LED enhances anti-inflammatory effect of luteolin (3',4',5,7-tetrahydroxyflavone) in vitro

Abstract

Neuroinflammation is a complex pathological process usually results from abnormal microglial activation, thus, intervention in a microglial stimulation pathway could be a promising approach for the treatment of neurodegenerative diseases. Luteolin is an important bioflavonoid possesses anti-inflammatory properties, which is widely studied over these years. Light emitting diode (LED) therapy is reported to be a potential therapeutic strategy for many diseases including neurodegenerative diseases. However, little is known about the anti-inflammatory effect of LED therapy on activated microglial cells, even less is known whether there is a synergistic anti-inflammatory effect exist in LED and luteolin therapy. In this study, we aimed to confirm the anti-inflammatory effect of luteolin and LED combination therapy in lipopolysaccharide (LPS)-stimulated BV2 microglial cells. We showed that luteolin inhibited LPS-induced cytotoxicity, tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) production through modulation of p38 and extracellular signal-regulated kinase (ERK) signaling in BV2 cells. In addition, LED therapy enhanced the anti-inflammatory effect of luteolin. These results suggest that a synergistic effect between luteolin and LED could be a new effective therapy in relieving neuroinflammation.

Keywords: ERK; IL-6; LED; Neuroinflammation; TNFα; luteolin; p38.

Figure 1

Effects of Luteolin and/or LED on cell viability. The MTT results showed that both luteolin (2.5-10 µM) and luteolin/LED could reverse the inhibitory effects induced by LPS. BV2 cells were treated with various concentrations of luteolin (2.5-10 µM) and/or LPS (10 ng/ml) for 6 h, with or without LED; cell viability was determined by MTT assay. The data represent the means ± standard deviation (SD) (*P<0.05 compared with unstimulated cells; #P<0.05 compared with LED treated cells). The results did not show significant difference between luteolin (2.5-10 µM) groups and luteolin/LED groups.

Figure 2

LED light enhances the effect of luteolin in inhibiting LPS-induced TNFα and IL-6 production. BV2 cells were incubated with luteolin (2.5-10 μM) and/or LPS (10 ng/ml) for 6 h, with or without LED light. ELISA was performed after collecting the supernatant in these groups. The results showed that luteolin (2.5 μM, 5 μM, 10 μM) could suppress the TNFα and IL-6 production levels induced by LPS, while LED could enhance the suppressive effects of luteolin. A. TNFα ELISA. *P<0.05, ^*P<0.05 compared with unstimulated cells; #P<0.05, ^#P<0.05 compared with LPS treated cells; ##P<0.05 compared luteolin/LED groups with luteolin groups in counterparts. B. IL-6 ELISA. *P<0.05, ^*P<0.05 compared with unstimulated cells; #P<0.05, ^#P<0.05 compared with LPS treated cells; ##P<0.05 compared luteolin/LED groups with luteolin groups in counterparts. Data are shown as means ± SEM.

Figure 3

ERK and p38 MAPK pathways are involved in the synergistic effects of LED and luteolin. BV2 cells were incubated with Luteolin (0-10 μM) and/or LPS (10 ng/ml) for 1 h, with or without LED light. The results showed that luteolin (5 μM, 10 μM) could inhibit ERK and p38 phosphorylation levels induced by LPS, while LED could enhance the inhibitory effects of luteolin. A. Western Blotting was performed after collecting the cell lysis in each groups, phospho-ERK (pERK), total ERK (tERK), phospho-p38 (pp38) and total p38 (tp38) were then tested. B. pERK/tERK ratio analysis. *P<0.05, ^*P<0.05 compared with unstimulated cells; #P<0.05, ^#P<0.05 compared with LPS treated cells; ##P<0.05 compared luteolin/LED groups with luteolin groups in counterparts. C. pp38/tp38 ratio analysis. *P<0.05, ^*P<0.05 compared with unstimulated cells; #P<0.05, ^#P<0.05 compared with LPS treated cells; ##P<0.05 compared luteolin/LED groups with luteolin groups in counterparts. Data are shown as means ± SEM.

Figure 4

ERK and p38 MAPK pathways are involved in the synergistic effects of LED and luteolin. BV-2 cells were pretreated with MAPK P38/ERK inhibitors for 1h (SB202190/PD98059, 20 μM), then incubated with LPS (10 ng/ml) and/or Luteolin (10 μM) for 6 h, with or without LED light. ELISA was performed after collecting the supernatant in these groups. The results showed that luteolin, SB202190 and PD98059 could suppress the TNF-α and IL-6 production levels induced by LPS. A. TNF-α ELISA. *P<0.05, ^*P<0.05 compared with unstimulated cells; #P<0.05, ^#P<0.05 compared with LPS treated cells. B. IL-6 ELISA. *P<0.05, ^*P<0.05 compared with unstimulated cells; #P<0.05, ^#P<0.05 compared with LPS treated cells. Data are shown as means ± SEM.

Figure 5

ERK and p38 MAPK pathways are involved in the synergistic effects of LED and luteolin. BV-2 cells were pretreated with MAPK P38/ERK inhibitors for 1 h (SB202190/PD98059, 20 μM), and then incubated with Luteolin and/or LPS for 1 h, with or without LED light. The results showed that luteolin (10 μM) and luteolin/LED combination could inhibit ERK and p38 phosphorylation levels induced by LPS, while SB202190/PD98059 could further inhibit ERK and p38 phosphorylation levels (A and C). Western Blotting was performed after collecting the cell lysis in each groups, p-ERK, ERK, p-P38 and p38 were then tested. (B and D) p-ERK/ERK ratio and p-P38/p38 ratio. Data are shown as means ± SEM.