The Recombinant Mouse UBC13 Inhibits Inflammatory Cytokines Secretion Through NF-κB Pathways And Its Mechanism

Objective:The aim of this study was to investigate the regulatory effect of recombinant UBC13 on the expression of inflammatory cytokines and its molecular mechanism in RAW264.7 cells.Method:The viability of cells was measured using MTT assay.In immunoregulation experiment,UBC13(20 μg/mL,10 μg/mL,5 μg/mL),cell and PBS groups were set up.qPCR method was used to detect the effects of different concentrations(20 μg/mL,10 μg/mL and 5 μg/mL)of UBC13 on the mRNA expression levels of IL-6,TNF-a,IL-1β and COX-2 at 3 h,6 h,9 h,12 h and 24 h.In anti-inflammatory experiment,the cell culture model was designed adding LPS(100 ng/mL)and UBC13(different concentrations)together,the UBC13(20 μg/mL,10 μg/mL,5 μg/mL),cell,PBS and LPS(100 ng/mL)groups were set up.IL-6,TNF-α,IL-1β,COX-2 and iNOS mRNA and protein expression levels were determined using qPCR,ELISA,Griess reagent and Western blot at 24 h.The protein expression levels of p65,IκBα and p-IκBα in the cytoplasmic,and the protein expression level of p65 in the nucleus were detected by Western blot at 24 h.Results:In immunoregulation experiment:The results of qPCR showed that the relative mRNA expression of IL-6,TNF-α,IL-1β and COX-2 decreased gradually at the whole stage of the experiment.Compared with the cell group,the relative mRNA expression of IL-6,TNF-α,IL-1β and COX-2 were significantly decreased in UBC13 treatment group(P<0.05)at 24 h in a dose-dependent manner.In anti-inflammatory experiment:The results of qPCR showed that,LPS treatment group obviously enhanced the relative mRNA expression levels of IL-6,TNF-a,IL-1β,COX-2 and iNOS.Compared with the LPS group,the relative genes expression of IL-6,TNF-a,IL-1β and iNOS were also significantly decreased in UBC13 treatment group at 24 h(P<0.05).Meanwhile,the relative genes expression of COX-2 had no significantly change(P>0.05).The results of ELISA assay and Greiss reagent showed that,the levels of IL-6,TNF-a and NO in LPS control was higher than those in cells control at 24 h(P<0.05).Whereas,the UBC13 treatment groups significantly decreased IL-6,TNF-a and NO expression(P<0.05),comparing with the LPS group.Meanwhile,the protein expression levels of IL-1β and COX-2 had no significantly change(P>0.05).Western blot results showed that,comparing with the cell control group,the protein expression levels of p65 and IκBα in the cytoplasmic were significantly decreased in LPS group at 24 h(P<0.05),and the protein expression levels of p-IκBα in the cytoplasmic and p65 in the nucleus were increased in LPS group at 24 h(P<0.05).The protein levels of cytoplasmic IκBα and cytoplasmic p65 in UBC13 treatment groups were significantly increased at 24h(P<0.05),and the protein levels of p-IκBα in the cytoplasmic and p65 in the nucleus were decreased in UBC13 treatment groups at 24h(P<0,05),comparing with the LPS group.Conclusion:In summary,the recombinant UBC13 inhibits inflammatory responses in LPS-stimulated RAW264.7 macrophages cell by suppressing TNF-a,IL-6 and NO expression through NF-κB pathways which suggested that UBC13 has anti-inflammatory effects in v.itro...