Research On Rapamycin In Ameliorating Hepatic Ischemia-reperfusion Injury Via Glycolytic Pathway And The Molecular Mechanism In Rat Models

Objective: To observe the changes of mammalian target of rapamycin(m TOR)signaling pathway and glycolytic pathway during hepatic ischemia-reperfusion injury in a rat model,and investigate the underlying mechanism of glycolytic pathway disorder in hepatic ischemia-reperfusion injury rats and the intervention of rapamycin.Methods: 72 SPF SD rats were divided into 3 groups randomly: Rapamycin pretreatment group(RPM group): Rats were injected intraperitoneally with rapamycin at 2.0mg/kg/d for 3 days before the surgical maneuver performed + 30 min ischemia.Control group(IRI group): Rats were injected intraperitoneally with 0.9% saline at 2.0ml/kg/d for 3 days before the surgical maneuver performed + 30 min ischemia.Sham group: Rats were injected intraperitoneally with 0.9% saline at 2.0ml/kg/d for 3 days before the surgical maneuver performed.Samples were harvested at 2h,6h,and 24 h after reperfusion.Tail venous blood was collected to measure blood glucose concentration.Serum alanine aminotransferase(ALT),total bilirubin(TBil),and insulin(INS)level and hepatic adenosine triphosphate(ATP),superoxide dismutase(SOD),glutathione(GSH),hexokinase 2(HK2),and phosphofructokinase 1(PFK1)content were assayed.Hepatic histologic features were observed.Hepatic m TOR,ribosome protein S6 kinase 1(S6K1),and protein kinase B(Akt)message ribonucleic acid(m RNA)expression and p-m TOR(Ser2448)?p-S6K1(Thr389)?p-Akt(Ser473)protein level were detected by using quantitative reverse transcription polymerase chain reaction(q RT-PCR)and western blot(WB),respectively.Results: Hepatic histological assay: At 2h,6h,and 24 h after reperfusion,hepatic histological lesion wasn't significant in Sham group.The hepatic lesion in Control group and RPM group was aggravated as compared with in Sham group.But the lesion in RPM group was mitigated as compared with in Control group.The hepatic damage increased gradually in both RPM group and Control group from 2h to 24 h after reperfusion.Blood glucose assay: There wasn't significant difference in the blood glucose level between 3 groups before the surgery performed.At 2h,6h,and 24 h after reperfusion,the blood glucose level in Control group and RPM group was higher than in Sham group.But the blood glucose level in RPM group was lower than in Control group.The blood glucose level decreased gradually in both RPM group and Control group from 2h to 24 h after reperfusion,but showed no statistically significance in Sham gourp.Serum parameters assay: At 2h,6h,and 24 h after reperfusion,serum ALT and TBil increased and INS decreased in Control group and RPM group as compared with Sham group,but the ALT and TBil was lower and INS was higher in RPM group than in Control group.The ALT,TBil,and INS increased gradually in both RPM group and Control group from 2h to 24 h after reperfusion.Hepatic ELISA assay: At 2h,6h,and 24 h after reperfusion,liver SOD,GSH,ATP,HK2,and PFK1 decreased in Control group and RPM group as compared with Sham group,but was higher in RPM group than in Control group.These indexes decreased gradually in both RPM group and Control group from 2h to 24 h after reperfusion,but showed no statistically significance in Sham group.Hepatic q RT-PCR and WB assay: At 2h,6h,and 24 h after reperfusion,relative m RNA expression of liver m TOR and S6K1 and protein content of liver p-m TOR(Ser2448)and pS6K1(Thr389)elevated in Control group as compared with RPM group and Sham group,and was lower in RPM group than in Sham group.Liver Akt m RNA expression and pAkt(Ser473)protein content declined in Control group as compared with RPM group and Sham group,and was higher in RPM group than in Sham group.Conclusion: Over-activation of m TOR,inhibition of Akt,and glycolytic pathway disorder showed in HIRI rats.Rapamycin can ameliorate liver injury and promote liver repair by improving glycolytic pathway and reducing oxidative stress.This protective effect of rapamycin may be induced by activating Akt via inhibition of m TOR signaling pathway.