Anat Cell Biol.  2015 Jun;48(2):95-103. 10.5115/acb.2015.48.2.95.

A single fraction from Uncaria sinensis exerts neuroprotective effects against glutamate-induced neurotoxicity in primary cultured cortical neurons

Affiliations
  • 1Department of Korean Medicine, School of Korean Medicine, Pusan National University, Yangsan, Korea. choibt@pusan.ac.kr
  • 2Division of Meridian and Structural Medicine, School of Korean Medicine, Pusan National University, Yangsan, Korea.

Abstract

We identified a neuroprotective single fraction among 62 ones of hexane extract from Uncaria sinensis (JGH43IA) and investigated its effects and mechanisms in primary cortical neurons. Pretreatment with JGH43IA showed a significantly increase cell viability in a dose-dependent manner with a decrease in the lactate dehydrogenase release. When we performed morphological assay and flow cytometry to determination of the type of cell death, pretreatment with JGH43IA showed a significant reduction of glutamate-induced apoptotic cell death. Then we explored the downstream signaling pathways of N-methyl-D-aspartate receptor (NMDAR) with calpain activation to elucidate possible pathways of neuroprotection by JGH43IA. Pretreatment with JGH43IA exhibited a significant attenuation of NMDAR GluN2B subunit activation and a decrease in active form of calpain 1 leading to subsequent cleavage of striatal-enriched protein tyrosine phosphatase (STEP). In addition, pretreatment with JGH43IA showed a marked increase of cAMP responsive element binding protein. These results suggest that JGH43IA may have neuroprotective effects through down-regulation of NMDAR GluN2B subunit and calpain 1 activation, and subsequent alleviation of STEP cleavage. This single fraction from U. sinensis might be a useful therapeutic agent for brain disorder associated with glutamate injury.

Keyword

Uncaria sinensis; Glutamates; N-Methyl-D-aspartate receptors; Calpain; STEP

MeSH Terms

Brain Diseases
Calpain
Carrier Proteins
Cell Death
Cell Survival
Down-Regulation
Flow Cytometry
Glutamates
Glutamic Acid
L-Lactate Dehydrogenase
N-Methylaspartate
Neurons*
Neuroprotective Agents*
Protein Tyrosine Phosphatases
Receptors, N-Methyl-D-Aspartate
Uncaria*
Calpain
Carrier Proteins
Glutamates
Glutamic Acid
L-Lactate Dehydrogenase
N-Methylaspartate
Neuroprotective Agents
Protein Tyrosine Phosphatases
Receptors, N-Methyl-D-Aspartate

Figure

  • Fig. 1 Effect of JGH43IA on the glutamate-induced toxicity. MTT (A) and LDH (B) assays of cell viability and toxicity in glutamate-treated primary cultured cortical neurons. Cortical neurons were pre-incubated with 0.01, 0.03, 0.07, 0.1, and 1 µg/ml JGH43IA for 24 hours, followed by exposure to 200 µM glutamate for 6 hours. MTT, 3-(4,5-dimetylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; LDH, lactate dehydreogenase. *P<0.05, **P<0.01, and ***P<0.001 as compared with to the glutamate-treated group; ††P<0.01 and †††P<0.001 as compared to the control group.

  • Fig. 2 Effect of JGH43IA on the glutamate-induced nuclear condensation. Detection of nuclear condensation (arrows) using Hoechst 33342 staining in glutamate-treated primary cultured cortical neurons. (A) Cortical neurons were pre-incubated with JGH43IA at the indicated concentration of 0.01 and 0.1 µg/ml for 24 hours, followed by exposure to 200 µM glutamate for 6 hours. Glu, glutamate. (B) Quantitative analysis of the histograms expressed as the percentage of apoptotic neurons in the total neurons observed under each microscopic field. Data are represented as the means±SEM of three independent experiments. *P<0.05 as compared with glutamate-treated group; ††P<0.01 as compared with control group.

  • Fig. 3 Effect of JGH43IA on the glutamate-induced DNA fragmentation. Detection of DNA fragmentation using TUNEL assay in glutamate-treated primary cultured cortical neurons. Cortical neurons were pretreated with JGH43IA 0.01 and 0.1 µg/ml for 24 hours, followed by treatment with glutamate 200 µM for 6 hours. (A) TUNEL-positive cells were stained with green and nuclei were counterstained with PI (red). Glu, glutamate; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling; PI, propidium iodide. (B) Quantitative analysis of the histograms expressed as the ratio of TUNEL-positive neuronal cells observed in the same field. **P<0.01 and *P<0.05 as compared with glutamate-treated group; ††P<0.01 as compared with control group. Data are expressed as the means±SEM from three independent experiments.

  • Fig. 4 Effect of JGH43IA on the types of glutamate-induced cell death. Flow cytometric analysis of the type of cell death in glutamate-treated cortical neurons. Cortical cells were pretreated with JGH43IA at concentration of 0.01 and 0.1 µg/ml for 24 hours, followed by exposure to 200 µM for 6 hours and then co-stained with FITC-Annexin V and PI; analysis was performed using a flow cytometer. (A) Representative flow cytometry analysis scatter-grams of Annexin V/PI staining. Glu, glutamate; PI, propidium iodide. (B, C) Quantitative analysis of the histograms expressed as the percentage of apoptotic or necrotic cortical neurons in total cortical neurons. **P<0.01 and *P<0.05 as compared to the glutamate-treated group; ††P<0.01 and †P<0.05 as compared to the control group. Data are represented as the means±SEM from three independent experiments.

  • Fig. 5 Effect of JGH43IA on the expression of neuronal survival and death related proteins. Western blot analysis of neuronal cell survival and death related proteins in glutamate-treated primary cultured cortical neurons. The pretreatment with JGH43IA 0.1 µg/ml for 24 hours, followed glutamate-treated with 300 µM for 15 minutes. Following preparation of cell lysates and equal amounts of cellular proteins were subjected to Western blot assays. Equal protein loading was confirmed by actin expression. STEP, striatal-enriched protein tyrosine phosphatase.


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