| ObjectiveAs we all know, gut is the biggest place where bacteria are located in our body. There are about 500-1000 species which amount to 100 trillions of bacteria in the gut. These bacteria are affecting the digestive, and immune system, and they are very important to one’s health. Among them, dead gram-negative bacteria can release endotoxin which makes the gut become the biggest endotoxic storeroom. Acute liver injury induced by intestinal endotoxin is one of the common causes of liver function failure. Injecting LPS, which is the main component of endotoxin, intraperitoneally to mice can mimic the process of intestinal endotoxin infection. In addition, D-GalN can deplete the UTP in liver specifically so that the synthesis of protein and nucleic acid are inhibited, which lead to the damage even death of hepatocytes. Therfore, injection of LPS together with D-GalN can specifically lead to the fulminant hepatic failure, and it is a perfect animal model to study the acute liver injury.Autophagy is a homeostatic process that takes place in all eukaryotic cells, and it involves the sequestration of cytoplasmic components in double-membraned vesicle called autophagosomes which subsequently fuse with lysosomes, and then their "cargoes" are delivered for degradation and recycling. Autophagy is a cellular pathway crucial for development, differentiation, survival and homeostasis, and its implication in human diseases has been highlighted during the last decade. Recent data show that autophagy is involved in major fields of hepatology. However, reports about the role of autophagy in acute liver injury are very few, and the results are not consistent.Wortmannin is a highly cell permeable antifungal antibiotic isolated from Penicillium funiculosum, and it has no antibacterial properties and acts specifically on fungi. Many reports have demonstrated that wortmannin can inhibit autophagy in vivo and vitro. The aim of the current study is to explore the changes of hepatocytes necrosis, apoptosis and autophagy in LPS/D-GalN induced acute liver injury, and the effect of pretreatment with wortmannin on hepatocytes necrosis, apoptosis and autophagy. The completion of this study can not only further clarify the molecular mechanism of acute liver injury induced by LPS/D-GalN, but also be helpful to develop drugs for therapy acute liver injury induced intestinal endotoxin.Methods1. To induce acute liver injury with LPS/D-GaINAcute liver injury was induced by intraperitoneal (IP) injection of LPS together with D-GalN which can increase the sensitivity of hepatocytes to LPS by depleting uridine triphosphate (UTP). Blood was collected from retro-orbital venous plexus at 3 h and 5 h after the injection of LPS/D-GalN. Then mice were executed by cervical dislocation and liver tissues were removed immediately for subsequent RNA, protein extraction and histological detection.(1) The ALT and AST levels in serum were determined by automatic biochemical analyzer.(2) Liver tissue sections were stained with hematoxylin and eosin (HE) to evaluate the liver tissue pathology, and stained with TUNEL reaction mixture to evaluate the apoptosis of the hepatocytes.(3) The expression of cleaved caspase-3 was detected by immunohistochemical (IHC) staining and western blot analysis.2. To monitor the change of autophagy in LPS/D-GalN induced acute liver injury(1) The expression of LC3B-II in liver tissues was detected by western blot analysis.(2) The formation of autophagosome was detected by immunofluorescence assay.3. To detect the secretion of inflammatory cytokines in LPS/D-GalN induced acute liver injury(1) The concentrations of serum inflammatory cytokines including TNF-α, IL-1β, MCP-1, IL-6, IL-10 were measured by flow cytometry or enzyme-linked immunosorbent assay (ELISA).(2) The mRNA expression of these cytokines in liver tissues was measured by RT-PCR.4. To explore the effect of wortmannin on liver injury and the level of inflammatory cytokines induced by LPS/D-GalNWortmannin was dissolved in dimethylsulfoxide (DMSO) and injected intraperitoneally to each treated mouse at 1 hour before the injection of LPS/D-GalN. Five hours later, the mice were executed by cervical dislocation and liver tissues were removed immediately for subsequent RNA, protein extraction and histological detection.(1) The serum ALT and AST levels were determined by the same automatic biochemical analyzer to explore the effect of wortmannin on the liver function.(2) Liver tissue sections were stained with hematoxylin and eosin (HE) and TUNEL reaction mixture to explore the effect of wortmannin on the necrosis and apoptosis of hepatocytes.(3) The expression of cleaved caspase-3 was detected by immunohistochemical (IHC) staining and western blot analysis to explore the effect of wortmannin on the activation of caspase-3.(4) The mRNA expression of inflammatory cytokines in liver tissues was also measured by RT-PCR to explore the effect of wortmannin on them.5. To explore the effect of wortmannin on the level of autophay in liver(1) The expression of LC3B-Ⅱ in liver tissues was detected by western blot analysis.(2) The formation of autophagosome was detected by immunofluorescence assay.6. To explore the effect of wortmannin on the activation of signal pathway.(1) The expression of phosphorylated ERK, JNK and p38 in MAPK signal pathway in liver tissues were detected by western blot assay.(2) The expression of phosphorylated p65 in NF-κB signal pathway in liver tissues was detected by western blot assay.Result1. LPS/D-GalN can induce the acute liver injury of mice and lead to the necrosis and apoptosis of hepatocytes(1) The serum ALT and AST levels were both increased at 5 hour after injection of LPS/D-GalN, while there was no obvious change at 3 hour.(2) The liver tissues displayed much more necrotic hepatocytes, internal hemorrhage, lymphocytes infiltration at 5 hour after injection, while there was only a little internal hemorrhage at 3 hour.(3) Massive TUNEL positive cells in liver tissues were observed at 5 hour after injection, while there was few at 3 hour. In addition, caspase-3 was obviously activated at 5 hour after injection while there was no obvious change at 3 hour.2. Autophagy in liver tissues was induced to increase in LPS/D-GalN induced acute liver injury.(1) The level of autophagy in liver tissues was obviously induced to increase at 5 hour after injection while there was no obvious change at 3 hour.(2) The autophagosome began to form at 3 hour after injection and increased strikingly at 5 hour.3. The secretion of inflammatory cytokines increased in LPS/D-GalN induced acute liver injury.(1) Levels of serum inflammatory cytokines including TNF-α、MCP-1、IL-1β、IL-6、 IL-10 were elevated both at 3 and 5 hour after injection.(2) The mRNA expression of these cytokines in liver tissues was increased both at 3 and 5 hour after injection too.4. Pretreatment with wortmannin could alleviate hepatocytes damage and suppress the increased levels of inflammatory cytokines in LPS/D-GalN induced acute injury.(1) Compared with the LPS/D-GalN group, the serum ALT and AST levels were obviously decreased in LPS/D-GalN and wortmannin co-treatment group.(2) Compared with the LPS/D-GalN group, the internal hemorrhage, lymphocytes infiltration in liver and hepatic pathological damage were distinctly improved in LPS/D-GalN and wortmannin co-treatment group.(3) Compared with the LPS/D-GalN group, the numbers of TUNEL positive cell and activation of caspase-3 were obviously decreased in LPS/D-GalN and wortmannin co-treatment group.(4) Compared with the LPS/D-GaIN group, the mRNA expression of cytokines except Mcpl in liver tissues was decreased in LPS/D-GalN and wortmannin co-treatment group.5. Pretreatment with wortmannin could effectively inhibit increased autophagy in LPS/D-GalN induced acute liver injury.(1) Compared with the LPS/D-GalN group, the increased autophagy were suppressed in LPS/D-GalN and wortmannin co-treatment group.(2) Compared with the LPS/D-GalN group, fewer autophagosomes were observed in LPS/D-GalN and wortmannin co-treatment group.6. Pretreatment with wortmannin could affect the activation of MAPK and NF-kB signal pathways in LPS/D-GaIN induced acute liver injury.(1) Compared with the LPS/D-GalN group, only phosphorylated ERK were obviously decreased in LPS/D-GalN and wortmannin co-treatment group.(2) Compared with the LPS/D-GalN group, phosphorylated p65 were also decreased in LPS/D-GalN and wortmannin co-treatment group.Conclusions& OriginalityIn the current study, we demonstrated that wortmannin could downregulate the expression of phosphorylated extracellular regulated protein kinase and p65, decrease the production and release of hepatic inflammatory cytokines, and then reduce hepatocytes apoptosis and necrosis. More importantly, we found that autophagy was induced to increase in LPS/D-GalN-induced acute liver injury, and pretreatment with wortmannin could effectively inhibit increased autophagy in acute liver injury. In conclusion, these results indicate that wortmannin plays a protective role in LPS/D-GalN induced hepatocytotoxity maybe by inhibiting autophagy and could be acted as a target for the treatment of acute liver injury.It is the first time to find that wortmannin can effectively alleviate liver injury in LPS/D-GalN induced acute liver injury, which can help to develop drug for the treatment of intestinal endotoxemia-induced acute liver injury In addition, we also found that autophagy perhaps plays an important role in LPS/D-GalN induced acute liver injury, which is significant for further exploring the mechanism and new therapy strategy of intestinal endotoxemia-induced acute liver injury. |
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