Effects And Mechanisms Of Dioscin Against Hepatic And Cerebral Ischemia/reperfusion Injure

Objective: To investigate the effects and potential mechanisms of dioscin against hepatic and cerebral ischemia/reperfusion(I/R) injure.Methods:(1) In the study of dioscin against hepatic I/R injure, 70% partial hepatic warm ischemia was induced in Wistar rats for 60 min followed by succedent reperfusion. In the prophylactic test, dioscin was administered intragastrically(i.g.) to the rats at the doses of 20, 40 and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats were received dioscin intragastrically(i.g.) at the dose 60 mg/kg once 2 h before I/R. The protective effect of dioscin was evaluated based on the levels of ALT, AST, histopathological examination and the survival of the rats. Moreover, the levels of SOD, MDA, CAT, GSH-Px, GSH, NO, iNOS and TNOS were detected. The effects of dioscin on nuclei were evaluated by DAPI, TUNEL and DAB staining assays. The methods of immunohistochemistry, western blotting and real-time PCR were used to detect the expression levels of protein/gene releated to inflammation and apoptosis.(2) In the study of dioscin against cerebral I/R injure, an in vitro oxygen-glucose deprivation and reoxygenation(OGD/R) model was used. The PC12 cells and primary cortical neurons were treated with dioscin(50, 100 and 200 ng/mL) for 12 h before OGD/R injure, and the effects of dioscin were assayed by MTT assay, LDH and NeuN detection. In vivo, a middle cerebral artery occlusion(MCAO) model was used. In the prophylactic test, dioscin was administered intragastrically(i.g.) to the rats at the doses of 20, 40 and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats were received dioscin intragastrically(i.g.) at the dose 60 mg/kg once 2 h before I/R. The protective effect of dioscin was evaluated based on TTC and TUNEL assays, histopathological examinations for detecting Neu N, GFAP and Kir4.1 and the survival of rats. Real-time PCR and western blotting were used to detect the expression levels of protein/gene releated to inflammation and HMGB-1/TLR4 signaling pathways. In addition, for elucidating whether blockade of the HMGB-1/ TLR4 signaling pathway was involved in the underlying molecular mechanism, siRNA and over-expressed genes of HMGB-1 and TLR4 were applied in vitro experiments, respectively.Results:(1) In the experiment to study the effect of dioscin against hepatic I/R injure, we found that dioscin significantly decreased serum ALT and AST activities, increased survival rate of rats and improved I/R-induced hepatocyte abnormality. In addition, dioscin obviously increased the levels of SOD, CAT, GSH-Px, GSH, decreased the levels of MDA, TNOS, iNOS, NO, and prevented DNA fragmentation caused by I/R injure. Further research indicated that dioscin markedly decreased the gene expressions of IL-1β, IL-6, TNF-α, ICAM-1, MIP-1α, MIP-2, Fas, FasL, down-regulated the protein expressions of NF-κB, AP-1, COX-2, HMGB-1, CYP2E1, Bak, p53, PARP, Caspase-3, Caspase-9, decreased the levels of JNK, ERK and p38 MAPKs phosphorylation, and up-regulated the levels of Bcl-2 and Bcl-x.(2) In the experiment to study the effect of dioscin against cerebral I/R injure, the results indicated that dioscin clearly protected PC12 cells and primary cortical neurons against OGD/R insult by increasing the cells viabilities and NeuN release, decreasing the LDH release. In addition, dioscin significantly prevented cerebral I/R injury by decreasing infarct volume, LDH activities, GFAP, kir4.1 and TUNEL positive cells, increasing Neu N positive cells and improving the histopathological examination, survival rates and neurological deficits. Further research demonstrated that dioscin-induced neuroprotection was accompanied by a significant inhibition in the expression and the nuclear to cytosolic translocation of HMGB-1, reflected by decreased TLR4 expression. Blockade of the TLR4 signaling pathway by dioscin inhibited NF-κB and AP-1 transcriptional activities, MAPKs and STAT3 phosphorylation, and pro-inflammatory cytokine responses, and up-regulated the levels of anti-inflammatory factors. In addition, siRNA and over-expressed genes of HMGB-1 and TLR4 were applied in vitro experiments, respectively, and the results further confirmed that diosicn showed an efficient neuroprotection because of its inhibiting effects on HMGB-1/TLR4 signaling and subsequent suppressing inflammation.Conclusions:(1) This paper suggested that dioscin has potent actions against hepatic I/R injure through suppression of inflammation, oxidative/nitrative stress and apoptosis.(2) Our data also suggested that dioscin conferred direct protective effects on the ischemic brain by inhibiting HMGB-1/TLR4 signaling and the subsequent immune and neuroinflammatory responses in vivo and in vitro models of ischemic brain. In short, dioscin has good biological activities in hepatic and brain I/R injury, it is worth in-depth exploration and research.