NADPH Oxidase Inhibitor Attenuates Lipopolysaccharide/D-galactosamine-induced Acute Liver Injury In Mice

Objective: the overproduction of reactive oxygen species(ROS)plays an important role in the development of acute liver injury.Nicotinamide adenine dinucleotide phosphate oxidase(NOX)is the main enzyme that catalyzes the production of ROS in inflammation,but the pathological significance of NOX in acute liver injury and the pharmacological effect of NOX inhibitors are not clear.In this paper,the effects of NOX and its inhibitors on acute liver injury induced by lipopolysaccharide(LPS)/D-galactosamine(D-Gal)in mice were studied.Methods: LPS(10 ?g/kg)/D-Gal(700 mg/kg)was injected intraperitoneally to duplicate the acute liver injury model in male BALB/c mice.In order to determine whether the ROS level changed in LPS/D-Gal-induced acute liver injury,we killed the mice at 0,2,4 and 6 hours after LPS/D-Gal injection respectively and collected liver tissue samples.The ROS level was detected by ROS fluorescent dye DHE.In order to find out whether the change of ROS is catalyzed by NOX,we injected NOX inhibitor apocynin 0.5 hours before LPS/D-Gal injection intraperitoneally.Six hours after LPS/D-Gal injection,we detected the change of ROS level in mouse liver,as well as oxidative damage markers: 8-hydroxy-2-deoxyguanosine(8-OH-dG),protein carbonyl(C=O)and thiobarbituric acid reactive substance(TBARS).To investigate whether NOX is involved in the development of liver injury,we also analyzed the plasma transaminase,liver histomorphology,tumor necrosis factor alpha(TNF-?)and survival rate of apocynin treated mice.Our previous studies have found that LPS/D-Gal-induced acute liver injury is closely related to a large number of apoptosis of hepatocytes.In order to further reveal whether NOX affects the occurrence and development of acute liver injury by regulating apoptosis of hepatocytes,we measured the activity of caspase-3,caspase-8 and caspase-9,which are the members of caspase family,in apocynin treated liver tissues of mice.In addition,western blot was used to analyze the expression of apoptosis marker proteins,such as cleaved caspase-3 and cleaved PARP,and TUNEL staining was used to analyze the apoptosis of hepatocytes.In order to further study the possible mechanism of NOX regulating hepatocyte apoptosis,we detected the activation of AMP activated protein kinase(AMPK)and c-Jun N-terminal kinase(JNK).In order to further explore whether inhibition of NOX has therapeutic clinical significance in acute liver injury,we injected apocynin intraperitoneally two hours after LPS/D-Gal injection,and then detected transaminase and survival rate.In addition,in the above model,we used N-acetyl-L-cysteine(NAC),an ROS scavenger,to inject intraperitoneally 2 hours after LPS/D-Gal injection.Through the determination of plasma transaminase and the analysis of liver histomorphology,we further confirmed the important role of ROS in the development of LPS/D-Gal-induced acute liver injury.Finally,in order to confirm the central role of JNK in regulating apoptosis in this model,we used SP600125,a JNK inhibitor,to inject intraperitoneally 2 hours after LPS/D-Gal injection,and detected the apoptosis of hepatocytes and transaminase.Results:(1)LPS/D-Gal treatment could increase the level of ROS in liver tissue of mice in a time-dependent manner.(2)The pretreatment of NOX inhibitor apocynin inhibited the production of ROS induced by LPS/D-Gal,and reduced the production of oxidative damage markers 8-OH-dG,C=O and TBARS.(3)Apocynin pretreatment inhibited the elevation of transaminase induced by LPS/D-Gal,alleviated the histological abnormality of liver,inhibited the production of TNF-?,and,more importantly,increased the survival rate of LPS/D-Gal-induced acute liver injury.(4)Apocynin pretreatment blocked the activation of caspase cascade reaction and reduced the number of TUNEL positive cells.Western blot analysis showed that LPS/D-Gal-induced expression of cleaved caspase-3 and cleaved PARP protein was also inhibited by apocynin pretreatment,and the phosphorylation of AMPK and JNK protein was also inhibited by apocynin pretreatment.(5)The results of apocynin post-treatment were similar to those of pre-treatment,such as reducing ROS production induced by LPS/D-Gal,reducing hepatocyte apoptosis,reducing transaminase and increasing survival rate of mice with acute liver injury.(6)The post-treatment of ROS scavenger NAC also inhibited the activation of AMPK and JNK,the activation of caspase cascade reaction,the decrease of hepatocyte apoptosis,the decrease of serum transaminase and the reduction of liver injury.(7)SP600125 post treatment inhibited JNK,decreased apoptosis of hepatocytes,decreased plasma transaminase and reduced liver injury.Conclusion: This study suggests that ROS derived from NOX can directly cause oxidative damage of tissue and cells in LPS/D-Gal-induced acute liver injury.On the other hand,in the late stage of acute liver injury,by activating AMPK/JNK pathway,it can enhance apoptosis signal and induce extensive apoptosis of hepatocytes.This may be an important mechanism of LPS/D-Gal-induced acute liver injury,and an NOX inhibitor apocynin may have potential application value in the treatment of liver diseases.