The hepatocytes with nuclear immunoreaction for acetylated-lysine were increased in number 1–3 h after the concomitant administration of LPS/GalN, but they were decreased. At 8 h, aberrant, extracellular acetylated-lysine expression was labeled and mainly restricted to pericentral inflammatory areas. At 10 h a distinct nuclear expression for acetylated-lysine was again increased in the hepatic cells, although some hepatocytes remained to express cytoplasmic acetylatedlysine in the pericentral areas. Administration of GL strongly inhibited the immunoreaction of acetylated-lysine in the LPS/GalN-induced liver injury. Immunohistochemical analysis for acetylated-lysine used in this experiment shows that administration of GL may Fruquintinib inhibit acetylation of lysine in HMGB1 in the liver remnants of the LPS/GalN-induced mice. The ability of GL to suppress ALT levels is observed when administered at 30nmin before or at 10 min and 60 min after LPS/GalN, but GLtreatment has little effect on ALT levels when administered 3 h after LPS/GalN injection. Thus, inhibitory effect of GL on LPS/GalN-induced liver injury might be due to binding directly to HMGB1 before its acetylation. Acetylated HMGB1 has been shown to be involved in regulating HMGB1 DNA binding properties along with its subcellular location. In vitro experiments have shown that lysine residues of HMGB1 between 27 and 43 represent functional nuclear localizing signals in macrophages. Also, recent mutation analysis for the abilities of HMGB1 protein to bind and bend DNA has reported the role of lysines 2 and 81 as target sites for acetylation in full-length HMGB1 and truncated tail-less protein,Farampator respectively. Serum HMGB1 released following liver ischemia/reperfusion in vivo is acetylated and hepatocytes exposed to oxidative stress in vitro also release acetylated HMGB1. Levels of acetylated HMGB1 increase with a concomitant decrease in total nuclear histone deacetylase activity, suggesting that suppression in HDAC activity contributes to the increase in acetylated HMGB1 release after oxidative stress in hepatocytes. Activation of HDAC1 is decreased in the nucleus of hepatocytes undergoing oxidative stress. In addition, HDAC1 knockdown with siRNA promotes HMGB1 translocation and release. Furthermore, HDAC4 is shuttled from the nucleus to cytoplasm in response to oxidative stress, resulting in decreased HDAC activity in the nucleus. On the contrary, in the present experiment we did not find a decrease in both the cytoplasmic and nuclear HDAC activities after injection of LPS/GalN. In addition, administration of GL did not induce a significant increase of nuclear HDAC activity. Phosphorylation of HMGB1 is another regulatory mechanism that influences its subcellular location. Recently, it was shown that HMGB1 phosphorylation by calcium/calmodulin protein kinase IV caused nuclear-to-cytoplasmic shuttling and release in LPS-stimulated macrophages. Damaged cells activate innate immunity and recruit inflammatory cells by collectively releasing danger signals known as the damage-associated molecular patterns. One such molecule is HMGB1, which has been implicated as an early mediator of organ damage in ischemia/reperfusion injury and hemorrhagic shock, as well as a late mediator of lethality in endotoxic shock. In most cases the cells actively secreting HMGB1 appear to be immune cells such as macrophages, natural killer cells, and dendritic cells. However, it is becoming increasingly clear that non-immune parenchymal cells also participate in active HMGB1 secretion.