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Figure legends
Figure 1. The schedule used in this study.
Figure 2. Luteolin protects against LPS-induced neuronal injury in the nigrostriatal system of the PD mouse model. (A) Representative immunohistochemical staining for tyrosine hydroxylase in the SN. Scale bar = 100μm. (B) Number of Th-positive cells within the SN was counted. (B) Abundance of DA, DOPAC, and HVA in the striatum measured by LC-MS/MS. N = 5 per group. **p < 0.01, ***p< 0.001 compared to the sham group. #p < 0.05, ##p < 0.01 compared to the LPS group.
Figure 3. Luteolin improved motor performance in the PD model.(A) In the pole test, the time for each mouse to completely turn its head downward (T-turn) and climb down to the ground (T-D) was recorded. (B) Using a rotarod test, the latency to fall off the rotating rod was recorded. N = 10 per group. ***p < 0.001 compared to the sham group, #p < 0.05, ##p< 0.01 compared to the LPS group.
Figure 4. Luteolin shifted microglial M1/M2 polarization and inhibited pro-inflammatory cytokine release in the PD model. (A) Relative mRNA expression of microglial M2 polarization phenotypic markers Arg-1, CD206, and IL-10. (B) Relative mRNA expression of microglial M1 polarization markers CD32, iNOS, and TNF-α. (C) Relative mRNA expression of IBA-1. (D) Pro-inflammatory cytokine levels in the mesencephalic tissue were measured using ELISA. N = 5 per group. ***p < 0.001 compared to the sham group; # p< 0.05, ##p < 0.01 compared to the LPS-only group.
Figure 5. Luteolin shifts microglial M1/M2 polarization and inhibited pro-inflammatory cytokine release in BV2 microglia cells challenged with LPS. (A) Relative mRNA expression of microglial M2 phenotypic markers Arg-1, CD206, and IL-10. (B) Relative mRNA expression of microglial M1 phenotypic markers CD32, iNOS, and TNF-α. (C) Relative mRNA expression of IBA-1. (D) Pro-inflammatory cytokines levels in BV2 cells were measured using ELISA. N = 5 per group. ***p< 0.001, **p < 0.01 compared to the Control (sham) group, #p < 0.05, ##p < 0.01 compared to the LPS-only group.
Figure 6. Luteolin pretreatment before LPS challenging in BV2 rescued neuronal injury in the co-cultured system. (A) A sketch of the co-culture system used. (B) Apoptosis detected by flow cytometry. (C) Bar graph showing the cellular apoptosis. (D) Cell viability was measured by CCK8. N = 4 per group. ***p < 0.001 compared to the control group, #p < 0.05, ##p< 0.01 compared to the LPS-only group.
Figure 7. Luteolin treatment reduces LPS-induced TLR4/NFkB signaling in vivo and in vitro model systems. (A) Relative TLR4 mRNA expression in mesencephalic tissue dissected from mice in the indicated treatment groups. N = 5 measurements per group. (B) Relative TLR4 mRNA abundance in cultured BV2. N = 5 measurements per group. (C) Representative immunofluorescent confocal images of TLR4 staining in BV2 cells. Scale bar = 20 μm. (D) Mean TLR4 fluorescence intensity in treated and untreated BV2 cells. N = 5 measurements per group. (E) Immunoblots (top ) and bar graph of immunoblot quantification (bottom ) for TLR4 in mesencephalic tissue. N = 3 per group. (F) Immunoblots (top ) and bar graph of immunoblot quantification (bottom ) for TLR4 in treated and untreated BV2 cells. N = 3 per group. (G) Immunoblots (top ) and bar graph of immunoblot quantification (bottom ) for phospho-p65 in mesencephalic tissue. N = 3 per group. (H) Immunoblots (top ) and bar graph of immunoblot quantification (bottom ) for phospho-p65 in treated and untreated BV2 cells. N = 3 per group. ***p< 0.001 compared to the control group, #p < 0.05, ##p < 0.01 compared to the LPS-only group.