Korean J Physiol Pharmacol.  2018 Nov;22(6):689-696. 10.4196/kjpp.2018.22.6.689.

Cyanidin-3-glucoside inhibits amyloid β₂₅₋₃₅-induced neuronal cell death in cultured rat hippocampal neurons

Affiliations
  • 1Department of Physiology, College of Medicine, Catholic Neuroscience Institute, The Catholic University of Korea, Seoul 06591, Korea. s-hyoon@catholic.ac.kr
  • 2College of Pharmacy, The Catholic University of Korea, Bucheon 14662, Korea.

Abstract

Increasing evidence implicates changes in [Ca²âº]i and oxidative stress as causative factors in amyloid beta (Aβ)-induced neuronal cell death. Cyanidin-3-glucoside (C3G), a component of anthocyanin, has been reported to protect against glutamate-induced neuronal cell death by inhibiting Ca²âº and Zn²âº signaling. The present study aimed to determine whether C3G exerts a protective effect against Aβ₂₅₋₃₅-induced neuronal cell death in cultured rat hippocampal neurons from embryonic day 17 fetal Sprague-Dawley rats using MTT assay for cell survival, and caspase-3 assay and digital imaging methods for Ca²âº, Zn²âº, MMP and ROS. Treatment with Aβ25-35 (20 µM) for 48 h induced neuronal cell death in cultured rat pure hippocampal neurons. Treatment with C3G for 48 h significantly increased cell survival. Pretreatment with C3G for 30 min significantly inhibited Aβ₂₅₋₃₅-induced [Zn²âº]i increases as well as [Ca²âº]i increases in the cultured rat hippocampal neurons. C3G also significantly inhibited Aβ₂₅₋₃₅-induced mitochondrial depolarization. C3G also blocked the Aβ₂₅₋₃₅-induced formation of ROS. In addition, C3G significantly inhibited the Aβ₂₅₋₃₅-induced activation of caspase-3. These results suggest that cyanidin-3-glucoside protects against amyloid β-induced neuronal cell death by reducing multiple apoptotic signals.

Keyword

Amyloid β₂₅₋₃₅; Cyanidin-3-glucoside; Hippocampal neurons; Mitochondrial membrane potential; Neuroprotection

MeSH Terms

Amyloid*
Animals
Anthocyanins
Caspase 3
Cell Death*
Cell Survival
Membrane Potential, Mitochondrial
Neurons*
Neuroprotection
Oxidative Stress
Rats*
Rats, Sprague-Dawley
Amyloid
Anthocyanins
Caspase 3

Figure

  • Fig. 1 Effects of C3G on Aβ25–35-induced cell death in cultured pure rat hippocampal neurons.Aβ25–35 (20 µM)-induced neuronal damage was measured through the reduction of MTT in viable cells at 11 days in culture. The bar graph shows the purple formazan product from pure hippocampal neurons after treatment. (A) Effects of C3G on Aβ25–35-induced cell death in the non-treated(control) (vehicle, n=5; Aβ, n=5), 5 µg/ml C3G (vehicle, n=4; Aβ, n=4), 10 µg/ml C3G (vehicle, n=4; Aβ, n=4), and 15 µg/ml C3G (vehicle, n=4; Aβ, n=4)-treated cells in the absence (vehicle) and presence of 20 µM Aβ25–35 for 48 h. (B) Representative phase contrast photomicrographs showing cultured pure rat hippocampal neurons 48 h following co-treatment of C3G with Aβ25–35 at 11 days in culture. Data are expressed as mean±S.E. of experiments. ##p<0.01 relative to control, *p<0.05 relative to control Aβ (ANOVA with Bonferroni test).

  • Fig. 2 Effects of C3G on Aβ25–35-induced [Ca2+]i increases.Reproducible [Ca2+]i increases were elicited by applying Aβ25–35 (20 µM) for 5 min at 35 min intervals (A). Pretreatment with C3G (10 µg/ml) for 30 min significantly inhibited Aβ25–35-induced [Ca2+]i responses (B). (C) Summary of the Aβ25–35-induced [Ca2+]i increases in non-treated (control, n=17) and C3G-pretreated (C3G, n=10) cells. Aβ25–35 induced response is presented as a percentage of the initial Aβ25–35 induced response (peak 2/peak 1). Data are expressed as mean±S.E. *p<0.05 relative to control (non-paired Student's t-test).

  • Fig. 3 Effects of C3G on Aβ25–35-induced [Zn2+]i increases.(A) Treatment with Aβ25–35 (20 µM) for 10 min increased [Zn2+]i increases (n=26). (B, C) Pretreatment with C3G (10 µg/ml) for 30 min significantly inhibited the Aβ25–35-induced [Zn2+]i responses (n=22). (C) Aβ25–35 induced response is normalized to the initial value measured before the addition of agonist. Data are expressed as mean±S.E. **p<0.01 relative to control (non-paired Student's t-test).

  • Fig. 4 Effects of C3G on Aβ25–35-induced mitochondrial depolarization.(A) Reproducible depolarization in mitochondrial membrane potential (MMP) was elicited by applying Aβ25–35 (20 µM) for 5 min at 35 min intervals. (B) Pretreatment with C3G (10 µg/ml) for 30 min significantly inhibited Aβ25–35-induced mitochondrial depolarization. (C) Summary of glutamate-induced mitochondrial depolarizations in non-treated (control, n=18) and C3G-pretreated (n=18) cells. Cells were preincubated with 10 µM rhodamine 123 for 15 min. Change in MMP was shown as a percentage of the maximal intensity of rhodamine 123 (3 µM FCCP). Data are expressed as mean±S.E. **p<0.01 relative to control (non-paired Student's t-test).

  • Fig. 5 Effects of C3G on the Aβ25–35-induced formation of intracelluar ROS.Treatment with Aβ25–35 (20 µM) for 10 min significantly increased superoxide formation (vehicle, n=19; Aβ, n=22). Pretreatment with C3G (10 µg/ml) for 30 min blocked Aβ25–35-induced superoxide formation (vehicle, n=16, Aβ, n=14). Superoxide formation was shown as a percentage of the initial intensity of dihydroxyehidine. Data are expressed as mean±S.E. #p<0.05 relative to vehicle (non-paired Student's t-test). *p<0.05 relative to respective control (non-paired Student t-test).

  • Fig. 6 Effects of C3G on Aβ25–35-induced activation of caspase-3.Treatment with Aβ25–35 (20 µM) for 24 hr increased the activation of caspase-3 (n=6). Co-treatment with C3G (10 µg/ml) for 24 hr significantly inhibited the Aβ25–35-induced activation of caspase-3 (n=4). Data are expressed as mean±S.E. ##p<0.01 relative to vehicle (non-paired Student's t-test) *p<0.05 relative to respective control (non-paired Student's t-test).


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