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Sulforaphane Ameliorates 3-Nitropropionic Acid-Induced Striatal Toxicity by Activating the Keap1-Nrf2-ARE Pathway and Inhibiting the MAPKs and NF-κB Pathways

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Abstract

The potential neuroprotective value of sulforaphane (SFN) in Huntington’s disease (HD) has not been established yet. We investigated whether SFN prevents and improves the neurological impairment and striatal cell death in a 3-nitropropionic acid (3-NP)-induced mouse model of HD. SFN (2.5 and 5.0 mg/kg/day, i.p.) was given daily 30 min before 3-NP treatment (pretreatment) and from onset/progression/peak points of the neurological scores. Pretreatment with SFN (5.0 mg/kg/day) produced the best neuroprotective effect with respect to the neurological scores and lethality among other conditions. The protective effects due to pretreatment with SFN were associated with the following: suppression of the formation of a lesion area, neuronal death, succinate dehydrogenase activity, apoptosis, microglial activation, and mRNA or protein expression of inflammatory mediators, including tumor necrosis factor-alpha, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase, and cyclooxygenase-2 in the striatum after 3-NP treatment. Also, pretreatment with SFN activated the Kelch-like ECH-associated protein 1 (Keap1)—nuclear factor erythroid 2-related factor 2 (Nrf2)—antioxidant response element (ARE) pathway and inhibited the mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB) pathways in the striatum after 3-NP treatment. As expected, the pretreatment with activators (dimethyl fumarate and antioxidant response element inducer-3) of the Keap1-Nrf2-ARE pathway decreased the neurological impairment and lethality after 3-NP treatment. Our findings suggest that SFN may effectively attenuate 3-NP-induced striatal toxicity by activating the Keap1-Nrf2-ARE pathway and inhibiting the MAPKs and NF-κB pathways and that SFN has a wide therapeutic time-window for HD-like symptoms.

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Acknowledgments

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and future Planning (NRF-2014R1A2A1A11051240).

Conflict of Interest

All the authors of this manuscript have no conflict of interest in this subject.

Authors’ Contributions

MJ performed the behavioral experiments, immunohistochemistry, PCR analysis, and Western blots and also prepared the figures. IHC conceived all experiments, analyzed the results, and wrote the manuscript. All authors have read and approved the final manuscript.

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Authors and Affiliations

  1. Department of Convergence Medical Science, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea

    Minhee Jang & Ik-Hyun Cho

  2. Department of Cancer Preventive Material Development, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea

    Minhee Jang

  3. Brain Korea 21 Plus Program, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea

    Ik-Hyun Cho

  4. Institute of Korean Medicine, College of Oriental Medicine, Kyung Hee University, Seoul, 130-701, Republic of Korea

    Ik-Hyun Cho

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  1. Minhee Jang
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Correspondence to Ik-Hyun Cho.

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Supplementary Figure 1

The effect of pre-, onset-, progression-, and peak-treatment with SFN in neurological score or body weight by the 3-NP treatment. (A and B) Pre-, onset-, and progression-treatment with SFN (2.5 or 5.0 mg/kg/day, i.p.) did not recover the reduced mean body weight stemming from 3-NP-treatment. (C-E) The peak-treatment with SFN (5.0 mg/kg/day, i.p.) did not show positive effects on the neurological scores (C), survival rate (D), and mean body weight (E) caused by the 3-NP-treatment. Neurological disorders in the sham control were not detected and the bar is displayed as N.D. (not detected) in graphs (C). The 4th and 8d refer to the 24 hours after the last (4th) 3-NP treatment and the 8 days after the last 3-NP treatment, respectively. Statistical significance: # P < 0.05 vs. sham-treated mice. (PDF 18.7 kb)

Supplementary Figure 2

Pretreatment with SFN reduced striatal lesions caused by 3-NP treatment. (A and B) The proportion of mice with striatal lesion (A) and mean striatal lesion area (B) were analyzed using sections stained with cresyl violet dye (Fig. 2c-g). Both items were decreased by pretreatment with 2.5 and 5.0 mg/kg/day of SFN in a dose-dependant manner. Striatal lesions in sham-treated and SFN alone-treated mice were not detected (N.D.). Statistical significance: * P < 0.01, ** P < 0.05 vs. 3-NP-treated mice; # P < 0.05 vs. sham-treated mice. (PDF 1.22 mb)

Supplementary Figure 3

Activation of astrocytes was not affected by treatment with 3-NP or SFN. (A-G) Striatal sections were stained with anti-GFAP antibody 24 hours after the last (4th) 3-NP-treatment. Insets display high magnification micrographs of the areas marked with squares (A-D). The astrocytes were not affected by treatment with 3-NP or SFN. Scale bar = 100 μm. Striatal tissues 12 hours after the last 3-NP-treatment were analyzed by real-time PCR analysis (E). Striatal tissues 24 hours after the last 3-NP-treatment were analyzed by Western blot analysis using a GFAP antibody (F and G). The mRNA and protein expression of GFAP were not affected by treatment with 3-NP or SFN. Statistical significance: # P < 0.01 vs. sham-treated mice. (PDF 1.22 mb)

Supplementary Table 1

PCR primer sequence for PCR analysis. (PDF 1.22 mb)

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Jang, M., Cho, IH. Sulforaphane Ameliorates 3-Nitropropionic Acid-Induced Striatal Toxicity by Activating the Keap1-Nrf2-ARE Pathway and Inhibiting the MAPKs and NF-κB Pathways. Mol Neurobiol 53, 2619–2635 (2016). https://doi.org/10.1007/s12035-015-9230-2

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