Nimbolide针对多种信号通路减少BV2小胶质细胞的神经炎症

智能文字提取功能测试中

Nimbolide是楝树植物中的一种柠檬类化合物，研究了它对用LPS激活的BV2小胶质细胞的神经炎症的影响。用LPS（100 ng/ml）刺激培养的BV2细胞，然后用Nimbolide（125、250和500 nM）处理。结果显示，Nimbolide特使LPS激活的BV2细胞中的TNFα、IL-6、IFNγ、NO/iNOS、PGE 2 /COX-2水平明显下降。进一步的实验显示，LPS诱导的磷脂酰-p65和磷脂酰-IκBα蛋白的表达增加，在Nimbolide的存在下减少。此外，Nimbolide减少了LPS诱导的NF-κB乙酰化、与共识位点结合的增加和转导，以及p38和JNK MAPKs的磷酸化。伴随着Nimbolide减少细胞ROS的产生，gp91phox蛋白水平也有所下降，同时通过提高HO-1和NQO-1的蛋白水平观察到抗氧化作用。据观察，用Nimbolide处理BV2小胶质细胞导致细胞质中的Nrf2水平下降，同时细胞核中的Nrf2水平上升。此外，用这种化合物处理导致Nrf2与ARE共识位点的结合增加，并伴随着ARE荧光素酶活性的增强。基因敲除实验显示，在转染了Nrf2 siRNA的细胞中，Nimbolide失去了抗炎活性。用Nimbolide处理导致SIRT-1的核积累，而SIRT-1的siRNA敲除导致Nimbolide的抗炎活性逆转。有人提出，Nimbolide通过导致NF-κB和MAPK途径的双重抑制的机制来减少BV2小胶质细胞的神经炎症。还提出，激活Nrf2抗氧化剂以及SIRT-1去乙酰化机制可能有助于其抗炎活性Preprints are preliminary reports that have not undergone peer review.Research Squareor referenced by the media as validated information. marked luciferase activity as shown in Fig. 6F. On pre-treating cells with nimbolide (125， 250 and 500nM)， a concentration-dependent inhibition of NF-KB luciferase activity was observed (Fig.6F). They should not be considered conclusive， used to inform clinical practice， Nimbolide targets multiple signalling pathways toreduce neuroinflammation in BV2 microglia Folashade O Katola University of Huddersfield School of Applied Sciences Olumayokun A Olajide (o.a.olajide@hud.ac.uk) University of Huddersfield https：//orcid.org/0000-0002-9254-8334 Research Article Keywords： Nimbolide， Neuroinflammation， NF-KB， MAPK， Nrf2， SIRT-1 Posted Date： November 1st， 2022 DOI： https：//doi.org/10.21203/rs.3.rs-2202062/v1 License： ce ① This work is licensed under a Creative Commons Attribution 4.0 International License.Read Full License Abstract Nimbolide， a limonoid compound found in the neem plant， was investigated for effects onneuroinflammation in BV2 microglia activated with LPS. Cultured BV2 cells were stimulated with LPS(100 ng/ml) followed by treatment with nimbolide (125， 250 and 500 nM). Results showed that nimbolidecaused significant reduction in the levels of TNFa， IL-6， IFNy， NO/iNOS，PGE2/COX-2 in LPS-activated BV2cells. Further experiments revealed that LPS-induced increased expression of phospho-p65 and phospho-lkBa proteins were reduced in the presence of nimbolide. Also，LPS-induced NF-KB acetylation， increasedbinding to consensus sites， and transactivation， as well as phosphorylation of p38 and JNK MAPKs werereduced by nimbolide. Reduction of cellular ROS generation by nimbolide was accompanied by reductionin gp91phox protein levels， while antioxidant effects were observed through elevation in protein levels ofHO-1 and NQ0-1. It was observed that treatment of BV2 microglia with nimbolide resulted in reducedlevels of cytoplasmic Nrf2 which was accompanied by increased levels in the nucleus. Furthermore，treatment with this compound resulted in increased binding of Nrf2 to ARE consensus sites accompaniedby enhanced ARE luciferase activity. Knockdown experiments revealed a loss of anti-inflammatoryactivity by nimbolide in cells transfected with Nrf2 siRNA. Treatment with nimbolide resulted in nuclearaccumulation of SIRT-1， while siRNA knockdown of SIRT-1 resulted in reversal of anti-inflammatoryactivity of nimbolide. It is proposed that nimbolide reduces neuroinflammation in BV2 microglia throughmechanisms resulting in dual inhibition of NF-KB and MAPK pathways. It is also proposed that activationof Nrf2 antioxidant as well as SIRT-1 deacetylation mechanisms may be contributing to its anti-inflammatory activity. Introduction Accumulating evidence continues to implicate microgliamediated neuroinflammation in the pathogenesisof neurodegenerative disorders， such as Alzheimer's disease (AD)， Parkinson's disease (PD)，muscularamyotrophic lateral sclerosis (ALS， and multiple sclerosis (MS) [1]. Microglia-mediatedneuroinflammation has also been linked to the neurological dysfunctions in parasitic diseases such ascerebral malaria [2]， African trypanosomiasis [3]， viral diseases such as viral encephalitis [4]， HIV [5]，aswell as early stages of neuropathic pain [68]. Consequently， inhibition of neuroinflammation remains aviable strategy in the search for novel neuroprotective drugs. Multiple cellular signalling processes have been linked to microglia-mediated neuroinflammation；evidence suggests that targeting critical steps involved in these signalling pathways is an importantstrategy for discovery of novel compounds for treating neurodegenerative and other CNS conditions. The NF-KB transcription factor remains one of the most investigated drug targets for suppressingmicroglia-mediated neuroinflammation. NF-KB is principally responsible for the regulation of genesassociated with critical processes involved in inflammatory responses. Considerable body of evidencesuggests that NF-KB activation in the CNS results in multi-cellular processes and gene transactivationwhich are implicated in the initiation and progression of neurodegenerative diseases [9]. Following microglia activation， NF-KB is translocated to the nucleus to induce transactivation of pro-inflammatorygenes and neurotoxic factors which promote neurodegeneration [9]. This has led to several studies toidentify novel inhibitors of NF-KB for suppressing neuroinflammation and neurodegeneration [10-12]. The mitogen-activated protein kinases (MAPKs) include c-Jun NH2-terminal kinase (JNK)， p38 MAPK，andextracellular signal-regulated kinase (ERK). Of the MAPKs， the p38 MAPK is known to contribute tomicroglia-mediated neuroinflammation [13-15]. In fact， studies have suggested that the deficiency ofMAPK-activated Protein Kinase 2 (an upstream kinase of p38) resulted in inhibition of pro-inflammatorymediator release and neurotoxicity [16]. Consequently， targeting microglia MAPKs activation is also avaluable pharmacological strategy for inhibiting neuroinflammation. Nimbolide (Fig. 1) is a tetranortriterpenoid compound found in Azadiractha indica (neem) plant. Thiscompound has been widely investigated as an anticancer natural product [17-20]. Nimbolide has alsobeen reported to produce antiplasmodial activity against Plasmodium falciparum， in vitro [21]. Studiesreported by Diddi et al. showed that nimbolide produced anti-inflammatory and anti-fibrotic effects inbleomycin-induced scleroderma [22]. Further evidence on the anti-inflammatory activity of this compoundwas provided in a study that showed its effects in reducing inflammatory mediator release inlipopolysaccharide (LPS)-stimulated RAW264.7 macrophages， and in dextran sulfate sodium (DSS)-induced acute colitis [23]. However， it is not clear if nimbolide could affect microglia-mediatedneuroinflammation. In this study we investigated effects of nimbolide on LPS-induced neuroinflammation in BV2 microglia，and explored the potential molecular mechanisms involved in its anti-inflammatory activity. Materials And MethodsDrugs and chemicals Nimbolide was purchased from Sigma， dissolved in dimethylsulfoxide (DMSO) to a concentration of0.05M and aliquots stored at -20 C. Lipopolysaccharide (LPS) derived from Salmonella enterica serotypetyphimurium was purchased from Caltag Medsystems (UK). Cell Culture BV2 mouse microglia cell line (ICLCATL03001) was purchased from Interlab Cell Line Collection (BancaBiologica e Cell Factory， Italy) and cultured in RPMI medium supplemented with 10% foetal bovine serum. Determination of BV2 microglia viability Cultured BV2 cells were incubated with or without LPS (100 ng/ml) in the presence of nimbolide (125，250 and 500 nM) for 24 h. Thereafter， XTT/PMS solution (25 ul) was added to the cells in 100 ul of culture medium and incubated for a further 2 h. Cell viability was determined by measuring absorbanceat 450nm. In separate experiments， LDH release by cells was determined by transferring 50 pl of cell supernatantsinto a 96-well plate， followed by the addition of 50 pl of CytoTox 96@ reagent (Promega) followed byincubation in the dark at room temperature for 30 min. Production of pro-and anti-inflammatory cytokines， NO andPGE Cultured BV2 microglia were treated with nimbolide (125， 250 and 500 nM)， followed by stimulation withLPS (100 ng/ml) for 24 h. Levels of pro-inflammatory (TNFa， IL-6， and IFNy) and anti-inflammatory (IL-10) cytokines in culture supernatants were measured using mouse ELISA kits (Biolegend)， according tothe manufacturer's instructions. Release of nitrite (as a measure of NO production) in culturesupernatants was determined using the Griess assay kit (Promega， UK)， according to the manufacturer'sinstructions. PGE2 levels were determined with a PGE2 enzyme immunoassay (EIA) kit (Arbor Assays，USA)， according to the manufacturer's instructions. Cellular generation of reactive oxygen species (ROS) Generation of intracellular ROS in BV2 microglia was evaluated using 2'，7'-dichlorofluorescin diacetate(DCFDA)-cellular reactive oxygen species detection assay kit (Abcam). BV2 microglia in 96-well plateswere washed and stained with 20 uM DCFDA for 45 min at 37C. Cells were then washed and pre-treatedwith nimbolide (125，250 and 500 nM) for 30 min， followed by stimulation with LPS (100 ng/ml) for 24 h.DCFDA fluorescence was measured in a POLARstar Optima microplate reader (BMG Labtech). Live imaging of ROS generation in BV2 was also carried out using Image-iT live Green Reactive OxygenSpecies Detection Kit (ThermoFisher). Cultured BV2 cells were washed with Hanks' Balanced SaltSolution (HBSS)， and stained with DCFDA for 45 min at 37℃. This was followed by treatment withnimbolide (125，250 and 500 nM) for 30 min， and stimulation with LPS (100 ng/ml) for a further 24 h.Fluorescence images were captured using EVOS FLoid cell imaging station. Immunoblotting Equal amounts of protein (20-30pg) from cell lysates or nuclear extracts were subjected to SDS-PAGEunder reducing conditions. This was followed by protein transfer to polyvinylidene fluoride (PVDF)membrane (Millipore)， which was then blocked for 1 h at room temperature， washed with Tris-bufferedsaline+0.1% Tween 20 (TBS-T) and incubated overnight at 4℃ with primary antibodies. Primaryantibodies used were rabbit anti-iNOS (Cell signalling， 1：1000)， rabbit anti-COX-2 (Cell signalling， 1：1000)，rabbit anti-phospho-p65 (Cell signalling， 1：1000)， rabbit anti-lkBa (Cell signalling， 1：1000)， rabbit anti-phospho-lkBa (Abcam， 1：5000)， rabbit anti-phospho-JNK (Cell signalling， 1：1000)， rabbit anti-total-JNK(Cell signalling， 1：1000)， rabbit antiphospho-p38a (Cell signalling， 1：1000)， rabbit anti-total-p38 (SantaCruz， 1：500)， rabbit anti-gp91phox (Abcam，1：5000)， rabbit anti-Keap1 (Santa Cruz，1：500)， mouse anti- Nrf2 (Santa Cruz， 1：500)， rabbit anti-H01 (Santa Cruz， 1：500)， rabbit anti-NQ01 (Santa Cruz， 1：500)， rabbitanti-acetyl-p65 (Cell Signalling， 1：1000)， rabbit anti-SIRT1 (Santa Cruz， 1：500)， rabbit anti-actin (Sigma，1：1000)， rabbit antilamin B1 (Santa Cruz，1：500). Then， membrane was washed with TBS-T followed byincubation with goat anti-rabbit (Alexa Fluor 680) secondary antibody (1：10000； Thermo Fisher， UK) atroom temperature for 1 h. Blots were detected using LI-COR Odyssey Imager. All western blot experimentswere carried out at least three times. Transcription factor assays An ELISA-based DNA binding assay was used to determine the effects of nimbolide on DNA binding ofNF-KB and Nrf2. Nuclear extracts were obtained from BV2 microglia treated with nimbolide (125， 250 and500 nM) for 24 h. These extracts were then used for DNA binding assays using TransAM Nrf2transcription factor ELISA Kit (Activ Motif， Belgium)， containing a 96-well plate to which anoligonucleotide containing the antioxidant responsive element (ARE) consensus binding site (5'-GTCACAGTGACTCAGCAGAATCTG-3) were immobilised. Similarly， nuclear extracts were obtained from BV2 microglia which were treated with nimbolide (125，250and 500 nM) prior to stimulation with LPS (100 ng/ml) for 1 h. Nuclear extracts were then analysed witha transcription factor assay kit (Abcam)， which has a double stranded DNA sequence containing the NF-KB response element (5'-GGGACTTTCC-3') immobilised onto the wells of a 96-well plate. Transient transfection and reporter gene assays To determine the effect of nimbolide on transactivation of NF-KB， a luciferase reporter gene assay wascarried out. Cultured BV2 cells at 60% confluence were transfected with Cignal NF-KB luciferase reporter(Qiagen) using magnetofection (OZ Biosciences)， as earlier described [24] and incubated for 20 h at 37°℃in a 5% CO2 incubator. At the end of the incubation， cells were stimulated with LPS (100 ng/ml) in thepresence or absence of nimbolide (125， 250 and 500 nM) for 6 h. Luciferase activities were evaluatedwith a Dual-Glo luciferase assay kit (Promega， UK). Luminescence was measured using POLARstarOptima microplate reader (BMG Labtech). Similar procedures were used in BV2 cells transfected withCignal Nrf2 luciferase reporter (Qiagen) and treated with nimbolide (125， 250 and 500 nM) for 24 h. Small interfering RNA-mediated knockdown of Nrf2 andSIRT-1 BV2 cells microglia were seeded out into 6-well plates at a concentration of 4×104 cells/ml and cultureduntil they were 60% confluence. This was followed by incubation in Opti-MEM for 2 h at 37℃. Complexescontaining Glial-Mag transfection reagent (1.8 pl； OZ Biosciences) and either Nrf2 siRNA (2 pl； Santa CruzBiotechnology)， SIRT1 siRNA (2 ul； Santa Cruz Biotechnology)， or control siRNA (Santa CruzBiotechnology) were then added to the cells. The culture plate was placed on a magnetic plate (OZBiosciences) for 30 min， followed by incubation at 37°℃ for a further 24-h period. Effects of Nrf2 or SIRT1knockdown on the anti-inflammatory effect of nimbolide were evaluated by treating cells with nimbolide(500 nM) followed by stimulation with LPS (100 ng/ml) for 24 h. Culture supernatants were collected and analysed for levels of nitrite using Griess assay， and TNFa levels determined using mouse ELISA kits(Biolegend) Statistical analysis All experiments were carried out at least 3 times. Experimental data are presented as mean±SEM.Results were analysed with one-way analysis of variance (ANOVA) followed by post-hoc Tukey multiplecomparisons test. Results were considered significant at p <0.05. Statistical analyses were done withGraphPad Prism Software version 9. Results Treatment with nimbolide did not affect the viability of LPS-stimulated BV2 microglia XTT cell viability assays to evaluate effects of 125， 250 and 500 nM of nimbolide on the viability of LPS-stimulated BV2 microglia revealed no cytotoxicity following 24 h incubation (Fig. 2A). Similarly， there wasno significant difference in LDH release in LPS-stimulated BV2 cells treated with nimbolide (125， 250 and500 nM)， in comparison with control (untreated and unstimulated) cells (Fig.2B). Effects of nimbolide on the production of pro-and anti-inflammatory cytokines Following incubation of BV2 microglia with LPS (100ng/ml) there were significant (p<0.0001) increasesin the secretion of pro-inflammatory cytokines TNFa (Fig. 3A)， IL-6 (Fig.3B)， and IFNy (Fig. 3C). However，pre-treating these cells with nimbolide (125， 250 and 500 nM) resulted in significant (p<0.0001) andconcentrationdependent reduction in the production of TNFa， IL-6， and IFNy in these cells. Furthermore，stimulation of BV2 cells caused a significant (p<0.001) suppression in the production of anti-inflammatory cytokine IL-10 (Fig. 3D). In the presence of nimbolide (125 nM)， an increase in IL-10 wasobserved， when compared with LPS stimulation. However， this increase was not statistically significant.On increasing the concentration of nimbolide to 250 and 500 nM， there were significant (p<0.0001)increases in the production of IL-10， in comparison with LPS stimulated cells (Fig. 3D). Nimbolide reduced iNOS-mediated LPS-induced increasedsecretion of NO Further investigations on the effects of nimbolide on pro-inflammatory mediator production revealed asignificant (p<0.001) and concentration-dependent reduction in the production of nitric oxide followingpre-treatment with 125， 250 and 500 nM of the compound prior to stimulation of BV2 microglia with LPS(Fig. 4A). Immunoblotting of lysates obtained from the cells showed significant reduction in theexpression of iNOS protein (Figs. 4B and 4C). Suppressive effects of nimbolide on LPS-induced increasedPGE2 production is mediated through COX-2 Incubation of BV2 microglia with LPS (100 ng/ml) for 24 h resulted in ~ 4.5-fold increase in the secretionof PGE2 (Fig.5A). However， in the presence of nimbolide (125 nM)， LPS-induced elevated PGE2production was significantly (p<0.0001) reduced from 100% to~44%. On increasing the concentrationsof nimbolide to 250 and 500 nM，PGE2 production was further reduced to 29% and 24%， respectively(Fig. 5A). Results of further studies on cell lysates revealed a concentration-dependent reduction in LPS-induced increased COX-2 protein expression as a result of pre-treatment with 125， 250 and 500 nMnimbolide (Figs. 5B and 5C). Inhibition of neuroinflammation by nimbolide is mediatedby targeting microglia NF-KB activation pathway Based on our results showing that nimbolide reduced the production of pro-inflammatory mediators inLPS-activated BV2 microglia， we proceeded to determine whether these effects were mediated throughNF-KB signalling. Immunoblotting analyses of cytoplasmic lysates from LPS-stimulated BV2 microgliarevealed significant elevation in the expression of phospho-p65 NF-KB protein. However， pre-treating thecells with nimbolide (125 and 250 nM) prior to LPS stimulation resulted in equivalent but significant (p<0.0001) reduction in the levels of phospho-p65 protein. On increasing the concentration of nimbolide to500 nM， phospho-p65 protein was further reduced by ~ 76%， in comparison with LPS stimulation alone(Figs. 6A & 6B). Similar results were obtained when lysates were analysed for protein levels of phospho-lkBa. Stimulationof BV2 microglia with LPS (100 ng/ml) resulted in ~ 5.7-fold increase in cytoplasmic levels of phospho-lkBa when compared with control (unstimulated) cells. Pre-treatment with nimbolide (125 and 250 nM)prior to LPS stimulation resulted in ~ 2.2 and ~2.5-fold reduction in expression， respectively incomparison to LPS stimulation alone. However， increasing the concentration of nimbolide to 500 nMcaused ~4.8-fold reduction in phospho-lkBa protein expression， when compared with LPS stimulation(Figs. 6C and 6D). Assessment of nuclear events revealed that following a 60-minute stimulation of BV2 microglia with LPS，there was significant (p<0.001) increase in binding of NF-KB to consensus sites in the nucleus (Fig. 6E).In the presence of nimbolide (125，250 and 500 nM)， there was concentration-dependent reduction inbinding of NF-KB to consensus sites (Fig. 6E). Furthermore， reporter gene assays were conducted to evaluate effects of nimbolide on the ability of NF-KB to activate the expression of genes encoding pro-inflammatory proteins such as the cytokines， iNOSand COX-2. LPS stimulation of BV2 cells transfected with NF-KB luciferase reporter plasmid resulted in Nimbolide induced deacetylation of p65 in LPS-activatedmicroglia Acetylation of lysine residues in NF-KB p65 has been shown to determine its activation or deactivation.Expectedly， LPS stimulation of BV2 cells resulted in increased expression of acetyl-p65 protein (lysine310) (Figs. 7A & 7B). In the presence of nimbolide (125， 250 and 500 nM)， a concentration-dependentreduction in acetylation of NF-KB p65 was observed. Nimbolide reduced protein levels of phospho-p38 andphospho-JNK in LPS-stimulated BV2 microglia It is known that the production of some pro-inflammatory cytokines following inflammatory stimuli ispartly linked to activation of MAPK， especially p38 and JNK. Experiments were therefore carried out toinvestigate whether nimbolide could have any effects on the activation of these proteins. Incubation of BV2 microglia with LPS (100 ng/ml) caused an increase in protein expression of phospho-p38， indicating activation. In the presence of 125 nM nimbolide， there was a slight reduction in LPS-induced increase in phospho-p38 protein (Figs. 8A &8B). On increasing the concentration of nimbolide to250 and 500 nM， there was significant (p<0.001) reduction in phospho-p38， in comparison with LPSstimulation alone (Figs. 8A &8B). Similarly， pre-treatment of BV2 microglia with nimbolide (125， 250 and500 nM) prior to LPS-stimulation resulted in significant reduction in phospho-JNK protein， whencompared with LPS stimulation alone (Figs. 8C&8D). Effects of nimbolide on cellular ROS generation and proteinlevels of (NOX2) gp91phox Results of experiments to evaluate effects of nimbolide on cellular generation of ROS followingstimulation with LPS are shown in Figs. 8A and 8B. Microplate detection of ROS revealed that stimulationof BV2 microglia with LPS (100 ng/ml) resulted in ~ 90% increase in ROS generation， an outcome thatwas markedly diminished in the presence of 125， 250 and 500 nM nimbolide (Fig. 9A). These effects wereconfirmed using fluorescence microscopy which revealed marked reduction in DCFDA fluorescence byLPS-activated microglia pre-treated with 125 nM nimbolide， while 250 and 500 nM concentrations of thecompound produced an almost complete inhibition of fluorescence (Fig.9B). In the presence of pro-inflammatory stimuli such as LPS， an increase in the expression of gp91phoxprotein results in cellular generation of ROS. Based on results showing inhibitory effects of nimbolide onLPS-induced increased ROS production， effects of the compound on protein levels of gp91phox were alsoevaluated. Results in Figs. 9C and 9D show an increase in expression of gp91phox protein following stimulation of BV2 microglia with LPS. However， in the presence of nimbolide (125， 250 and 500 nM)， aconcentration-dependent and significant (p<0.001) reduction in the levels of gp91phox protein wasobserved. Nimbolide directly increased protein levels of HO-1 andNQ0-1 antioxidant proteins in BV2 microglia Following results showing antioxidant activity by nimbolide through gp91phox-mediated reduction in ROSgeneration in LPS-stimulated microglia， further experiments were conducted to determine direct effects ofthe compound on levels of HO-1 and NQ0-1 antioxidant proteins in the cells. Incubation of BV2 microgliawith 125 nM nimbolide did not affect basal levels of HO-1 (~1.05 fold-increase). Nevertheless， increasingthe concentration of the compound to 250 and 500 nM caused~1.5-and ~2.3-fold increase in theexpression of HO-1 (Figs. 10A & 10B). Interestingly， incubating the cells with 125 nM of nimbolideresulted in significant (p<0.05)~ 1.4-fold increase in NQ0-1 protein expression. In the presence of higherconcentrations (250 and 500 nM) of the compound， there were~ 1.6-and ~2.4-fold increases in NQ0-1protein levels， respectively (Figs. 10C & 10D). Nimbolide directly activates Nrf2 Nrf2 is a transcription factor that that regulates antioxidant responses through the expression of genesencoding proteins such as HO-1 and NQ0-1. Encouraged by the results demonstrating increase inexpression of these proteins by nimbolide， experiments were conducted to determine effects of thecompound on mechanisms involved in the activation of Nrf2. Results in Figs. 10A and 10B show that treating BV2 microglia with nimbolide (125，250 and 500 nM)resulted in concentration-dependent and significant (p<0.01) decrease in protein levels of cytoplasmicNrf2， accompanied by corresponding concentration-dependent and significant (p<0.001) reduction inKeap-1 protein (Figs. 11C & 11D). These outcomes indicate that nimbolide treatment inducedtranslocation of Nrf2 from the cytoplasm due to degradation of Keap-1. In the presence of nimbolide (125 nM)， there was an insignificant (p<0.05) increase in expression of Nrf2in the nucleus， when compared with untreated cells. However， on treating the cells with 250 and 500 nMof the compound， significant (p<0.01) increase in nuclear expression of Nrf2 was observed (Figs. 11E &11F). Transcription factor assays revealed that significant (p<0.01) increase in binding to consensussites was achieved with 500 nM of nimbolide while the increase observed with lower concentrations ofthe compound were not significant (p<0.05) (Fig.12A). Results in Fig. 11B show that nimbolidetreatment produced significant (p<0.001) and concentration-dependent elevation in ARE luciferaseactivity， suggesting an increase in the ability of the compound to produce direct increase in the ability ofNrf2 to activate the expression of genes encoding antioxidant proteins. Nrf2 is required for the anti-inflammatory activity ofnimbolide in BV2 microglia Following the observation that nimbolide produced direct enhancement of Nrf2 activation in BV2microglia， experiments were conducted to explore the role of Nrf2 induction in the anti-inflammatoryactivity of the compound. Results in Fig. 13A shows that in cells transfected with control siRNA，significant (p<0.001) LPS-induced elevation in NO production was reduced (p<0.05) by nimbolide (500nM) pretreatment. On the other hand， in Nrf2 siRNA-transfected cells， pre-treatment with nimbolide prior tostimulation with LPS did not result in significant (p<0.05) reduction in NO production. Similar results(Fig.13B) were observed in assays to determine TNFa production. siRNA knockdown of Nrf2 in BV2 cellsresulted in the ability of nimbolide (500 nM) to reduce LPS-induced increase in TNFa production. Nimbolide increased nuclear SIRT-1 protein Studies have linked deacetylation of NF-KB p65 to nuclear SIRT-1， and results from this study have shownthat nimbolide prevented LPS-induced acetylation of NF-KB p65 at lysine 310. Interestingly， western blotanalyses revealed that incubation of BV2 microglia with nimbolide (125， 250 and 500 nM) resulted insignificant (p<0.001) and concentration-dependent increase in nuclear SIRT-1 protein expression(Figs. 14A & 14B). These results prompted experiments to determine if nimbolide-induced increase inSIRT-1 protein in the nucleus may be contributing to its anti-inflammatory activities. Results of siRNAexperiments in Figs. 14A and 14B show that siRNA-mediated knockdown of SIRT1 resulted in the loss ofanti-inflammatory activity of nimbolide as measured by production of NO and TNFa. Discussion Nimbolide is a compound found in Azadiractha indica， a plant that has been linked with in vivo and invitro anti-inflammatory effects [25-29]. Earlier， nimbolide was shown to reduce the expression of pro-inflammatory cytokines IL-6， IL-8， IL-12， and TNFa in LPS-stimulated RAW 264.7 and IL10-/-peritonealmacrophages [23]. In a separate study， nimbolide was reported to reduce levels of pro-inflammatorycytokines (IL-1B， IL-6， IL-12， TNFa， TGFB) and chemokines (MIP-1a and MIP-1β)， while increasing levels ofanti-inflammatory cytokines (IL-4，IL-10， and IL-13) in lung tissues of mice and in RAW 264.7 and A549cells [30]. In this study we showed that nimbolide inhibited neuroinflammation through reduction of LPS-induced increased production of pro-inflammatory cytokines TNFa， IL-6， and IFNy， as well as increasingproduction of IL-10. Inducible nitric oxide synthase-mediated excessive production of NO in microglia is a critical contributorto neuron-damaging neuroinflammation， in response to inducers such as lipopolysaccharide andcytokines. Consequently， targeting iNOS-mediated NO production is a critical strategy in reducingneuroinflammation. This study found that inhibition of iNOS protein， accompanied by suppression of NOproduction in LPS-activated BV2 microglia contributed to inhibition of neuroinflammation by nimbolide. It is noteworthy that this compound has shown similar profiles in RAW 264.7 macrophages stimulated withLPS and in cerulein-induced pancreatic inflammation in mice [30， 31]. Inhibition of neuroinflammation bynimbolide was also evidenced by a reduction in protein expression of COX-2， with accompanyingreduction in the secretion of PGE，. Nimbolide belongs to a chemical group of natural products known as terpenoids， a structurally diversegroup of compounds [32] which have been widely reported to be valuable as inhibitors ofneuroinflammation in vivo and in vitro [33]. Examples of terpenoids which have been shown to producesimilar anti-neuroinflammatory effects include artemisinin [34]， carnosol [35]， carnosic acid [36]，parthenolide [37] and thymoquinone [38， 39]. The NF-KB transcription factor promotes neuroinflammation through positive regulation of genesencoding pro-inflammatory cytokines such as TNFa and IL-6， as well as iNOS and COX-2. Since nimbolidereduced the production of some pro-inflammatory mediators which are known to be under the regulatorycontrol of NFKB， investigations were carried out to determine whether the compound produced anti-inflammatory activity in the microglia by targeting NF-KB activation. Activation of this pathway involvesphosphorylation of IkBa-NF-KB p65 complex， followed by proteasomal degradation of IkBa andtranslocation of p65 subunit to the nucleus [40， 41]. This study found that nimbolide blocksphosphorylation of lkBa and NF-KB p65 sub-units， which suggests that the compound may be acting inthe cytoplasm to target events resulting in translocation of NF-KB to the nucleus. However， this study didnot establish whether nimbolide could act on upstream targets involving LPS-activated toll-like receptor(TLR) adapters， MyD88 and TRIF. Following release from the cytoplasm， free NF-KB binds to promoter and enhancer regions containing KBsites in the nucleus. Interestingly， transcription factor assay results showed that in the presence ofnimbolide， there was decreased binding of NF-KB in nuclear extracts to GGGACTTTCC consensus sites.The impact of nimbolide on DNA binding capacity of NF-KB is probably a reflection of its action in thecytoplasm by preventing translocation of the transcription factor to the nucleus. This effect may alsoaccount for the diminished transactivation of NF-KB observed in LPS-stimulated microglia treated withnimbolide. Similar NF-KB inhibitory activity has been reported for nimbolide in LPS-stimulated A549 cells and lungtissues [30]， as well as in studies employing intestinal epithelial cells and macrophages [23]. Inhibition ofNF-KB activation by nimbolide in BV2 microglia reported in this study appears to suggest that thecompound targets the transcription factor in both peripheral and CNS immune cells， as well as non-immune cells such as epithelial cells. The MAPKs mediate a variety of cellular responses. However， p38 MAPK has been linked to microgliaactivation and neuroinflammation [13-16]. Similarly， JNK has been shown to phosphorylate c-Jun andATF-2 proteins resulting in increased transcriptional activity of the AP-1 and thus upregulation of cytokineproduction [42]. Results of this study showed that nimbolide inactivated both p38 and JNK MAPKs through inhibition of their phosphorylation. The observed effects on these MAPKs suggest that theirinhibition by nimbolide may in fact contribute to its ability to reduce the production of pro-inflammatorymediators. This proposal is justified by reports linking activation of p38 to mRNA stability of COX-2， TNF-a， IL-6， and iNOS genes [42， 43]. Binding of LPS to TLR4 activates the MyD88-dependent activation of TRAF-6 which in turn catalyses thephosphorylation of TAK-1. Activated TAK-1 then initiates phosphorylation steps resulting in the activationof NF-KB and MAPK pathways [44]. Dual inhibition of LPS-induced activation of NF-KB and MAPKactivation by nimbolide could therefore be explained by potential targeting of one or more of theseupstream signals. In the microglia， NADPH oxidase (NOX) are a group of five enzymes which catalyse the generation ofROS. Specifically， NOX2 (gp91phox) is known to be highly expressed in the microglia [45]. Reports fromseveral studies have indicated that NOXmediated generation of ROS in microglia is linked to both NF-KBand MAPK activation [46]. In this study we showed that LPS-induced increased ROS generation andgp91phox protein levels in BV2 microglia were reduced in the presence of nimbolide， which suggests thatthis compound could be promoting anti-inflammatory effects through mechanisms involving attenuationof oxidative stress. Nrf2 is a transcription factor involved in redox signalling， as well as in antioxidant and anti-inflammatoryresponses. Under physiological conditions， Nrf2 is complexed in the cytoplasm by Keap1. However， inresponse to oxidative stress， Nrf2 dissociates from Keap-1 and translocates into the nucleus to activatethe transcription of antioxidant genes. Results showing that nimbolide reduced LPS-induced increasedoxidative stress in the microglia prompted experiments which showed that the compound increasedlevels of antioxidant proteins (HO-1 and NQ0-1)， while activating Nrf2 through direct actions on bindingto antioxidant response elements in the nucleus. We used RNAi to further demonstrate loss of anti-inflammatory activity by nimbolide following knockdown of Nrf2 in BV2 cells. These are interestingresults which suggest that nimbolide possibly inhibits neuroinflammation and oxidative stress through itsability to enhance Nrf2 activation to promote antioxidant response. Our findings appear to reflect studiesreported by Rojo et al.(2010，2018)， who showed that Nrf2 knockout in mice resulted in an increase inmicroglia activation accompanied by elevated levels of COX-2， iNOS， IL-6， and TNFa [47， 48]. The sirtuin SIRT1 inhibits NF-KB signalling through deacetylation of lysine residue 310 on the RelA/p65subunit [49]. Based on the role of acetylation at 310 in the transcriptional activity of NF-KB [50]， weconducted experiments which showed that nimbolide attenuated LPS-induced acetylation of p65 sub-unitin BV2 microglia. We further showed that treatment of BV2 microglia with nimbolide resulted in activationof SIRT-1 as shown by its nuclear accumulation. Furthermore， activation of SIRT-1 appears to contributeto anti-inflammatory activity of nimbolide as shown by the loss of this activity in SIRT-1 siRNA-transfected cells stimulated with LPS. The outcome of this study reveals that nimbolide is a natural product which inhibits neuroinflammationin BV2 microglia through as-yet-unknown mechanisms resulting in dual inhibition of NF-KB and MAPK pathways. We further report that activation of Nrf2 antioxidant mechanisms， and possibly SIRT-1deacetylation mechanisms contribute to its anti-inflammatory activity in BV2 microglia. Declarations Funding This study was partly funded through the 'Development of Nutritional Products Promoting HealthyAgeing' sandpit by the University of Huddersfield. Folashade O Katola received partial fee waiverscholarship from the School of Applied Sciences， University of Huddersfield. Data availability The datasets generated during and/or analysed during the current study are available from thecorresponding author on reasonable request. Competing interests All authors certify that they have no affiliations with or involvement in any organization or entity with anyfinancial interest or non-financial interest in the subject matter or materials discussed in this manuscript. Author Contributions Conceptualisation： [Olumayokun A Olajide and Folashade O Katola]， Methodology： [Folashade O Katola]，Formal analysis and investigation： [Folashade O Katola]， Writing-original draft preparation： [Folashade OKatola and Olumayokun A Olajide]， Writing-review and editing：[Olumayokun A. Olajide]， Fundingacquisition： [Olumayokun A Olajide]， Resources： [Olumayokun A Olajide]， Supervision： [Olumayokun AOlajide]. All authors read and approved the final manuscript. References 1. Xu L， He D， Bai Y (2016) Microglia-mediated inflammation and neurodegenerative disease. MolNeurobiol. 53： 6709-6715. https：//doi.org/10.1007/s12035-015-9593-4 2. Velagapudi R， Kosoko AM， Olajide OA (2019) Induction of neuroinflammation and neurotoxicity bysynthetic hemozoin. Cell Mol Neurobiol 39：1187-1200. https：//doi.org/10.1007/s10571-019-00713-4 3. Rodgers J， Steiner l， Kennedy PGE (2019) Generation of neuroinflammation in human Africantrypanosomiasis. Neurol Neuroimmunol Neuroinflamm 2019，6：e610.https：//doi.org/10.1212/NXI.0000000000000610 4. Chen Z， Zhong D， Li G (2019) The role of microglia in viral encephalitis： a review. JNeuroinflammation.16：76. https：//doi.org/10.1186/s12974-019-1443-2 5. Garden GA (2002) Microglia in human immunodeficiency virus-associated neurodegeneration. Glia.40：240-251. https：//doi.org/10.1002/glia.10155 ( 6 . Mika J， Zychowska M， Popiolek-Barczyk K， Rojewska E， Przewlocka B (2013) Importance of glial activation in neuropathic pain. Eur J Pharmacol. 716：106-119.https：//doi . org/10.1016/j.ejphar.2013.01.072 ) ( 7. Burke NN， Kerr DM， Moriarty O， Finn DP Roche M (2014) Minocyc l ine modulates neuropathic painbehaviour and cortical M1-M2 microglial gene expression in a rat model of depression. Brain Behav Immun. 42：147-156. https：//doi . org/10.1016/ j .bbi.2014.06.015 ) ( 8. Popiolek-Barczyk K， Mika J (2016) Targeting the microglia l signaling pathways： New insights in themodulation of neuropathic pain. Curr Med Chem. 23：2908-2928. https：//doi.org/10.2174/0929867323666160607120124 ) ( 9. Srinivasan M， Lahiri DK (2015) Significance of NF-kB as a pivotal therapeutic target in theneurodegenerative pathologies of Alzheimer's disease and mul t iple sclerosis. Expert Opin Ther Targets. 19：471-487. https：//doi.org/10.1517/14728222 . 2014.989834 ) 10. Seo EJ， Fischer N， Efferth T (2018) Phytochemicals as inhibitors of NF-kB for treatment ofAlzheimer's disease. Pharmacol Res. 129：262-273. https：//doi.org/10.1016/j.phrs.2017.11.030 ( 11 . Thawkar BS， Kaur G (2019) Inhibitors of NF-kB and P2X7/NLRP3/Caspase 1 pathway in microglia：Novel therapeutic opportunities in neuroinflammation induced early-stage Alzheimer's disease. JNeuroimmunol . 326：62 - 74. https：//doi . org/10.1016/j.jneuroim.2018.11 . 010 ) 12. Zhou W， Hu W (2013) Anti-neuroinflammatory agents for the treatment of Alzheimer's disease. FutureMed Chem. 5：1559-1571. https：//doi.org/10.4155/fmc.13.125 13. Kim， E.K.， Choi， E (2015) Compromised MAPK signaling in human diseases： an update. Arch Toxicol.89：867-882.https：//doi.org/10.1007/s00204-015-1472-2 14. Lee JK， Kim NJ (2017) Recent advances in the inhibition of p38 MAPK as a potential strategy for thetreatment of Alzheimer's disease. Molecules. 22：1287. https：//doi.org/10.3390/molecules22081287 15. Munoz L， Ammit AJ (2010) Targeting p38 MAPK pathway for the treatment of Alzheimer's disease.Neuropharmacology. 58：561-568. https：//doi.org/10.1016/j.neuropharm.2009.11.010 16. Culbert AA， Skaper SD， Howlett DR， et al.(2006) MAPK-activated protein kinase 2 deficiency inmicroglia inhibits pro-inflammatory mediator release and resultant neurotoxicity. Relevance toneuroinflammation in a transgenic mouse model of Alzheimer disease. J Biol Chem. 281：23658-23667.https：//doi.org/10.1074/jbc.M513646200 17. Gupta SC， Prasad S， Reuter S， Kannappan R， Yadav VR， Ravindran J， Hema PS， Chaturvedi MM， NairM， Aggarwal BB (2010) Modification of cysteine 179 of IkappaBalpha kinase by nimbolide leads todown-regulation of NF-kappaB-regulated cell survival and proliferative proteins and sensitization oftumor cells to chemotherapeutic agents. J Biol Chem. 285：35406-35417.https：//doi.org/10.1074/jbc.M110.161984 18. Kavitha K， Vidya Priyadarsini R， Anitha P Ramalingam K， Sakthivel R， Purushothaman G， Singh AK，Karunagaran D， Nagini S (2012) Nimbolide， a neem limonoid abrogates canonical NF-kB and Wntsignaling to induce caspase-dependent apoptosis in human hepatocarcinoma (HepG2) cells. Eur JPharmacol. 681：6-14.https：//doi.org/10.1016/j.ejphar.2012.01.024 19. Roy MK， Kobori M， Takenaka M， Nakahara K， Shinmoto H， Isobe S， Tsushida T (2007)Antiproliferative effect on human cancer cell lines after treatment with nimbolide extracted from anedible part of the neem tree (Azadirachta indica). Phytother Res. 21：245-250.https：//doi.org/10.1002/ptr.2058 20. Wang L， Phan DD， Zhang J， Ong PS， Thuya WL， Soo R， Wong AL， Yong WP Lee SC， Ho PC， Sethi G，Goh BC (2016) Anticancer properties of nimbolide and pharmacokinetic considerations to accelerateits development. Oncotarget. 7：44790-44802. https：//doi.org/10.18632/oncotarget.8316 ( 21 . Rochanakij S， Thebtaranonth Y， Yenjai C， Yuthavong Y (1985) Nimbolide， a constituent ofAzadirachta indica， inhibits Plasmodium falciparum in culture. Southeast Asian J Trop Med Public Health. 16：66-72. ) ( 22. Diddi S， Bale S， Pulivendala G， Godugu C (2019) Nimbolide ameliorates fibrosis and inflammation inexperimental murine model of bleomycin-induced scleroderma. Inflammopharmacology. 27：139-149. https：//doi.org/10.1007/s10787-018-0527-4 ) ( 23. Seo JY， Lee C， Hwang SW， Chun J， Im JP Kim JS (2016) Nimbolide inhibits nuclear factor-kB pathwayin intestinal epithelial cells and macrophages and alleviates experimental colitis in mice. Phytother Res. 30：1605-1614.https：//doi.org/10.1002/ptr.5657 ) 24. Olajide OA， Iwuanyanwu VU， Adegbola OD， Al-Hindawi AA (2022) SARS-CoV-2 spike glycoprotein S1induces neuroinflammation in BV-2 microglia. Mol Neurobiol. 59：445-458.https：//doi.org/10.1007/s12035-021-02593-6 25. Lee JW， Ryu HW， Park SY， et al. (2017) Protective effects of neem (Azadirachta indica A. Juss.) leafextract against cigarette smoke- and lipopolysaccharide-induced pulmonary inflammation. Int J MolMed. 40：1932-1940. https：//doi.org/10.3892/ijmm.2017.3178 26. llango K， Maharajan G， Narasimhan S (2013) Anti-nociceptive and anti-inflammatory activities ofAzadirachta indica fruit skin extract and its isolated constituent azadiradione. Nat Prod Res.27：1463-1467. https：//doi.org/10.1080/14786419.2012.717288 27. Schumacher M， Cerella C， Reuter S， Dicato M， Diederich M (2011) Anti-inflammatory， pro-apoptotic，and anti-proliferative effects of a methanolic neem (Azadirachta indica) leaf extract are mediated viamodulation of the nuclear factor-kB pathway. Genes Nutr. 6：149-160.https：//doi.org/10.1007/s12263-010-0194-6 28. Manogaran S， Sulochana N， Kavimani S (1998) Anti-inflammatory and antimicrobial activities of theroot， bark and leaves of Azadirachta indica. Anc Sci Life. 18：29-34. 29. Okpanyi SN， Ezeukwu GC (1981) Anti-inflammatory and antipyretic activities of Azadirachta indica.Planta Med. 41：34-39. https：//doi.org/10.1055/s-2007-971670 30. Pooladanda V， Thatikonda S， Bale S， et al. (2019) Nimbolide protects against endotoxin-inducedacute respiratory distress syndrome by inhibiting TNF-a mediated NF-kB and HDAC-3 nucleartranslocation. Cell Death Dis. 10：81. https：//doi.org/10.1038/s41419-018-1247-9 31. Bansod S， Godugu C (2019) Nimbolide ameliorates pancreatic inflammation and apoptosis bymodulating NF-kB/SIRT1 and apoptosis signaling in acute pancreatitis model. Int Immunopharmacol. 2021， 90：107246.https：//doi.org/10.1016/j.intimp.2020.107246 32. Nahar L， Sarker SD (2019) Chemistry for pharmacy students： general， organic and natural productchemistry. John Wiley & Sons. 33. Olajide OA， Sarker SD (2020) Alzheimer's disease： natural products as inhibitors ofneuroinflammation. Inflammopharmacology. 28：1439-1455. https：//doi.org/10.1007/s10787-020-00751-1 34. Zhu C， Xiong Z， Chen X， et al. (2012) Artemisinin attenuates lipopolysaccharide-stimulatedproinflammatory responses by inhibiting NF-kB pathway in microglia cells. PLoS One. 7： e35125.https：//doi.org/10.1371/journal.pone.0035125 35. Foresti R， Bains SK， Pitchumony TS， de Castro Bras LE， Drago F， Dubois-Rande JL， Bucolo C，Motterlini R (2013) Small molecule activators of the Nrf2-H0-1 antioxidant axis modulate hememetabolism and inflammation in BV2 microglia cells. Pharmacol Res. 2013，76：132-148.https：//doi.org/10.1016/j.phrs.2013.07.010 36. de Oliveira MR， de Souza ICC， Furstenau CR (2018) Carnosic acid induces anti-inflammatory effectsin paraquat-treated SH-SY5Y cells through a mechanism involving a crosstalk between the Nrf2/HO-1 Axis and NF-kB. Mol Neurobiol.55：890-897. https：//doi.org/10.1007/s12035-017-0389-6 37. Khare P Datusalia AK， Sharma SS (2017) Parthenolide， an NF-kB inhibitor ameliorates diabetes-induced behavioural deficit， neurotransmitter imbalance and neuroinflammation in type 2 diabetesrat model. Neuromolecular Med. 19：101-112. https：//doi.org/10.1007/s12017-016-8434-6 38. Velagapudi R， Kumar A， Bhatia HS， et al. (2017) Inhibition of neuroinflammation by thymoquinonerequires activation of Nrf2/ARE signalling. Int Immunopharmacol. 48：17-29.https：//doi.org/10.1016/j.intimp.2017.04.018 39. Velagapudi R， El-Bakoush A， Lepiarz l， Ogunrinade F，Olajide OA (2017) AMPK and SIRT1 activationcontribute to inhibition of neuroinflammation by thymoquinone in BV2 microglia. Mol Cell Biochem.435：149-162. https：//doi.org/10.1007/s11010-017-3064-3 40. Karin M， Ben-Neriah Y (2000) Phosphorylation meets ubiquitination： the control of NF-[kappa]Bactivity. Annu Rev Immunol. 18：621-63. https：//doi.org/10.1146/annurev.immunol.18.1.621 41. Hayden MS， Ghosh S (2004) Signaling to NF-kappaB. Genes Dev. 18：2195-2224.https：//doi.org/10.1101/gad.1228704 42. Kaminska B， Gozdz A， Zawadzka M， Ellert-Miklaszewska A， Lipko M (2009) MAPK signaltransduction underlying brain inflammation and gliosis as therapeutic target. Anat Rec (Hoboken).292：1902-1913.https：//doi.org/10.1002/ar.21047 43. Clark AR， Dean JL， Saklatvala J (2003) Post-transcriptional regulation of gene expression bymitogen-activated protein kinase p38. FEBS Lett. 546：37-44. https：//doi.org/10.1016/s0014-5793(03)00439-3 44. Singh S， Sahu K， Singh C， Singh A (2022) Lipopolysaccharide induced altered signaling pathways invarious neurological disorders. Naunyn Schmiedebergs Arch Pharmacol.395：285-294.https：//doi.org/10.1007/s00210-021-02198-9 45. Nauseef WM (2008) Biological roles for the NOX family NADPH oxidases. J Biol Chem. 283：16961-16965. https：//doi.org/10.1074/jbc.R700045200 46. Simpson DSA， Oliver PL (2020) ROS generation in microglia： understanding oxidative stress andinflammation in neurodegenerative disease. Antioxidants (Basel). 9：743.https：//doi.org/10.3390/antiox9080743 47. Rojo Al， Innamorato NG， Martin-Moreno AM， De Ceballos ML， Yamamoto M， Cuadrado A (2010) Nrf2regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease. Glia.58：588-598.https：//doi.org/10.1002/glia.20947 48. Rojo Al， Pajares M， Garcia-Yague AJ， Buendia l， Van Leuven F， Yamamoto M， Lopez MG， Cuadrado A(2018) Deficiency in the transcription factor NRF2 worsens inflammatory parameters in a mousemodel with combined tauopathy and amyloidopathy. Redox Biol. 18：173-180.https：//doi.org/10.1016/j.redox.2018.07.006 49. Yeung F， Hoberg JE， Ramsey CS， Keller MD， Jones DR， Frye RA， Mayo MW (2004) Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J. 23： 2369-2380.https：//doi.org/10.1038/sj.emboj.7600244 50. Calao M， Burny A， Quivy V，Dekoninck A， Van Lint C (2008) A pervasive role of histoneacetyltransferases and deacetylases in an NF-kappaB-signaling code. Trends Biochem Sci. 33：339-349. https：//doi.org/10.1016/j.tibs.2008.04.015 Figures Figure 1 Figure 2 Nimbolide did not affect BV2 cell viability. XTT assay results showed that there was no significantdifference in the viability of LPS-stimulated BV2 cells treated with nimbolide (125， 250 and 500nM) whencompared with untreated cells (A). There was no significant difference in LDH released from BV2microglia treated with nimbolide (125， 250 and 500 nM) when compared with control (untreated) cells(B). Data are expressed as mean + SEM for at least three independent experiments. Statistical analysis wasperformed using one way ANOVA with post-hoc Tukey test (multiple comparisons) ***p<0.001， versus celllysis control. **** 日 D performed using One way ANOVA with post-hoc Tukey test (multiple comparisons) **p<0.01， ***p<0.001，versus LPS control. B Figure 4 Nimbolide decreased the release of nitric oxide (NO) through the suppression of inducible nitric oxide(iNOS) protein expression in BV2 microglia stimulated with LPS. BV2 cells were stimulated with LPS (100 ng/ml) in the presence or absence of nimbolide (125， 250 and 500 nM) for 24 h. Culture supernatantsand cell lysates were collected to evaluate levels of nitrite using Griess assay (A) and iNOS protein usingimmunoblotting (B) respectively. Densitometric analysis of iNOS immunoblotting (C). Data are expressedas mean ± SEM for at least three independent experiments. Statistical analysis was performed using oneway ANOVA with post-hoc Tukey test (multiple comparisons). ***p<0.001， versus LPS control. Figure 5 A Enzyme immunoassay showing reduction of PGE2 production (A) and immunoblotting showing inhibitionof COX-2 protein expression (B) by nimbolide (125，250 and 500 nM) in LPS-activated BV2 microglia.Densitometric analysis of COX-2 immunoblotting (C). Cells were pre-treated with nimbolide (125， 250 and500 nM) followed by stimulation of LPS (100 ng/ml) for 24 h. Data are expressed as mean ± SEM for atleast three independent experiments. Statistical analysis was performed using one way ANOVA with post-hoc Tukey test (multiple comparisons).***p<0.001 versus LPS control. Figure 6 Nimbolide inhibits NF-kB activation in BV2 microglia. Nimbolide (125， 250 and 500 nM) reduced levels ofphospho-p65 (A and B) and phospho-lkBa (C and D) in lysates of BV2 microglia stimulated with LPS (100ng/ml). 6E： Reduction in LPS-induced increased binding of p65 to consensus sites by nimbolide. BV2cells were treated with nimbolide (125， 250 and 500 nM) then stimulated with LPS (100 ng/ml) for 30 minand NF-kB transcription factor assay was carried out on nuclear extracts. 6F： Nimbolide inhibited LPS-induced NF-kB-dependent gene expression in BV2 cells. Transfected cells were treated with nimbolide(125，250 and 500 nM) and stimulated with LPS (100 ng/ml) for 6 h， followed by luciferase reporter geneassay. Data are expressed as mean± SEM for at least three independent experiments. Statistical analysis wasperformed using one way ANOVA with post-hoc Tukey test (multiple comparisons).***p<0.001，****p<0.0001 versus LPS control. Figure 7 Figure 7 Nimbolide interferes with acetylation of p65 sub-unit following activation with LPS. Nimbolide-treatedBV2 microglia were stimulated with LPS (100 ng/ml). Western blotting on nuclear extracts showedreduction in protein expression of acetyl-p65 (A). Densitometric analysis of blots (B). Densitometricvalues are expressed as mean ± SEM for at least three independent experiments. Statistical analysis wasperformed using one way ANOVA with post-hoc Tukey test (multiple comparisons).***p<0.001，****p<0.0001 versus LPS control. way ANOVA with post-hoc Tukey test (multiple comparisons). **p<0.01，***p<0.001， ****p<0.0001 versusLPS control. A B **** Figure 9 or without nimbolide (125，250 and 500 nM) for 24 h. Fluorescence detection of ROS generation wasdone using Polar star Optima plate reader (A) and EVOSQ Floid@ cell imaging station (B). Data areexpressed as mean ± SEM for at least three independent experiments. Statistical analysis was performedusing one way ANOVA with post-hoc Tukey test (multiple comparisons). ****p<0.0001 versus LPS control. Nimbolide increased protein levels of HO-1 (A and B) and NQ0-1 (C and D) in BV2 microglia. Cells weretreated with nimbolide (125， 250 and 500 nM) for 24 h. Thereafter， lysates were analysed usingimmunoblotting. Densitometric data are expressed as mean ± SEM for at least three independentexperiments. Statistical analysis was performed using one way ANOVA with post-hoc Tukey test (multiplecomparisons). ns (not significant)， *p<0.05， **p<0.01，****p<0.0001， versus untreated control. Nimbolide activates Nrf2 antioxidant mechanisms in BV2 microglia. Cells were treated with nimbolide(125，250 and 500 nM) for 24 h. Immunoblotting of cytoplasmic extracts revealed disappearance of Nrf2(A and B) and Keap-1 (C and D). Results of western blotting of nuclear extracts showing accumulation ofNrf2 (E and F). Densitometric values are expressed as mean+ SEM for at least three independentexperiments. Statistical analysis was performed using one way ANOVA with post-hoc Tukey test (multiplecomparisons). ns (not significant)， **p<0.01， ****p<0.0001， versus untreated control. Figure 12 Figure 12 Figure 13 Effects of Nrf2 knockdown on anti-inflammatory effects of nimbolide. Control siRNA- and Nrf2 siRNA-transfected BV2 cells were pre-treated with nimbolide (500 nM) prior to stimulation with LPS (100 ng/ml)for 24 h. Culture supernatants were analysed for nitrite (A) and TNFa (B). Western blot experiments onnuclear extracts to determine knockout efficiency (C). Data are expressed as mean ± SEM for at leastthree independent experiments. Statistical analysis was performed using one way ANOVA with post-hocTukey test (multiple comparisons). *p<0.05，**p<0.01，***p<0.001， nimbolide + LPS in Nrf2 siRNA-transfected cells versusnimbolide+LPS in control siRNA-transfected cells. Figure 14 Nimbolide activates SIRT-1 in BV2 microglia. BV2 cells were treated with nimbolide， followed byimmunoblotting of nuclear extracts (A). Densitometric analysis of blots (B). Densitometric values areexpressed as mean ± SEM for at least three independent experiments. Statistical analysis was performedusing one way ANOVA with post hoc Tukey test (multiple comparisons). ***p<0.001， ****p<0.0001versusuntreated control. A B Control siRNA + + SIRT-1 siRNA SIRT-1 一 Figure 15 Effects of SIRT-1 knockdown on anti-inflammatory effects of nimbolide. Control siRNA-and SIRT-1 siRNA-transfected BV2 cells were pre-treated with nimbolide (500 nM) prior to stimulation with LPS (100 ng/ml)for 24 h. Culture supernatants were analysed for nitrite (A) and TNFa (B). Western blot experiments onnuclear extracts to determine knockout efficiency (C). Data are expressed as mean ± SEM for at leastthree independent experiments. Statistical analysis was performed using one way ANOVA with post-hocTukey test (multiple comparisons). *p<0.05，**p<0.01， ***p<0.001， nimbolide + LPS in SIRT-1 siRNA-transfected cells versusnimbolide+LPS in control siRNA-transfected cells. Page /