Glycyrrhetinic Acid Attenuates Liver Ischemia/Reperfusion Injury Via Inhibition Of HMGB1-TLR4 Signaling Pathway In Mice

Aims: To explore the effect of glycyrrhetinic acid on liver ischemia/reperfusion(IR)injury and the possible underlying mechanisms.Methods: The male C57BL/6 mice were randomly divided into 4 groups(6 mice per group),including the control group,glycyrrhetinic acid(100 mg/kg)group,IR group and IR + glycyrrhetinic acid(100 mg/kg)group.The experimental model of hepatic IR injury(70%)was performed by blocking the blood of portal vein and hepatic artery that supply the left and middle hepatic lobes.After 90 minutes,the blood supply was restored by removing the atraumatic clamp.6 hours later,all mice were sacrificed for blood and liver samples.Automatic biochemical analyzer was used to determine the levels of alanine aminotransferase(ALT)and aspartate aminotransferase(AST)in serum.Hematoxylin and eosin(HE)staining was performed to evaluate the pathological changes of hepatic tissue.Apoptosis of hepatocytes were assessed by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL)staining and the activity of caspase-3 in liver tissue was analyzed by spectrophotometer.Immunofluorescence(IF)staining and flow cytometry(FCM)were used to measure the number of neutrophils recruited in liver parenchyma.The qRT-PCR(quantitative reverse transcription polymerase chain reaction)was used to detect the mRNA levels of IL-1?,TNF-? and toll-like receptor 4(TLR4).Enzyme linked immunosorbent assay(ELISA)was used to detect the protein levels of IL-1? and TNF-? in serum.Immunohistochemistry(IHC)technique was used to determine the level of high mobility group box-1 protein(HMGB1)in extracellular matrix.Western blot was used to detect the expression of molecules in TLR4 signaling pathways,including TLR4,phospho-IRAK1,phospho-ERK,phospho-JNK,phospho-P38 and phospho-NF-κB.Results: Compared with the control group,the levels of serum ALT and AST in the IR group were notably increased,and a large number of congestion,necrosis and edema in ischemic liver tissues were observed in the IR group.Moreover,the number of TUNEL-positive hepatocytes and the activity of caspase-3 in the IR group were significantly increased as compared to the control group.The neutrophils infiltration and the mRNA,protein levels of IL-1? and TNF-? in the IR group were all increased compared with the control group.The expression of released HMGB1 and the levels of TLR4,phospho-IRAK1,phospho-ERK,phospho-JNK,phospho-P38 and phospho-NF-κB in the IR group were all significantly increased as compared to the control group.After glycyrrhetinic acid pretreatment,compared with the IR group,the levels of aminotransferases were notably decreased,and the necrosis and edema of hepatocytes were significantly reduced,and the number of TUNEL-positive hepatocytes as well as the activity of caspase-3 were reduced.The neutrophils infiltration and the mRNA,protein levels of IL-1? and TNF-? were decreased.Additionly,the expression of released HMGB1 and the levels of phospho-IRAK1,phospho-P38 and phospho-NF-κB in the IR group were all reduced compared with the IR group.There were no significant difference in TLR4 expression and phospho-ERK and phospho-JNK levels between the IR group and the IR + glycyrrhetinic acid group.Conclusion: Glycyrrhetinic acid had a protective effect on hepatic IR injury in mice.This protective effect of glycyrrhetinic acid might be associated with the decrease of hepatocytes apoptosis,down-regulation of the HMGB1 expression in extracellular matrix and inhibition of the HMGB1/TLR4 signaling pathway,which resulted in lower production of inflammatory cytokines and infiltration of neutrophils.