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.