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

Kim HN, Jang JY, Choi BT - Anat Cell Biol (2015)

Bottom Line: Pretreatment with JGH43IA showed a significantly increase cell viability in a dose-dependent manner with a decrease in the lactate dehydrogenase release.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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Korean 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.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: We performed Hoechst 33342 staining and TUNEL assay in order to characterize the types of neuronal death by JGH43IA treatment. Results of Hoechst staining indicated that treatment with glutamate alone exhibited high levels of condensed chromatin and apoptotic bodies. But pretreatment with JGH43IA showed a significant decrease of these apoptotic features at both concentrations of 0.01 and 0.1 µg/ml (Fig. 2). DNA fragmentation was also evaluated by TUNEL assay as another method for detection of apoptosis. TUNEL-positive cells increased to 29.6% in total cells after exposure to glutamate alone. However, pretreatment with JGH43IA resulted in significantly decreased of TUNEL-positive cells to nearly 19.7% and 9.4% at a concentration of 0.01 and 0.1 µg/ml, respectively (Fig. 3).


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

Kim HN, Jang JY, Choi BT - Anat Cell Biol (2015)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488647&req=5

Figure 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.
Mentions: We performed Hoechst 33342 staining and TUNEL assay in order to characterize the types of neuronal death by JGH43IA treatment. Results of Hoechst staining indicated that treatment with glutamate alone exhibited high levels of condensed chromatin and apoptotic bodies. But pretreatment with JGH43IA showed a significant decrease of these apoptotic features at both concentrations of 0.01 and 0.1 µg/ml (Fig. 2). DNA fragmentation was also evaluated by TUNEL assay as another method for detection of apoptosis. TUNEL-positive cells increased to 29.6% in total cells after exposure to glutamate alone. However, pretreatment with JGH43IA resulted in significantly decreased of TUNEL-positive cells to nearly 19.7% and 9.4% at a concentration of 0.01 and 0.1 µg/ml, respectively (Fig. 3).

Bottom Line: Pretreatment with JGH43IA showed a significantly increase cell viability in a dose-dependent manner with a decrease in the lactate dehydrogenase release.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).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.

View Article: PubMed Central - PubMed

Affiliation: Department of Korean 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.

No MeSH data available.


Related in: MedlinePlus