| Background: Kupffer Cells (KCs) play a key role in hepatic ischemiareperfusion injury (IRI) after transplantation. The classical NF-κB signalingpathway is an important pathway for activation of KCs. How to inhibit theactivation of KCs by the classical NF-κB signaling pathway and protectbasal function of KCs is still a problem. Recently, it is reported that NBDpeptides could interfere the formation of IκB kinase complex (IKK) anddown-regulate the classical NF-κB signaling pathway, but not affect NF-κBbasal function. These findings interest us to investigate the effects of NBDpeptides on hepatic IRI.Part1: NBD peptides inhibit the LPS induced activation of KCsObjective: To isolate KCs in SD rats in order to observe whetherNBD peptides could inhibit LPS-induced activation of the classical NF-κBsignaling pathway and protect NF-κB basal function, and to investigate themechanism of the impact of NBD peptides on the classical NF-κB signaling pathway in vitro.Methods: To isolate KCs in SD rats and to employ ELISA to detectthe impact of2h pre-treatment of WT NBD with different concentrationson the levels of TNF-α and IL-6given either LPS stimulation or not, and toprove whether WT NBD could inhibit LPS-induced inflammation andprotect NF-κB basal function as well as investigate the effectiveconcentration. Using the given effective concentration for further research,KCs were divided into six groups randomly:①LPS group (LPSstimulation for24h),②Mut NBD pre-treatment group (to add Mut NBD2h prior to stimulation with LPS for24h),③WT NBD pre-treatment group(to add WT NBD2h prior to stimulation with LPS for24h),④WT NBDpost-treatment group(to add WT NBD2h after LPS stimulation),⑤normal group,⑥normal+WT NBD group (cultured with WT NBD for24h). The methods were as follows: Immunofluorescence for expression ofNF-κB/p65and IκBα; EMSA for NF-κB activity；Western Blot for theexpression of P-IKK-2and IκBα；Real-time PCR for TNF-α mRNA.Results: Under the LPS stimulation, pre-treatment of WT NBD couldinhibit TNF-α and IL-6released from KCs in a concentration-dependentmanner; while under the normal condition, WT NBD of variousconcentrations had no function with TNF-α and IL-6. Using theconcentration of100μg/ml for further research, the results were as below:the NF-κB activity, NF-κB translocation rates, expression of P-IKK-2, levels of TNF-α mRNA were higher in group①,②,③and④, and lowerin group⑤and⑥; group②and④had no statistic difference from group①(P>0.05); group③was significantly lower than group①(P<0.05);and group⑤had no difference from group⑥(P>0.05). On contrary, theexpression of IκBα was lower in group①,②,③and④, and higher ingroup⑤and⑥; group②and④had no statistic difference from group①(P>0.05); group③was significantly higher than group①(P<0.05); andgroup⑤had no difference from group⑥(P>0.05).Conclusion: It is a first report that the pre-treatment of WT NBD caninhibit LPS-induced activation of NF-κB in KCs through lowering thephosphorylation of IKK complex and attenuating the degradation of IκBα,and protect NF-κB basalfunction.Part2: NBD peptides protect IRI after liver transplantation in rats bydown-regulating the classical NF-κB signaling pathwayObjective: To perform SD-SD orthotopic liver transplantation in orderto observe whether the pre-treatment of NBD peptides could attenuate IRIand protect NF-κB basal function in healthy rats, and to examine themechanism of NBD peptides on IRI.Methods: To perform orthotopic liver transplantation in rats in orderto observe the impact of different concentrations of NBD peptidespre-treatment with donor rats two hours before the surgery on ALT three hours after transplantation, and to prove whether NBD peptidespre-treatment could protect IRI as well as investigate the effectiveconcentration. Using the given effective concentration for further research,the rats were divided into four groups randomly:①NBD pre-treatment+IRI group,②IRI group,③normal+NBD group (healthy rats dealt withNBD peptides by intraperitoneal injection but without any surgery),④normal group (healthy rats dealt with the same volume of saline as thecontrol group). Group①and②: to harvest the sample3h,6h, and24hafter transplantation; Group③and④: to harvest the sample24h afterinjection. The methods were as follows: Beckman CX7for ALT levels, HEfor the change of pathology, TUNEL for apoptosis, ELISA for TNF-α,immunohistochemical for the expression of ICAM-1and iNOS, EMSA forNF-κB activity, Western Blot forthe expressionofP-IKK-2and IκBα.Results: NBD peptides could lower the levels of ALT3h aftertransplantation in a concentration-dependent manner. Using theconcentration of8mg/kg for further research, the results were as follows:the levels of ALT and TNF-α, NF-κB activity, expression of P-IKK-2inboth group①and②increased and reached the peak value6h aftertransplantation, but group①were significantly lower than group②ateach time point (P<0.05). Group③and④had lower levels and there wasno difference (P>0.05). On contrary, the expression of IκBα in both group①and②reduced and reached the minimum value6h after transplantation, but group①were significantly higher than group②at each time point(P<0.05). Group③and④had higher level and there was no difference(P>0.05). Further more, the degree of damage, apoptosis, expression ofICAM-1and iNOS in group①were significantly lower than group②6hafter transplantation.Conclusion: It is also a first report that pre-treating donors with NBDpeptides can protect liver IRI after transplantation through down regulatingclassical NF-κB pathway, and have no impact on NF-κB basal function invivo.Part3: NBD peptides inhibit the activation of KCs by the classicalNF-κB signaling pathway in liver grafts.Objective: To isolate KCs in donor liver at each time point afteroperation in order to observe the change of the classical NF-κB signalingpathway in KCs and prove the inhibited activation of the KCs by theclassical NF-κB signaling pathway in vivo.Methods: To isolate KCs from donor liver in group①and②3h,6hand24h after transplantation and detect NF-κB activation by EMSA, theexpression of P-IKK-2and IκBα by Western Blot,and the TNF-α mRNAlevel by Real-time PCR.Results: The NF-κB activity, expression of P-IKK-2, TNF-α mRNAlevel in group①were significantly lower than group②at each time point (P<0.05), while the expression of IκBα was significantly higher (P<0.05).Conclusion: Pre-treatment of NBD peptides inhibit the activation ofKCs by the classical NF-κB pathway in ischemia reperfusion. It is likelythat the mechanism of the protection of NBD peptides is partially due to theregulation of KCs function. |
