Role of Pellino-1 Overexpression on Ubiquitin of Tumor Necrosis Factor Receptor-associated Factors 3 and MAPK Signaling Pathway in Endotoxin-tolerant Kupffer Cells / 中山大学学报(医学科学版)

ABSTRACT

[Objective]To investigate the effect of pellino-1 gene overexpression by lentivirus vector on the ubiquitin of tumor necrosis factor receptor-associated factors 3 (TRAF3) and the activation of mitogen-activated protein kinases (MAPK) signaling pathway in endotoxin-tolerant kupffer cells (KCs).[Methods]Isolated KCs of C57BL/6 mouse were randomly divided into two groupscontrol group,which transfected with control lentivirus vector for 48 h,then pretreated with 10 ng/mL lipopolysaccharide (LPS)for 24 h,and next treated with 300 ng/mL LPS;overexpression group,which transfected with pellino-1 gene overexpression lentivirus vector for 48 h,then pretreated with 10 ng/mL lipopolysaccharide(LPS)for 24 h,and next treated with 300 ng/mL LPS. The protein expressions of pellino-1,K48-Ub,TRAF3,p38,p-p38,c-Jun N-terminal kinase(JNK)and p-JNK were analyzed by Western blot. The level of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and interleukin-10(IL-10)in superna-tants were measured by enzyme linked immunosorbent assay(ELISA).[Results]Compared with control group,the protein expres-sions of pellino-1,K48-Ub,p-JNK and p-p38 and the levels of IL-1β(P < 0.05),TNF-α(P < 0.05)in supernatants was in-creased in overexpression group,while the protein levels of TRAF3 and the levels of IL-10(P < 0.05)in supernatants were de-creased.[Conclusion]Overexpression of pellino-1 can promote TRAF3 K48 ubiquitination degradation,decrease the protein expres-sion of TRAF3,activate the downstream MAPK signaling pathway,and thus impair the formulation of endotoxin tolerance.