The Role And Mechanism Of Tumor Necrosis Factor-a In The Early Stage Of Vascular Injury In Sepsis

Objective: To investigate the level of endothelial nitric oxide synthase(e NOS)expression and phosphorylation in inflammatory mediators and the mechanism;To study the effect of inflammatory mediators on the aortic diastolic function in septic mice and screen potential therapeutic targets;Methods: TNF-?-treated BAECs were pretreated with or without PKC-? inhibitors(RO-32-0432)?PKC-? small interfering RNA(si RNA),LPS-induced mice were pretreated by PKC-? inhibitors(RO-32-0432)?Etanercept and D609;Western blot was used to detected the level of e NOS? PKC-? and phosphorylation in BAECs and mice;the diastolic function of the thoracic aorta was measured by vascular tension test system.Results: BAECs were typical "pebble-like" growth in the light microscope,the ? factor and CD31 expression were positive.Compared with control group,e NOS Thr497 and p-PKC-? 638 increased significantly(P < 0.05)in a time-dose-dependent manner in TNF-? treated BAECs,thus the levels of e NOS protein?e NOS Ser1179?e NOS Ser635 and PKC-?protein did not change(P < 0.05).IL-1? and IL-6 had no effect on e NOS protein expression and phosphorylation(P < 0.05)within 40ng/ml in BAECs.After the pretreated of RO-32-0432 and PKC-? si RNA in BAECs with or without TNF-? treatment,there was no significant change on e NOS protein and e NOS Thr497(P < 0.05).Compared with control groups,the e NOS Thr495 and the p-PKC-? 638 significantly raised in LPS-treated mice(P < 0.05),and have no obvious changes in RO-32-0432 and Etanercept pretreated in LPS-treated mice(P < 0.05).Thoracic aorta diastolic function was reduced significantly in LPS-induced mice,Etanercept and RO-32-0432 pretreatment could significantly weaken the thoracic aorta diastolic function in LPS-induced mice(P < 0.05);The pretreatment of D609 showed no difference compared with LPS group(P <0.05),the results of transgenic mice were identical with wild type mice.Conclusion: The experiment results showed that TNF-? up-regulated the e NOS Thr497 by PKC-? pathway,which may lead to the vascular dysfunction,suggesting that the inflammatory mediators played a very important role in the vascular injury of sepsis.In animal experiments,PKC-? inhibitors can significantly reduce vascular diastolic dysfunction,indicating PKC may be a potential target for the treatment of sepsis vascular injury.