Nicotine alleviates MPTP-induced nigrostriatal damage through modulation of JNK and ERK signaling pathways in the mice model of Parkinson's disease

Abstract

Introduction: Nicotine (Nic) has previously been proven to reduce neurodegeneration in the models of Parkinson's disease (PD). The present study is intended to investigate the detailed mechanisms related to the potential neuroprotective effects of Nic in vivo. Methods: We established a PD model using 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced C57BL6 mice (25 mg/kg/d, 5 d, i.p.) to investigate the neuropharmacological modulation of Nic pretreatment (2.5 mg/kg/d, 5 d, i.p., 30 min before MPTP injection) from the perspectives of neurobehavioral assessment, the pathological alterations, microglial cell inflammation and MAPK signaling pathways in specific brain regions. Results: The open field test, elevated plus maze, rotarod and traction test suggested that Nic pretreatment could significantly improve MPTP-induced motor impairment and had an anxiolytic effect. Nic was found to improve neuroapoptosis, enhance tyrosine hydroxylase activity, and reduce the accumulation of the phosphorylated α-synuclein in the substantia nigra and striatal regions of PD mice by TUNEL and immunohistochemical assays. Immuno-fluorescent method for labeling Iba1 and CD68 indicated that Nic remarkably alleviates the activation of microglia which represents the M1 polarization state in the mice brain under MPTP stimulation. No significant difference in the expression of p38/MAPK pathway was found in the nigrostriatal regions, while Nic could significantly inhibit the elevated p-JNK/JNK ratio and increase the declined p-ERK/ERK ratio in the substantia nigra of MPTP-exposed brains, which was further confirmed by the pretreatment of CYP2A5 inhibitor to decline the metabolic activity of Nic. Discussion: The molecular signaling mechanism by which Nic exerts its neuroprotective effects against PD may be achieved by regulating the JNK and ERK signaling pathways in the nigra-striatum related brain regions.

Keywords: MAPK pathway; Parkinson’s disease; neuroprotective effect; nicotine; nigrostriatal region.

FIGURE 1

Effects of nicotine on locomotor performance and anxiety-like behavior in MPTP-treated mice. (A) Schematic diagram of the construction of a mouse model based on MPTP-induced neurological damage. (B) Representative locomotor paths of each group of mice in the open field test. (C) Percentage of horizontal motion distance in the central region in the open field test. (D) Mean latency to fall time in traction test. (E) Representative locomotor paths of each group of mice in the elevated plus maze test. (F) Open arm horizontal movement time in elevated plus maze. (G) Mean latency to fall time in the rotarod test. Data are expressed as mean ± SEM from five mice in each group. Significant differences were determined by an unpaired Student’s t-test and one-way ANOVA followed by Tukey’s multiple comparison post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2

Effects of nicotine on apoptosis in the substantia nigra and striatal brain regions of MPTP-treated mice. Representative image of TUNEL signals (green panel), cell nuclei (blue panel) and co-localization (merge panel) in brain sections from the substantia nigra (A) and striatal regions (B) of mice. Statistical results on the status of apoptotic cells in TUNEL in the substantia nigra (C) and the striatal regions (D). Scale bar at 50 μm. Data are expressed as mean ± SEM from five mice in each group, every point on the graph is an average from four fixed fields of view for each mouse. Significant differences were determined by an unpaired Student’s t-test and one-way ANOVA followed by Tukey’s multiple comparison post hoc test *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 3

Effects of nicotine on dopaminergic neurons and p-α-syn expression in the substantia nigra and striatal brain regions of MPTP-treated mice. (A) Immunohistochemical staining results of TH-positive neurons in the midbrain. Scale bar at 300 μm. (B) Statistical map of TH-positive neurons density. (C) Immunohistochemical staining results of TH expression in striatal regions. Scale bar at 100 μm. (D) Statistical map of TH expression density. (E) Immunofluorescence histochemical staining results of tissue sections in the substantia nigra. Scale bar at 100 μm. (F) Statistical results of p-α-syn fluorescence intensity to TH ratio in the substantia nigra. (G) Immunofluorescence histochemical staining results of the striatal tissue sections. Scale bar at 100 μm. (H) Statistical results of p-α-syn fluorescence intensity to TH ratio in the striatal regions. Data are expressed as mean ± SEM from five mice in each group, every point on the graph is an average from four fixed fields of view for each mouse. Significant differences were determined by an unpaired Student’s t-test and one-way ANOVA followed by Tukey’s multiple comparison post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4

Nicotine inhibits the activation of MPTP-treated mouse substantia nigra and striatal microglia iba1 and the pro-inflammatory factor CD68. (A) Immunofluorescence histochemical staining in the substantia nigra. (B) Statistical results of colocalization of Iba1 with CD68 in the substantia nigra. (C) Immunofluorescence histochemical staining in the striatal regions. (D) Statistical results of colocalization of Iba1 with CD68 in the striatal regions. (E,F) Statistical results of microglia branching in the substantia nigra and striatal regions. (G,H) Statistical results of microglial process lengths in the substantia nigra and striatal regions. Scale bar at 20 μm. Data are expressed as mean ± SEM from five mice in each group, every point on the graph is an average from four fixed fields of view for each mouse. Significant differences were determined by an unpaired Student’s t-test and one-way ANOVA followed by Tukey’s multiple comparison post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 5

Effect of nicotine on the expression of pro-inflammatory factors iNOS, IL-6 in the substantia nigra and striatum of mptp-treated mice. Representative images and quantification of iNOS, IL-6 immunoblot analysis in substantia nigra (A) and striatal tissue (D). (B,C) Statistical results of iNOS, IL-6 in the substantia nigra. (E,F) Statistical results of iNOS, IL-6 statistics in the striatum. Data are expressed as mean ± SEM from four mice in each group. Significant differences were determined by an unpaired Student’s t-test and one-way ANOVA followed by Tukey’s multiple comparison post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001.

FIGURE 6

Effects of nicotine on the MPTP-induced expression of MAPK signaling pathways. Representative pictures and quantification for immunoblot analysis of p-JNK/JNK, p-ERK/ERK, p-p38/p38 from the substantia nigra (A) and striatal tissues (E) are shown. (B,C,D) Statistical results of p-JNK/JNK, p-ERK/ERK, p-p38/p38 in the substantia nigra. (F,G,H) Statistical results of p-JNK/JNK, p-ERK/ERK, p-p38/p38 statistics in the striatum. (I,J) Concentration-time curve of nicotine and cotinine in plasma of saline and MOP-pretreatment mice. Data are expressed as mean ± SEM from four or five mice in each group. Significant differences were determined by an unpaired Student’s t-test and one-way ANOVA followed by Tukey’s multiple comparison post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.