| BackgroundSulforaphane(SFN),a natural dietary isothiocyanate,has very strong anti-cancer,antioxidant,and anti-inflammatory activities.SFN is a highly promising chemopreventive agent against multiple diseases and that it can reduce microglia-mediated neuroinflammation or oxidative stress response.Although it has been known that SFN plays an important role in the Nrf2-mediated inflammation,SFN can activate Nuclear factor E2-related factor 2(Nrf2),which can counteract oxidative stress via upregulating the gene expression of antioxidant response element(ARE),then triggering the transcription of cytoprotective genes.Actually,what involed in microglia-mediated neuroinflammation about SFN’s role in other pathways is still unclear.As the resident immune cells of the central nervous system(CNS),microglia areserve as a bridge between the CNS and immune system.More and more evidence shows that inflammatory response has taken part in the pathogenesis of neurodegenerative diseases such as Alzheimer’s disease,Huntington’s disease,and Parkinson’s disease.Lipopolysaccharide(LPS)is an important component of the outer membrane of Gram-negative bacteria and cyanobacteria,it has been widely used to induce an immune response in microglia.When microglia is activated,it can include the overexpression of pro-inflammatory mediators such as tumor necrosis factor-alpha(TNF-a),interleukin-6(IL-6),interleukin-1 beta(IL-1β),inducible NO synthase(iNOS).MAPK and Nuclear transcription factor-kappa B(NF-kB)signaling pathway is a significant component of LPS-induced pro-inflammatory responses.Many neurological diseases are closely related to inflammation.When comparing the different effects of pro-inflammatory mediators,TNF-a acts as a candidate in the induction of neuronal damage.TNF-a is a pleiotropic cytokine that can initiate apoptosis via binding to TNF receptor(TNFR)1 or 2,or necroptosis.Plenty of evidence shows that that necroptosis is induced by receptor interacting protein 3(RIP3)dependent on RIP1，or independent of RIP1 under certain circumstances,leading to the recruitment and phosphorylation of mixed lineage kinase domain like(MLKL).Therefore,RIPK3 and MLKL are the key hallmarks of necroptosis in the lack of a validated definitive molecular marker.ObjectiveThis study is to investigate the effects of sulforaphane in LPS-induced pro-inflammatory responses of BV-2 microglia,thus reducing the expression of pro-inflammatory mediators including TNF-a,IL-lβ,IL-6,iNOS and suppressing the release of TNF-a,IL-6 in culture medium,and to explore the underlying mechanisms of SFN in microglia-mediated neuronal damage associated with MAPK/NF-kB signaling pathways.Methods and materials1.CCK8 viability assay detected the viability of BV-2 microglia and primary cortical neurons.2.mRNA expression of TNF-a,IL-1β,IL-6 and iNOS were detected by qPCR.3.ELISA detected the amounts of TNF-a,IL-1β,IL-6 and iNOS in culture mediumofBV-2microglia.4.Immunofluorescence was used to observe the morphological changes of neurons.5.TUNEL detected the apoptosis of primary cortical neurons.6.Western blot analysis detected the protein expression of TNF-α IL-1β,IL-6,iNOS in BV-2 microglia;Western blot analysis detected the protein expression and phosphorylated levels of p38,JNK,ERK1/2,p65 in BV-2 microglia;Western blot analysis detected the protein expression of caspase-3,Bax,RIPK3,MLKL in primary cortical neurons.Results1.CCK-8 assay results showed SFN has a negligible effect on BV-2 viability at a range of concentrations up to 15 μM.SFN pre-treatment inhibited the activation of BV-2 microglia in a concentration-dependent manner.2.The Western blot analysis and mRNA expression of TNF-a,IL-1β,IL-6 and iNOS were obviously decreased in the groups pre-treated with SFN compared to the LPS-treated group in a concentration-dependent manner.A similar result was only observed in the amounts of TNF-a,IL-6 in the culture medium.SFN suppressed LPS-stimulated production of pro-inflammatory mediators in BV-2 microglia and the release of TNF-a,IL-6 in culture medium.3.Western blot analysis results showed that LPS-induced phosphorylation of p3 8,JNK,ERK1/2 and NF-kB p65 was obviously reduced in BV-2 microglia after pretreatment with SFN.The LPS-induced phosphorylation of MAPK(p38,JNK and ERK1/2)and NF-kB p65 was markedly inhibited by their inhibitors SB203580,U0126,SP600125 and PDTC,respectively.SFN inhibited MAPK and p65 activation in LPS-activated BV-2 microglia.4.Western blot analysis results showed that the protein expression of TNF-α,IL-6,IL-1β and iNOS was markedly inhibited by SB203580,SP600125,and PDTC respectively in the presence or absence of SFN compared with LPS treatment.U0126 without SFN obviously inhibited the protein expression of IL-1β and iNOS,but not TNF-a or IL-6 in LPS-activated BV-2 microglia.And,the protein expression of TNF-a and IL-6 was still markedly inhibited in the groups treated with SFN alone or with U0126.5.Western blot analysis results showed that the phosphorylation level of NF-kB p65 was significantly decreased after pretreatment with each of the three MAPK inhibitors,suggesting that the MAPK signaling pathway is the upstream of NF-kB p65.6.CCK-8 assay results showed that SFN was not cytotoxic to neurons up to 5μM.SFN did not directly attenuate the impact of microglia-mediated insult on neuron viability.7.CCK-8 assay results showed that SFN attenuated microglia-mediated neurotoxicity indirectly(through an effect on the microglia)rather than directly(through an effect on the neurons).Immunofluorescence showed that the number of MAP2-positive neurons decreased strongly in the LPS-CM treated neurons,suggesting that neuronal synapses were damaged compared with Control-CM treated group;In SFN+LPS-CM-treated neurons,however,the synaptic morphology of neurons was maintained,accompaning notable increasing in the number of neurons compared to LPS-CM treated group.SFN indirectly attenuated the cell viability and the morphology of neurons in microglia-mediated neural damage.8.Western blot analysis results showed that the protein expression of the apoptotic proteins caspase-3 and Bax were unchanged,SFN+LPS-CM had significantly lower expression of RIPK3 and MLKL.TUNEL assay showed that the neurons in SFN+LPS-CM group displayed a negligible decrease in the percentage of apoptosis compared to LPS-CM group,and there was no obvious apoptosis in LPS-CM group neurons compared to Control-CM group.SFN indirectly suppressed microglia-mediated neuronal necroptosis.9.Western blot analysis results showed that the protein expression of RIPK3 and MLKL but not caspase-3 and Bax was suppressed by inhibitors in the presence or absence of SFN compared with LPS-CM treatment.10.Western blot analysis results showed that the protein expression of RIPK3 and MLKL was markedly inhibited by SB203580,SP600125,and PDTC respectively in the presence or absence of SFN compared with LPS-CM treatment group.In contrast,there was no obvious effect of U0126 alone on the expression of RIPK3 and MLKL,but the expression of RIPK3 and MLKL was strongly inhibited by SFN alone or SFN+ U0126.SFN inhibited microglia-mediated neuronal necroptosis through p38,JNK,and NF-kB p65 but not ERK1/2 signaling pathways.Conclusions1.SFN inhibited LPS-stimulated activation of BV-2 microglia.2.SFN suppressed LPS-induced expression of pro-inflammatory mediators in BV-2 microglia and the release of TNF-a,IL-6 in culture medium BV-2 microglia.3.SFN inhibited MAPK and p65 activation in LPS-induced BV-2 microglia.4.MAPK and NF-kB inhibitors suppressed the expression of pro-inflammatory mediators,the MAPK signaling pathway is the upstream of NF-kB p65.5.SFN did not attenuate the impact of microglia-mediated damage on neuron viability directly.6.SFN attenuated the cell viability and the morphology of neurons in microglia-mediated neural damage indirectly.7.SFN indirectly attenuated microglia-mediated neuronal necroptosis.8.MAPK and NF-kB inhibitors suppressed microglia-mediated neuronal necroptosis but not apoptosis.9.SFN suppressed microglia-mediated neuronal necroptosis through p38,JNK,and NF-kB p65 but not ERK1/2 signaling pathways. |
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