静脉注入胆红素对新生大鼠脾磷酸化TAK1和磷酸化IKK蛋白表达的影响

Effects of intravenous infusion of bilirubin on the expression of phospho-TAK1 and phospho-IKK in neonatal rats' splenocytes

目的建立新生大鼠高胆红素血症模型,探讨胆红素对脾磷酸化转化生长因子-β激活激酶1（TAK1）和磷酸化核因子κB抑制因子激酶（IKK）蛋白表达的影响。方法清洁级7~8 d新生SD大鼠,雌雄不限,体质量12.0~15.0 g,采用随机抽签法分为生理盐水对照组（Ⅰ组）、脂多糖（LPS）对照组（Ⅱ组）、15 mg/kg胆红素对照组（Ⅲ组）、15 mg/kg胆红素＋LPS组（Ⅳa组）、30 mg/kg胆红素＋LPS组（Ⅳb组）和50 mg/kg胆红素＋LPS组（Ⅳc组）,共6组。每组设置2 h、5 h、24 h三个时间点。每个时间点8只。颈静脉注射不同剂量胆红素（15 mg/kg、30 mg/kg和50 mg/kg）建立新生SD大鼠高胆红素血症模型。1 h时腹腔注入LPS 1 mg/kg。在注入胆红素后2 h、5 h和24 h时处死大鼠,免疫组织化学法测定脾磷酸化TAK1和磷酸化IKK蛋白的表达。结果 LPS单独作用能促进脾磷酸化TAK1和磷酸化IKK蛋白的表达（P〈0.05）;胆红素单独作用及与LPS共同作用均能够抑制脾磷酸化TAK1和磷酸化IKK蛋白的表达（P〈0.05）,随着胆红素浓度的升高,抑制作用增强。在注入胆红素2 h和5 h时,磷酸化TAK1和磷酸化IKK蛋白表达水平与血浆总胆红素浓度呈负相关（P〈0.01）。在2 h、5 h和24 h时,磷酸化IKK与磷酸化TAK1 IOD（SUM）值呈正相关（P〈0.01）。结论胆红素可通过调节免疫细胞TLRs信号通路中磷酸化TAK1和磷酸化IKK蛋白的表达而影响免疫功能。

Objective To investigate the effects of bilimbin on phospho-transforming growth factor-~l activated kinase-I （TAK1） and phospho-inhibitor of nuclear factor r,B kinase （IKK） of splenocytes in the hyperbilirubinemia animal model. Methods Seven-day-old Sprague Dawley rats （clean grade）, male or female, weighting 12.0-15.0 g, were randomly assigned to 6 groups. There were saline group（ I ）, lipopolysaccharide Control group （LPS, II）, 15 mg/kg bilirubin control （free-LPS） group （III）, 15 mg/kg group （IVa）, 30 mg/kg group （IVb） and 50 mg/kg group （IVc）, and then subsequently divided into 2 h, 5 h and 24 h subgroiips in each groups, and 8 rats were in each subgroups. Newborn Spragne Dawley rats were administered at various doses ofbilirubin （15 mg/kg, 30 mg/kg and 50 mg/kg respectively） intravenously; 1 h after the injection ofbilirubin or saline, all groups were administered LPS intraperitoneally. Blood samples were obtained for measurement of plasma bilirubin concentrations at 2 h, 5 h and 24 h following bilirubin administration. Phospho-TAKl and phospho-IKK were determined by imrnunohistochemistry. Results LPS could stimulate the expression of phospho-TAK1 and phospho-IKK （P〈0.05）. Bilirubin could inhibit the expression ofphospho-TAK1 and phospho-IKK in response to stimulation of LPS （P〈0.05）. The inhibition strengthened with increasing concentration of bilirubin. There was negative correlation between the expression of phospho-TAKl and phospho-IICK and the concentration of bilirubin （P〈0.01）. There was positive correlation between the expression of phospho-TAK1 and phosphoJKK respectively at 2 h, 5 h and 24 h （P〈0.01）. Conclusion Bilirubin could affect immune function by regulating phosphorylation of TAK1 and IKK in immune ceils at TLRs signal pathway.