Sulforaphane Attenuates Microglia-Mediated Neuronal Damage By Down-Regulating The ROS/Autophagy/NLRP3 Signal Axis In Aβ-Activated Microglia

BackgroundMicroglia are resident immune cells in the central nervous system.Previous studies have suggested that the phagocytosis of Aβ by microglia will lead to lysosome membrane permeability(LMP),and then activate NLRP3 inflammasome,leading to an inflammatory cascade reaction and microglia-mediated neuronal damage.Inhibition of NLRP3 inflammasome activation in AD transgenic mice can down-regulate the release of IL-1β and promote the transformation of microglia from M1 to M2 phenotype,thus improving spatial memory impairment and behavioral abnormalities.Sulforaphane(SFN)has strong antioxidant,anti-inflammatory and anticancer effects,and plays protective role in a variety of neurodegenerative diseases such as Alzheimer’s disease(AD).However,whether SFN can inhibit the activation of NLRP3 inflammasomes in Aβ-induced primary microglia and its potential mechanism remains unclear.ObjectiveThe purpose of this study was to investigate whether SFN could inhibit Aβ-induced lysosome membrane permeability and NLRP3 inflammasome activation in microglia,thereby regulating M1/M2 phenotype transformation and reducing microglia-mediated neuronal damage.The role of reactive oxygen species(ROS)and autophagy was further discussed.Methods and materials1.The effects of Aβ on the activity and toxicity of primary microglia were detected by CCK8 and LDH.2.The effects of Aβ or conditioned medium(CM)obtained from Aβ-activated microglia on the activity of neuron were detected by CCK8.3.The content of Aβ in CM was detected by dot blotting.4.qPCR and WB were used to detect the expression level of NLRP3,caspase-1 and IL-1β in Aβ-induced microglia and the hippocampus of AD mice.5.The expression of LC3 and P62 in Aβ-induced microglia and the hippocampus of AD mice were detected by WB.6.The lysosomal membrane permeability of microglia was observed by acridine orange staining.7.Flow cytometry was used to detect ROS,CD 16/32 and CD206 in Aβ-induced microglia.Results1.The results of CCK8 and LDH suggested that SFN reduced the microglia toxicity induced by Aβ.2.CCK8 results suggested that SFN inhibited the direct toxicity of Aβ to neurons and the indirect toxicity of Aβ mediated by microglia.3.The results of dot blotting,qPCR and flow cytometry indicated that SFN inhibited the indirect toxicity of Aβ to neurons,which was not related to the decrease of Aβ content in microglial CM,but was related to the inhibition of microglial inflammatory response and M1 transformation.4.The results of qPCR and WB indicated that SFN inhibited the activation of NLRP3 inflammasome in Aβ-activated microglia.5.The results of WB and acridine orange staining suggested that SFN inhibited the iniation of autophagic block and autophagic lysosomal membrane permeability in AP-activated microglia,just like 3-MA(autophagy initiation inhibitor).Autophagic block was upstream of NLRP3 inflammasome activation.6.Flow cytometry and WB results suggest that SFN,like NAC(ROS inhibitor),can inhibit autophagy/NLRP3 signal axis in Aβ-activated microglia by reducing intracellular ROS.ConclusionsSFN indirectly attenuated microglia-mediated neurotoxicity.The intrinsic mechanism behind this effect was that SFN inhibited the activation of cytostatic autophagy and NLRP3 inflammasome as well as the production of pro-inflammatory cytokines and M1 polarization.This mostly related to the decrease of intracellular ROS in Aβ-activated microglia.