Effects of 2-APB on Store-operated Ca^(2+) Channel Currents of Hepatocytes after Hepatic Ischemia/Reperfusion Injury in Rats

The effects of hepatic ischemia/reperfusion (I/R) injuries on hepatocellular viability and store-operated calcium current (Isoc) in isolated rat hepatocytes and the effects of 2-APB on store-operated calcium current (Isoc) in isolated rat hepatocytes after hepatic ischemia/reperfusion injuries were studied. Hepatic ischemia and reperfusion injury model was established and whole cell patch-clamp techniques were used to investigate the effects of 2-APB on Isoc. The results showed that ischemia/reperfusion injuries could significantly reduce hepatocellular viability and further increase Isoc in hepatocytes and 2-APB (20, 40, 60, 80, 100 μmol/L) produced a concentration-dependent decrease of Isoc with IC 50 value of 64.63±10.56 μmol/L (n=8). It was concluded that ischemia/reperfusion injuries could reduce hepatocellular viability, probably through increased Isoc in hepatocytes and 2-APB had a protective effect on ischemia/reperfusion-induced liver injury, probably though inhibiting Isoc.

Significance of Rosiglitazone Inhibiting TLR4 Expression in Partial Hepatic Ischemia/Reperfusion of Mice

The effect of rosiglitazone as the ligand of peroxisome proliferator-activated receptor γ （PPARγ） inhibiting the TLR4 expression in ischemic lobes in partial hepatic ischemia/reperfusion injury （IRI） in BABL/C mice and the action of rosiglitazone inhibiting the TLR4 receptor-mediated inherent immune response were investigated. The model of the mouse partial hepatic ische- mia/reperfusion injury was established. All the animals were randomly divided into 3 groups： rosiglitazone group, vehicle （dimethylsulphoxide, DMSO） group and sham operation group. The hepatic samples were collected when mice were sacrificed 0, 2, 4 and 6 h after reperfusion following 1 h ischemia to analyze the acute phase of hepatic IRI. The dynamic expression of TlR4 mRNA was de- tected quantitatively by real-time-PCR, and the levels of TNF-α, IL-10 and ALT in portal vein were determined in all groups. After restoration of blood supply, the expression of TLR4 mRNA in ischemic lobes was detected in 0, 2, 4 and 6 h after reperfusion following 1 h ischemia in rosiglitazone group and vehicle group. The most intensive expression of TLR4 mRNA was present at 4 h after reperfusion in ischemic lobes in vehicle group. As compared with vehicle group, the expression of TLR4 mRNA in ischemic lobes in rosiglitazone group was significantly decreased at 4 h after reper- fusion. The level of IL-10 in portal vein was markedly up-regulated in rosiglitazone group as compared with vehicle group. Contrarily, the levels of TNF-α and ALT in portal vein were markedly down-regulated in rosiglitazone group as compared with vehicle group at every time point in mouse partial hepatic IRI model. Rosiglitazone could alleviate the hepatic IRI by inhibiting TLR4 receptor-mediated inherent immune response.

Peptidomic Analysis Reveals that Novel Peptide LDP2 Protects Against Hepatic Ischemia/Reperfusion Injury

Background and Aims:Hepatic ischemia/reperfusion(I/R)injury has become an inevitable issue during liver transplantation,with no effective treatments available.However,peptide drugs provide promising regimens for the treatment of this injury and peptidomics has gradually attracted increasing attention.This study was designed to analyze the spectrum of peptides in injured livers and explore the potential beneficial peptides involved in I/R injury.Methods:C57BL/6 mice were used to establish a liver I/R injury animal model.Changes in peptide profiles in I/R-injured livers were analyzed by mass spectrometry,and the functions of the identified peptides were predicted by bioinformatics.AML12 cells were used to simulate hepatic I/R injury in vitro.After treatment with candidate liver-derived peptides(LDPs)1–10,the cells were collected at various reperfusion times for further study.Results:Our preliminary study demonstrated that 6 h of reperfusion caused the most liver I/R injury.Peptidomic results suggested that 10 down-regulated peptides were most likely to alleviate I/R injury by supporting mitochondrial function.Most importantly,a novel peptide,LDP2,was identified that alleviated I/R injury of AML12 cells.It increased cell viability and reduced the expression of inflammation-and apoptosis-related proteins.In addition,LDP2 inhibited the expression of proteins related to autophagy.Conclusions:Investigation of changes in the profiles of peptides in I/R-injured livers led to identification of a novel peptide,LDP2 with potential function in liver protection by inhibiting inflammation,apoptosis,and autophagy.

The gut microbial metabolite, 3,4-dihydroxyphenylpropionic acid, alleviates hepatic ischemia/reperfusion injury via mitigation of macrophage pro-inflammatory activity in mice

Hepatic ischemia/reperfusion injury(HIRI) is a serious complication that occurs following shock and/or liver surgery. Gut microbiota and their metabolites are key upstream modulators of development of liver injury. Herein, we investigated the potential contribution of gut microbes to HIRI.Ischemia/reperfusion surgery was performed to establish a murine model of HIRI. 16 S r RNA gene sequencing and metabolomics were used for microbial analysis. Transcriptomics and proteomics analysis were employed to study the host cell responses. Our results establish HIRI was significantly increased when surgery occurred in the evening(ZT12, 20:00) when compared with the morning(ZT0, 08:00);however, antibiotic pretreatment reduced this diurnal variation. The abundance of a microbial metabolite3,4-dihydroxyphenylpropionic acid was significantly higher in ZT0 when compared with ZT12 in the gut and this compound significantly protected mice against HIRI. Furthermore, 3,4-dihydroxyphenylpropionic acid suppressed the macrophage pro-inflammatory response in vivo and in vitro. This metabolite inhibits histone deacetylase activity by reducing its phosphorylation. Histone deacetylase inhibition suppressed macrophage pro-inflammatory activation and diminished the diurnal variation of HIRI. Our findings revealed a novel protective microbial metabolite against HIRI in mice. The potential underlying mechanism was at least in part, via 3,4-dihydroxyphenylpropionic acid-dependent immune regulation and histone deacetylase(HDAC) inhibition in macrophages.