CDDO-Me Distinctly Regulates Regional Specific Astroglial Responses to Status Epilepticus via ERK1/2-Nrf2, PTEN-PI3K-AKT and NFκB Signaling Pathways

Abstract

2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) is a triterpenoid analogue of oleanolic acid. CDDO-Me shows anti-inflammatory and neuroprotective effects. Furthermore, CDDO-Me has antioxidant properties, since it activates nuclear factor-erythroid 2-related factor 2 (Nrf2), which is a key player of redox homeostasis. In the present study, we evaluated whether CDDO-Me affects astroglial responses to status epilepticus (SE, a prolonged seizure activity) in the rat hippocampus in order to understand the underlying mechanisms of reactive astrogliosis and astroglial apoptosis. Under physiological conditions, CDDO-Me increased Nrf2 expression in the hippocampus without altering activities (phosphorylations) of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), phosphatidylinositol-3-kinase (PI3K), and AKT. CDDO-Me did not affect seizure activity in response to pilocarpine. However, CDDO-Me ameliorated reduced astroglial Nrf2 expression in the CA1 region and the molecular layer of the dentate gyrus (ML), and attenuated reactive astrogliosis and ML astroglial apoptosis following SE. In CA1 astrocytes, CDDO-Me inhibited the PI3K/AKT pathway by activating PTEN. In contrast, CDDO-ME resulted in extracellular signal-related kinases 1/2 (ERK1/2)-mediated Nrf2 upregulation in ML astrocytes. Furthermore, CDDO-Me decreased nuclear factor-κB (NFκB) phosphorylation in both CA1 and ML astrocytes. Therefore, our findings suggest that CDDO-Me may attenuate SE-induced reactive astrogliosis and astroglial apoptosis via regulation of ERK1/2-Nrf2, PTEN-PI3K-AKT, and NFκB signaling pathways.

Keywords: 3CAI; AKT; CDDO-Me; PI3K; SC79; U0126.

Figure 1

Effects of 2-Cyano-3,12-dioxo-oleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me) on nuclear factor-erythroid 2-related factor 2 (Nrf2) expression and seizure activity in response to pilocarpine. (A,B) Effect of CDDO-Me on Nrf2 expression in response to pilocarpine. CDDO-Me increases Nrf2 expression under physiological conditions. Pilocarpine-induced status epilepticus (SE) decreases Nrf2 expression, which is attenuated by CDDO-Me. (A) Representative Western blots of Nrf2 expression. (B) Quantification of Nrf2 expression based on Western blot data. Open circles indicate each individual value. Horizontal and vertical bars indicate mean value and S.E.M., respectively (mean ± S.E.M.; *,# p < 0.05 vs. control animals and vehicle, respectively, one-way ANOVA followed by Newman–Keuls post hoc test; n = 7, respectively). (C,D) Effect of CDDO-Me on seizure activity in response to pilocarpine. CDDO-Me does not influence seizure activity induced by pilocarpine. (C) Representative electroencephalogram (EEG) traces and frequency-power spectral temporal maps in response to pilocarpine. (D) Quantification of total EEG power (seizure intensity) in response to pilocarpine. Open circles and vertical bars indicate mean value and S.E.M., respectively (mean ± S.E.M.; repeated one-way ANOVA; n = 7, respectively).

Figure 2

Effects of CDDO-Me on Nrf2 expression and astroglial responses following SE. CDDO-Me ameliorates the reduced Nrf2 expression in CA1 astrocytes and ML astrocytes following SE. CDDO-Me also attenuates reactive CA1 astrogliosis and ML astroglial apoptosis induced by SE. (A) Representative photos of GFAP and Nrf2 in the hippocampus. Abbreviations: CA1, CA1 pyramidal cell layer; SR, stratum radiatum; SLM, stratum lacunosum-moleculare; ML, molecular layer of the dentate gyrus; DGL, dentate granule cell layer. (B,C) Quantification of the fluorescent intensities of GFAP and Nrf2 in CA1 (B) and molecular layer regions (C). Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (*,# p < 0.05 vs. control animals and vehicle, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively). (D) Representative photos of apoptosis of ML astrocytes. (E) Quantification of the fraction of TUNEL-signals in ML astrocytes. Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (# p < 0.05 vs. vehicle, two-tailed Student t-test; n = 7, respectively).

Figure 3

Effects of CDDO-Me on activities (phosphorylations) of PTEN, PI3K, and AKT following SE. PTEN, PI3K, and AKT expressions and their phosphorylations are unaffected by SE. CDDO-Me reduces PTEN, PI3K, and AKT phosphorylations without changing their expression levels under the post-SE condition. (A) Representative Western blots of PTEN, PI3K, and AKT expressions and their phosphorylations. (B) Quantification of PTEN, PI3K, and AKT expressions and their phosphorylations based on Western blot data. Open circles indicate each individual value. Horizontal and vertical bars indicate mean value and S.E.M., respectively (mean ± S.E.M.; *,# p < 0.05 vs. control animals and vehicle, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively).

Figure 4

Effects of 3CAI and SC79 on Nrf2 expression and astroglial responses following SE. 3CAI attenuates reactive CA1 astrogliosis but deteriorates ML astroglial apoptosis, which are reversed by SC79. Neither 3CAI nor SC79 affects Nrf2 expression in CA1 astrocytes and ML astrocytes following SE. (A) Representative photos of GFAP and Nrf2 in the hippocampus. Abbreviations: CA1, CA1 pyramidal cell layer; SR, stratum radiatum; SLM, stratum lacunosum-moleculare; ML, molecular layer of the dentate gyrus; DGL, dentate granule cell layer. (B,C) Quantification of the fluorescent intensities of GFAP and Nrf2 in CA1 (B) and molecular layer regions (C). Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (* p < 0.05 vs. vehicle, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively). (D) Representative photos of apoptosis of ML astrocytes. (E) Quantification of the fraction of TUNEL-signals in ML astrocytes. Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (* p < 0.05 vs. vehicle, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively).

Figure 5

Effects of CDDO-Me on NFκB phosphorylation following SE. NFκB-S311 and -S468 phosphorylations are increased in reactive CA1 astrocytes, but only-S468 phosphorylation is elevated in ML astrocytes, which were abrogated by CDDO-Me. (A) Representative photos of NFκB-S311 phosphorylation in the hippocampus. Abbreviations: CA1, CA1 pyramidal cell layer; SR, stratum radiatum; SLM, stratum lacunosum-moleculare; ML, molecular layer of the dentate gyrus; DGL, dentate granule cell layer. (B) Quantification of the fluorescent intensity of NFκB-S311 phosphorylation in CA1 and molecular layer regions. Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (*,# p < 0.05 vs. control animals and vehicle, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively). (C) Representative photos of NFκB-S468 phosphorylation in the hippocampus. (D) Quantification of the fluorescent intensity of NFκB-S468 phosphorylation in CA1 and molecular layer regions. Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (*,# p < 0.05 vs. control animals and vehicle, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively).

Figure 6

Effects of CDDO-Me and U0126 co-treatment on Nrf2 expression and ERK1/2 activity (phosphorylation) following SE. Under physiological conditions, CDDO-Me increases ERK1/2 phosphorylation and Nrf2 expression. CDDO-Me also attenuates SE-induced reductions in ERK1/2 phosphorylation and Nrf2 expression. U0126 co-treatment abolishes the CDDO-Me-induced upregulation of ERK1/2 phosphorylation and Nrf2 expression under physiological and post-SE conditions. (A) Representative Western blots of Nrf2, ERK1/2, and p-ERK1/2. (B,D) Quantification of ERK1/2 (B), p-ERK1/2 (C), and Nrf2 (D) levels based on Western blot data. Open circles indicate each individual value. Horizontal and vertical bars indicate mean value and S.E.M., respectively (mean ± S.E.M.; *,# p < 0.05 vs. control animals and vehicle, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7).

Figure 7

Effects of CDDO-Me and U0126 co-treatment on Nrf2 expression and astroglial responses following SE. U0126 co-treatment does not affect the abolishment of reactive CA1 astrogliosis by CDDO-Me. However, U0126 co-treatment inhibits the upregulation of Nrf2 expression induced by CDDO-Me in CA1 astrocytes and ML astrocytes. In addition, U0126 abrogates the protective effect of CDDO-Me against SE-induced ML astroglial apoptosis. (A) Representative photos of GFAP and Nrf2 in the hippocampus. Abbreviations: CA1, CA1 pyramidal cell layer; SR, stratum radiatum; SLM, stratum lacunosum-moleculare; ML, molecular layer of the dentate gyrus; DGL, dentate granule cell layer. (B,C) Quantification of the fluorescent intensities of GFAP and Nrf2 in CA1 (B) and molecular layer regions (C). Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (*,# p < 0.05 vs. vehicle and CDDO-Me, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively). (D) Representative photos of apoptosis of ML astrocytes. (E) Quantification of the fraction of TUNEL-signals in ML astrocytes. Open circles indicate each individual value. Horizontal bars indicate mean value. Vertical bars indicate S.E.M. (*,# p < 0.05 vs. vehicle and CDDO-Me, respectively, one-way ANOVA followed by Newman-Keuls post hoc test; n = 7, respectively).

Figure 8

Scheme of the effects of CDDO-Me on reactive CA1 astrogliosis and ML astroglial apoptosis. CDDO-Me attenuates reactive CA1 astrogliosis by activating PTEN, which inhibits PI3K/AKT activities. CDDO-Me also abates reactive CA1 astrogliosis by reducing NFκB S311 and S468 phosphorylation. In addition, CDDO-Me ameliorates ML astroglial apoptosis via upregulation of ERK1/2-mediated Nrf2 expression and inhibition of NFκB S468 phosphorylation. Therefore, CDDO-Me mitigates both reactive astrogliosis and astroglial degeneration induced by SE.