Molecular Mechanisms Of Lipopolysaccharide-induced Acute Apoptotic Liver Injury In Mice

Mice were co-injected with lipopolysaccharide (LPS) and D-galactosamine (GalN) to induce acute apoptotic liver injury. Tumor necrosis factor alpha (TNF-α) plays an important role in GalN/LPS-evoked acute apoptotic liver damage. In vitro studies have showed that nuclear factor kappa B (NF-κB) activation protected against TNF-α-mediated hepatocellular apoptosis, whereas exogenous reactive oxygen species (ROS) and endogenous oxidative stress aggravated TNF-α-triggered hepatocellular apoptosis. Accordingly, the present study aimed to investigate the different effects of pyrrolidine dithiocarbamate (PDTC), a hibitor of NF-κB activity, and two kinds of antioxidants, melatonin (MT) and N-acetylcysteine (NAC),on LPS-induced acute apoptotic liver damage, and to clarify different roles of NF-κB activation and ROS production in GalN/LPS-induced acute apoptotic liver injury.1. Effects of PDTC on GalN/LPS-induced acute apoptotic liver injury in miceObjective The present study was to investigate the effects of PDTC on LPS-induced acute apoptotic liver injury in mice and to clarify the role of NF-κB activation in GalN/LPS-induced acute apoptotic liver injury. Methods All mice were randomly divided into six groups. Mice in GalN/LPS group were co-injected with GalN (600 mg/kg, i.p.) and LPS (20μg/kg, i.p.). Mice in PDTC+GalN/LPS group were injected with two doses of PDTC, one (100 mg/kg, i.p.) at 24 h before LPS and the other at 2 h before LPS (20μg/kg, i.p.). Mice in control groups were treated with LPS (20μg/kg, i.p.), GalN (600 mg/kg, i.p.), PDTC (100 mg/kg, i.p.) or saline. Ten mice each group were observed for animal survival within 72 h after LPS treatment. Six mice in each group were sacrificed 1.5 h after LPS for collecting blood and isolating livers. The expression of hepatic TNF-αmRNA was determined by reverse transcription and polymerase chain reaction (RT-PCR). Hepatic NF-κB binding activity was measured using electrophoretic mobility shift assay (EMSA). Serum TNF-αlevel was analyzed by enzyme-linked immunosorbent assay (ELISA). Twelve mice in each group were sacrificed 8 h after LPS treatment. Serum was collected for measurement of alanine aminotransferase (ALT) and nitrate plus nitrite. Livers were dissected for measurements of glutathione (GSH) content, caspase-3 activity and hepatocellular apoptosis and histological examination. Results Co-injection of GalN and LPS markedly increased serum ALT activity. Histopathological examination of liver sections revealed that GalN/LPS induced hepatic congestion and necrosis, and massive macrophages infiltration, increased the number of TUNEL-positive cells in mouse liver. GalN/LPS treatments significantly increased hepatic caspase-3 activity, led to 90% mortality within 72 h and with severe congestion and necrosis in the liver of all the dead mice. PDTC pretreatment significantly inhibited GalN/LPS-induced hepatic NF-κB activation and TNF-αexpression. In contrast, PDTC aggravated GalN/LPS-triggered hepatocellular apoptosis, increased serum ALT activity, exacerbated hepatic hemorrhage and necrosis, and accelerated death. Conclusion PDTC aggravates GalN/LPS-induced acute apoptotic liver injury via inhibiting NF-κB-mediated anti-apoptotic effects.2. Effects of MT on GalN/LPS-induced acute apoptotic liver injury in miceObjective The present study was to investigate the effects of MT on GalN/LPS-induced acute apoptotic liver injury and to elucidate the roles of ROS in GalN/LPS-induced acute apoptotic liver injury. Methods Mice were randomly divided into three groups. Mice in GalN/LPS group were co-injected with GalN (600 mg/kg, i.p.) and LPS (20μg/kg, i.p.). Mice in MT+GalN/LPS group were administered with three doses of MT, one (5.0 mg/kg, i.p.) at 0.5 h before GalN/LPS, another (2.5 mg/kg, i.p.) at 1.0 h after GalN/LPS, and the other (2.5 mg/kg, i.p.) at 2.0 h after GalN/LPS treatments. The control mice were given with normal saline. Mice were sacrificed at 8 h after GalN/LPS. Blood serum was collected for measurements of ALT, TNF-α, and nitrate plus nitrite. Livers were excised to determine hepatic GSH content and antioxidant enzyme activity. Hepatocellular apoptosis were analyzed by measurement of DNA fragmentation and caspase-3 activity. Results Serum ALT activities were significantly increased 8 h after GalN/LPS, massive hemorrhage being observed in histological sections of liver from GalN/LPS-treated mice. MT markedly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, MT significantly improved GalN/LPS-evoked hepatic congestion. Additional experiment showed that MT distinctly attenuated GalN/LPS-triggered hepatic apoptosis, measured by inhibition of caspase-3 activities and reduction of DNA fragment. Moreover, MT significantly increased hepatic glutathione peroxidase (GSH-Px) and glutathione reductase (GSH-Rd) activities and attenuated hepatic GSH depletion in GalN/LPS-treated mice. Conclusion MT protects mice against GalN/LPS-evoked acute apoptotic liver injury.3. Effects of NAC on GalN/LPS-induced acute apoptotic liver injury in miceObjective The present study was to investigate the effects of NAC on GalN/LPS-induced acute apoptotic liver injury and to elucidate the roles of ROS in GalN/LPS-induced acute apoptotic liver injury. Methods All female ICR mice were randomly divided into four groups. Except for NS group, all mice were treated with GalN (600 mg/kg, i.p.) and LPS (20μg/kg, i.p.). Mice in NAC/GalN/LPS group were injected with NAC(150 mg/kg,i.p.)at 0.5 h before GalN/LPS administrations. Mice in buthionine sulfoximine (BSO)/NAC/GalN/LPS group were challenged with BSO (100+100 mg/kg,i.p.) plus NAC (150 mg/kg,i.p.) before GalN/LPS treatments. The control mice were given with saline. Some mice were killed at 1.5 h after GalN/LPS administrations. Serum was collected for determinations of TNF-αlevel by ELISA. The remaining mice were sacrificed at 8 h after GalN/LPS. Blood was collected by drawing the eyeball. Serum ALT acitivity and NO production were measured. Livers were dissected for measurements of caspase-3 activity and GSH content. Hepatocellular apoptosis were analyzed by DNA laddering. Liver sections were examined by HE stain. Results There was a significant increase in serum ALT activity of GalN/LPS-treated mice as compared with the controls. Massive hemorrhage was also observed in histological sections of liver from GalN/LPS-treated mice. NAC markedly attenuated GalN/LPS-induced elevation of serum ALT. In parallel, NAC significantly improved GalN/LPS-evoked hepatic congestion. Another experiment indicated that NAC distinctly attenuated GalN/LPS-triggered hepatic apoptosis, measured by inhibition of caspase-3 activities and attenuation of DNA fragmentation. Moreover, NAC significantly attenuated hepatic GSH depletion in GalN/LPS-treated mice. Conclusion NAC prevents mice from GalN/LPS-evoked acute apoptotic liver injury.Taken together, these results indicate that NF-κB activation plays a protective role in LPS-induced acute apoptotic liver injury, whereas ROS release exerts its detrimental effects in GalN/LPS-induced acute apoptotic liver damage.