The Role Oftoll-like Receptor 4/NF-κBsignaling Pathwaysin Protective Effects Of Isoflurane Preconditioning On Myocardium After Ischemic-reperfusion Injury In Rat

Objective:The study is to investigate whether the protection of isoflurane preconditioning against myocardial ischemia/reperfusion(I/R) injury is mediated by toll-like receptor 4(TLR4)/NF-κB signaling pathway, and to provide theoretical basis for clinical therapy to myocardial ischemia/reperfusion injury.Methods:Forty SD rats were randomly divided into five groups(8 each):Sham-operated group(Group S),ischemia/reperfusion group(Group IR), isoflurane preconditioning group(Group ISO), TAK-242 inhibitor group(Group T), isoflurane preconditioning and TAK-242 inhibitor group(Group ISOT). After adaptive feeding for a week, each experimental group respectively for processing.In Group S,the animal served as untreated controls;in Group IR,the animals only served myocardial ischemia reperfusion; in Group ISO, the animals inhaled 1.0MAC isoflurane for 30 min, stopped in 15 min;in Group T, gived the tail vein injection of TLR4 receptor inhibitor TAK-242 10mg/kg;in Group ISOT, tail vein injection of TAK-242 10 mg/kg 1 minute before inhaling isoflurane.Then the myocardial ischemia reperfusion injury model was established by cutting on the left side of the 3-4 ribs,and then ligating the left anterior descending coronary artery of rat using 5-0 line for 30 minutes,then reperfusion for 2h in each experimental group.ECG was used to detect ST changes before and after myocardial ischemia.And the ST segment elevation as a marker of ischemia success.Blood samples were taken from femoral artery at the end of 120 min reperfusion, ELISA was employed to determine proinflammatory cytokines production of IL-6 and TNF-α in sera of these animals. Then the rats werekilled and the myocardial tissues were taken instantly.Myocardial specimens were obtained by light microscope and myocardial infarct size was measured by using HE staining. RT-PCR and Western blot analysis was used to assay the transcriptional level and protein level of Toll-like receptor 4(TLR4) in cardiac muscle tissue with pathological change,respectively.Western bloting were used to detect the cardiomyocyte apoptosis and protein expressin of NF-κB and Caspase-3.Results:Compared with Group IR(39.42±7.49),the infarct size in other experimental groups were significantly reduced(P<0.01), Compared with Group ISO(20.43±1.75) and Group T(22.62±1.64), the infarct size was significantly reduced in Group ISOT(16.59±3.04)(P<0.01).And there was no difference between Group ISO(20.43±1.75)and Group T(22.62±1.64)(P>0.05).Compared with Group IR, the rest of the groups of IL-6 and TNF-α expression were significantly lower(P<0.01); compared with Group T, IL-6 and TNF-α expression were significantly higher in Group ISO(P<0.01); Compared with group T,IL-6 and TNF-α expression were significantly lower in Group ISOT(P<0.01) and IL-6 and TNF-α expressed the lowest in Group ISOT. Compared with Group IR, TLR4 m RNA and protein expression levels were significantly lower in the rest of the groups(P<0.01); Compared with Group ISO, T, TLR4 m RNA and protein levels were down-regulated significantly(P<0.01) in Group ISOT;.Compared with Group ISO,TLR4 m RNA and protein levels were down-regulated significantly(P<0.01) in Group T.Compared with group IR, the rest of the groups of NF-κB protein expression decreased significantly(P<0.01);compared with Group T and Group ISOT,NF-κB protein expression decreased significantly in Group ISO(P<0.01).Compared with group IR, the rest of the groups of Caspase-3 expression were significantly lower(P<0.01);there was no significant difference between Group ISO and Group T.Conclusion:TLR4/NF-κB signaling plays an important role in myocardial ischemia-reperfusion injury; This may be the important signal pathway of isoflurane preconditioning on myocardium against ischemia-reperfusion injury.