N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes

Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.

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Main Authors: Aquino,P.E.A., Siqueira,E.A. de, Paes,L.C.F., Magalhães,E.P., Barbosa,T.M., Carvalho,M.A.J. de, Azul,F.V.C. Serra, Lustosa,I. Rosal, Mottin,M., Sampaio,T.L., Martins,A.M.C., Silveira,E.R., Viana,G.S.B.
Format: Digital revista
Language:English
Published: Associação Brasileira de Divulgação Científica 2022
Online Access:http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2022000100670
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spelling oai:scielo:S0100-879X20220001006702022-10-26N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytesAquino,P.E.A.Siqueira,E.A. dePaes,L.C.F.Magalhães,E.P.Barbosa,T.M.Carvalho,M.A.J. deAzul,F.V.C. SerraLustosa,I. RosalMottin,M.Sampaio,T.L.Martins,A.M.C.Silveira,E.R.Viana,G.S.B. Temporal lobe epilepsy Voltage-dependent anion channel Mitochondrial transmembrane potential Oxidative stress Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.info:eu-repo/semantics/openAccessAssociação Brasileira de Divulgação CientíficaBrazilian Journal of Medical and Biological Research v.55 20222022-01-01info:eu-repo/semantics/articletext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2022000100670en10.1590/1414-431x2022e12381
institution SCIELO
collection OJS
country Brasil
countrycode BR
component Revista
access En linea
databasecode rev-scielo-br
tag revista
region America del Sur
libraryname SciELO
language English
format Digital
author Aquino,P.E.A.
Siqueira,E.A. de
Paes,L.C.F.
Magalhães,E.P.
Barbosa,T.M.
Carvalho,M.A.J. de
Azul,F.V.C. Serra
Lustosa,I. Rosal
Mottin,M.
Sampaio,T.L.
Martins,A.M.C.
Silveira,E.R.
Viana,G.S.B.
spellingShingle Aquino,P.E.A.
Siqueira,E.A. de
Paes,L.C.F.
Magalhães,E.P.
Barbosa,T.M.
Carvalho,M.A.J. de
Azul,F.V.C. Serra
Lustosa,I. Rosal
Mottin,M.
Sampaio,T.L.
Martins,A.M.C.
Silveira,E.R.
Viana,G.S.B.
N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
author_facet Aquino,P.E.A.
Siqueira,E.A. de
Paes,L.C.F.
Magalhães,E.P.
Barbosa,T.M.
Carvalho,M.A.J. de
Azul,F.V.C. Serra
Lustosa,I. Rosal
Mottin,M.
Sampaio,T.L.
Martins,A.M.C.
Silveira,E.R.
Viana,G.S.B.
author_sort Aquino,P.E.A.
title N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
title_short N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
title_full N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
title_fullStr N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
title_full_unstemmed N-Methyl-(2S, 4R)-trans-4-hydroxy-L-proline, the major bioactive compound from Sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
title_sort n-methyl-(2s, 4r)-trans-4-hydroxy-l-proline, the major bioactive compound from sideroxylon obtusifolium, attenuates pilocarpine-induced injury in cultured astrocytes
description Glial cells have been implicated in temporal lobe epilepsy in humans and in its models. Astrocytes are lost in several brain regions after acute seizures induced by pilocarpine and may suffer hyperplasia at subsequent time points. This study investigated the effect of N-methyl-(2S,4R)-trans-4-hydroxy-L-proline (NMP) on astrocytes exposed to cytotoxic concentrations of pilocarpine. Astrocytes were incubated with pilocarpine (half maximal inhibitory concentration (IC50)=31.86 mM) for 24 h. Afterwards, they were treated with NMP at concentrations ranging from 3.12 to 100 μg/mL for 24 h. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cytoplasmic reactive oxygen species (ROS) and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry using 2',7'-dichlorofluorescein diacetate (DCFH-DA) and rhodamine-123 (Rho123), respectively. Expression of glial fibrillary acidic protein (GFAP) and voltage-dependent anion channel-1 (VDAC-1) were measured by western blot. Pilocarpine significantly decreased cell viability and mitochondrial potential and increased ROS concentration significantly by 6.7 times compared to the control. NMP concentrations ≥25 µg/mL protected astrocytes against pilocarpine-induced injury in a concentration-dependent manner. Concomitantly, NMP reduced cytoplasmic ROS accumulation to 27.3, 24.8, and 12.3% in the groups treated with 25, 50, and 100 µg/mL NMP, respectively. NMP also protected mitochondria from pilocarpine-induced depolarization. These effects were associated with improvement of pilocarpine-induced GFAP and VDAC-1 overexpression, which are important biomarkers of astrocyte dysfunction. In conclusion, the improvement of ROS accumulation, VDAC-1 overexpression, and mitochondrial depolarization are possible mechanisms of the NMP protective action on reactive astrocytes.
publisher Associação Brasileira de Divulgação Científica
publishDate 2022
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X2022000100670
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