All animal experiments were approved by the Affiliated Nanhua Hospital (2024-KY-059). C57BL/6 mice (20–22 g) aged 6–8 weeks were kept with free access to water and food. Mice were trained to adapt a behavioral device for five consecutive days and mice that exhibited poor motor ability in this adjustment were not included in further study. A total of 40 animals were enrolled in this experiment and randomly allocated to each group. On day 6, mice were anesthetized with a mixture of xylazine (7361-61-7; Merck, Rahway, NJ, USA) (10 mg/kg, i.p.) and ketamine (H20023609; Jiuxu Pharmaceutical Co., LTD, Jinhua, Zhejiang, China) (90 mg/kg, i.p.). Following this, mice were injected with lipopolysaccharide (LPS) (5 µg dissolved in 2 µL PBS, C0221A, Beyotime, Shanghai, China) or saline vehicle following stereotaxic coordinates measured from the bregma (Lateral: 1.3 mm; Posterior: 2.8 mm; Ventral to the surface of the dura mater: 4.5 mm). The needle was retained in the left side of the substantia nigra for a period of 10 minutes. The day after LPS injection, mice were intra-peritoneally injected with 40 mg/kg/d luteolin (Catalog: L409168; Aladdin, Shanghai, China), or saline for consecutive 9 days. Subsequently, mice were subjected to behavioral tests to evaluate motor performance on days 16 and 17. Afterward, mice were sacrificed by decapitation, and samples were collected for further analysis. The procedure is given in Fig. 1.

Fig. 1.

The schedule used in this study. LPS, lipopolysaccharide.

BV2 microglia or PC12 neuron-like cells were purchased from Procell Life Science & Technology (Wuhan, Hubei, China) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, Anaheim, CA, USA) supplemented with 10% fetal bovine serum (FBS) and 1% streptomycin/penicillin. All cell lines were validated and tested negative for mycoplasma contamination. BV2 and PC12 were both cultured in a 5% CO₂ incubator at 37 °C. BV2 were pretreated with luteolin (40 µM) or saline solution for 3 h after which BV2 were challenged with either LPS (1 µg/mL) or saline (control) for 12 h. For establishing the indirect co-culture system, PC12 was incubated in a 24-well plate for 48 h, BV2 was then transferred to a 0.4 µm pore-sized Transwell insert and co-cultured with PC12 (PC12:BV2 = 2:1) for another 48 h.

The animals were subjected to rotarod test to examine the strength and coordination. Each mouse was placed on an accelerated rotating rod (30 revolutions per minute) and the latency period to fall from the rod was recorded. If animal did not fall from the rod within 300 seconds, a maximum of 300 seconds was recorded. Each experiment was repeated three times and the mean value was calculated.

A ball wrapped with gauze was fixed on a rough rod (diameter: 0.8 cm; Height: 60 cm). Mice were placed on the ball with its head vertically up. The time it took to completely turn its head downward was recorded as T-turn, and the time it took to climb down to the ground was recorded as T-D. If the mouse fell off the pole, the data was not recorded. When mice stayed on the pole for more than 120 seconds, a maximum value of 120 seconds was recorded.

Animal were transcardially perfused with saline followed by infusion of 4% paraformaldehyde through the left ventricle. Following this, the brain tissue was dissected from the skull, and tissue was placed in 4% paraformaldehyde (PFA) and left overnight at 4 °C, after which the brain tissues were preserved in 30% sucrose solution until the tissue sank. The fixed tissue was sliced using a sliding microtome and brain sections were rinsed and subsequently incubated overnight with primary antibodies (anti-tyrosine hydroxy lase, 1:400; Abcam, Cambridge, UK, cat: ab75875). For immunohistochemistry, sections were incubated with anti-tyrosine hydroxylase (1:400; Abcam, Cambridge, UK, cat: ab75875), followed by enzyme-conjugate IgG (1:50; Beyotime, Shanghai, China) secondary antibodies.

For immunofluorescence, BV2 was incubated with primary antibody against TLR4 (1:100; Santa Cruz, Dallas, TX, USA, cat: sc-293072). After several washings, cells were incubated with goat anti-mouse secondary antibodies CY3 (1:8000; Abcam, Cambridge, UK) in a dark room. The photos were taken using a confocal fluorescence microscope (Leica, Heidelberg, Germany). Immunohistochemistry (IHC) stained cells were examined using a brightfield microscope (Leica, Heidelberg, Germany).

Appropriate amounts of tissue were exhaustively chopped and homogenized. Following this, tissues were then transferred into a centrifuge tube, and 3 mL of 10% sodium carbonate solution and 10 mL ethyl acetate were added to the tube. Tissues were subsequently homogenized by shaking for 10 min at 4 °C. After centrifugation (6000 r/min for 10 min), the upper layer organic phase was transferred into a pear-shaped bottle and then subjected to rotary evaporation to dry at 40 °C. The residue was dissolved in 1 mL 50% acetonitrile solution and this solution was cooled for 30 min and centrifuged at 16,000 r/min for 5 min. An appropriate amount of the supernatant was filtered through a 0.22 µm membrane and then analyzed by liquid chromatography–mass spectrometry (LC-MS/MS). The devices used in this study are a Waters Acuity UPLC liquid chromatography (Waters, Milford, MA, USA), an AB SCIEX 5500 Qtrap-MS mass spectrography (AB SCIEX, Framingham, MA, USA), and an Acquity UPLC HSS T3 chromatographic column (2.1 µm $\times$ 1.8 mm $\times$ 100 mm; Waters, USA).

Trizol reagent (Sigma-Aldrich, St. Louis, MO, USA) was applied to extract total RNA from BV2 microglial cells or mesencephalic tissue. A PrimeScript RT reagent kit (Takara, Kusatsu, Japan) was then applied to reverse transcribed RNA into cDNA. RT-qPCR was performed using a 7300 Plus Real-time PCR System (Thermo Fisher, Waltham, MA, USA) and an SYBR Green kit (Takara, Japan). Thermocycling was conducted using the following conditions: Denaturation at 95 °C for 15 s, 40 cycles at 95 °C for 10 s, 60 °C for 30 s. Relative quantitative values were calculated using the 2^{–Δ⁢Δ⁢Cq} method. Each 20 µL reaction mixture contained 10 µL PCR mixture, 1 µL cDNA template, 5 pmol primer, and a proper amount of water. Primers applied in this experiment are outlined in Table 1.

The levels of Interleukin (IL)-6, IL-1$\beta$, and tumor necrosis factor-alpha (TNF-$\alpha$) proteins in the mesencephalic tissue and BV2 were determined using Enzyme-linked Immunosorbent Assay (ELISA) following the manufacturer’s manual (Beyotime, China).

PC12 apoptosis when co-cultured with BV2 cells was determined by flow cytometry. PC12 were fixed and permeabilized, cells were subsequently stained with propidium iodide and Annexin (AP101, Multi Sciences, Hangzhou, Zhejiang, China) following the manufacturer’s manual. Stained cell preparations were run and analyzed on a flow cytometer (CytoFLEX, Beckman, Pasadena, CA, USA).

Cells were seeded into a 96-well plate at 1 $\times$ 10⁴ cells per well. A proper amount of Cell Counting kit-8 (CCK-8; Beyotime, C0039, China) buffer was added to each well, and cells were cultured at 37 °C for 2 hours. A microplate reader was used to acquire absorbance at 450 nm.

Radio immunoprecipitation assay (RIPA, Beyotime, Shanghai, China) buffer containing Phenylmethanesul fonyl fluoride (PMSF) was applied to homogenize tissues and cells. Proteins were resolved on 10% liquid chromatography-mass spectrometry (SDS-PAGE), and subsequently transferred to the polyvinylidene fluoride (PVDF) membrane. After blocking for 1 hr, membranes were incubated with primary antibody TLR4 (1:100; Santa Cruz, USA, cat: sc-293072), phospho-nuclear factor kappa-gene binding (NF-$\kappa$B) p65 (1:800; CST, Danvers, MA, USA, cat: #3033), $\beta$-actin (1:1000; HuaBio, Hangzhou, Zhejiang, China, cat: ET1701-80), and TATA-binding protein (TBP) (1:1000; HuaBio, China, cat: HA500518). Membranes were subsequently incubated with peroxide-labeled secondary antibodies. Immune complexes were detected using enhanced chemiluminescence (Millipore, Burlington, MA, USA), and results were recorded using a chemiluminescence imaging system (Biorad, Berkeley, CA, USA).

All data presented are expressed as mean $\pm$ SEM. Data were analyzed using one-way ANOVA with Tukey’s post-test. Data were analyzed using SPSS 22 software (IBM Corp., Armonk, NY, USA). p 0.05 is considered statistically significant.

To assess the neuroprotective responses of luteolin in the outlined PD model, we counted the number of Th positive cells in the substantia nigra (SN) using immunohistochemical staining (Fig. 2A). We found that LPS injection lead to 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 abundance of dopamine (DA) and its metabolites, homovanillic acid (HVA) and dihydroxy-phenyl acetic acid (DOPAC) in the striatum using LC-MS/MS. As shown in Fig. 2C, mice injected with LPS lead to decreased abundance of DA (p 0.001), HVA (p = 0.0016), and DOPAC (p 0.001) 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.

Fig. 2.

Luteolin mitigates 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 = 100 µm. (B) Th positive cells number within the SN was examined. (C) Abundance of DA, HVA, and DOPAC 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. LPS, lipopolysaccharide; PD, Parkinson’s disease; DA, dopamine; HVA, homovanillic acid; SN, substantia nigra; DOPAC, dihydroxy-phenyl acetic acid. LC-MS/MS, liquid chromatography–mass spectrometry.

Mice were subjected to pole and rotarod tests to assess grip strength and coordination, respectively. LPS injection showed a poorer motor ability 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.

Fig. 3.

Luteolin improves motor performance in the PD model. (A) The time for each mouse to turn its head completely downward (T-turn) and climb down to the ground (T-D) was recorded in the pole test. (B) The latency to fall off the rotating rod was recorded in the rotarod test. N = 10 per group. ***p 0.001 compared to the sham group, #p 0.05, ##p 0.01 compared to the LPS group.

Microglial polarization is vital for neuroinflammation and neuronal degeneration in PD. Thus, we measured relative mRNA abundance of several M1/M2 polarization markers in the midbrain using RT-qPCR. LPS injection reduced anti-inflammatory M2 phenotype markers like Arginase-1 (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 injection elevated the abundance of pro-inflammatory M1 markers such as CD32 (p 0.001), inducible nitric oxide synthase (iNOS) (p 0.001), and TNF-$\alpha$ (p 0.001) (Fig. 4B). Luteolin treatment significantly increased the level 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 to LPS-only injected mice (Fig. 4A), and reduced the abundance of M1 markers CD32 (p = 0.04), iNOS (p = 0.006), and TNF-$\alpha$ (p = 0.012) (Fig. 4B). Luteolin treatment in this PD model also restrained microglial hyperactivation, as evidenced by lower ionized calcium-binding adapter molecule 1 (IBA-1) mRNA level when compared to LPS-only group (p = 0.007, Fig. 4C).

Fig. 4.

Luteolin shifts M1/M2 polarization and inhibits the release of pro-inflammatory cytokines in the PD model. (A) Relative mRNA abundance of microglial M2 polarization phenotypic markers Arg-1, CD206, and IL-10. (B) Relative mRNA abundance of microglial M1 polarization markers CD32, iNOS, and TNF-$\alpha$. (C) Relative mRNA abundance of IBA-1. (D) Pro-inflammatory cytokine abundance 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, ###p 0.001 compared to the LPS-only group.

We next measured the abundance of indicated pro-inflammatory cytokines in the midbrain using ELISA. LPS injection resulted in an elevated level of pro-inflammatory cytokine release in the midbrain; specifically, IL-6 (p 0.001), TNF-$\alpha$ (p 0.001), and IL-1$\beta$ (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 (IL-6: p = 0.009; TNF-$\alpha$: p = 0.009; IL-1$\beta$: p 0.001) 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.

We next measured relative mRNA levels of M1/M2 phenotypic markers in BV2. As expected, following LPS challenge, BV2 cells exhibited decreased levels 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 levels of M1 markers CD32 (p 0.001), iNOS (p 0.001), and TNF-$\alpha$ (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 the abundance of M1 markers CD32 (p = 0.002), iNOS (p = 0.031), and TNF-$\alpha$ (p = 0.02) compared to mice injected with LPS alone (Fig. 5B). Luteolin pretreatment prior to LPS challenging strikingly limited microglial activation, as evidenced by lower IBA-1 mRNA abundance when compared to LPS-only group (p = 0.027, Fig. 5C). We further observed that luteolin pretreatment reduced LPS-induced pro-inflammatory cytokines release in protein extracts of BV2. Specifically, we measured lower IL-6 (p = 0.038), TNF-$\alpha$ (p = 0.028), and IL-1$\beta$ (p = 0.003) abundance in BV2 cells following both luteolin and LPS administration (Fig. 5D). These data clearly support an anti-inflammatory effect of luteolin in BV2 cells treated with LPS.

Fig. 5.

Luteolin shifts microglial M1/M2 polarization and limited the release of pro-inflammatory cytokines in BV2 microglia cells challenged with LPS. (A) Relative mRNA abundance of microglial M2 phenotypic markers Arg-1, CD206, and IL-10. (B) Relative mRNA abundance of microglial M1 phenotypic markers CD32, iNOS, and TNF-$\alpha$. (C) Relative mRNA abundance of IBA-1. (D) Pro-inflammatory cytokines abundance in BV2 cells were detected using ELISA. N = 5 per group. **p 0.01, ***p 0.001 compared to the Control (sham) group, #p 0.05, ##p 0.01 compared to the LPS-only group. ELISA, Enzyme-linked Immunosorbent Assay

We established a microglia/neuron indirect co-culture system to explore the neuroprotective actions of luteolin-treated microglia on dopaminergic neurons (Fig. 6A). A 0.4 µm pore-sized membrane was used to separate BV2 and PC12 cells. After treatment with LPS, luteolin, or LPS combined with luteolin, BV2 cells were then transferred to the co-culture system and co-cultured with PC12 for an additional 48 h. After this, PC12 apoptosis was measured by flow cytometry (Fig. 6B), and cell survival was measured using a CCK-8 assay. For the analysis of apoptosis, pretreatment of BV2 cells with luteolin significantly decreased the apoptotic response of PC12 cells (p = 0.002) compared to BV2 cells challenged with LPS-only (Fig. 6C). For the CCK-8 cell viability analysis, pretreatment with luteolin in BV2 prior to LPS administration remarkably increased cell viability of PC12 (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.

Fig. 6.

Luteolin pretreatment before LPS challenging in BV2 rescues neuronal injury in the co-cultured system. (A) A sketch of the co-culture system used. (B) Cellular apoptosis examined by flow cytometry. (C) Bar graph showing the cellular apoptosis. (D) Cell viability was examined by Cell Counting kit-8 (CCK-8). N = 4 per group. ***p 0.001 compared to the control group, #p 0.05, ##p 0.01 compared to the LPS-only group. PI, propidium iodide; UL, up left; UR, up right; LL, low left; LR, low right.

TLR4/NF-$\kappa$B signaling plays a pivotal role in microglial-mediated neuroinflammation. Thus, we next evaluated whether the anti-inflammatory potential of luteolin was associated with TLR4/NF-$\kappa$B signaling in both in vivo and in vitro experimental systems. Increased relative TLR4 mRNA abundance was observed upon LPS challenge in mesencephalic tissue (p 0.001; Fig. 7A) dissected from LPS-treated PD model mice, and in LPS-treated cultured BV2 cells (p 0.001; Fig. 7B). This rise in TLR4 expression was inhibited by luteolin treatment both in vivo (p = 0.016, Fig. 7A) and in vitro (p = 0.004; Fig. 7B). We also examined the intensity of TLR4 staining in BV2 cells using immunofluorescence microscopy (Fig. 7C). Compared to the control group, BV2 cells challenged with LPS resulted in a higher intensity of TLR4 staining (p 0.001; Fig. 7D). In contrast, luteolin pretreatment prior to LPS challenge decreased the fluorescence intensity of TLR4 (p = 0.002; Fig. 7D). Immunoblot analysis also indicated that luteolin treatment significantly decreased LPS-induced increases in TLR4 within dissected mesencephalic tissue (p = 0.029; Fig. 7E) and cultured BV2 cells (p = 0.016; Fig. 7F).

Fig. 7.

Luteolin treatment reduces LPS-induced TLR4/NF-$\kappa$B 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. NF-$\kappa$B, nuclear factor kappa-gene binding; TBP, TATA binding protein.

LPS treatment was also observed to significantly increase the abundance of phosphorylated (activated) NF-$\kappa$B subunit p65 both in vivo (p 0.001; Fig. 7G) and in vitro (p 0.001; Fig. 7H). Luteolin treatment strikingly downregulated LPS-induced, phosphorylated p65 abundance in the PD mouse model (p = 0.024; Fig. 7G) and cultured BV2 cells (p = 0.0035; Fig. 7H). These data provide evidence that the anti-inflammatory activity of luteolin observed in both the in vivo PD model, and in vitro cultured microglial cells is likely associated, in part, with diminished TLR4/NF-$\kappa$B signaling.