Mangiferin Attenuates LPS/D-GalN-Induced Acute Liver Injury by Promoting HO-1 in Kupffer Cells

Abstract

Acute liver injury and its terminal phase, hepatic failure, trigger a series of complications, including hepatic encephalopathy, systematic inflammatory response syndrome, and multiorgan failure, with relatively high morbidity and mortality. Liver transplantation is the ultimate intervention, but the shortage of donor organs has limited clinical success. Mangiferin (MF), a xanthone glucoside, has been reported to have excellent anti-inflammatory efficacy. Here, a lipopolysaccharide (LPS)/D-galactosamine (D-GalN)-induced acute liver injury mouse model was established to investigate the protective role of MF and the underlying mechanisms of action. Pretreatment with MF improved survival, decreased serum aminotransferase activities, and inhibited hepatic TNF-α production in LPS/D-GalN-challenged mice. Through Kupffer cell (KC) deletion by GdCl₃ and KC adoptive transfer, KCs were confirmed to be involved in these beneficial effects of MF. MF reduced LPS-mediated TNF-α production via the suppression of the TLR4/NF-κB signaling pathway in vitro. MF promoted HO-1 expression, but the knockdown of HO-1 prevented TNF-α inhibition, suggesting that the damage-resistance effects of HO-1 occurred via the suppression of TNF-α synthesis. When HO-1-silenced KCs were transferred to the liver with KC deletion, the protective effect of MF against LPS/D-GalN-induced acute liver injury was reduced, illustrating the role of KC-derived HO-1 in the anti-injury effects of MF. Collectively, MF attenuated acute liver injury induced by LPS/D-GalN via the inhibition of TNF-α production by promoting KCs to upregulate HO-1 expression.

Keywords: TNF-α; acute liver injury; heme oxygenase-1 (HO-1); lipopolysaccharide/D-galactosamine (LPS/D-GalN); mangiferin.

Figure 1

MF improved survival and pathological liver injury induced by LPS/D-GalN. Mice were pretreated orally with vehicle (PBS) or MF (30, 100, or 150 mg/kg, respectively) at 1 and 7 h prior to LPS/D-GalN challenge. Survival rates of mice (A) were monitored for 48 h after LPS/D-GalN challenge. Serum ALT and AST activities (B) and hepatic tissue pathological changes (C) were assessed at 6 h after LPS/D-GalN challenge. Hepatic TNF-α mRNA (D) and protein (E) were determined by RT-qPCR and ELISA at 1.5 h after LPS/D-GalN challenge. Data are presented as mean ± SD. n = 10 (for survival rate analysis) or 6; *P < 0.05 and **P < 0.01.

Figure 2

MF upregulated HO-1 expression and activity in LPS/D-GalN-challenged mice. Mice were pretreated orally with vehicle (PBS) or MF (30, 100, or 150 mg/kg, respectively) at 1 and 7 h prior to LPS/D-GalN challenge. Hepatic tissue HO-1 mRNA (A) and protein (B) expression and activity (C) were determined at 6 h after LPS/D-GalN challenge. Data are presented as mean ± SD. n = 6, *P < 0.05, and **P < 0.01.

Figure 3

Inhibition of HO-1 removed the protective effect of MF against LPS/D-GalN-induced acute liver injury. Mice were pretreated orally with MF (150 mg/kg) at 1 and 7 h with or without ZnPP IX (40 mg/kg) i.p. 30 min prior to LPS/D-GalN challenge. (A) Survival rates of mice were monitored for 48 h after LPS/D-GalN challenge. (B) Serum ALT and AST activity were assessed at 6 h after LPS/D-GalN challenge. Hepatic TNF-α mRNA (C) and protein (D) expression were determined by RT-qPCR and western blotting, respectively, at 1.5 h after LPS/D-GalN. Data were presented as mean ± SD. n = 6, *P < 0.05, **P < 0.01.

Figure 4

KCs are involved in the protective effect of MF on LPS-mediated acute liver injury in D-GalN-sensitized mice. Mice were injected i.p. with GdCl₃ 2 days before LPS/D-GalN challenge, KCs were i.v.-infused into mice at 24 h prior to LPS/D-GalN challenge, and MF pretreatment was given orally at 1 and 7 h prior to LPS/D-GalN challenge. (A) Survival rates of mice were monitored for 48 h after LPS/D-GalN challenge. (B) Serum ALT and AST activities were assessed at 6 h after LPS/D-GalN challenge. Hepatic TNF-α mRNA (C) and protein (D) expression were determined by RT-qPCR and ELISA, respectively, at 1.5 h after LPS/D-GalN challenge. Data are presented as mean ± SD. n = 6, **P < 0.01.

Figure 5

KCs are involved in MF-mediated upregulation of HO-1 in LPS-D-GalN-treated mice. Mice were injected i.p. with GdCl₃ 2 days before LPS/D-GalN challenge, KCs were i.v.-infused into mice at 24 h prior to LPS/D-GalN challenge, and MF pretreatment was given orally at 1 and 7 h prior to LPS/D-GalN challenge. Hepatic tissues HO-1 mRNA (A) and protein (B) expression and activity (C) were determined at 6 h after LPS/D-GalN challenge. Data are presented as mean ± SD. n = 6, **P < 0.01.

Figure 6

KC-derived HO-1 is critical for the effect of MF against LPS/D-GalN-induced acute liver injury in mice. KCs transfected with HO-1-specific siRNA or non-targeting control were i.v.-infused into mice with KC deletion by GdCl₃ at 24 h prior to LPS/D-GalN challenge. Mice were pretreated with MF orally at 1 and 7 h prior to LPS/D-GalN challenge. Survival rates (A) were evaluated for 48 h following LPS/D-GalN challenge. Serum ALT and AST activities (B) were determined at 6 h after LPS/D-GalN challenge. mRNA (C) and protein (D) expression of TNF-α were measured by RT-qPCR and ELISA. Data are presented as mean ± SD. n = 6, **P < 0.01.

Figure 7

MF inhibited LPS-activated TLR4 signaling and HO-1 upregulation in KCs. KCs were transfected with both pAP1-Luc or pNF-κB-Luc and the pRL-TK Renilla plasmid for 24 h, pretreated with PBS or MF (10⁻⁵ M) 30 min prior to PBS or LPS challenge (100 ng/mL). (A) RNA was extracted from KCs at 3 h after LPS challenge and TNF-α mRNA was assayed by real-time RT-PCR. (B) Cell culture supernatant was obtained 3 h after LPS stimulation to determine TNF-α levels by using ELISA. (C) AP1 and NF-κB activities were measured at 30 min after LPS challenge. (D) The expression of TLR4 on the surface of KCs was analyzed at 3 h after LPS by using flow cytometry. HO-1 mRNA (E) and protein (F) expression and activity (G) were determined at 3 h after LPS challenge. Data are presented as mean ± SD. n = 6, **P < 0.01.

Figure 8

HO-1 mediated the suppression of MF on the LPS-induced inflammatory responses in KCs. HO-1 specific siRNA and non-targeting control siRNA were transfected into freshly isolated primary KCs for 24 h. After transfection, the cells were pretreated PBS or MF (10⁻⁵ M) 30 min prior to PBS or LPS challenge (100 ng/mL). HO-1 mRNA (A) and protein (B) expression and activity (C) were determined at 3 h after LPS challenge. Total RNA was extracted from KCs at 3 h after LPS challenge and TNF-α mRNA expression was assayed by real-time RT-PCR (D). Cell culture supernatant was obtained at 3 h after LPS challenge and TNF-α protein expression was determined by ELISA (E). The expressions of TLR4 on the surface of KCs was analyzed at 3 h after LPS challenge by flow cytometry (F). Data are presented as mean ± SD. n = 6, **P < 0.01.