Proanthocyanidin B2 attenuates high-glucose-induced neurotoxicity of dorsal root ganglion neurons through the PI3K/Akt signaling pathway

Abstract

High glucose affects primary afferent neurons in dorsal root ganglia by inhibiting neurite elongation, causing oxidative stress, and inducing neuronal apoptosis and mitochondrial dysfunction, which finally result in neuronal damage. Proanthocyanidin, a potent antioxidant, has been shown to have neuroprotective effects. Proanthocyanidin B2 is a common dimer of oligomeric proanthocyanidins. To date, no studies have reported the neuroprotective effects of proanthocyanidin B2 against high-glucose-related neurotoxicity in dorsal root ganglion neurons. In this study, 10 µg/mL proanthocyanidin B2 was used to investigate its effect on 45 mM high-glucose-cultured dorsal root ganglion neurons. We observed that challenge with high levels of glucose increased neuronal reactive oxygen species and promoted apoptosis, decreased cell viability, inhibited outgrowth of neurites, and decreased growth-associated protein 43 protein and mRNA levels. Proanthocyanidin B2 administration reversed the neurotoxic effects caused by glucose challenge. Blockage of the phosphatidylinositol 3 kinase/Akt signaling pathway with 10 µM LY294002 eliminated the protective effects of proanthocyanidin B2. Therefore, proanthocyanidin B2 might be a potential novel agent for the treatment of peripheral diabetic neuropathy.

Keywords: apoptosis; cell viability; diabetes; growth-associated protein 43; high glucose; nerve regeneration; neural regeneration; neurons; neuropathy; reactive oxygen species.

Figure 1

Effect of proanthocyanidin B2 (ProB2) on the neurite length of single neurons under normal culture conditions. (A–C) ProB2 effects on neuronal morphology under normal conditions (immunofluorescent staining). (A) Control group. (B) ProB2 group: Neurites were denser and longer than that in control group after ProB2 incubation. (C) ProB2 + LY294002 group: The trend of neurite elongation induced by ProB2 was inhibited by PI3K inhibitor LY294002 preincubation. Arrows show neurons. Scale bar: 50 µm. (D) Quantitative analysis of the total neurite length of each neuron. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. **P < 0.01, ***P < 0.001.

Figure 2

Effect of proanthocyanidin B2 (ProB2) on the intracellular reactive oxygen species (ROS) in dorsal root ganglion neurons with high glucose toxicity. (A–E) ProB2 effects on ROS levels in neurons under high glucose conditions (fluorescent microscopy). (A) Control group. (B) Mannitol group: ROS fluorescence intensity was similar to the control group with mannitol incubation. (C) High glucose group: increased ROS fluorescence intensity after high glucose incubation. (D) High glucose + ProB2 group: downward trend of ROS fluorescence intensity after ProB2 incubation in dorsal root ganglion neurons with high glucose challenge. (E) High glucose + ProB2 + LY294002 group: inhibitory effect of PI3K inhibitor LY294002 on ProB2 inhibited ROS generation. Arrows show neuronal cell bodies with different fluorescence intensities in each group. Scale bar: 50 µm. (F) Quantification of fluorescent density of ROS in dorsal root ganglion neurons. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. **P < 0.01, ***P < 0.001.

Figure 3

Effect of proanthocyanidin B2 (ProB2) on apoptosis of dorsal root ganglion neurons with high glucose toxicity. (A–E) ProB2 effect on neuronal apoptosis under high glucose conditions (Hoechst 33342 staining). (A) Control group: Normal DRG neuronal nuclei with Hoechst 33342 staining. (B) Mannitol group: Similar Hoechst 33342 staining condition to the control group with mannitol incubation. (C) High glucose group: shrunken nuclei and condensed or fragmented chromatin in apoptotic neurons after high glucose incubation. (D) High glucose + ProB2 group: A downward trend of apoptosis of DRG neurons after ProB2 incubation. (E) High glucose + ProB2 + LY294002 group: Inhibitory effect of PI3K inhibitor LY294002 on ProB2 inhibited apoptosis. Arrows show the shrunken nuclei and condensed or fragmented chromatin in apoptotic neurons. Scale bar: 50 µm. (F) Quantification of the apoptotic rate. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. **P < 0.01, ***P < 0.001.

Figure 4

Effect of proanthocyanidin B2 (ProB2) on the cell viability of dorsal root ganglion neurons incubated in high glucose. Cell viability was detected by Cell Counting Kit-8. Control group: neurobasal medium; mannitol group: 20 mM mannitol; high glucose group: 45 mM; high glucose + ProB2 group: 45 mM high glucose with 10 µg/mL ProB2; high glucose + LY294002 + ProB2 group: 45 mM high glucose with PI3K inhibitor LY294002 (10 µM) and 10 µg/mL ProB2. The cell viability in the control group was set as 1, and the results of other groups were normalized accordingly. The cell viability in experimental groups was compared with the value of the control group. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. ***P < 0.001.

Figure 5

Effect of proanthocyanidin B2 (ProB2) on the neurite length of a single neuron in dorsal root ganglion neurons with high glucose toxicity. (A–E) ProB2 effect on neuronal morphology under high glucose conditions (immunofluorescent staining). (A) Control group. (B) Mannitol group: Similar neurite length as the control with mannitol incubation. (C) High glucose group: Neurites were sparse and short after high glucose incubation. (D) High glucose + ProB2 group: Neurites were denser and longer after ProB2 incubation compared with the high glucose group. (E) High glucose + ProB2 + LY294002 group: The trend of Neurite elongation induced by ProB2 was inhibited by PI3K inhibitor LY294002 preincubation. Arrows show neurites. Scale bar: 50 µm. (F) Quantitative analysis of the total neurite length of each neuron. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. ***P < 0.001.

Figure 6

Effect of proanthocyanidin B2 (ProB2) on growth-associated protein 43 (GAP-43) mRNA levels in dorsal root ganglion neurons detected by real time-polymerase chain reaction. Control group: neurobasal medium; mannitol group: 20 mM mannitol; high glucose group: 45 mM; high glucose + ProB2 group: 45 mM high glucose with 10 µg/mL ProB2; high glucose + ProB2 + LY294002 group: 45 mM high glucose with PI3K inhibitor LY294002 (10 µM) and 10 µg/mL ProB2. GAP-43 mRNA level in the control group was set as 1, and experimental group results were normalized accordingly. GAP-43 mRNA levels in experimental groups were compared with the level in the control group. Mannitol incubation showed similar GAP-43 mRNA levels with the controls. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. ***P < 0.001.

Figure 7

Effect of proanthocyanidin B2 (ProB2) on growth-associated protein 43 (GAP-43) protein expression levels in the dorsal root ganglion neurons incubated in high glucose detected by western blot assay. Control group: Neurobasal medium; mannitol group: 20 mM mannitol; high glucose group: 45 mM; high glucose + ProB2 group: 45 mM high glucose with 10 µg/mL ProB2; high glucose + ProB2 + LY294002 group: 45 mM high glucose with PI3K inhibitor LY294002 (10 µM) and 10 µg/mL ProB2. (A) Western blot bands of GAP-43. (B) Quantification of GAP-43 levels. The GAP-43 protein level in the control group was set as 1, and experimental group results were normalized accordingly. The data are expressed as the mean ± SD (n = 5; one-way analysis of variance followed by the Student-Newman-Keuls test). ***P < 0.001. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase.

Figure 8

Effect of ProB2 on growth-associated protein 43 (GAP-43)-immunoreactive (IR) neurons in the dorsal root ganglion neurons incubated with high glucose detected by double fluorescence staining. (A–C) ProB2 effects on the number of GAP-43-IR neurons under high glucose conditions (double fluorescence staining). (A) Control group. (B) Mannitol group: similar proportion of GAP-43-IR neurons with the control group with mannitol incubation. (C) High glucose group: a decreased proportion of GAP-43-IR neurons after high glucose incubation. (D) High glucose + ProB2 group: the number of GAP-43-IR neurons after ProB2 incubation was greater than in the high glucose group. (E) High glucose + ProB2 + LY294002 group: GAP-43-IR neurons after ProB2 incubation were inhibited by PI3K inhibitor LY294002 preincubation. Arrows show GAP-43-IR neurons. Scale bar: 50 µm. (F) Numbers of GAP-43-IR neurons. The data are expressed as the mean ± SD (n = 5), and were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls test. **P < 0.01, ***P < 0.001.