Oleanolic Acid Alleviates Hepatic Ischemia Reperfusion Injury Based On Inhibiting The Positive Feedback Loop Of HMGB1-TLR4-neutrophils

Objective:To explore the protective effects and the possible mechanism of oleanolic acid on hepatic ischemia-reperfusion(IR) injury in mice.Methods:Balb/c male mice were randomly divided into 4 groups:sham-operated group(Control group), oleanolic acid group(OA group),hepatic ischemia-reperfusion group(IR group) and oleanolic acid pretreated group(OA+IR group). Mice underwent anesthesia and then exposure the portal triad to block artery of the left and median lobes using microvascular clamp to approach 70% nonlethal segmental hepatic ischemia. After 3 hours of ischemia and 6 hours of repercussion, mice were euthanized. Mice were gavaged with oleanolic acid(OA, 100 mg / kg) 3times interval 8 hours, and the last time is at 30 minutes before surgery.After 6 hours of reperfusion, the samples of serum were collected to analyze the activity of ALT and AST; the liver histopathologic changeswere evaluated by hematoxylin-eosin(H&E) staining; hepatocyte apoptosis were determined by TUNEL and Western blot of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-7 and cleaved caspase-9. The expression of HMGB1 was assayed using immunohistochemistry(IHC). The mRNA expression of toll like receptor 4(TLR4), tumor necrosis factor-alpha(TNF-? ? and Interleukin-1beta(IL-1?) were detected by quantitative reverse transcription PCR(q RT-PCR). The inflammatory factors of TNF-?and IL-1? levels in the serum were measured by Enzyme-linked immunosorbent assay(ELISA). The activation of p-MAPKs(p-ERK,p-JNK and p-p38) proteins in hepatic tissue were detected using Western blot. The recruitment of neutrophils was analyzed by flow cytometry(FCM).Results:Compared OA+IR group with IR group, pretreatment with OA markedly reduced the activity levels of ALT and AST in serum, observably alleviated the liver tissue pathology damage degree and inhibited hepatocyte apoptosis, prevented HMGB1 releasing from nucleus into extracellular microenvironment, vastly down-regulated the activation of p-MAPKs( p-ERK, p-JNK and p-p38) proteins, lowered the mRNA transcription and protein synthesis of TNF-? and IL-1?, observably decreased the recruitment of neutrophils.Conclusion:Oleanolic acid alleviates hepatic ischemia reperfusion injury. The potential protective mechanism may be based on inhibiting the positive feedback loop of HMGB1-TLR4-neutrophils.