Natural carbazole alkaloid murrayafoline A displays potent anti-neuroinflammatory effect by directly targeting transcription factor Sp1 in LPS-induced microglial cells

Neuroinflammation is a leading cause for neurological disorders. Carbazole alkaloids, isolated from the medic-inal plants of Murraya species (Rutaceae), have exhibited wide pharmacological activities particularly for neu-roinflammation. However, its underlying cellular targets and molecular mechanisms still remain unclear. In current study, we found that murrayafoline A (MA), a carbazole alkaloid obtained from Murraya tetramera, potently inhibited the production of neuroinflammation mediators, such as nitric oxide (NO), TNF-alpha, IL-6 and IL-1 beta in LPS-induced BV-2 microglial cells. Then, we performed thermal proteome profiling (TPP) strategy to identify Specificity protein 1 (Sp1) as a potential cellular target of MA. Moreover, we performed surface plasmon resonance (SPR), cellular thermal shift assay (CETSA) and drug affinity responsive target stability (DRATS) as-says to confirm the direct interaction between MA and Sp1. Furthermore, we downregulated Sp1 expression in BV2 cells using siRNA transfection, and observed that Sp1 knockdown significantly antagonized MA-mediated inhibition of neuroinflammation mediator production. Meanwhile, Sp1 knockdown also markedly reversed MA-mediated inactivation of IKK beta/NF-kappa B and p38/JNK MAPKs pathways. Finally, in vivo studies revealed that MA significantly suppressed the expression of Iba-1, TNF-alpha, and IL-6, while increased the number of Nissl bodies in the brains of LPS-induced mice. Taken together, our study demonstrated that MA exerted obvious anti-neuroinflammation effect by directly targeting Sp1, thereby inhibiting NF-kappa B and MAPK signaling pathways. Our findings also provided a promising direction of pharmacological targeting Sp1 for anti-neuroinflammation therapeutics as well as novel agent development.