静脉注入胆红素对新生大鼠脾磷酸化p38丝裂原活化蛋白激酶蛋白表达和细胞凋亡的影响

Effects of intravenous infusion of bilirubin on the expression of phospho-p38 mitogen-activated protein kinase and the apoptosis in splenocytes of neonatal rats

目的建立新生大鼠高胆红素血症模型，探讨静脉注入胆红素对脾磷酸化p38丝裂原活化蛋白激酶（p38MAPK）（p—p38MAPK）蛋白表达和细胞凋亡的影响。方法选用清洁级7～8d新生SD大鼠，雌雄不限，体质量12．0～15．0g，随机抽签法分为空白对照组（I组）、脂多糖（LPS）对照组（Ⅱ组）、15mg／kg胆红素对照组（Ⅲ组）、15mg／kg胆红素＋LPS组（1Va组）、30mg／kg胆红素＋LPS组（1Vb组）和50mg／kg胆红素＋LPS组（IVC组），共6组，每组设置2h、5h、24h3个时间点，在实验过程中200只乳鼠有部分死亡，最终纳入统计数据的乳鼠共144只，每个时间点8只。颈静脉注射不同剂量胆红素（15mg／kg、30mg／kg和50mg／kg）建立新生SD大鼠高胆红素血症模型，1h时腹腔注入LPS1mg／kg。在注入胆红素后2h、5h处死大鼠，取脾脏，采用免疫组织化学法检测脾p-p38MAPK蛋白的表达，TUNEL法检测脾细胞凋亡情况。结果1．胆红素对脾P—p38MAPK蛋白表达的影响：低、中水平范围的胆红素可抑制p-p38MAPK的表达（Ⅳa组2h、5h的q值分别为20．93、10．37，P均〈0．01；Ⅳb组2h、5h的q值分别为79．97、14．79，P均〈0．01），水平越高，抑制作用越明显，持续时间越长；高水平范围的胆红素激活p-p38MAPK表达（ⅣC组2h、5h、24h的q值分别为32．55、19．23、27．72，P均〈0．01）。2．胆红素对脾细胞凋亡的影响：LPS单独作用使脾细胞凋亡增加（q=54．62，P〈0．01），低水平范围胆红素对脾细胞凋亡无明显影响（q=43．92，P〉0．05）；低、中水平范围的胆红素与LPS共同作用可减少脾细胞凋亡（qⅣa=4．48，P〈0．01；qⅣb=2．07，P〈0．05）；高水平范围的胆红素可增加细胞凋亡（q=5．08，P〈0．01）。结论低、中水平胆红素可抑制p-p38MAPK表达的趋势，高水平则促进p-p38MAPK表达，且随着水平的升高，作用时间延长。高水平胆红素对脾细胞有毒性作用，细胞凋亡增多。提示胆红素对免疫细胞的影响可能与调节TLRs信号通路中p38MAPK的磷酸化及诱导细胞的凋亡有关。

Objective To explore the effects of bilirubin on myeloid differentiation factor phospho-p38 mitogeM-activated protein kinase （p-p38MAPK） and apoptosis in splenocytes of neonatal rats. 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 blank control group （ Ⅰ ） , lipopolysaccharide （LPS） control group （ 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 subgroups in each groups. Some of the 200 newborn rats died amid the experiment, finally, a total of 144 cases were involved in the analysis of results, and 8 rats in each subgroups. Newborn Sprague Dawley rats were administered at various doses of bilirnbin （15 mg/kg,30 mg/kg and 50 mg/kg, respectively） intravenously; 1 h after injection,the rats were administered LPS intraperitoneally at a dose of 1 mg/kg;p-p38MAPK were detected by immunohistochemistry;Apoptosis in splenocytes was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling methods at 2 h 5 h and 24 h after the injection of bilirubin. Results 1. Expression of p-p38MAPK in each group：bilirnbin in low-mid concentrations of range inhibited LPS-induced p38MAPK activation （qⅣa = 20.93, 10.37 ,respectively at 2 h,and 5 h,all P 〈0.01 ;qlVb =79.97,14.79 ,all P 〈0.01 ）. The inhibition strengthened with increasing concentration of bilirubin. The effect was observed at 2 h, strengthened at 5 h, disappeared at 24 h. Bilirubin in the high concentrations of range stimulated the expression of p-p38MAPK （qⅣc = 32.55,19.23,27.72, respectively at 2 h,5 h and 24 h, all P 〈 0.01 ）, observed at 5 h, reduced at 24 h. 2. Effects of bilirubin on apoptosis in splenocytes： LPS could increased the apoptosis index （AI） of splenocytes（q = 54.62,P 〈 0.01 ） ;The AI of splenocytes had no significant change in low concentrations of range of bilirnbin （q = 43.92,P 〉 0.05）. Low-mid concentration of bilirnbin with LPS reduced the AI of splenocytes （ q Ⅳ a = 4.48, P 〈 0.01 ; q Ⅳ b = 2.07, P 〈 0.05 ）, while high concentration of bilirnbin with LPS increased the AI of splenocytes （ q = 5.08, P 〈 0.01 ）. Conclusions Bilirnbin in low-mid concentrations of range could inhibit/he expression of LPS-induced p38MAPK, while bilirubin in high concentrations of range stimulated the expression. As the concentration of bilirubin elevated,its inhibition was prolonged. Bilirubin in high concentrations of range bilirubin could induce apoptosis in splenocytes. The immune dysfunction in neonatal hyperbilirubinemia may have something to do with the regulation of phosphorylation of p38MAPK and activation of apoptotic pathways.