Baicalein inhibits macrophage lipid accumulation and inflammatory response by activating the PPARγ/LXRα pathway

Abstract

Lipid accumulation and inflammatory response are two major risk factors for atherosclerosis. Baicalein, a phenolic flavonoid widely used in East Asian countries, possesses a potential atheroprotective activity. However, the underlying mechanisms remain elusive. This study was performed to explore the impact of baicalein on lipid accumulation and inflammatory response in THP-1 macrophage-derived foam cells. Our results showed that baicalein up-regulated the expression of ATP binding cassette transporter A1 (ABCA1), ABCG1, liver X receptor α (LXRα), and peroxisome proliferator-activated receptor γ (PPARγ), promoted cholesterol efflux, and inhibited lipid accumulation. Administration of baicalein also reduced the expression and secretion of TNF-α, IL-1β, and IL-6. Knockdown of LXRα or PPARγ with siRNAs abrogated the effects of baicalein on ABCA1 and ABCG1 expression, cholesterol efflux, lipid accumulation as well as pro-inflammatory cytokine release. In summary, these findings suggest that baicalein exerts a beneficial effect on macrophage lipid accumulation and inflammatory response by activating the PPARγ/LXRα signaling pathway.

Keywords: LXRα; PPARγ; baicalein; inflammatory response; lipid accumulation.

Graphical abstract

Figure 1

effects of baicalein on cell viability and intracellular lipid accumulation. (A) Chemical structure of baicalein. (B) THP-1 macrophage-derived foam cells were treated with the indicated concentrations of baicalein for 24 h or with 100 μM baicalein for various times. MTT assay was employed to evaluate cell viability. (C–D) THP-1 macrophage-derived foam cells were incubated with PBS or 100 μM baicalein for 24 h. (C) The cells were fixed and then stained with Oil Red O to assay intracellular lipid deposition. The magnification of each panel is 200×. (D) Intracellular TC, FC, and CE amounts were detected by HPLC. Data are expressed as mean ± SD from three independent experiments. *P < 0.05 vs 0 μM or 0 h groups.

Figure 2

Baicalein facilitates ABCA1- and ABCG1-dependent cholesterol efflux. (A–D) THP-1 macrophage-derived foam cells were exposed to PBS or 100 μM baicalein for 24 h. (A) The mRNA and protein levels of ABCA1 and ABCG1 were determined by qRT-PCR and western blot, respectively. (B) Cholesterol efflux to apoA-I and HDL was analyzed by the liquid scintillation counting method. (C) Detection of CD36 and SR-A expression using qRT-PCR and western blot. (D) Representative fluorescent images of Dil-ox-LDL uptake. The magnification of each panel is 200×. All data are presented as the mean ± SD from three independent experiments. **P < 0.01 vs. control group.

Figure 3

involvement of LXRα in the induction of ABCA1 and ABCG1 expression by baicalein. (A) THP-1 macrophage-derived foam cells were incubated with PBS or 100 μM baicalein for 24 h, followed by measurement of LXRα expression using qRT-PCR and western blot. (B–E) THP-1 macrophage-derived foam cells were transfected with scrambled siRNA or LXRα siRNA for 48 h and then incubated with or without 100 μM baicalein for 24 h. (B) Cell lysates were subjected to western blot analysis to assess the protein levels of LXRα. (C–D) The expression of ABCA1 and ABCG1 were evaluated using qRT-PCR and western blot. (E) Cholesterol efflux to apoA-I and HDL was detected by the liquid scintillation counting method. Data are expressed as mean ± SD from three independent experiments. **P < 0.01 vs control group; ^#P < 0.05, ^##P < 0.01 vs baicalein group.

Figure 4

involvement of PPARγ in the baicalein-induced enhancement of LXRα, ABCA1, and ABCG1 expression. (A–B) THP-1 macrophage-derived foam cells were treated with or without 100 μM baicalein for 24 h. (A) PPARγ expression was analyzed by qRT-PCR and western blot. (B) Western blot analysis of PPARγ levels in the cytoplasm and nucleus. (C–E) THP-1 macrophage-derived foam cells were transfected with scrambled siRNA or PPARγ siRNA for 48 h, followed by treatment with or without 100 μM baicalein for 24 h. (C) Detection of PPARγ protein expression using western blot. (D) The expression of LXRα, ABCA1, and ABCG1 was determined by qRT-PCR and western blot. (E) The liquid scintillation counting method was used to measure the efflux of cholesterol onto apoA-I and HDL. The results are shown as the mean ± SD from three independent experiments. **P < 0.01 vs. control group; ^#P < 0.05, ^##P < 0.01 vs. baicalein group.

Figure 5

Baicalein inhibits inflammatory response through the PPARγ/LXRα signaling pathway. (A–F) THP-1 macrophages were incubated with or without 50 μg/ml ox-LDL for 48 h, transfected with or without siRNAs against LXRα or PPARγ for 48 h, and then treated with 100 μM baicalein for 24 h. (A, C–E) The mRNA levels of TNF-α, IL-1β, and IL-6 were determined using qRT-PCR. (B, D–F) The cell culture supernatant was collected and then subjected to ELISA for detecting the levels of TNF-α, IL-1β, and IL-6. Data are the mean ± SD from three independent experiments. ***P < 0.001 vs. control group; ^##P < 0.01 vs. ox-LDL group; ^&&P < 0.01 vs. ox-LDL + baicalein group.