| Objective1.To investigate autophagy phenomenon in normal liver and to investigate the level of autophagy variation of the liver during ischemia-reperfusion injury 2.To explore the protective effect of astilbin in hepatic ischemia-reperfusion injury(HIRI).So as it can provide reliable theoretical basis to reduce hepatic ischemia re perfusion injury in clinical work.Methods1.Fifty-four SD rats were divided into Sham group(control group),HIRI group(ischemia-reperfusion group)and astilbin group,to establish the model of rat HIRI.2.Groups of rats fasting,water before 12 h.The ischemia perfusion rats which treated with astilbin(40mg/kg*d),Prior to surgery seven days of astilbin,1 times per one day for 7 days.The Sham group and HIRI intraperitoneal injection of saline group was given the same volume.After rats anesthesia was successful,underwent liver ischemia reperfusion surgery.Establishing the liver ischemia reperfusion model,the rat was intraperitoneal injection of 1%sodium pentobarbital anesthesia(50ug/g)for anesthesia.In the center of the abdominal longitudinal incision into the abdomen,fully exposed after intra-abdominal viscera,free out of the first porta hepatis.Using nondestructive microscopic vascular clamp clip liver left,middle vascular stem for 45 minutes,making the middle and the left of hepatic lobe to achieve 70%of the liver tissue ischemia.Incision gauze covering physiological saline,achieving loosening vascular clamp after getting the time,then closed abdomen.3.After liver were reperfused with blood(in 4 h,8 h,16 h),collecting the specimens of blood and liver tissues.Serum alanine aminotransferase(ALT)and aspertate aminotransferase(AST)were detected by automatic biochemical analyzer.The changes of liver cell microstructure were observed under the optical microscope.Western Blot analysised the expression of LC3,Beclin-1 in the liver tissue.The number of autophagosome was observed under electron microscope.Results1.54 healthy SD rats were able to wake up immediately after the operation of HIRI,to maintain effective heartbeat and breathing,and to turn over.2.Changes of serum levels of ALT and AST:Compared with those in sham group,ALT and AST levels of serum in the other two groups increased significantly(P<0.05).Those peaked in 8h after I/R,and decreased in 16h after I/R.Compared with those in HIRI group,levels of serum AST and ALT in astilbin group on pre treatment were significantly reduced(P<0.05).The level of ALT,AST in HIRI group raised and in astilbin group decreased obviously.3.Hepatic tissue HE staining:liver cells in sham group were normal,liver cell cords arranged in hepatic lobule,hepatic central vein,hepatic portal structures showed clear.In 4h after I/R,mild swelling of liver cells were around the central vein of liver,and there were no degeneration and necrosis of liver cells in HIRI group.There showed no obvious injury in astilbin group.In 8h after I/R,there was severe local edema,ballooning degeneration,hepatic sinusoid narrowing,congestion in liver cells in central vein of liver,and there was severe edema,inflammatory cell infiltration around the hepatic portal area in HIRI group.There were liver cells with mild edema,no degeneration and necrosis in astilbin group.In 16h after I/R,postoperative liver cell edema improved,part of liver cells occurred coagulative necrosis in HIRI group.There was mild edema,basical normal morphology in astilbin group.4.The change of the level autophagy protein expression in liver cells,by western blot detection autophagy related liver protein Beclin 1 and LC3-?/LC3-?,protein expression of ischemia-reperfusion group and astilbin group increased(in 4 h,8 h,16 h)significantly(P<0.05),compared with shame group;And that showed that Beclinl and LC3-?/LC3-?peaked at 8h after reperfusion,in 16h gradually reduce in ischemia-reperfusion group and astilbin group.Compared with ischemia-reperfusion group,autophagy related liver protein Beclin 1 and LC3-?/LC3-? of astilbin treatment group significantly reduced(P<0.05).The results of Western Blot displayed that expression of LC3-?/LC3-? and Beclin-1 in astilbin group was significantly reduced.5.The number of three groups of rat liver autophagosome(8h)was observed under electron microscope.The results showed that,compared to the average number of autophagosome shame group(0.69±0.04),the average number of autophagosome ischemia-reperfusion group(7.12±0.60)increased obviously,with statistical significance(P<0.05).The number of autophagosome in astilbin group also decreased.Compared to the average number of autophagosome ischemia-reperfusion group(7.12±0.60),the average number of autophagosome astilbin group reduced and was statistically significant(P<0.05).ConclusionThe results suggest that serum ALT and AST levels of the cats increased after I/R,and astilbin preconditioning before I/R can reduce serum ALT and AST levels.The liver tissue HE staining results show that astilbin can relieve the liver iscnemia reperfusion injury in rats.The leveis of autophagy related liver protein Beclin 1 and LC3-?/LC3-? expression of liver tissue and the comparative analysis of the liver cell apoptosis rates in the astilbin group and HIRI group show that astilbin can inhibit the expression of Beclin 1 and LC3-?/LC3-? of the I/R rat liver,inhibit apoptosis,and then reduce the hepatic ischemia-reperfusion injury.The number of autophagosome in astilbin group also decreased.Compared to the average number of autophagosome ischemia-reperfusion group,the average number of autophagosome astilbin group reduced and was statistically significant.Pretreatment can reduce HIRI and its mechanism may be associated with inhibiting the autophagy.The results for Astilbin protects liver ischemia-reperfusion injury provides a new theoretical basis,which have a important clinical value. |
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