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A Review Study of Substance Abuse Status in High School Students, Isfahan, Iran

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Official websites use. Share sensitive information only on official, secure websites. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The aim of the present study was to investigate the neuroprotective effects of Melissa officinalis, a major antioxidant plant, against neuron toxicity in hippocampal primary culture induced by 3,4-methylenedioxymethamphetamine MDMA or ecstasy, one of the most abused drugs, which causes neurotoxicity. Melissa officinalis has revealed neuroprotective effects against apoptosis induced by MDMA in the primary neurons of hippocampal culture, which could be due to its free radical scavenging properties and monoamine oxidase MAO inhibitory effects. Previous studies on the neurotoxic properties of MDMA are focused mostly on the damage of serotonergic and dopaminergic neurons, which are situated mainly in the midbrain 2 , 3 However, apoptotic changes in hippocampal neurons in MDMA-treated cultures have also been detected 4. These changes could be due to an oxidative stress event resulting from chronic ecstasy exposure 5. Oxidative stress incidents produce reactive oxygen species ROS , including hydrogen peroxide which is involved in neurotoxic events related to some neurodegenerative diseases 6. Therefore, extreme production of ROS might cause protein and lipid oxidation leading to neuronal death and apoptosis 7. Natural antioxidants from flora are wellknown to maintain the human organism safe from free radicals and protect it from some diseases 8. It is known that lemon balm or Melissa officinalis L. Lamiaceae extracts contain some compounds such as flavonoids and phenolic acids 9 that may scavenge these free radicals and prevent apoptosis. The neuroprotective effects of this plant were investigated using an in vitro cellular model with the PC12 cell line, which shows some characteristics of neurons In a previous study, we found that high doses of ecstasy correlate with an increase in caspase-3 activity and apoptosis in primary culture of hippocampal neurons 4. In the present work, we studied whether apoptotic neuronal death induced by ecstasy is abolished by treatment with the antioxidant plant Melissa officinalis. Buffers, culture plates, and other cell culture materials except media , rabbit anti-microtubuleassociated protein-2 MAP2 polyclonal antibody, Hoechst , propiedium iodide PI , Mowiol , caspase-3 colorimetric assay kit and 3- 4, 5-dimethylthiazolyl -2, 5-diphenyltetrazolium bromide MTT were purchased from Sigma USA. Anthos microplate reader and fluorescence microscope were respectively from Biochrom UK and Nikon Japan. All procedures were performed in accordance with institutional guidelines for animal care and use. Aerial parts of cultivated flowering plant Melissa officinalis L. Dried leaves were ground to a fine powder. The powdered leaves 50g were macerated in distilled water ml at room temperature for 24 hours. Subsequently, the mixture was filtered using Watman filter paper. Dissociated hippocampal neurons were prepared from day old Wistar rats using a method described previously Briefly, pregnant female rats were anesthetized and killed by cervical dislocation and subjected to caesarean section to obtain fetal brains. Culture method was used with some modifications. The meninges were removed from cerebral hemispheres and the hippocampi were dissected, minced into small pieces, and digested with 0. Fetal bovine serum FBS was used to inactivate the trypsin. The 4, 5-dimethylthiazolyl -2, 5-diphenyl tetrazolium MTT assay was used to evaluate the reduction-oxidation status of living cells and mitochondrial activity, reflecting cell survival due to the formation of formazan The plates were read using an Anthos microplate reader at a wavelength of nm and a reference of nm. The assay was performed according to a previously described method 16 using the caspase-3 colorimetric assay kit. The amount of P-nitroanilide was continuously monitored over a minute period through the use of a plate reader. Absorbance was measured at nm, normalized to absorbance of control groups, and expressed as percent of control. Each experiment was run in triplicate. Viable neurons with cell membrane integration could pump PI out hence late apoptotic and necrotic cells not, here are presented as PI positive neurons. Hoechst staining 0. PI positive neurons were counted under a fluorescence microscope Nikon, Japan at an excitation wavelength of Hoechst and nm PI. Data were analyzed using SPSS software version One-way analysis of variance was used to determine overall significance. Assessment of neuronal purity was performed using an antibody specific to the neuronal marker MAP2, followed by nuclear counterstaining with Hoechst dye. Neuronal purity according to quantification of cells stained with Hoechst A and microtubule-associated proteinpositive neurons B. So this dose of MDMA was chosen for the rest of the experiments. Different concentrations of Melissa officinalis were added in order to find lethal concentration MDMA group. Neurons were also treated just with the same dose of Melissa as sham B. The cytotoxicity assay demonstrated that high concentrations of Melissa officinalis aqueous extract decreased neuronal viability. An in vitro cytotoxicity assay using the same method as ours also indicated that Melissa is toxic against a series of cancer cell lines Lemon balm has a high percentage of aldehydes which are used as anti-infectious agents Elevated amounts of these agents in high doses of Melissa could be the main cause of decline in neuronal viability, although another study is running in order to establish an exact dose response of this plant extract in the hippocampi of rats in vitro Unpublished. In the present study low concentrations of MDMA increased mitochondrial activity of hippocampal primary cultures, resembling cell viability, and conversely high doses of it lowered neuronal viability leading to apoptosis which is approved by caspase-3 activity assay and was discussed in our previous study 4. A previous study using the PC12 cell line which resembles some characteristics of neurons, reported increase in cell survival after pretreatment with both aqueous and methanolic extract of Melissa against H 2 O 2 toxicity Considering that oxidative stress is induced by high doses of MDMA in hippocampi 5 , we can assume that the neuroprotective effects of Melissa in the MDMA plus Melissa group arise from its free radical scavenging properties. However, we could not show this procedure by fractionation of Melissa extract or find the best fraction to be used for this protection. The protective effect of Melissa in neuronal viability was in agreement with the results of caspase-3 activity and PI staining assessments. Caspase-3 is a key element in many apoptotic pathways and the termination of this process has been confirmed? Another possible factor in the neuroprotective quality of Melissa could be its monoamine oxidase MAO inhibition 12 which may interfere with the monoamine transporter inhibition property of MDMA Melissa officinalis has revealed neuroprotective effects against apoptosis induced by MDMA in the primary neurons of hippocampal culture which could be due to its free radical scavenging properties and MAO inhibitory effects. These results propose the potential use of this plant for central nervous system disorders and as a neuroprotective agent to prevent neurodegenerative diseases, although more research has to be done in order to determine the exact fraction of Melissa and the molecular mechanisms involved in this neuroprotection. We thank Dr. There is no conflict of interest in this article. As a library, NLM provides access to scientific literature. Cell J. Neuroprotective Properties of Melissa Officinalis L. Find articles by Gholamreza Hassanzadeh. Parichehr Pasbakhsh , Ph. Find articles by Parichehr Pasbakhsh. Mohammad Akbari , Ph. Find articles by Mohammad Akbari. Saeed Shokri , Ph. Find articles by Saeed Shokri. Mohammadhosein Ghahremani , Ph. Find articles by Mohammadhosein Ghahremani. Gholamreza Amin , Ph. Find articles by Gholamreza Amin. Iraj Kashani , Ph. Find articles by Iraj Kashani. Abolfazl Azami Tameh , Ph. Find articles by Abolfazl Azami Tameh. Any use, distribution, reproduction or abstract of this publication in any medium, with the exception of commercial purposes, is permitted provided the original work is properly cited. Open in a new tab. Similar articles. Add to Collections. Create a new collection. Add to an existing collection. Choose a collection Unable to load your collection due to an error Please try again. Add Cancel.

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