Ellagic Acid Produces Neuroprotection Against LPS-induced Dopamine Neurotoxicity Via The Inhibition Of Microglial NLRP3 Inflammasome Activation

Objective: Investigate the effects and mechanisms of ellagic acid(EA)on lipopolysaccharide(LPS)-induced dopaminergic(DA)neuronal damage.Methods: 1.In vivo.SD rats(200–250 g)were randomly divided into five groups: sham,EA(50 mg/kg),LPS,LPS+EA(10 mg/kg)and LPS+EA(50 mg/kg).DA neuron injury model was induced by stereotaxic unilateral injection of LPS(10 μg)into substantia nigra.After seven daily intragastric administration of EA,rat behavior changes were analyzed by rotarod test.Animals were then subjected to biochemical analysis.Immunohistochemistry staining was performed to detect the expression of TH.Fluorescence double labeling was applied to observe the colocalization phenomenon of Iba-1 and NLRP3.Western blot assay was used to measure the protein expressions of NLRP3,caspase-1,pro-caspase-1,TNF-α,IL-1β and IL-18.2.In vitro.(1)To observe whether EA directly protects DA neurons,MN9 D cells were randomly divided into 5 groups: control,EA(1 μM),6-OHDA,6-OHDA+EA(0.1 μM),6-OHDA+EA(1 μM).Cells were pretreated with EA for 30 min and then treated with 6-OHDA for 24 h.MTT detect DA neuronal damage,immunocytochemistry and Western Blot detect TH expression.(2)Above experiment found that EA has no direct protective effect on DA neurons,and EA can inhibit microglia activation.We speculated EA-elicited neuroprotection may through regulating microglia.Based on this,BV-2 cells were randomly divided into 5 groups,control,EA(1 μM),LPS(100 ng/mL),LPS+EA(0.1 μM),LPS+EA(1 μM).After 30 min of EA pretreatment,LPS were added for 24 h.Immunocytochemistry detect microglia activation,Western Blot detect protein changes of Iba-1,NLRP3,pro-caspase-1 and caspase-1,ELISA detect the level of TNF-α,IL-1β and IL-18.(3).To determine the role of NLRP3 inflammasome in the antiinflammatory action of EA,siRNA NLRP3 transfection was used,BV-2 cells divided into 8 groups,control,EA(1 μM),LPS(100 ng/ml),LPS+EA(1 μM),NLRP3 siRNA,NLRP3 siRNA + EA(1 μM),NLRP3 siRNA + LPS(100 ng/ml),NLRP3 siRNA + LPS+EA(1 μM).Induction of BV-2 cells with LPS after 30 min pretreatment with ellagic acid,and then ransfer Microglia-conditioned Medium to MN9 D cells.The effect of NLRP3 on EA-mediated neuroprotection was observed by Western Blot.Results: 1.In vivo.Compared with the sham group,LPS reduced the time stad on rods in rats,EA(50 mg/kg)treatment significantly increased dwell time in rats and improved LPS-induced neuronal damage.The result of the pathological analysis also suggested that EA inhibited microglial and NLRP3 inflammasome activation.In addition,EA decreased LPS-induced protein expressions of TNF-α,IL-1β and IL-18.2.In vitro.1)In MN9 D cells,6-OHDA caused MN9 D cells damage compared with the control,after treatment with EA(1 μM)didn’t generate direct neuroprotection on DA neurons.2)In BV-2 cells,compared with the blank group,LPS induced BV-2 cell activation,Iba-1 and NLRP3 protein expression increased,and inflammatory factors(TNF-α,IL-1β,and IL-18)released increased.After administration of EA(1 μM),the expression of Iba-1 and NLRP3 inflammasome and the release of inflammatory cytokines were significantly reduced.3)After selective silencing of NLRP3 in microglia,regulation of EA on BV-2 cells disappeared.Moreover,after translocation of bv-2 supernatant to MN9 D cells,the supernatant of BV-2 cells treated with LPS caused the damage of MN9 D cells.After EA(1 μM)treatment,MN9 D cell TH protein expression increased.After selective silencing of NLRP3 in microglia,the anti-inflammatory and neuroprotective effects of EA disappeared.Conclusion: EA conferred neuroprotection against LPS-induced DA neuronal damage,which may be related on inhibition of microglial NLRP3 inflammasome signaling activation.