TM4Counter-regulates Nur77-IKKβ-NF-κB Pathway And Inhibits The High-fat Induced Chronic Inflammation

Part1The construction and verification of TM4(UBAC2) gene recombinant eukaryotic and lentivirus expression vectorObjective:To construct recombinant eukaryotic and lentivirus expression vector of TM4(UBAC2) gene.Methods:1) The full-length ORF of human TM4and mouse genes was amplified from human TM4plasmid and mouse cDNA via PCR, respectively.2) TM4(h) and TM4(m) genes were further subcloned into eukaryotic expression vector pcDNA. The sequence of TM4gene in pcDNA-TM4recombinant plasmids was confirmed by sequencing, enzyme-cutting, and further cell transfection.3) The verified plasmids were transfected into HEK293cells by Lipofectamine2000.4) TM4lentiviral vector plasmids were harvested with a virus packaging system, and the virus titers were determined. Then,293TN cells were infected by TM4lentiviral vectors.5) The transfection/infection efficiency was assayed under immunofluoresence microscopy. The expression of TM4in HEK293and293TN cells was identified by western-blot and real-time PCR6) TM4(h) lentiviral vector plasmid was in turn transfected into THP-1cells, and TM4(m) lentiviral vector plasmid was transfected into3T3-L1cells to evaluate the MOI.Results:1) pcDNA-TM4(h) and pcDNA-TM4(m) recombinant eukaryotic expression plasmid vectors were successfully constructed. pcDNA-TM4recombinant plasmids can stably express TM4protein.2) The recombinant lentivirus vectors expressing TM4(h) and TM4(m) genes were constructed successfully.3) The titer of TM4(h) and TM4(m) virus suspension was1.56X104ifu/ul and1.67×104ifu/ul, respectively. The MOI for THP-1and3T3-L1cells was8and18.4, respectively.Conclusions:Human/mouse recombinant TM4eukaryotic and lentivirus expression vector was successfully constructed. Part2TM4counter-regulates Nur77-IKKβ-NF-kB pathway and inhibits the high-fat induced inflammationObjectives:To investigate the interaction of TM4and Nur77-IKKβ-NF-kB pathway and the impact of TM4on chronic low-grade inflammation.Methods:The THP-1/3T3-L1cells were treated with control, empty vector lentivirus, and EGFP-labeled TM4lentivius, respectively. The3groups were further treated with the mixture of palmitic acid and oleic acid (1mM,1:1) or control. Finally, the expression of TM4, Nur77, IKKβ, and NF-kB p65were detected under real-time PCR and western blot.Results:1) After96h of lentivirus infection, the proportion of cells with green fluorescent was near100%. TM4expression was significantly enhanced in the TM4lentivirus infected group.2) TM4expression was down-regulated while the expression of inflammatory genes including Nur77, IKKβ, NF-kB p65and TNF-a was up-regulated, after palmitic/oleic acid intervention.3) The expression of Nur77, IKKβ, NF-kB p65and TNF-a, as well as palmitic/oleic acid induced inflammation could be significantly inhibited by TM4overexpression. Conclusions:TM4counter-regulates the Nur77-IKKβ-NF-κB inflammatory pathway, and could inhibit high-fat induced inflammation. Part3TM4overexpression alleviates the chronic inflammation and insulin resistance of high-fat-diet fed db/db mouseObjective:To study the impact of TM4overexpression during the development of diabetes, obesity and insulin resistance in high-fat-diet fed db/db mouse.Methods:Thirty db/db mice aged2weeks were randomized into3groups. They were treated with nature saline, empty lentivirus vector, and TM4(m) lentivirus vector respectively, and were fed with a high-fat-diet for10weeks. Weight, food intake, fasting plasma glucose, and blood pressure were measured every week. In week10, intraperitoneal glucose tolerance test (IPGTT) was performed. Before sacrifice, blood sample was collected and fasting insulin, lipids, as well as inflammatory cytokines were determined. After sacrifice, skeletal muscle, liver, and epididymis fat tissues were collected, in which the expression of TM4, Nur77, IKKβ, and NF-kB was examined by real-time PCR and western blot. HE stained sections of liver and epididymis fat tissues were evaluated under microscopy.Results:1) According to real-time PCR and western blot, TM4expression in skeletal muscle, liver, and epididymis fat tissues of the TM4(m) lentivirus treated mice was significantly enhanced.2) After10weeks of TM4(m) lentivirus infection, the weight, fasting plasma glucose, fasting plasma insulin, HOMA-IR, systolic BP, mean BP, TC, TG, and LDL-c were significantly lower than control.3) On IPGTT, the Omin,30min,60min,120min glucose and area under curve (AUC) of the TM4(m) lentivirus treated mice were significantly lower than control. 4) On HE staining, a large number of lipid droplets accumulated in the liver cells of empty lentivirus and control group, while no evident ballooning degeneration was observed in the TM4lentivirus group. The average size of epididymis adipocytes of the TM4lentivirus infected mice was significantly smaller than control.5) Nur77, IKKβ, and phosphorylated NF-kB expression in the skeletal muscle, liver, as well as epididymis fat tissues was significantly inhibited. The serum inflammatory cytokines (TNF-α, IL-1β, MCP-1) were significantly lower than control.Conclusions:Glucose intolerance, obesity, hypertension, fatty liver, and chronic low-grade inflammation could be alleviated by TM4overexpression in db/db mice, which could be partially interpreted by the inhibition of Nur77-IKKβ-NF-kB pathway.