Protective Mechanism Of Pterostilbene In Hepatic Ischemia-Reperfusion Injury

Aim:To explore the mechanism of the natural phenolic compound pterostilbene(PTE)in liver ischemic reperfusion protection.Methods:A total of 40 C57 mice were divided into control group,model group and treatment group and establish a 70% liver ischemic reperfusion(ischemic 60 min)model,then isolated primary LSECs by perfusion and digestion.Hepatic structural disruption was observed by HE staining.Ultrastructure of hepatic sinus endothelial cells was observed by transmission electron microscopy(TEM).Scanning electron microscopy(SEM)observed the structure of LSECs fenestrae.The apoptosis of LSECs was detected by flow cytometry.CCK8 assay was used to detect the activity of LSECs.Reactive oxygen species(ROS)production and transmission electron microscopy(TEM)were used to observe mitochondrial oxidative stress damage in LSECs.Western Blot detected the expression level of heme oxygenase 1(HO-1)in LSECs.Result:1.Protective effect of PTE treatment in mouse liverThe levels of ALT and AST in the I/R group were significantly higher than sham operation group,while the levels of ALT and AST in the PTE treatment group were significantly reduced,which indicated that PTE had a significant protective effect in mouse liver.The results of HE staining of liver tissue of mice showed that the morphological boundary of hepatocytes in the sham operation group was complete without any pathological changes.In I/R group,karyopyknosis,severe necrosis and increased pathological damage were observed.Mice treated with PTE showed smaller areas of hepatocyte death,with significantly less necrosis compared with the I/R group.2.Protective effect of PTE treatment in LSECsThe ultrastructure of liver tissue was observed by transmission electron microscopy.In the sham operation group,the endothelial cells of hepatic sinuses were arranged around the cavity of hepatic sinuses in a flat shape,and a large number of fenestrae,thin inner subcutaneous membrane,lack of organized basement membrane,and few collagenous fibers were observed.In I/R group,compared with the sham operation group,the number of fenestrae was significantly reduced,and the inner subcutaneous membrane hyperplasia became thicker,with continuous basement membrane.PTE pretreatment group: the number of fenestrae increased significantly.3.Effect of PTE treatment in LSECs fenestraeScanning electron microscopy(SEM)was used to detect primary hepatic sinuses endothelial cells.The results showed that the normal hepatic sinuses endothelial cells had obvious fenestrae.However,fenestrae in I/R group were significantly reduced.Compared with the I/R group,the number of fenestrae in the PTE treatment group was significantly increased.These results indicated that PTE could improve the fenestrae of hepatic sinusoidal endothelial cells after ischemia reperfusion,facilitate the recovery of liver microcirculation,and alleviate liver ischemia reperfusion injury.4.Effect of PTE treatment in LSECs viabilityAfter OGD/R,CCK8 assay results showed that compared with the control group,OGD/R group significantly decreased cell viability,while PTE(10,15,20,40,60 and 80 μmol/L)treatment group significantly increased cell viability.Flow cytometry showed that the apoptosis rate was 2.56% in the control group and 17.82% in the OGD/R group.Compared with the control group,the percentage of apoptotic cells increased significantly after OGD/R treatment,but decreased to 9.28%,8.53% and 4.88% in the PTE treatment group(10,20 and 40 μmol/L).The apoptosis rate was significantly decreased.5.Protective effect of PTE in mitochondria of LSECsCompared with the control group,ROS content of hepatic sinusoidal endothelial cells in OGD/R group was significantly increased.Compared with OGD/R group,ROS level in PTE treatment group was significantly decreased.The morphology of mitochondria was observed by transmission electron microscopy(TEM).The mitochondria in the control group were round or oval with abundant mitochondrial cristae.After OGD/R,Obvious damage of mitochondria was observed,mitochondrial crest fracture or cavitation.After PTE treatment,mitochondrial morphology was relatively complete and mitochondrial cavitation was reduced.The indicates PTE can improve mitochondrial oxidative stress damage.6.Effect of PTE treatment in LSECs nitric oxide synthase protein expressionCompared with the control group,the expression of HO-1 protein was significantly decreased after OGD/R,and was significantly up-regulated after PTE pretreatment.Conclusion:PTE alleviates oxidative stress injury of hepatic sinusoidal endothelial cells,inhibits apoptosis of hepatic sinusoidal endothelial cells,and protects liver from ischemia reperfusion injury by inducing heme oxygenase-1 expression.