Protective Action Of Reduced Glutathione Against Toxicity Of 6-hydroxy-dopamine On Bone Marrow Stromal Cells

Idiopathic Parkinson`s disease (PD) is a normal neurodegenerative disorder of the midaged which is characterized by progressive degeneration of the dopaminergic (DA)neurons of the nigrostriatal pathway and formation of lewy body.Although levodopa is still considered as the gold standard of antiparkinsonism drug therapy,chronic levodopa treatment is associated with the development of adverse events in the majority of patient,such as motor fluctuations,dyskinesias,and neuropschiatric problems.Cell transplantation is one of the hottest field in PD treatment. BMSCs can differentiate into neural stem cells,neurons,glial cells, and secrete several neurotrophic factor,such as brain-derived neurotrophic factor(BDNF),glial cell line-derived neurotrophic factor(GDNF).It supply a new approach to solve the source of donator for cell transplantation.6hydroxydopamine(6-OHDA) which has a similar structure to dopamine (DA) and is often intaken by dopaminergic neurons by mistake as dopamine transmitter,can produce hydroxy radical,induce cell oxidative stress reaction and inhibit mitochondrial oxidation-respiration chain complexⅠandⅣetc, to selectively destroy dopaminergic neurons andcause dopaminergic neurons death.Reducedglutathione (GSH) is a physio-synthesis peptide in human cytoplasm,which is composed of glutamate,cysteine,glycine,include active sulfhydryl, widespread distribute in all organs of body and play an important role in maintaining cell biological functions. As the most important anti-oxidant in the cerebral,GSH has many functions including free radical scavenging ,mitochondria protection etc. N-acetyl-l-cysteine(NAC) is also a clinical commonly used anti-oxidant,which has active sulfhydryl ,is precursor of GSH,can convert to GSH ,and also has many functions including free radical scavenging, mitochondria protection etc. As a commonly used drug in animal model of PD, 6-OHDA whether has toxicity on bone marrow stromal cells which are not dopaminergic cells. Anti-oxidant GSH or its precursor NAC whether can protect BMSCs against toxicity of 6-OHDA. Our research focus on these questions.Part One: Protective action of reduced glutathione against toxicity of 6-hydroxy-dopamine on bone marrow stromal cellsAim: To investigate the toxicity of 6-hydroxy-dopamine on bone marrow stromal cells and protective action of reduced glutathione against it.Methods: Bone marrow stromal cells were separated from bone marrow of femur,shin and humerus of SD Rats weight between 60 and 90 gram,and were cultured to the third generation. Toxicity of 6-hydroxy-dopamine on them was determined by MTT assay, at the same time protection of reduced glutathione on them was determined under the same conditions. In MTT assay,BMSCs were divided into four groups: 1) PBS control group,add equivalent PBS; 2) GSH group(terminal concentrations : 0.5,1,1.5,2.25,3,6,12 mg/ml) ; 3) 6-OHDA group(terminal concentrations:0.05,0.1 mg/ml);4) GSH +6-OHDA group,6-OHDA(0.05 mg/ml)+ GSH(0.5,1 mg/ml), 6-OHDA(0.1 mg/ml)+ GSH(0.5,1 mg/ml).Results: By MTT assay , reduced glutathione induced a increase in bone marrow stromal cells viability (p<0.05).6-hydroxy-dopamine induced a decrease in cell viability of bone marrow stromal cells (p<0.05). Reduced glutathione induced a increase in cell viability of bone marrow stromal cells treated with 6-hydroxy-dopamine (p<0.05).Conclusions: 6-hydroxy-dopamine of certain concentration have toxicity on bone marrow stromal cells. Reduced glutathione can protect bone marrow stromal cells against toxicity of 6-hydroxy-dopamine on them. Part Two: Action of n-acetyl-l-cysteine and oxidized glutathione on bone marrow stromal cellsAim: To investigate the action of n-acetyl-l-cysteine(NAC) and oxidized glutathione (GSSG) on bone marrow stromal cells.Methods: Bone marrow stromal cells were separated from bone marrow of femur,shin and humerus of SD Rats weight between 60 and 90 gram,and were cultured to the third generation. Action of n-acetyl-l-cysteine and oxidized glutathione on them were determined by MTT assay. In MTT assay,BMSCs were divided into three groups: 1) PBS control group,add equivalent PBS; 2)NAC group(terminal concentrations: 0.0375,0.075,0.15,0.3,0.6,1.2,2.4,4.8,9.6 mg/ml);3)GSSG group(terminal concentrations : 0.05,0.1,0.25,0.5,1,1.5,3,6,12mg/ml).Results: By MTT assay , n-acetyl-l-cysteine induced a increase in bone marrow stromal cells viability (p<0.05). When GSSG is 0.05mg/ml , ratio of cell viability of GSSG group is higher than PBS control group (p<0.05).As GSSG concentration increases, cell viability ratio decreases gradually .When GSSG≥1.5mg/ml,ratio of cell viability is lower than PBS control group (p<0.05).Conclusions: At certain concentration,n-acetyl-l-cysteine can increase cell viability of bone marrow stromal cells . At lower concentration, oxidized glutathione can increase cell viability of bone marrow stromal cells . At higher concentration, oxidized glutathione can decrease cell viability of bone marrow stromal cells .Overall Conclusions:1. 6-hydroxy-dopamine of certain concentration have toxicity on bone marrow stromal cells.2.Reduced glutathione can protect bone marrow stromal cells against toxicity of 6-hydroxy-dopamine on them.3. At certain concentration,n-acetyl-l-cysteine can increase cell viability of bone marrow stromal cells .4.At lower concentration, oxidized glutathione can increase cell viability of bone marrow stromal cells . At higher concentration, oxidized glutathione can decrease cell viability of bone marrow stromal cells .