Protective Effect of Ferulic Acid on Lipopolysaccharide-Induced BV2 Microglia Inflammation via AMPK/mTOR Signaling Pathway

Abstract

In neurodegenerative diseases, microglial activation and neuroinflammation are essential for the control and progression of neurodegenerative diseases. Mitigating microglium-induced inflammation is one strategy for hindering the progression of neurodegenerative diseases. Ferulic acid (FA) is an effective anti-inflammatory agent, but its potential role and regulation mechanism in neuroinflammatory reactions have not been fully studied. In this study, the neuroinflammation model was established by lipopolysaccharide (LPS), and the inhibitory effect of FA on neuroinflammation of BV2 microglia was studied. The results showed that FA significantly reduced the production and expression of reactive oxygen species (ROS), tumor necrosis factor-α (TNF-α), leukocyte-6 (IL-6) and interleukin-1β (IL-1β). We further studied the mechanism of FA's regulation of LPS-induced BV2 neuroinflammation and found that FA can significantly reduce the expression of mTOR in BV2 microglia induced by LPS, and significantly increase the expression of AMPK, indicating that FA may have an anti-inflammatory effect by activating the AMPK/mTOR signaling pathway to regulate the release of inflammatory mediators (such as NLRP3, caspase-1 p20 and IL-1β). We further added an autophagy inhibitor (3-MA) and an AMPK inhibitor (compound C, CC) for reverse verification. The results showed that FA's inhibitory effects on TNF-α, IL-6 and IL-1β and its regulatory effect on AMPK/mTOR were destroyed by 3-MA and CC, which further indicated that FA's inhibitory effect on neuroinflammation is related to its activation of the AMPK/mTOR autophagy signaling pathway. In a word, our experimental results show that FA can inhibit LPS-induced neuroinflammation of BV2 microglia by activating the AMPK/mTOR signaling pathway, and FA may be a potential drug for treating neuroinflammatory diseases.

Keywords: AMPK/mTOR signaling pathway; BV2 microglia; NLRP3 inflammasome; autophagy; ferulic acid; neuroinflammation.

Figure 1

The effects of LPS and FA on BV2 microglial viability. (A) Effects of different concentrations of LPS on cell viability. (B) Effects of different concentrations of FA on cell viability. Data are means ± standard deviations (SDs) (n = 3).

Figure 2

FA inhibited the LPS-induced ROS and NLRP3 (green) expression in a dose-dependent manner. (A) The expression of ROS (20× magnification) was observed under a fluorescence microscope. DHE staining results. Scale bar = 50 μM. (B) The expression of NLRP3 (50× magnification) in BV2 microglia was observed via inverted fluorescence microscope. (C) This figure shows the results of fluorescence statistics of NLRP3. The nuclei were stained with Hoechst 33,342 (blue). Scale bar = 20 μM. Data are means ± standard deviations (SDs) (n = 3). ** p < 0.01, vs. control; ^## p < 0.01, vs. LPS.

Figure 3

Effects of FA on the production of LPS-activated inflammatory cytokines in BV2 microglia BV2 microglia pretreated with different doses of FA for 2 h and then treated with 1 μg/mL of LPS for 12 h. (A–D) The expression levels of IL-1β, IL-6, TNF-αand NLRP3 were detected by reverse transcription-real-time quantitative PCR (qPCR). (E–G) The expression levels of IL-6, IL-1β and TNF-α in the supernatants left by BV2 microglia were determined by ELISA. Data are means ± standard deviations (SDs) (n = 3). ** p < 0.01, vs. control; ^## p < 0.01, ^# p < 0.05, vs. LPS.

Figure 4

FA inhibits pro-inflammatory mediators and associated key targets in LPs-induced BV2 microglia. (A–C) NLRP3, IL-1β and caspase-1 p20 were detected by Western blot. FA restored the autophagy of BV2 microglia inhibited by LPS. (D,E) Autophagy-related proteins P62 and LC3 were detected by Western blot. (F–I) Western blot analysis showed the expression of P-AMPK/GAPDH, AMPK/GAPDH, P-mTOR/GAPDH and mTOR/GAPDH proteins. (N,O) The expression of NLRP3 (50× magnification) in BV2 microglia was observed via inverted fluorescence microscope. The nuclei were stained with Hoechst 33,342 (blue). Scale bar = 20 μM. Data are means ± standard deviations (SDs) (n = 3). ** p < 0.01, * p < 0.05, vs. control; ^## p < 0.01, ^# p < 0.05, vs. LPS.

Figure 5

Autophagy inhibitors (3-MA) block the protective effects of FA: restoring autophagy and inhibiting inflammation levels. (A) ROS was detected via DCF-DA fluorescent probe. (B–E) The qPCR kit was used to detect autophagy-related proteins P62 and LC3; and NLRP3 and IL-1β. (F–I) The expression of NLRP3 and P62 (50× magnification) in BV2 microglia was observed via inverted fluorescence microscope. The nuclei were stained with Hoechst 33,342 (blue). Scale bar = 20 μM. (N–P) Molecular docking results of ferulic acid with LC3, NLRP3 and P62 proteins. Data are means ± standard deviations (SDs) (n = 3). ** p < 0.01, * p < 0.05, vs. control; ^## p < 0.01, ^# p < 0.05, vs. LPS; ^▲▲ p < 0.01, vs. FA + LPS.

Figure 6

Autophagy inhibitors (3-MA) undermine all beneficial effects of FA. (A–I) NLRP3, IL-1β and caspase-1 p20 were detected by Western blot. Autophagy-related proteins LC3 and P62 and pathway proteins P-AMPK/GAPDH, AMPK/GAPDH, P-mTOR/GAPDH and mTOR/GAPDH were expressed. Data are means ± standard deviations (SDs) (n = 3). ** p < 0.01, * p < 0.05, vs. control; ^## p < 0.01, ^# p < 0.05, vs. LPS; ^▲▲ p < 0.01, ^▲ p < 0.05 vs. FA + LPS.

Figure 7

AMPK inhibitors destroy all beneficial effects of FA. (A–I) NLRP3, IL-1β and caspase-1 p20 were detected via Western blot. Autophagy-related proteins LC3 and P62 and pathway proteins P-AMPK/GAPDH, AMPK/GAPDH, P-MTOR/GAPDH and mTOR/GAPDH were expressed. Data are means ± standard deviations (SDs) (n = 3). ** p < 0.01, * p < 0.05, vs. control; ^## p < 0.01, ^# p < 0.05, vs. LPS; ^▲▲ p < 0.01, ^▲ p < 0.05 vs. FA + LPS.