Mechanisms Of IL-6-mediated MiR-674-5p/5-LO Activation In Concanavalin A-induced Acute Mouse Liver Injury

Background and objective:Development of hepatitis is a complex pathological process, in which abnormal secretion of cytokines and inflammatory mediator triggered by pathological immune response plays an important role. As a group of lipid, Arachidonic acid (AA) and related metabolites were found to play an important role in liver pathology and physiology. Enzymes such as 5-lipoxygenase (5-LO) involved in the metabolic pathway of AA were expected as key roles to understand the liver injury, the mechanism however is still not fully illustrated. In the present study, we employed Concanavalin A (Con A) induced mouse liver injury model mediated by T cells to mimic autoimmune hepatitis and to explore the post-transcriptional regulatory mechanisms of generation of some key cytokines and inflammatory mediators and to provide experimental basis for elucidating the acute phase mechanism of immunological hepatitis.Methods:Male Balb/c mice were randomly divided into 5groups,6 mice/group.Con A (20 mg/kg) was adminnistratered through the tail vein, and mice were sacrificed at 2 h, respectively. Saline in the corresponding volume served as control group. miRNACURYTM LNA Chip platform was applied to detect microRNAs (miRNAs) expression pattern of mice, and qRT-PCR was used to comfirm the differentially expressed miRNAs (change> 1.5-fold, P<0.05). Further miRNA profiling was conducted followed by bioinformatics analysis and prediction to speculate possible "miRNA-target genes". In order to validate the regulatory mechanisms of "cytokines/miRNA/5-LO pathway" in Con A induced acute liver injury in vitro, miRNA mimics were transfected into the HepG2 cells with interleukin 6 (IL-6) or tumor necrosis factor α (TNF-α) treatment individually, and the expression of target protein was detected by Western-Blot.Results:Our previous studies found that the serum IL-6 and TNF-αand the metabolity of AA cysteinyl-leukotrienes (cysLTs) levels increased at the early stage of Con A induced liver injury, implying that there may be a correlative relationship between the two inflammatory cytokines and cysLTs. After pretreatment with cyclosporin A, the Con A induced liver injury was attenuated and the increase in cysLTs was inhibited with the downregulation of 5-LO expression. All the results demonstrated that 5-LO pathway directly involves in Con A induced acute liver injury.9 miRNAs were screened out to differentially express more than 1.5-fold in Con A group via miRNA microarray analysis and qRT-PCR validation. Afterwards,8 bioinformatics softwares including TargetScan were used to match the miRNAs with potential targets in the 5-LO pathway one to one, and mmu-miR-674-5p was found to exhibit a negative post-transcriptional regulation on 5-LO gene. Western-blot analysis demonstrated that miR-674-5p mimic-transfected Hepa 1-6 cells showed significantly downregulated expression of 5-LO with 1 h of IL-6 treatment or 4 h of TNF-α treatment (P＜0.01). Meanwhile, miR-674-5p mimic-transfected HepG2 cells also showed significantly downregulated expression of 5-LO with 4 h of IL-6 treatment (P< 0.05). These in vitro results further demonstrated the rapid regulation effect of IL-6 associated miR-674-5p downregulation on 5-LO upregulation.Conclusions:These findings suggested that miR-674-5p downregulation mediated by IL-6 upregulated the expression of 5-LO, leading to the increase of cysLTs in Con A induced mouse acute liver injury.