Nimbolide is a compound found in Azadiractha indica, a plant that has been linked with in vivo and in vitro anti-inflammatory effects [25–29]. Earlier, nimbolide was shown to reduce the expression of pro-inflammatory cytokines IL-6, IL-8, IL-12, and TNFα in LPS-stimulated RAW 264.7 and IL10−/− peritoneal macrophages [23]. In a separate study, nimbolide was reported to reduce levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-12, TNFα, TGFβ) and chemokines (MIP-1α and MIP-1β), while increasing levels of anti-inflammatory cytokines (IL-4, IL-10, and IL-13) in lung tissues of mice and in RAW 264.7 and A549 cells [30]. In this study we showed that nimbolide inhibited neuroinflammation through reduction of LPS-induced increased production of pro-inflammatory cytokines TNFα, IL-6, and IFNγ, as well as increasing production of IL-10.
Inducible nitric oxide synthase-mediated excessive production of NO in microglia is a critical contributor to neuron-damaging neuroinflammation, in response to inducers such as lipopolysaccharide and cytokines. Consequently, targeting iNOS-mediated NO production is a critical strategy in reducing neuroinflammation. This study found that inhibition of iNOS protein, accompanied by suppression of NO production 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 with LPS and in cerulein-induced pancreatic inflammation in mice [30, 31]. Inhibition of neuroinflammation by nimbolide was also evidenced by a reduction in protein expression of COX-2, with accompanying reduction in the secretion of PGE2.
Nimbolide belongs to a chemical group of natural products known as terpenoids, a structurally diverse group of compounds [32] which have been widely reported to be valuable as inhibitors of neuroinflammation in vivo and in vitro [33]. Examples of terpenoids which have been shown to produce similar anti-neuroinflammatory effects include artemisinin [34], carnosol [35], carnosic acid [36], parthenolide [37] and thymoquinone [38, 39].
The NF-κB transcription factor promotes neuroinflammation through positive regulation of genes encoding pro-inflammatory cytokines such as TNFα and IL-6, as well as iNOS and COX-2. Since nimbolide reduced the production of some pro-inflammatory mediators which are known to be under the regulatory control of NFκB, investigations were carried out to determine whether the compound produced anti-inflammatory activity in the microglia by targeting NF-κB activation. Activation of this pathway involves phosphorylation of IκBα-NF-κB p65 complex, followed by proteasomal degradation of IκBα and translocation of p65 subunit to the nucleus [40, 41]. This study found that nimbolide blocks phosphorylation of IκBα and NF-κB p65 sub-units, which suggests that the compound may be acting in the cytoplasm to target events resulting in translocation of NF-κB to the nucleus. However, this study did not 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-κB binds to promoter and enhancer regions containing κB sites in the nucleus. Interestingly, transcription factor assay results showed that in the presence of nimbolide, there was decreased binding of NF-κB in nuclear extracts to GGGACTTTCC consensus sites. The impact of nimbolide on DNA binding capacity of NF-κB is probably a reflection of its action in the cytoplasm by preventing translocation of the transcription factor to the nucleus. This effect may also account for the diminished transactivation of NF-κB observed in LPS-stimulated microglia treated with nimbolide.
Similar NF-κB inhibitory activity has been reported for nimbolide in LPS-stimulated A549 cells and lung tissues [30], as well as in studies employing intestinal epithelial cells and macrophages [23]. Inhibition of NF-κB activation by nimbolide in BV2 microglia reported in this study appears to suggest that the compound 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 microglia activation and neuroinflammation [13–16]. Similarly, JNK has been shown to phosphorylate c-Jun and ATF-2 proteins resulting in increased transcriptional activity of the AP-1 and thus upregulation of cytokine production [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 their inhibition by nimbolide may in fact contribute to its ability to reduce the production of pro-inflammatory mediators. This proposal is justified by reports linking activation of p38 to mRNA stability of COX-2, TNF-α, IL-6, and iNOS genes [42, 43].
Binding of LPS to TLR4 activates the MyD88-dependent activation of TRAF-6 which in turn catalyses the phosphorylation of TAK-1. Activated TAK-1 then initiates phosphorylation steps resulting in the activation of NF-κB and MAPK pathways [44]. Dual inhibition of LPS-induced activation of NF-κB and MAPK activation by nimbolide could therefore be explained by potential targeting of one or more of these upstream signals.
In the microglia, NADPH oxidase (NOX) are a group of five enzymes which catalyse the generation of ROS. Specifically, NOX2 (gp91phox) is known to be highly expressed in the microglia [45]. Reports from several studies have indicated that NOXmediated generation of ROS in microglia is linked to both NF-κB and MAPK activation [46]. In this study we showed that LPS-induced increased ROS generation and gp91phox protein levels in BV2 microglia were reduced in the presence of nimbolide, which suggests that this compound could be promoting anti-inflammatory effects through mechanisms involving attenuation of oxidative stress.
Nrf2 is a transcription factor involved in redox signalling, as well as in antioxidant and anti-inflammatory responses. Under physiological conditions, Nrf2 is complexed in the cytoplasm by Keap1. However, in response to oxidative stress, Nrf2 dissociates from Keap-1 and translocates into the nucleus to activate the transcription of antioxidant genes. Results showing that nimbolide reduced LPS-induced increased oxidative stress in the microglia prompted experiments which showed that the compound increased levels of antioxidant proteins (HO-1 and NQO-1), while activating Nrf2 through direct actions on binding to 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 interesting results which suggest that nimbolide possibly inhibits neuroinflammation and oxidative stress through its ability to enhance Nrf2 activation to promote antioxidant response. Our findings appear to reflect studies reported by Rojo et al. (2010, 2018), who showed that Nrf2 knockout in mice resulted in an increase in microglia activation accompanied by elevated levels of COX-2, iNOS, IL-6, and TNFα [47, 48].
The sirtuin SIRT1 inhibits NF-κB signalling through deacetylation of lysine residue 310 on the RelA/p65 subunit [49]. Based on the role of acetylation at 310 in the transcriptional activity of NF-κB [50], we conducted experiments which showed that nimbolide attenuated LPS-induced acetylation of p65 sub-unit in BV2 microglia. We further showed that treatment of BV2 microglia with nimbolide resulted in activation of SIRT-1 as shown by its nuclear accumulation. Furthermore, activation of SIRT-1 appears to contribute to 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 neuroinflammation in BV2 microglia through as-yet-unknown mechanisms resulting in dual inhibition of NF-κB and MAPK pathways. We further report that activation of Nrf2 antioxidant mechanisms, and possibly SIRT-1 deacetylation mechanisms contribute to its anti-inflammatory activity in BV2 microglia.