Effect Of TAT-NBD On Inflammation Of Rat’s Cortical Astrocytes By Inhibiting Bilirubin-Induced Nuclear Factor-B Activation

ObjectiveTo observe the effect of TAT-NBD on neuro-inflammation by inhibting the bilirubin-induced nuclear factor-κB (NF-κB) activation of rat’s cortical astrocytes.To study the effect of TAT-NBD on bilirubin induced brain injury and on improving learning and memory ability, may through inhibiting the activation of NF-κB and GFAP overexpression。MethodsAstrocytes were separated from the cortical of newborn SD rat. After identification and purification, second-generation astrocytes were randomly divided into control group, bilirubin group and TAT-NBD intervention group. Cellular morphology were observed by immunofluorescence, the cell survival rate and apoptosis situation were assessed with a modified MTT assay and Annexin V-FITC/PI assay, the expression and activity of NF-KBp65 protein was detected by Western blot and EMS A, and the concentration of IL-1β、TNF-α and IL-6 in the supernatants were investigated with ELISA.5-day Sprague-Dawley rat pups(10-15g) were randomly divided into three groups:control group, bilirubin encephalopathy group and TAT-NBD intervention group. After modeling, the expression of NF-κB and GFAP were observed by immunofluorescence in brain slice of 24h, weight was daily recorded within 3 days. The survival and mortality rate were calculated in 40 days. Turn rods and avoid dark evading experiment were performed at the age of 28 days to test its movement and spatial memory ability.ResultsCompared with the astrocytes in control group, the expression of NF-κBp65 protein in bilirurin-treated cells significantly increased (p<0.01), reaching the peak level at the time points of 2h and 12h (p<0.05). Consistent with the expression of NF-κB, the levels of IL-1β> TNF-α and IL-6 showed a time-dependent manner. TAT-NBD intervention inhibited dramatically bilirubin-induced NF-κBp65 activation as well as the releasing of IL-1β, TNF-α and IL-6 (p<0.01). As time on, the viability of astrocyte cells decreased after bilirubin exposure, whereas the survival rate of TAT-NBD intervention group was significantly higher than that of bilirubin group and lower than that of the control group (p<0.01). TAT-NBD can keep the normal cell morphology, reduce apoptosis and injury of cells.24h after modeled, the overexpression of GFAP and NF-κBp65 in bilirubin encephalopathy group as well as the nuclear transfer and activation of NF-κB. Bodyweight gain in TAT-NBD intervention group was higher than bilirubin encephalopathy group but no significant difference (p>0.05). The acute mortality rate of TAT-NBD intervention group was dramaticalliy lower than bilirubin encephalopathy group (p<0.01), long-term survival situation of TAT-NBD intervention group was better than bilirubin encephalopathy group but significant difference. The staying time on turn bar in balance exercise experiment as well as the latency in avoid dark evading experiment of TAT-NBD intervention group rats were longer than that of bilirubin group (p<0.05), shorter than that of control group respectively (p<0.01).ConclusionIn vitro model, bilirubin-induced rat’s cortical astrocytes inflammation is obviuosly related to the over expression and activation of NF-κB. TAT-NBD does show strong anti-inflammatory effects by inhibiting the activation of NF-κB and releasing of inflammatory cytokines, which might be useful in prophylaxis of bilirubin-induced brain injury.Early intraperitoneal administration of TAT-NBD could markedly reduce the overexpression of GFAP and NF-κB as well as the nuclear transfer and activation of NF-κB, meanwhile improving foragaing ability and long-term survival situation, reducing the acute mortality rate, and most importantly improving learning and memory ability as well as balance exercise capacity.