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.