Research Of ?-arrestin 2 In Inflammation Of MC3T3-E1 Cells Induced By Palmitic Acid

BackgroundThe increasing incidence of obesity with high therapy cost and severe health problem has informed the world of its threaten to all human beings. More and more studies report that obesity may induce chronic inflammation and inflammation factors like IL-6 and TNF-? can be produced during this chronic inflammation.Previous studies report that dietary fatty acids, especially palmitic acid, could induce chronic inflammation in adipocytes, which is associated with insulin resistance.M. Muluke find that in the diet-induced obesity mouse model, except high level of FFAs in the circulation, the concentration of FFAs in bone tissue increases as well. Osteoblasts are involved in the rebuilding in bone marrow microenvironment,originating from bone marrow like adipocytes. Palmitic acid, the most common saturated fatty acids, is capable of promoting to the production of inflammatory cytokines in osteoblasts.?-arrestin2, a recently discovered multifunctional adapter protein which is expressed in a variety of cells like osteoblasts, can negatively regulate inflammation in body. However, what the role ?-arrestin2 can play in inflammation of osteoblasts induced by palmitic acid has not been reported. ObjectiveTo explore the change of ?-arrestin2 expression in MC3T3-E1 cells induced by palmitic acid,and the role of ?-arrestin2 in inflammation in MC3T3-E1 cells induced by palmitic acid. Materials and MethodsSection1 After starvation for 12 h, MC3T3-E1 cells are incubated with 0?mol/L, 100?mol/L, 200?mol/L, 300?mol/L, 400?mol/L, 500?mol/L palmitic acid for another 12 h. RT-qPCR is used to test ?-arrestin2 mRNA relative expression.Western blot is used to detect the ?-arrestin2, TAK1, p-TAK1 protein relative level.then the ratio of p-TAK1/ TAK1 is calculated.The level of IL-6 and TNF-? is also detected by ELISA.Section2 Infect the MC3T3-E1 cells with ?-arrestin2 overexpression lentiviral vector.The 4 groups including in this study are as follows: eGFP group, palmitic acid+?-arrestin2-eGFP group, palmitic acid+eGFP group and ?-arrestin 2-eGFP group. Western blot is used to detect the ?-arrestin2, TAK1, p-TAK1 protein relative level in each group. Then the ratio of p-TAK1/ TAK1 is calculated.The level of IL-6 and TNF-? is also detected by ELISA in each group. Results1.After incubation with different concentrations of palmitic acid for 12 h, the ratio of p-TAK1/TAK1 increases starting from 100?mol/L palmitic acid(p<0.05),and significantly increases starting from 400?mol/L palmitic acid(p<0.001). IL-6 and TNF-? relative level increases starting from 300?mol/L palmitic acid(p<0.05), and significantly increases starting from 500?mol/L palmitic acid(p<0.001).The relative expression of ?-arrestin2 mRNA and ?-arrestin2 protein decreases starting from 300?mol/L palmitic acid(p<0.05), and significantly decreases starting from 400?mol/L palmitic acid(p<0.001).2. Compared with the eGFP group,?-arrestin2 protein relative expression in ?-arrestin2-eGFP group increases(p<0.05), while p-TAK1/TAK1 ratio decreases(p<0.05), IL-6 and TNF-? level is lower than that in eGFP group(p<0.05); Compared with the palmitic acid+eGFP group,?-arrestin2 protein relative expression in palmitic acid+?-arrestin2-eGFP group is significantly higher(p<0.001), the p-TAK1/TAK1 ratio and IL-6 and TNF-? levels in palmitic acid+?-arrestin2-eGFP group is significantly lower than the latter(p <0.001). Conclusions1. Palmitic acid can induce the increase of inflammation factors in MC3T3-E1 cells.2. ?-arrestin2 can inhibit TAK1 phosphorylation and the expression of inflammation factors induced by palmitic acid.