A Small Molecule (B022) Targeting Nf-?b-inducing Kinase Ameliorates Liver Injury And Inflammation

Protein kinases,as necessary components in signal pathway transduction,are involved in the regulation of many physiological and pathological processes.Under pathological conditions,certain protein kinases are over-activated,so protein kinases have become potential drug targets for the treatment of many human diseases.As of 2017,38 small-molecule protein kinase inhibitors had been approved by the Food and Drug Administration?FDA?.NF-?B-inducing kinase?NIK?,as a core component of noncanonical NF-?B signaling pathway,is associated with a lot of physiological processes,such as formation of lymphatic organs and survival and maturation of B cells and so on.In addition,abnormal elevated NIK expression is linked to many pathological processes including liver damage,inflammation,fibrosis and cirrhosis.Whether inhibition of NIK activity ameliorates liver injury and inflammation remains unclear.In this paper,we synthesized an ATP-competitive small-molecule inhibitor?B022?of NIK and studied whether B022 can inhibit NIK-induced noncanonical NF-?B signaling pathway,NIK-induced inflammatory gene expression and whether B022 can alleviate NIK-or CCl₄-induced liver injury,inflammation and oxidative stress.In Hepa1 cells,B022 drastically suppresses NIK-induced p100-to-p52 processing in a dose-dependent manner,B022 also dose-dependently suppresses NIK-induced NF-?B luciferase reporter activity.In both Hepa1 cells and primary hepatocytes isolated from WT C57 BL/6 male mice?8 weeks?,B022 inhibits NIK-induced expression of proinflammatory cytokines,chemokines and nitric oxide synthase?iNOS?by qPCR assay in both dose-dependent and time-dependent manners.We also confirm that NIK overexpression in primary hepatocytes isolated from WT C57 BL/6 male mice?8 weeks?drasmatically triggers Raw264.7 cells to express iNOS and IL-1?,whereas B022 significantly suppresses those effects.Furthermore,B022 is able to ameliorate both NIK-and CCl₄-induced liver injury,inflammation and oxidative stress.In liver-specific NIK transgenic mice,intravenous administration of B022 ameliorates NIK-induced liver injury,as revealed by decreased NIK-induced noncanonical NF-?B signaling pathway,prevented death in mice,decreased liver weight,decreased blood ALT activity,decreased oxidative stress,decreased hepatocytes apoptosis,reduced inflammatory gene expression and reduced immune cell infiltration into the liver.More importantly,B022 ameliorates CCl₄-induced liver injury,inflammation and oxidative stress in mice via blocking CCl₄-induced noncanonical NF-?B signaling pathway,decreasing blood ALT activity,decreasing oxidative stress,decreasing hepatocytes apoptosis,reducing inflammatory gene expression and reducing immune cell infiltration into the liver.In summary,in this study,B022,an inhibitor of NIK,ameliorated NIK-induced liver inflammation,both in vitro and in vivo.B022 protected mice from NIK-induced liver failure and subsequent death.Moreover,it reduced CCl4-induced liver inflammation and injury in mice.Our data suggest that further efforts toward the development of B022 analogs could yield therapeutic drugs for the treatment of liver inflammation,oxidative stress,and injury.