DYN KO mice. Sal = saline, MA = methamphetamine 35 mg/kg, i.p., GRe = ginsenoside Re 20 mg/kg, i.p., Nor-B = Nor-binaltorphimine 3 or 6 mg/kg, i.p., L733 = L-733,060 5 or 10 mg/kg, i.p., WT = wild type, DYN KO = prodynorphin knockout. Each value is the mean ± SEM of six animals (one-way ANOVA followed by Fisher’s LSD pairwise comparisons)

Fig. 10

A schematic depiction ginsenoside Re (GRe)-mediated dopaminergic neuroprotective effects against MA insult. Treatment with a toxic dose of MA resulted in initial oxidative burdens, a perturbed redox status (a failure to compensate for reduced GPx) followed by the decreases in dynorphin level and prodynorphin mRNA expression. This decrease led to imbalance between dynorphin and substance P and to enhance substance P mRNA and substance P-immunoreactivity (SP-IR). At that time, SP-IR was co-localized in the Iba-1-immunoreactive microglia, NeuN-immunoreactive neurons, and GFAP-immunoreactive astrocytes. More importantly, GRe did not affect MA-induced astrocytic expression (i.e., GFAP expression), but GRe significantly attenuated against MA-induced microgliosis (i.e., Iba-1-immunoreactive-immunoreactivity and Iba-1 expression). Neurokinin 1 receptor activation, genetic depletion of dynorphin, or κ-opioid receptor antagonism (i.e., nor-binaltorphimine) potentiated this microgliosis, as well as pro-apoptotic changes. This morbid phenomenon contributed to dopaminergic impairment. Therefore, we propose that GRe attenuates MA-induced neurotoxicity via upregulation of prodynorphin-mediated κ-opioid receptor and downregulation of substance P-mediated NK1 receptor