| Objective To explore the Intervention effects of sodium para-aminosalicylate(PAS-Na) on concentrations of glutamate (Glu), glutamine (Gln), glycine(Gly) and γ-aminobutyric acid (GABA) et al amino acid neurotransmitters inbasal ganglia primary neurons and astrocyte of rats induced by manganese(Mn).Methods Neurons: Neurons in basal ganglia of rats cultured for8dayswere randomly exposed to:(1)0,25,50,100,200μmol/L manganese chloride(MnCl2)for24h,(2)0,5,50,500,5000μmol/L PAS-Na for24h; morphologyof basal ganglia neurons was observed, and the effect of MnCl2or PAS-Na onthe survival of basal ganglia neurons was determined by3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) analysis.(3) Basal ganglia neurons were randomly divided into divided into8groups: thecontrol group, Mn-exposed group, low, medium and high doses PAS-Na (L-PAS,M-PAS and H-PAS) intervention and control groups. Neurons of the control,L-PAS, M-PAS and H-PAS control groups were cultured only with incubatemedium for24h. Neurons of Mn-exposed, L-PAS, M-PAS and H-PAS intervention groups were exposed to culture medium plus Mn (MnCl250μmol/L) for the first24h. And then neurons of L-PAS, M-PAS, H-PAS interventionand control groups were cultured with mediums plus PAS-Na (50,150and450μmol/L) for another24h. Other groups cultured only with incubate mediumfor24h. Cell viability, lactate dehydrogenase (LDH), glutathione peroxidase(GSH-Px) and catalase (CAT) activit y kit, DNA damage and concentrations ofamino acid neurotransmitters of intracellular and extracellular neurons, such asGABA, Glu, Gln and Gly et al, were determined by MTT, kit, single cell geleletrophoresis(SCGE) and high performance liquid chromatography (HPLC).Astrocyte: After purified and identification, astrocyte in basal ganglia of ratswere randomly exposed to:(1)0,125,250,500,1000μmol/L MnCl2for24h,(2)0,5,50,500,5000μmol/L PAS-Na for24h; morphology of basal ganglia astrocytewas observed, and the effect of MnCl2or PAS-Na on the survival of basalganglia astrocyte was determined by MTT analysis.(3) Basal ganglia astrocytewere randomly divided into divided into8groups: the control group,Mn-exposed group, low, medium and high doses PAS-Na (L-PAS, M-PAS andH-PAS) intervention and control groups. Astrocyte of the control, L-PAS, M-PAS and H-PAS control groups were cultured only with incubate medium for24h. Astrocyte of Mn-exposed, L-PAS, M-PAS and H-PAS intervention groupswere exposed to culture medium plus Mn (MnCl2500μmol/L) for the first24h.And then astrocyte of L-PAS, M-PAS, H-PAS intervention and control groupswere cultured with mediums plus PAS-Na (50,150and450μmol/L) for another24h. Other groups cultured only with incubate medium for24h. Cell viability,DNA damage and concentrations of amino acid neurotransmitters ofintracellular and extracellular neurons, such Glu, Gln, Gly and GABA, et al, were determined by MTT, kit, SCGE and HPLC.Results Neurons:①Mn induced the morphology injury andconcentration-dependently descreased of cell viavility of neurons in the basalganglia;②A high concentrations (5000μmol/L) of PAS-Na may inducedmorphology injury and decreased the cell viavility of neurons in basalganglia;③The neurons of Mn-exposed group cell bodies swelling, shrinkage,projection decreased; Compared with the Mn-exposed group, L-, M-, H-PASintervention group reduced the above morphology injury. Cell viability ofMn-exposed group was lower than that of the control group; the cell viability ofL-PAS, M-PAS and H-PAS intervention groups were higher than that ofMn-exposed group (P＜0.05). The leakage rate of LDH in the Mn-exposedgroup was higher than the control group; The leakage rate of LDH of L-, M-PASintervention groups were lower than the Mn-exposed group (P＜0.05). CAT andGSH-Px activity of Mn-exposed group were higher than those in the controlgroup; CAT activity of M-, H-PAS group and GSH-Px activity of M-PAS higherthan those of the Mn-exposed group (P <0.05). Tail DNA ratio and Olive tailmoment of Mn-exposed group were higher than those in the control group;comet tail DNA ratio and Olive tail moment of L-, M-, H-PAS interventiongroups lower than Mn-exposed group (P<0.05). In the intracellular,concentrations of Glu and Gln contents of Mn-exposed group were lower thanthose of control group, GABA and Gly contents of Mn-exposed group werehigher than those of control group; Glu and Gly contents of L-, M-, H-PASintervention groups and GABA content of H-PAS intervention group were lowerthan those of Mn-exposed group (P<0.05). In the extracellular, Glu and Glncontents of Mn-exposed group were lower than those of control group and the Gly content of Mn-exposed group was higher than that of control group; Glycontent of L-, M-, H-PAS intervention groups and Glu content of M-PASintervention group were lower than those of Mn-exposed group (P<0.05).Astrocytes:①T he concentration above250μmol/L Mn induced the morphologyinjury and concentration-dependently decreased of cell viavility of astrocyte inthe basal ganglia;②The morphology and cell viavility of astrocyte in PAS-Na–exposed groups were similar to those of the control group;③The astrocyteofMn-exposed group cell bodies swelling and shrinkage; Compared with theMn-exposed group, L-, M-, H-PAS intervention groups reduced the abovemorphology injury. Cell viability of Mn-exposed group were lower than that ofthe control group; the cell viability of M-PAS and H-PAS intervention groupswere higher than that of Mn-exposed group (P＜0.05). Tail DNA ratio and Olivetail moment of Mn-exposed group were higher than those in the control group;comet tail DNA ratio of L-, M-, H-PAS intervention groups and Olive tailmoment of H-PAS intervention group were lower than Mn-exposed group(P<0.05). In the intracellular, concentrations of Gln contents of Mn-exposedgroup were lower than those of control group, Glu and Gly contents ofMn-exposed group were higher than those of control group; Glu and Glycontents of L-, M-, and H-PAS groups were lower than those of Mn-exposedgroup; Gln contents of H-PAS intervention group were higher than that of Mn-exposed group (P<0.05). In the extracellular, Gln contents of Mn-exposedgroup was lower than that of the control group and the Glu, and Gly contents ofMn-exposed group were higher than those of the control group; Gly and Glucontents of L-, M-, H-PAS intervention groups were lower than those ofMn-exposed group; Gln contents of L-、M-、H-PAS intervention groups were higher than that of Mn-exposed group (P <0.05).Conclusion In vitro, Mn has damage effects on neurons and astrocyte inbasal ganglia of rats and caused DNA damage and abnormal changes in aminoacid neurotransmitters levels; PAS-Na has intervention effects on these DNAdamage and changes of amino acid neurotransmitters induced by Mn. |
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