Emodin Attenuates Lipopolysaccharide-Induced Acute Liver Injury via Inhibiting the TLR4 Signaling Pathway in vitro and in vivo

Abstract

Aims: Emodin is an anthraquinone with potential anti-inflammatory properties. However, the possible molecular mechanisms and protective effects of emodin are not clear. The objective of this study was to investigate the possible molecular mechanisms and protective effects of emodin on lipopolysaccharide (LPS)-induced acute liver injury (ALI) via the Toll-like receptor 4 (TLR4) signaling pathway in the Raw264.7 cell line and in Balb/c mice. Methods: This study established an inflammatory cellular model and induced an ALI animal model. TLR4 was overexpressed by lentivirus and downregulated by small interfering RNA (siRNA) technology. The mRNA and protein levels of TLR4 and downstream molecules were detected in cells and liver tissue. The tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 levels in supernatant and serum were determined by ELISA. The distribution and expression of mannose receptor C type 1 (CD206) and arginase 1 (ARG1) in the liver were tested by immunofluorescence. Mouse liver function and histopathological observations were assessed. Results: Administration of emodin reduced the protein and/or mRNA levels of TLR4 and its downstream molecules following LPS challenge in Raw264.7 cells and in an animal model. Additionally, emodin suppressed the expression of TNF-α and IL-6 in cell culture supernatant and serum. The inhibitory effect of emodin was also confirmed in RAW264.7 cells, in which TLR4 was overexpressed or knocked down. Additionally, ARG1 and CD206 were elevated in the emodin groups. Emodin also decreased serum ALT and AST levels and alleviated the liver histopathological damage induced by LPS. Conclusion: Emodin showed excellent hepatoprotective effects against LPS-induced ALI, possibly by inhibiting TLR4 signaling pathways.

Keywords: TLR4; acute liver injury; emodin; lipopolysaccharide; signaling pathway.

FIGURE 1

In vitro cytotoxicity of emodin. The cytotoxicity of emodin was evaluated by the CCK8 assay. (A) A CCK-8 assay of RAW264.7 cell viability after emodin treatment. (B) Morphologies of Raw264.7 cells. (C) Morphologies of Raw264.7 cells after emodin treatment for 24 h. Data are shown as the mean ± SD. N = 3.

FIGURE 2

Inflammatory model establishment. (A–D) Effect of LPS stimulation on the expression of TRAF-6 according to western blotting. Data are shown as the mean ± SD. ^∗P < 0.05 vs either time point 0 or LPS untreated group.

FIGURE 3

Evaluation of TNF-α and IL-6 expression in cell supernatants by ELISA. (A,B) Effect of emodin on the expression of TNF-α and IL-6 according to ELISA. Data shown are the mean ± SD. ^#P < 0.05 vs normal group; ^∗P < 0.05, ^∗∗P < 0.01 vs model group.

FIGURE 4

Effect of emodin on TLR4 and downstream molecules after LPS stimulation. (A–C) The mRNA levels of TLR4, MyD88, TIRAP, IRF-5, TRAF-6, TRIF, IRF-3, AP-1, and NF–κB were detected by RT-PCR. (D) The protein levels of the above molecules were detected by western blotting. Data are shown as the mean ± SD. ^#P < 0.05 compared to the normal group. ^∗P < 0.05, ^∗∗P < 0.01 vs model group; ^∆P < 0.05, ^{∆ ∆}P < 0.01 vs DEX group.

FIGURE 5

Effect of emodin on TLR4 and downstream molecules after TLR4 knock-down. Expression of TLR4 was observed with a fluorescence microscope after siRNA was introduced into cells for 48 h, and then the cells were treated with different drug intervention for 24 h. (A–C) The levels of TLR4 and downstream molecules MyD88, TIRAP, IRF-5, TRAF-6, TRIF, IRF-3, AP-1, and NF-κB after transfection were confirmed by real-time quantitative PCR. (D) Expression of protein was assayed by western blotting. Data are shown as the mean ± SD. ^$P < 0.05 compared with the normal group; ^#P < 0.05 compared with the Si-TLR4 group; ^∗P < 0.05, ^∗∗P < 0.01, compared with the Si-TLR4-LPS group; ^∆P < 0.05, ^{∆ ∆}P < 0.01, compared with the DEX group. si-TLR4: TLR4 was knocked down in Raw264.7 cells by siRNA. si-NC: SiRNA negative control group.

FIGURE 6

Effect of emodin on TLR4 and downstream molecules after TLR4 overexpression. Overexpression of TLR4 in Raw264.7 cells via the lentiviral vector. Lentivirus vector interferes with Raw264.7 cells for 72 h to mediate TLR4 overexpression. (A–C) The levels of TLR4 and downstream molecules MyD88, TIRAP, IRF-5, TRAF-6, TRIF, IRF-3, AP-1, and NF-κB after transfection were confirmed by real-time quantitative PCR. (D,E) The expression of TLR4 was observed with a fluorescence microscope after lentivirus was introduced into Raw264.7 cells for 48 and 72 h. (F) Expression of protein was assayed by western blotting. Data are shown as the mean ± SD. ^$P < 0.05 compared with the normal group; ^#P < 0.05 compared with the Lv-TLR4 group; ^∗∗P < 0.05, ^∗P < 0.01, compared with the Lv-TLR4-LPS group. ^∆P < 0.05, ^{∆ ∆}P < 0.01, compared with the DEX group. Lv-TLR4: TLR4 was overexpressed in Raw264.7 cells by lentivirus vector. Lv-NC: lentivirus vector negative control group.

FIGURE 7

Effect of emodin on the expression of TNF-α and IL-6 in mouse serum. Effect of (A,B) emodin on the expression of TNF-α, IL-6 in mice serum was measured by ELISA. Data are shown as the mean ± SD. ^#P < 0.05 vs normal group; ^∗P < 0.05, ^∗∗P < 0.01 vs model group.

FIGURE 8

Effects of emodin on TLR4 signaling pathways in a mouse model. Effect of emodin on the expression of TLR4 and downstream molecule expression in a mouse model. (A–C) The mRNA levels of TLR4, MyD88, TIRAP, IRF-5, TRAF-6, TRIF, IRF-3, AP-1, and NF-κB were detected by RT-PCR. (D) The protein levels of above molecules were detected by western blotting. Data are shown as the mean ± SD. ^#P < 0.05 compared with the normal group. ^∗P < 0.05, ^∗∗P < 0.01 vs model group; ^∆P < 0.05, ^{∆ ∆}P < 0.01, compared with the DEX group.

FIGURE 9

Effect of emodin on liver morphology. (A–F) Pathological changes were observed in the liver tissues with H&E staining (original magnification×100). The (A) emodin (80 mg/kg) group, (B) emodin (40 mg/kg) group, (C) emodin (20 mg/kg) group, (D) DEX group, (E) model group, and (F) normal group. In the liver, interstitial cells showed obvious edema. (A–C) Liver tissues from mice treated with different concentrations of emodin groups looked healthier compared with those from model mice infected with LPS.

FIGURE 10

Effect of emodin on M2 macrophages in the liver by IF. The expression of mannose receptor ARG1 (green) and mannose receptor CD206 (red). Nuclei were visualized by DAPI (blue) staining. (A/G) Emodin (80 mg/kg) group, (B/H) emodin (40 mg/kg) group, (C/I) emodin (20 mg/kg) group, (D/J) DEX group, (E/K) model group, and (F/L) normal group (ARG1: A–F,M, CD206: G–L,N) figures were captured at 400X magnification. Data are shown as the mean ± SD. ^#P < 0.05 the model group vs the normal group. ^∗P < 0.05, ^∗∗P < 0.01 vs model group.