erks and mitochondria related pathways are essential for glycyrrhizic acid mediated CORD-Papers-2021-10-25 (Version 1)

Title: ERKs and mitochondria-related pathways are essential for glycyrrhizic acid-mediated neuroprotection against glutamate-induced toxicity in differentiated PC12 cells
Abstract: The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamate-enhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondria-related pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases.
Published: 7/25/2014
Journal: Braz J Med Biol Res
DOI: 10.1590/1414-431x20143760
DOI_URL: http://doi.org/10.1590/1414-431x20143760
Author Name: Wang, D
Author link: https://covid19-data.nist.gov/pid/rest/local/author/wang_d
Author Name: Guo, T Q
Author link: https://covid19-data.nist.gov/pid/rest/local/author/guo_t_q
Author Name: Wang, Z Y
Author link: https://covid19-data.nist.gov/pid/rest/local/author/wang_z_y
Author Name: Lu, J H
Author link: https://covid19-data.nist.gov/pid/rest/local/author/lu_j_h
Author Name: Liu, D P
Author link: https://covid19-data.nist.gov/pid/rest/local/author/liu_d_p
Author Name: Meng, Q F
Author link: https://covid19-data.nist.gov/pid/rest/local/author/meng_q_f
Author Name: Xie, J
Author link: https://covid19-data.nist.gov/pid/rest/local/author/xie_j
Author Name: Zhang, X L
Author link: https://covid19-data.nist.gov/pid/rest/local/author/zhang_x_l
Author Name: Liu, Y
Author link: https://covid19-data.nist.gov/pid/rest/local/author/liu_y
Author Name: Teng, L S
Author link: https://covid19-data.nist.gov/pid/rest/local/author/teng_l_s
sha: 41df61dc89201d699b23532cbd09da787afa7e84
license: cc-by-nc
license_url: https://creativecommons.org/licenses/by-nc/4.0/
source_x: PMC
source_x_url: https://www.ncbi.nlm.nih.gov/pubmed/
pubmed_id: 25075574
pubmed_id_url: https://www.ncbi.nlm.nih.gov/pubmed/25075574
pmcid: PMC4143205
pmcid_url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143205
url: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4143205/
has_full_text: TRUE
Keywords Extracted from Text Content: mitogen-activated protein/extracellular signal-regulated kinase kinase glycyrrhizic acid PI3K] PD98059 AKT PC12 LY294002 Cyto C ERKs GA phosphoinositide 3-kinase cell glutamate Bax caspase 3 Glycyrrhiza Radix protein kinase B glutamate-induced DPC12 cells cytochrome C extracellular signal-regulated kinases mitochondria glutamate-exposed DPC12 cells hippocampal slices B-cell lymphoma 2 FBS green sodium ferulate tetraethylbenzimidazolylcarbocyanine iodide PC12 LY294002 Cell Signaling GA Dulbecco Jilin Province MEK P,0.05 cell stress-activated c-Jun caspase 3 T-AKT rat primary neurons mitochondrial Glycyrrhiza Radix T-ERKs lung epithelial cells extracellular high-mobility glutamate-caused cell anti-inflammatory (6) nuclear WEHI-3 mouse leukemia cells kinase-P38 h. HS, Invitrogen 78.7±4.4 G. glabra mitochondrial membrane Santa Cruz glyceraldehyde-3phosphate dehydrogenase 71.7±5.7 Cells mitogen-activated protein/extracellular signal-regulated kinase kinase glutamate-induced cell total-ERKs Figure 1A Figure 2 19.6±8.9 caspases Red horseradish 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide GE phosphoinositide 3-kinase phospho-ERKs Bcl-xL nerve growth factor 102.3±12.7 Biotechnology DPC12 cells penicillin PC12 cell P-ERKs extracellular signal-regulated kinases PC12 cells streptomycin 12.5 mM. monolayers collagen 71.1±11.3 Bcl-2 3-morpholinosydnonimine-induced cell bovine serum PD98059 neural glutamate-induced MMP Cyto C Jilin UK PI3K cancer P-AKT 98.9±6.8 cytoplasm Glutamate phosphor-AKT horse serum DMH-induced colon ERK central nervous system 85.3±1.9 77.3±0.6 Glycyrrhizic acid excitatory cytochrome C total-AKT Wistar rats P-ERK JC-1 Mitochondrial membrane G. inflata cytosol glycyrrhizic acid CRL-1721 Figure 4A sodium dodecyl sulfate-polyacrylamide AKT ATCC brain nucleus Figure 4C neuronal ERKs dimethyl sulfoxide DPC12 cell glutamate Bax Glu cytoplasmic extracts Caspases MMP glutamate-induced cells Figure 1 . mitochondria Cell saline Bcl-2/Bax excitatory amino membranes P.R. China Jilin Province
Extracted Text Content in Record: First 5000 Characters:The present study focuses on the neuroprotective effect of glycyrrhizic acid (GA, a major compound separated from Glycyrrhiza Radix, which is a crude Chinese traditional drug) against glutamate-induced cytotoxicity in differentiated PC12 (DPC12) cells. The results showed that GA treatment improved cell viability and ameliorated abnormal glutamate-induced alterations in mitochondria in DPC12 cells. GA reversed glutamate-suppressed B-cell lymphoma 2 levels, inhibited glutamateenhanced expressions of Bax and cleaved caspase 3, and reduced cytochrome C (Cyto C) release. Exposure to glutamate strongly inhibited phosphorylation of AKT (protein kinase B) and extracellular signal-regulated kinases (ERKs); however, GA pretreatment enhanced activation of ERKs but not AKT. The presence of PD98059 (a mitogen-activated protein/extracellular signal-regulated kinase kinase [MEK] inhibitor) but not LY294002 (a phosphoinositide 3-kinase [PI3K] inhibitor) diminished the potency of GA for improving viability of glutamate-exposed DPC12 cells. These results indicated that ERKs and mitochondriarelated pathways are essential for the neuroprotective effect of GA against glutamate-induced toxicity in DPC12 cells. The present study provides experimental evidence supporting GA as a potential therapeutic agent for use in the treatment of neurodegenerative diseases. The excitatory amino acid glutamate serves as a fast excitatory neurotransmitter in the central nervous system (1) , and excitotoxicity and oxidative toxicity are considered to be two major pathways responsible for glutamateinduced toxicity (2, 3) . Excessive release of glutamate leading to excitotoxicity is responsible for neuronal injury associated with many acute and chronic brain diseases, such as brain ischemia, traumatic brain injury, and neurodegenerative disorders (4, 5) . Glycyrrhiza Radix, a crude Chinese traditional drug derived from Glycyrrhiza uralensis, G. glabra and G. inflata, exhibits various pharmacological activities including anti-inflammatory (6) , anti-obesity (7), anti-virus (8) , and anti-oxidative (9) effects. Glycyrrhizic acid (GA), a major component of Glycyrrhiza Radix, possesses antiinflammatory and anti-viral effects (10, 11) . Its structure is shown in Figure 1A . A previous study demonstrated that GA exerts a neuroprotective effect against 6-hydroxydopamine-induced PC12 cell death via the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway (12) . The protective effect of GA was also confirmed in rat primary neurons and hippocampal slices (13) . However, the protective effects of GA against glutamate-induced differentiated PC12 (DPC12) cell damage and the underlying mechanisms have not been described. Several signaling pathways are involved in the regulation of glutamate-induced neurotoxicity (14, 15) . Alteration of mitochondrial function is one of the factors Correspondence: L.S. Teng, School of Life Sciences, Jilin University, Changchun City 130012, Jilin Province, China. Fax: + +86-431-8516-8637. E-mail: tenglesheng@jlu.edu.cn responsible for cell damage (16) . Bax and B-cell lymphoma 2 (Bcl-2), located in mitochondria, are believed to be central regulators of mitochondrial function (17) . Additionally, activation of both AKT and extracellular signal-regulated kinases (ERKs) is considered to contribute to cell proliferation and apoptosis (18, 19) . Previous studies demonstrated that inhibition of phosphorylation of both AKT and ERKs is involved in glutamate-induced cell damage (20, 21) . Our study aimed to examine the effect of GA on attenuating glutamate cytotoxicity and its underlying mechanisms in DPC12 cells. Our results showed that GA improved cell viability and restored mitochondrial membrane potential dissipation. GA also normalized the expression of apoptosis-related proteins, including Bcl-2, Bax, cleaved caspase 3, and cytochrome C (Cyto C). Furthermore, GA pretreatment remarkably enhanced phosphorylation of ERKs but not AKT. Collectively, our findings demonstrated that ERKs and mitochondriarelated pathways are essential for GA-mediated neuroprotection against glutamate-induced toxicity in DPC12 cells. This study provides evidence that supports the use of GA as a potential therapeutic agent for neurodegenerative diseases. Cell culture PC12 cells (CRL-1721, passages ,10; obtained from ATCC) were grown in monolayers in collagen I-coated flasks with Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with 10% horse serum (HS, Invitrogen), 5% fetal bovine serum (FBS, Invitrogen), penicillin (100 U/mL), and streptomycin (100 mg/mL, Invitrogen), under a humidified atmosphere containing 5% CO 2 and 95% air at 376C. The culture medium was changed every 3 days. PC12 cells were differentiated with 20 ng/mL nerve growth factor for 48 h, which was added to the culture medium containing 1% FBS and 1% HS. Cells were ready for treatment when they reached 80% confluence. Cell viability was measured by a quantitative col
Keywords Extracted from PMC Text: tetraethylbenzimidazolylcarbocyanine iodide stress-activated c-Jun glutamate-caused cell MMP Glutamate cytochrome C P-ERK GA cancer PI3K inhibitor LY294002 PD98059 P<0.05 neural Cell CRL-1721 rat primary neurons Figure 4A 77.3±0.6 Bcl-2 glutamate-induced cell streptomycin DMH-induced colon mitogen-activated G. glabra glutamate-induced PC12 cells glutamate-induced PC12 cell Red membranes monolayers Glycyrrhizic acid penicillin dimethyl sulfoxide caspases UK signal-regulated kinase kinase DPC12 cells nucleus × central nervous system DPC12 cell Caspases cytosol ATCC MEK bovine serum Bax PC12 excitatory amino cytoplasmic extracts excitatory Bcl-2/Bax Figure 1A green cell T-ERKs mitochondria B-cell lymphoma 2 glyceraldehyde-3-phosphate dehydrogenase Santa Cruz JC-1 brain 71.7±5.7 102.3±12.7 Figure 4D nuclear mitochondrial membrane Wistar rats anti-inflammatory (6) sodium dodecyl sulfate-polyacrylamide total-AKT 71.1±11.3 glutamate P-AKT P-ERKs PI3K GE phospho-ERKs P<0.01 extracellular signal-regulated kinases neuronal phosphoinositide 3-kinase collagen 78.7±4.4 cells phosphor-AKT Bcl-xL caspase 3 FBS Figure 2 Figure 4C h. Dulbecco's modified saline WEHI-3 mouse leukemia cells ERKs mitochondrial kinase-P38 Signaling CO2 155.5±10.6 T-AKT horse serum Cells sodium ferulate G. inflata Biotechnology Cyto C 85.3±1.9 Glycyrrhiza Radix 3-morpholinosydnonimine-induced cell 98.9±6.8 HS, Invitrogen hippocampal slices mitochondrial DPC12 cells 2 μM JC-1 6.25-12.5 μM GA for 3 horseradish lung epithelial cells ERK AKT cytoplasm
Extracted PMC Text Content in Record: First 5000 Characters:The excitatory amino acid glutamate serves as a fast excitatory neurotransmitter in the central nervous system (1), and excitotoxicity and oxidative toxicity are considered to be two major pathways responsible for glutamate-induced toxicity (2,3). Excessive release of glutamate leading to excitotoxicity is responsible for neuronal injury associated with many acute and chronic brain diseases, such as brain ischemia, traumatic brain injury, and neurodegenerative disorders (4,5). Glycyrrhiza Radix, a crude Chinese traditional drug derived from Glycyrrhiza uralensis, G. glabra and G. inflata, exhibits various pharmacological activities including anti-inflammatory (6), anti-obesity (7), anti-virus (8), and anti-oxidative (9) effects. Glycyrrhizic acid (GA), a major component of Glycyrrhiza Radix, possesses anti-inflammatory and anti-viral effects (10,11). Its structure is shown in Figure 1A. A previous study demonstrated that GA exerts a neuroprotective effect against 6-hydroxydopamine-induced PC12 cell death via the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway (12). The protective effect of GA was also confirmed in rat primary neurons and hippocampal slices (13). However, the protective effects of GA against glutamate-induced differentiated PC12 (DPC12) cell damage and the underlying mechanisms have not been described. Several signaling pathways are involved in the regulation of glutamate-induced neurotoxicity (14,15). Alteration of mitochondrial function is one of the factors responsible for cell damage (16). Bax and B-cell lymphoma 2 (Bcl-2), located in mitochondria, are believed to be central regulators of mitochondrial function (17). Additionally, activation of both AKT and extracellular signal-regulated kinases (ERKs) is considered to contribute to cell proliferation and apoptosis (18,19). Previous studies demonstrated that inhibition of phosphorylation of both AKT and ERKs is involved in glutamate-induced cell damage (20,21). Our study aimed to examine the effect of GA on attenuating glutamate cytotoxicity and its underlying mechanisms in DPC12 cells. Our results showed that GA improved cell viability and restored mitochondrial membrane potential dissipation. GA also normalized the expression of apoptosis-related proteins, including Bcl-2, Bax, cleaved caspase 3, and cytochrome C (Cyto C). Furthermore, GA pretreatment remarkably enhanced phosphorylation of ERKs but not AKT. Collectively, our findings demonstrated that ERKs and mitochondria-related pathways are essential for GA-mediated neuroprotection against glutamate-induced toxicity in DPC12 cells. This study provides evidence that supports the use of GA as a potential therapeutic agent for neurodegenerative diseases. PC12 cells (CRL-1721, passages <10; obtained from ATCC) were grown in monolayers in collagen I-coated flasks with Dulbecco's modified Eagle's medium (Invitrogen, USA) supplemented with 10% horse serum (HS, Invitrogen), 5% fetal bovine serum (FBS, Invitrogen), penicillin (100 U/mL), and streptomycin (100 μg/mL, Invitrogen), under a humidified atmosphere containing 5% CO2 and 95% air at 37°C. The culture medium was changed every 3 days. PC12 cells were differentiated with 20 ng/mL nerve growth factor for 48 h, which was added to the culture medium containing 1% FBS and 1% HS. Cells were ready for treatment when they reached 80% confluence. Cell viability was measured by a quantitative colorimetric assay with 3-(4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich, USA) as reported previously (22). Briefly, PC12 cells were seeded onto 96-well plates at 1×104 cells/well. After differentiation, cells were pretreated with 6.25-12.5 μM GA for 3 h and exposed to 20 mM glutamate for 24 h. In separate experiments, cells were pretreated with 10 μM LY294002 (PI3K inhibitor) or 10 μM PD98059 [a mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitor] for 30 min, followed by treatment with 6.25-12.5 μM GA for 3 h and exposure to 20 mM glutamate for 24 h. Treated cells were incubated with MTT solution (0.5 mg/mL) for 4 h at 37°C in the dark. One hundred microliters of dimethyl sulfoxide were used to dissolve crystals. Absorbance was measured spectrophotometrically using a microplate reader (Bio-Rad, USA) at 540 nm. The viability of treated cells are reported as a percentage of that from corresponding control cells. 5,5′,6,6′-Tetrachloro-1,1′,3,3′ tetraethylbenzimidazolylcarbocyanine iodide (JC-1, Sigma-Aldrich) staining was used to measure MMP changes. PC12 cells were seeded onto six-well plates at 1×105 cells/well. After differentiation, cells were pretreated with 6.25 or 12.5 μM GA for 3 h and cotreated with glutamate for another 12 h. Next, cells were incubated with 2 μM JC-1 at 37°C for 10 min. After three washes with phosphate-buffered saline, fluorescent color in each group was examined using a fluorescence microscope (20×; Axio Observer Z1, CCD camera; Carl Zeiss, Germany
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