AbstractHyperactivation of microglia results in neuroinflammation and consequently neurodegeneration. Estrogen receptor modulators and natural products have been identified to be effective in the modulation of neuroinflammatory responses. AC-186 is a selective non-steroidal estrogen receptor β agonist that has been shown to exhibit neuroprotective effects in AD and PD models. However, it is undetermined whether this compound exerts its neuroprotective effects through inhibitory action against neuroinflammation. Nimbolide is a limonoid tetranortriterpenoid derived from leaves and flowers of Azadirachta indica. This bioactive natural product is found to have anti-inflammatory activities in diverse experimental models. Notwithstanding, the inhibitory activity of nimbolide on neuroinflammation remains undiscovered. In this study, the anti-neuroinflammatory activities of AC-186, its novel synthetic analogues (FK-03, FK-06, FK-07) and nimbolide were determined in LPS-stimulated BV-2 microglia. MTT, XTT and LDH assays were used to determine the effects of the investigated compounds on BV-2 cell viability. The levels of cytokines (TNFα, IL-6, IFN-γ, IL-10), nitrite and PGE2 were determined using ELISA, the Griess assay and the EIA, respectively. ROS generation was detected using Cellular ROS assay kit and Image-iT™ LIVE Green ROS Detection Kit. The protein levels of iNOS, COX-2, phospho-NF-B-p65, phospho- IBα, total-IBα, phospho-IKK, TAK-1, TRAF-6, TLR4, phospho-p38, phospho-JNK, gp91phox, p22phox, p67phox, phospho-p40phox, p47phox, Keap1, Nrf2, HO-1, NQO1, SIRT1, acetyl-NF-B-p65, ERβ, MAP-2 were determined using immunoblotting. NF-B DNA binding was investigated using NF-B p65 Transcription Factor Assay Kit while Nrf2 DNA binding was evaluated using TransAM Nrf2 DNA binding ELISA. The transcriptional activation of NF-B, ARE and ERE was determined using reporter gene assay. Further evaluation to elucidate the involvement of Nrf2 and SIRT1 in the inhibitory activities of the investigated compound(s) on neuroinflammation was performed in Nrf2-knockdown and SIRT1-knockdown BV-2 cells, respectively. The neuroprotective effects of the investigated compound(s) were evaluated in HT-22 neuronal cells. Results showed that AC-186 (0.625 μM, 1.25 μM, 2.5 μM, 5 μM) and its novel synthetic analogues, FK-03 (0.375 μM, 0.75 μM, 1.5 μM, 3 μM), FK-06 (12 μM, 24 μM, 48 μM, 96 μM) and FK-07 (3 μM, 6 μM, 12 μM, 24 μM) reduced the levels of nitrite released from LPS-induced BV-2 cells without affecting the viability of the cells. Experimental outcomes demonstrated that AC-186 and FK-03 prevented nitrite secretion via the inhibition of iNOS protein expression in LPS-activated BV-2 microglial cells. Intriguingly, pre-treatment with AC-186 and FK-03 significantly decreased the levels of pro-inflammatory (TNFα, IL-6 and IFN-γ) and elevated the levels of anti-inflammatory (IL-10) cytokines in LPS-induced BV-2 cells. Findings showed that AC-186 and FK-03 limited COX-2-mediated PGE2 secretion and NADPH oxidase-regulated ROS generation in LPS-challenged BV-2 microglial cells. Further investigations revealed that AC-186 and FK-03 impede LPS-induced neuroinflammation in BV-2 microglia by targeting NF-B signalling pathway. Additional findings showed that AC-186 suppressed the release of pro-inflammatory mediators by downregulating p38 MAPK signalling pathway in LPS-treated BV-2 cells. Investigations revealed that AC-186 inhibited neuroinflammation by repressing the protein levels of upstream targets, TAK-1, TRAF-6 and TLR-4 in LPS-induced BV-2 microglia. Furthermore, studies revealed that AC-186 dysregulated Keap1, promoted the nuclear translocation of Nrf2 and its binding to ARE, and upregulated the expression of antioxidant proteins, HO-1 and NQO1. The inhibitory activity of AC-186 on LPS-induced neuroinflammation was reversed in Nrf2-knockdown BV-2 cells. Mechanistic findings showed that AC-186 attenuated the acetylation of NF-B-p65 and enhanced SIRT1 activation in BV-2 cells. Interestingly, the anti-inflammatory effects of AC-186 was not abolished in SIRT1-knockdown LPS-stimulated BV-2 cells. Additionally, AC-186 increased ERβ protein expression and ERE transcriptional activity in BV-2 microglial cells. Other studies showed that AC-186 and its synthetic analogue, FK-03 inhibited neurotoxicity in H2O2-challenged HT-22 neuronal cells. Taken together, the inhibition of neuroinflammation by AC-186 is exerted by targeting NF-B, p38 MAPK, TAK-1, TRAF-6, TLR-4, Keap1/Nrf2/ARE/HO-1/NQO1, SIRT1, ERβ signalling pathways. Similarly, FK-03 can be said to dysregulate brain inflammatory responses via modulating NF-B signalling pathway. Investigation on the anti-neuroinflammatory activity of the natural product, nimbolide in LPS-challenged BV-2 microglia was also performed. Experimental outcome revealed that nimbolide (125 nM, 250 nM, 500 nM) did not alter BV-2 cell viability in the presence of LPS (100 ng/ml). Pre-treatment with nimbolide markedly diminished levels of pro-inflammatory (TNFα, IL-6 and IFN-γ) and increased levels of anti-inflammatory (IL-10) cytokines in LPS-stimulated BV-2 cells. More studies revealed that nimbolide blocked iNOS-regulated NO production, COX-2-regulated PGE2 production and NADPH oxidase-6
controlled ROS generation in LPS-activated BV-2 cells. Mechanistic findings showed that nimbolide exerts its anti-neuroinflammatory effects by dysregulating NF-B, p38 MAPK, JNK MAPK signalling pathways. Also, nimbolide negatively regulated Keap1, increased Nrf2 nuclear translocation and its binding to ARE, and enhanced the expression of HO-1 and NQO1. More so, the modulatory activity of nimbolide on neuroinflammation is suggested to be mediated by Nrf2. Further investigations revealed that nimbolide suppressed the expression of acetyl-NF-B-p65 and upregulated SIRT1 expression. Findings showed that the downregulation of neuroinflammation by nimbolide is independent of SIRT1 activity. Intriguingly, nimbolide was observed to significantly dampen high ROS levels and prevent neuronal death in H2O2-activated HT-22 neuronal cells via improving MAP-2 protein expression. Altogether, nimbolide can be said to modulate LPS-induced neuroinflammation in BV-2 cells by inhibiting NF-B, p38 MAPK, JNK MAPK signal transductions and activating SIRT1 and Keap1/Nrf2/ARE/HO-1/NQO1 antioxidant mechanism.
|Date of Award
|9 Sep 2022
|Olumayokun Olajide (Main Supervisor) & Karl Hemming (Co-Supervisor)