Results
Luteolin treatment protects against LPS-induced
dopaminergic neuronal loss in a PD animal model
To evaluate the potential neuroprotective effects of luteolin in the
outlined PD model, we measured Th-positive cells in the Substantia Nigra
using immunohistochemical staining (Fig. 2A). We observed that LPS
injection resulted in a remarkable decrease of dopaminergic neurons in
the SN (p < 0.001, Fig 2B), and that luteolin treatment
attenuated LPS-induced dopaminergic neuronal loss when LPS-only treated
mice are compared to the LPS+luteolin group (p = 0.02; Fig. 2B).
We also measured the content of dopamine (DA) and its metabolites in the
striatum using LC-MS/MS. As shown in Figure 2C, when compared to the
sham group, LPS injection resulted in decreased DA (p <
0.001), DOPAC (p < 0.001), and HVA (p = 0.0016)
abundance within the striatum. In contrast, treatment with luteolin
effectively prevented DA and metabolite loss in the PD model (DA:p = 0.007; DOPAC: p = 0.02; HVA: p = 0.016; Fig.
2C). These data indicated that luteolin alleviates LPS-induced
dopaminergic neuronal injury in vivo .
Luteolin treatment results in functional improvement in
the PD model
Mice were subjected to pole and rotarod tests to assess grip strength
and coordination, respectfully. Mice injected with LPS exhibited a
poorer performance in the pole test (T-turn: p < 0.001;
T-D: p < 0.001, Fig. 3A) and rotarod test (p< 0.001, Fig. 3B) when compared to sham control animals.
Luteolin treatment significantly improved motor performance when
compared to LPS-only injected animals in both the pole (T-turn: p= 0.025; T-D: p = 0.007, Fig. 3A) and rotarod test (p =
0.003, Fig. 3B). These data suggest that luteolin treatment can improve
motor function in our PD mouse model.
Luteolin treatment shifted microglial M1/M2 polarization
and restrained pro-inflammatory cytokine release in the PD model
Microglial polarization is vital for neuroinflammation and neuronal
degeneration in PD. Thus, we measured relative mRNA levels of several
M1/M2 polarization markers in the midbrain using RT-qPCR. LPS injection
reduced anti-inflammatory M2 phenotype markers such as Arg-1 (p< 0.001), CD206 (p < 0.001), and IL-10
(p < 0.001) when sham control animals were compared to
LPS-injected mice (Fig. 4A). In contrast, LPS promoted expression of
pro-inflammatory M1 markers such as CD32 (p < 0.001),
iNOS (p < 0.001), and TNF-α (p <
0.001) (Fig. 4B). Luteolin treatment significantly increased the
expression of anti-inflammatory M2 markers Arg-1 (p = 0.006),
CD206 (p = 0.005), IL-10 (p = 0.029), when luteolin+LPS
mice were compared LPS-only injected mice (Fig. 4A), and reduced the
expression of M1 markers CD32 (p = 0.04), iNOS (p =
0.006), and TNF-α (p = 0.012) (Fig. 4B). Luteolin treatment in
this PD model also inhibited microglial activation, as evidenced by
lower IBA-1 mRNA expression when compared to LPS-only group. (p =
0.007, Fig. 4C).
We next measured the abundance of pro-inflammatory cytokines in the
midbrain using ELISA. LPS injection resulted in an elevated level of
pro-inflammatory cytokine release in the midbrain; specifically, TNF-α:
(p < 0.001), IL-1β (p < 0.001), and
IL-6 (p < 0.001) abundance increased in response to LPS
injection (Fig. 4D). This rise in inflammatory cytokines was
significantly blunted in response to luteolin treatment (TNF-α: p= 0.009; IL-1β: p < 0.001; IL-6: p = 0.009) when
LPS injected mice were compared to those receiving both luteolin and LPS
(Fig. 4D). These findings support a potential anti-inflammatory effect
for luteolin in this LPS-induced PD mouse model.
Luteolin treatment shifts microglial M1/M2 polarization
and restrains pro-inflammatory cytokine release in BV2 microglial
cells challenged with LPS
We next measured relative mRNA levels of M1/M2 phenotypic markers in BV2
microglial cells. As expected, following LPS challenge, BV2 cells
exhibited decreased expression of the M2 markers Arg-1 (p< 0.001), CD206 (p < 0.001), and IL-10
(p < 0.001) when compared to the control group (Fig.
5A). Coordinately, we measured increased expression of M1 markers CD32
(p < 0.001), iNOS (p < 0.001), and
TNF-α (p < 0.001) following LPS challenge (Fig. 5B).
Luteolin pretreatment significantly increased expression of M2 markers
Arg-1 (p = 0.035), CD206 (p = 0.009), and IL-10 (p= 0.025) (Fig. 5A), and inhibited expression of M1 markers CD32
(p = 0.002), iNOS (p = 0.031), and TNF-α (p = 0.02)
compared to mice injected with LPS alone (Fig. 5B). Luteolin
pretreatment prior to LPS challenging significantly inhibited microglial
activation, as evidenced by lower IBA-1 mRNA expression when compared to
LPS-only group. (p = 0.027, Fig. 5C). In addition, we observed
that luteolin pretreatment reduced LPS-induced pro-inflammatory
cytokines release in protein extracts of BV2. Specifically, we measured
lower TNF-α (p = 0.028), IL-1β (p = 0.003), and IL-6
(p = 0.038) levels in BV2 cells following both luteolin and LPS
administration (Fig. 5D). These data clearly support an
anti-inflammatory effect of luteolin in BV2 cells challenged with LPS.
Luteolin treatment in BV2 attenuated neuronal injury in
the co-culture system
We established a microglia/neuron co-culture system to explore the
potential indirect neuroprotective effects of luteolin-treated microglia
on dopaminergic neurons (Fig. 6A). A 0.4 μm pore-sized membrane was used
to separate BV2 and SH-SY5Y cells. After treatment with LPS, luteolin,
or LPS combined with luteolin, BV2 cells were transferred to the
co-culture system and co-cultured with SH-SY5Y for an additional 48 h.
After this, SH-SY5Y apoptosis was measured by flow cytometry (Fig 6B),
and cell survival was measured using a CCK8 assay. In the analysis of
apoptosis, pretreatment of BV2 cells with luteolin significantly reduced
the apoptotic response of SH-SY5Y cells (p = 0.002) compared to
BV2 cells treated with LPS-only (Fig. 6C). In the CCK8 cell viability
analysis, pretreatment with luteolin in BV2 prior to LPS administration
remarkably increased cell viability of SH-SY5Y (p = 0.027) when
compared to LPS-only cells (Fig. 6D). These in vitro results
suggest that luteolin treatment of BV2 cells robustly ameliorates
inflammation-induced neuronal injury.
Luteolin treatment reduces LPS-induced activation of
TLR4/ NFkB signaling
in the PD model and BV2 cells.
TLR4/NFkB signaling plays a pivotal role in microglial-mediated
neuroinflammation. Thus, we next evaluated whether the anti-inflammatory
potential of luteolin was associated with TLR4/NFkB signaling in bothin vivo and in vitro experimental systems. Increased
relative TLR4 mRNA abundance was observed upon LPS challenge in
mesencephalic tissue (p < 0.001; Fig. 6A) dissected
from LPS-treated PD model mice, and in LPS-treated cultured BV2 cells
(p < 0.001; Fig. 6B). This rise in TLR4 expression was
inhibited by luteolin treatment both in vivo (p = 0.016, Fig. 6A)
and in vitro (p = 0.004; Fig. 6B). We also examined the
intensity of TLR4 staining in BV2 cells using immunofluorescence
microscopy (Fig. 6C). Compared to the control group, BV2 cells
challenged with LPS resulted in a higher intensity of TLR4 staining
(p < 0.001; Fig. 6D). In contrast, luteolin
pretreatment prior to LPS challenge decreased the fluorescence intensity
of TLR4 (p = 0.002; Fig. 6D). Immunoblot analysis also indicated
that luteolin treatment significantly decreased LPS-induced increases in
TLR4 within dissected mesencephalic tissue (p = 0.029; Fig. 6E)
and cultured BV2 cells (p = 0.016; Fig. 6F).
LPS treatment was also observed to significantly increase the abundance
of phosphorylated (activated) NFkB subunit p65 both in vivo (p
< 0.001; Fig. 6G) and in vitro (p < 0.001;
Fig. 6H). Luteolin treatment significantly downregulated LPS-induced,
phosphorylated p65 abundance in the PD mouse model (p = 0.024;
Fig. 6G) and cultured BV2 cells (p = 0.0035; Fig. 6H). These data
provide evidence that the anti-inflammatory activity of luteolin
observed in both the in vivo PD model, and in vitrocultured microglial cells is likely associated, in part, with diminished
TLR4/NFkB signaling.