Aspirin Protects Dopaminergic Neurons Against Lipopolysaccharide-induced Neurotoxicity In Primary Midbrain Cultures

Part IAspirin protects rat primary dopaminergic neurons induced by lipopolysaccharidesObjective:To investigate the effects of aspirin (ASA) on the protection of rat primary dopaminergic neurons induced by lipopolysaccharides (LPS). and to explore the possible underlying mechanisms.Methods:We co-cultured the primary neuron-glia cells. The co-cultured cells were divided into 5 groups:control group, LPS group (lOng/ml). LPS+ASA (0.01.0.1. 1mmol/L) group. The positive cells of tyrosine hydroxylase (TH) and the TH protein expression were detected by immunohistochemistry and western-blot. Further, we developed different primary cell cultures:neuron-enriched cultures, neuron-astrocyte cultures and neuron-enriched cultures supplemented by 10% microglia. They were randomly divided into 4 groups:the control group, ASA (lmmol/L) group, ASA (1mmol/L)+LPS (1Ong/ml) gruoup, LPS (lOng/ml) group. The number of the survival TH positive cells was evaluated by immunocytochemistry.Results:The number of TH positive neurons in the group of LPS was less than that in control group; the adding of different dose of ASA significantly reversed these changes caused by LPS. Morphological observation showed relatively short neuronal dendrites, and 1mmol/L ASA successfully reversed the change. Western-blot showed that the protein of TH in ASA+LPS group was more than that in LPS group (p<0.05). In the three different cell component cultures, we counting the number of TH positive neurons after immunostanning. it showed that LPS did not affect the DA neuron survival in neuron-enriched cultures; in the neuron-microglia cultures, The number of TH positive neurons in the group of LPS was less than that in control group, and lmmol/L ASA successfully reversed the change.Conclusion:ASA can protect embryonic rat mesencephalic DA-ergic neurons against LPS induced neurotoxicity in a dose-dependent manner, and this effect is likely to mediate through microglia activation.Part IIEffect of ASA on LPS-induced production and release of reactive oxygen species and proinflammatory and anti-inflammatory factorsObjective:To investigate the regulation of ASA on the production of inflammatory factors and reactive oxygen species (ROS) in primary neuron-glia co-cultures, and to explore the mechanisms underlying the neuroprotective role.Methods:Primary cultured cells were dissociated from embryonic rat mesencephalon and the cell cultures were treated by addition of ASA (0.01-lmmol/L) and LPS (10ng/ml). The production of superoxide was determined by measuring the SOD-inhibitable reduction of WST-1, intracellular ROS were determined by using a DCFH-DA assay. The production of nitric oxide (NO) was assessed as the accumulation of nitrite in the supernatants using Griess reagent; TNF-a. IL-10 and TGF-β1 concentrations in cell supernatants were measured using a commercial ELISA kit.Results:The production of ROS, NO, TNF-a significantly increased after stimulated by LPS, pretreatment with 0.01-1 mmol/L ASA significantly reversed these changes caused by LPS (p<0.05). The amounts of IL-10 and TGF-β1 were prominently increased after addition of ASA.Concludion:ASA acted on microglia principally by down-regulating the production of ROS and pro-inflammatory factors and up-regulating the production of anti-inflammatory cytokines to exert its neuroprotective role.Part IIINADPH oxidase plays an important role in ASA-mediated protection against LPS-induced neurodegenerationObjective:To investigate the effects of ASA on the activity of nicotinamide adenine dinucleotide phosphate oxidase (NADPH oxidase, PHOX) in the LPS induced neurotoxicity.Methods:We developed the embryonic rat mesencephalic neuron-glia culture, and the cultured cells were randomized into groups of control, LPS (10ng/ml), ASA(1mmol/L)+LPS(10ng/ml), apocynin(APO,0.25mmol/L)+LPS(10ng/ml). The survival number of TH positive cells was examined by immunocytochemistry. The western-blot was used to detect the levels of P47^phox on the cytomembrane.Results:The numbers of TH positive cells in ASA+LPS group and APO+LPS group were more than that in LPS group (p<0.05). Western-blot showed that the cytomembrane protein of P47^phox in ASA+LPS group and APO+LPS group were lower than that in LPS group.Conclusion:Pretreatment with ASA could protect rat primary DA neurons induced by LPS. The effect may be contributed to ASA-blocked activity of PHOX induced by LPS.