Discussion
In this study, we explored the neuroprotective effects of luteolin during inflammation-induced dopaminergic injury using both in vivo and in vitro model systems. The results showed that luteolin treatment induced a functional improvement in this response, and protected against dopaminergic neuronal loss. Luteolin treatment also promoted dopaminergic neuronal survival when SH-SY5Y neuronal cells were co-cultured with BV2 microglia challenged with LPS-only. Moreover, luteolin treatment shifted microglial M1/M2 polarization towards an anti-inflammatory M2 phenotype and blunted pro-inflammatory cytokine release in both in vivo and in vitro model systems. Mechanistically, our findings indicate that luteolin treatment deactivated TLR4 and downstream NFkB signaling. The effect of this resulted in an improved inflammatory microenvironment and reduced neuronal loss.
Imbalances in microglial polarization status are a key factor in the initiation of neurodegenerative diseases. Moreover, there is the possibility of mutual transformation between M1 and M2 microglial phenotypes corresponding to specific treatments. Therefore, we recognize the potential therapeutic value in agents that, in situations where phenotypical M1 microglia are over-activated, promote the transformation of M1-type microglia into M2 type and maintain a relative balance of M1/M2 cells. Luteolin has a wide range of effects in vivo , including anti-inflammatory effects, antioxidant action, and estrogen-like effects [12-14]. In accordance with our findings, other studies indicate that luteolin functions in a neuroprotective capacity during response to dopaminergic injury by limiting inflammatory response [15]. However, the mechanism(s) that govern such response remain unclear. We found that in LPS-treated PD mouse model and cultured BV2 cells, luteolin intervention inhibited the activation of M1-type microglia, reduced the production and release of pro-inflammatory cytokines, and promoted the activation of anti-inflammatory M2-type microglia resulting in a restoration of the M1/M2 ratio. These effects reduced the neuronal damage that occurs through inflammatory response. In our animal model, we further demonstrated that luteolin significantly blunts both dopaminergic neuronal injury and motor function in inflammation-induced PD mice. Because of limitations stemming from the blood-brain barrier, classic anti-inflammatory drugs are of limited benefit in the treatment of neurological diseases. Luteolin is a highly active natural polyphenol that crosses the blood-brain barrier, thus luteolin is potentially a more effective drug for the prevention and treatment of inflammatory responses mounted within the central nervous system [16].
Although the anti-inflammatory effects of luteolin have been extensively studied, its downstream effector targets remain unclear. Consistent with our results, previous studies have determined that LPS triggers the activation of TLR4 and downstream NFκB signaling [17, 18]. Using TLR4 KO mice, prior studies indicate that damage-associated response does not stimulate microglia through the TLR4 pathway [19, 20]. This implies that TLR4 deficiency may shape microglia polarization towards the M2 phenotype while inhibiting the M1 phenotype. TLR4 is a type of pattern recognition receptor and once activated, can prompt NFkB activation through nuclear translocation [21]. Activated NFkB subsequently promotes the expression of a variety of pro-inflammatory factors, activates anti-apoptotic genes, and promotes the activation of cell proliferation. Specifically, active NFkB can promote the expression of IL-1β, IL-6, TNF-α, iNOS, and other inflammatory factors, and regulate microglial polarization [22]. In addition, NFkB binds to the TLR4 gene promoter and promotes its expression [23]. Thus, harnessing TLR4/NFkB signaling is a crucial mechanism during the induction of a neuroinflammatory response. In this study, we found that luteolin can decrease TLR4 and p65 phosphorylation, indicating that TLR4/NFκB pathway is, at least in part, a target for luteolin in limiting neuroinflammation and dopaminergic neuron degeneration.
We note some limitations in this study. For example, the concentration of luteolin in mouse brain tissue is unclear. More reliable methods for judging drug distribution are needed to better understand how luteolin reaches the brain. Tissue distribution of luteolin may be considered using in vivo imaging of animals or through HPLC approaches. In addition, the anti-inflammatory effect of luteolin in TLR4-overexpressing microglial cells requires further examination.