The Role And Mechanism Of Gut-derived Lipopolysaccharide In Process Of Acetaminophen-Induced Hepatotoxicity

Objective: To investigate the effects and molecular mechanism of gut-derived LPS in process of APAP-induced liver injury.Methods:(1) Before this study, mice were first treated with broad-spectrum antibiotics in their drinking water for 4 weeks to remove the gut-derived LPS. After that mice were intraperitoneally injected with APAP for 48 hours. And then we tested the impact of gut-derived LPS on APAP-induced mortality, levels of the serum ALT and AST, hepatocyte apoptosis, levels of hepatic glutathione(GSH) and the lipid peroxidation product malondialdehyde(MDA) in liver tissues, expression of antioxidant genes like glutathione peroxidase-1(Gpx-1), superoxide dismutase-2(Sod2), c-glutamylcysteine ligase catalytic subunit(GCL) in both antibiotics-treated mice and controls.(2) Antibiotics-treated mice were pretreated with exogenous LPS by intraperitoneal injection(250μg/kg) once a day for two days. After that mice were intraperitoneally injection with APAP for 12 or 24 hours. And then we tested the impact of gut-derived LPS on APAP-induced mortality, levels of the serum ALT and AST, hepatocyte apoptosis, levels of hepatic GSH and the lipid peroxidation product MDA in liver tissues, expression of antioxidant genes like Gpx-1, Sod2, GCL in both LPS pretreatment mice or not.(3) We then detected the expression levels of Nuclear factor erythroid 2-related factor(Nrf2) and autophagy special marker LC3-II between antibiotics-treated mice and controls. In contrast, we also detected the expression levels of Nrf2 and LC3-II between LPS pretreated mice or not.(4) In order to verify whether autophagy was required for the protective function of gut–LPS against APAP-induced liver injury, antibiotics-treated mice pretreated with exogenous LPS were intraperitoneally injected with Chloroquine(CQ) to suppress the autophagy one hour before APAP injection. After that we tested the expression levels of Nrf2 and LC3-II.Results:(1) The mortality rate in APAP-treated antibiotics-treated mice was higher than that of APAP-treated controls during the period time of the observation. We also found that antibiotics-treated mice showed higher levels of the serum ALT and AST than controls. Histological analysis of liver sections by hematoxylin-eosin stain(HE) also revealed that the presence of APAP-induced necrosis in antibiotics-treated mice was more severe than that in controls. Taken these data for consideration, it suggested that gut-derived LPS alleviate APAP-induced liver injury and mortality in mice. DNA fragmentation, certified as a particular feature of APAP-induced hepatocyte death, was detected more aggravated in antibiotics-treated mice as compared to the controls after APAP injection by TUNEL staining and levels of hepatic GSH, known as a significant role in detoxification of the acetaminophen metabolite, were lower in antibiotics-treated mice than controls and levels of MDA were obviously augmented in antibiotics-treated mice when compared to controls. We next tested the hepatic expression levels of some key genes involved in this process between antibiotics-treated mice and controls by Q-PCR analysis. We found that the m RNA abundance of antioxidant genes like Gpx-1, Sod2 and GCL was reduced in antibiotics-treated mice as compared to controls.(2) Exogenous LPS significantly protected antibiotics-treated mice from APAP-induced liver injury, which was supported by attenuating the elevation of serum ALT and AST levels and an obvious reduction in liver necrosis and enhancing the expression of hepatic GSH, reducing the levels of MDA in liver tissues and increasing the expression of several antioxidant genes).(3) Hepatic expression levels of Nrf2 and LC3-II, a definitive marker of autophagy formation, were markedly attenuated in antibiotics-treated mice after APAP injection. We also found that the accumulation levels of nuclear Nrf2 were also lower in antibiotics-treated mice than controls. In contrast, when antibiotics-treated mice were pretreated with exogenous LPS resulted in a significant elevation in hepatic expression levels of Nrf2 and LC3-II as well as the nuclear Nrf2 accumulation levels.(4) We found that the serum levels of ALT and AST and necrosis area in hematoxylin-eosin(HE) staining liver sections in CQ treated mice were obviously higher than those without CQ treatment mice. The antioxidant function of LPS was also dramatically eliminated in CQ treated mice confirmed by the reduced the levels of GSH and hepatic MDA expression was also elevated even though there was no remarkable difference between the two groups of mice. Furthermore, the expression levels of nuclear Nrf2 and the several antioxidant genes were all eliminated in CQ treated mice.Conclusion: In this study, it has been found that when gut-derived LPS is reduced by antibiotics, the liver injury induced by APAP overdose was more serious in mice. In contrast, we found exogenous LPS pretreatment can significantly suppress the liver injury in vivo and also has a protective role against APAP-induced hepatotoxicity in vitro. In addition, we demonstrated that LPS alleviated APAP-induced hepatotoxicity in vivo and in vitro by increasing the nuclear abundance of Nrf2, which can be regulated by the up-regulation of autophagy, thereby reinforcing the antioxidant capacity in the liver. These results may be a quite good explanation for what a role the gut-derived LPS played on the process of APAP-induced liver injury.