Neuroprotective Effects of Ginsenoside Rb1 on High Glucose-Induced Neurotoxicity in Primary Cultured Rat Hippocampal Neurons

<div><p>Ginsenoside Rb1 is one of the main active principles in traditional herb ginseng and has been reported to have a wide variety of neuroprotective effects. Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, so the present study aimed to observe the effects of ginsenoside Rb1 on ER stress signaling pathways in high glucose-treated hippocampal neurons. The results from MTT, TUNEL labeling and Annexin V-FITC/PI/Hoechst assays showed that incubating neurons with 50 mM high glucose for 72h decreased cell viability and increased the number of apoptotic cells whereas treating neurons with 1 μM Rb1 for 72h protected the neurons against high glucose-induced cell damage. Further molecular mechanism study demonstrated that Rb1 suppressed the activation of ER stress-associated proteins including protein kinase RNA (PKR)-like ER kinase (PERK) and C/EBP homology protein (CHOP) and downregulation of Bcl-2 induced by high glucose. Moreover, Rb1 inhibited both the elevation of intracellular reactive oxygen species (ROS) and the disruption of mitochondrial membrane potential induced by high glucose. In addition, the high glucose-induced cell apoptosis, activation of ER stress, ROS accumulation and mitochondrial dysfunction can also be attenuated by the inhibitor of ER stress 4-phenylbutyric acid (4-PBA) and anti-oxidant N-acetylcysteine(NAC). In conclusion, these results suggest that Rb1 may protect neurons against high glucose-induced cell injury through inhibiting CHOP signaling pathway as well as oxidative stress and mitochondrial dysfunction.</p> </div

Effects of ginsenoside Rb1 on mitochondrial function in hippocampal neurons exposed to high glucose.

<p>(A) Representative photographs of JC-1 staining in different groups. (a) control group; (b) high glucose group; (c) ginsenoside Rb1+high glucose group; (d) NAC + high glucose group;(e) 4-PBA + high glucose group. Magnification 400× ; Scale bar = 34μm. (B) Quantitative analysis of the Δψm among groups. All values are denoted as means ±S.D from six independent photographs shot in each group. Data are expressed as percentage of control group. The experiments were repeated three times independently. **P<0.01, as compared to the control group; ^##P<0.01, as compared to the high glucose group.</p

Effects of ginsenoside Rb1 on apoptosis induced by high glucose in hippocampal neurons.

<p>(A) Morphological photomicrographs of TUNEL staining. Nuclei of all cells appear blue, while TUNEL-positive (apoptotic) cells appear green. (a) control group; (b) high glucose group; (c) ginsenoside Rb1+high glucose group; (d) NAC + high glucose group;(e) 4-PBA + high glucose group. Magnification 200× ; Scale bar = 50 μm. (B) The percentage of TUNEL-positive cells in total cultured hippocampal neurons. (C) Morphological photomicrographs of Annexin V-FITC/PI assay. Nuclei of all cells appear blue, while Annexin V(+) /PI(−)(apoptotic) cells appear green. (a) control group; (b) high glucose group; (c) ginsenoside Rb1+high glucose group; (d) NAC + high glucose group;(e) 4-PBA + high glucose group. Magnification 400×; Scale bar = 34μm. (D) The percentage of Annexin V(+) /PI(−) cells in total cultured hippocampal neurons. The experiments were repeated three times independently. **P<0.01, as compared to the control group; ^##P<0.01, as compared to the high glucose group.</p

Effects of ginsenoside Rb1 on intracellular ROS accumulation induced by high glucose in hippocampal neurons.

<p>(A) Representative photographs of DCF staining in different groups. (a) control group; (b) high glucose group; (c) ginsenoside Rb1+high glucose group; (d) NAC + high glucose group;(e) 4-PBA + high glucose group. Magnification 400× ; Scale bar = 34μm. (B) DCF fluorescence intensity was quantitatively analyzed. All values are denoted as means ±S.D from six independent photographs shot in each group. Data are expressed as percentage of control group. The experiments were repeated three times independently. *P<0.05, as compared to the control group; **P<0.01, as compared to the control group; ^##P<0.01, as compared to the high glucose group.</p

Effects of ginsenoside Rb1 on ER stress-mediated apoptotic signaling pathways in high glucose-treated hippocampal neurons.

<p>(A) Immunoreactive bands of p-PERK, PERK, CHOP, Bcl-2 and actin using specific antibody. (B) Quantitative analysis of relative protein level. Results are expressed as the means ± SD from 3 independent experiments.β-actin was used as the loading controls. (C)Histogram indicating the fold changes in mRNA levels by real-time PCR compared to the respective control after normalization to GAPDH. The results represent the mean ± S.D of three independent experiments. *P<0.05, as compared to the control group; **P<0.01, as compared to the control group; ^##P<0.01, as compared to the high glucose group.</p

Consort diagram.

<p>Consort diagram.</p

A. <i>Wuchereria bancrofti</i> antigen and antibody levels over time.

<p>Circulating <i>Wuchereria bancrofti</i> antigen levels in HIV+/FIL+ individuals at baseline and 1 year. <b>B</b>. BMA-specific IgG in HIV+/FIL+ subjects at baseline and 1 year. <b>C</b>. Total IgG4 levels in HIV+/FIL+ subjects at baseline and 1 year.</p

HIV viral load by over time by individual.

<p>HIV VL at baseline and 1 year in HIV+ /FIL+ individuals (<b>A</b>) and HIV+/FIL- individuals (<b>B</b>).</p

CD4 count and viral load comparisons at baseline and 1 year.

<p>Comparison of CD4 and viral load (VL) at baseline (<b>A</b> CD4 [count/dL]), <b>B</b> log VL [log₁₀ RNA Copies]) and at 1 year (<b>C</b> CD4 count, <b>D</b> log VL) between HIV+/FIL+ cases and HIV+/FIL- controls.</p

Demographics.

<p>Abbreviations: HIV: Human Immunodeficiency Virus; FIL: Filaria (<i>W</i>. <i>bancrofti</i> antigen) positive; GM: geometric mean; SD: standard deviation; WMW: Wilcoxon-Mann-Whitney; ART: highly active antiretroviral therapy, N/A: Not available; %: percent. Hgb: Hemoglobin (gm/dL); WBC: White blood cell count (x 10³/mm³); EOS: eosinophils; Plts: platelet count (x10³/uL); ALT: alanine transaminase (SGPT, IU/L); AST: aspartate transaminase (SGOT, IU/L); VL: viral load; Ag: antigen.</p><p>^ 52 subjects were enrolled, but 1 subject did not have baseline HIV viral load value done. These statistics are on all 52 except for the viral load.</p><p>* In addition to the 28 subjects who got ART throughout, there were 2 (3.8%) subjects who got ART only part of the time during the study. Fisher’s exact test excluded those 2 subjects.</p><p>^$ Measured on only n = 12 in the HIV+/FIL+ group and n = 39 in the HIV+/FIL- group.</p><p>Demographics.</p