中华实用儿科临床杂志
中華實用兒科臨床雜誌
중화실용인과림상잡지
Journal of Applied Clinical Pediatrics
2014年
12期
931-935
,共5页
梁小明%陈昌辉%邵天伟%骆娟%邹福兰%李茂军%唐彬秩
樑小明%陳昌輝%邵天偉%駱娟%鄒福蘭%李茂軍%唐彬秩
량소명%진창휘%소천위%락연%추복란%리무군%당빈질
胆红素%高胆红素血症%p38丝裂原活化蛋白激酶%凋亡%大鼠,新生
膽紅素%高膽紅素血癥%p38絲裂原活化蛋白激酶%凋亡%大鼠,新生
담홍소%고담홍소혈증%p38사렬원활화단백격매%조망%대서,신생
Bilirubin%Hyperbilirubinemia%p38 mitogen-activated protein kinases%Apoptosis%Rat,newborn
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-p38 MAPK)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)、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(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,1h时腹腔注入LPS 1 mg/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、24 h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.
目的 建立新生大鼠高膽紅素血癥模型,探討靜脈註入膽紅素對脾燐痠化p38絲裂原活化蛋白激酶(p38MAPK)(p-p38 MAPK)蛋白錶達和細胞凋亡的影響.方法 選用清潔級7～8d新生SD大鼠,雌雄不限,體質量12.0～15.0g,隨機抽籤法分為空白對照組(Ⅰ組)、脂多糖(LPS)對照組(Ⅱ組)、15 mg/kg膽紅素對照組(Ⅲ組)、15 mg/kg膽紅素+LPS組(Ⅳa組)、30 mg/kg膽紅素+LPS組(Ⅳb組)和50 mg/kg膽紅素+LPS組(Ⅳc組),共6組,每組設置2h、5h、24h3箇時間點,在實驗過程中200隻乳鼠有部分死亡,最終納入統計數據的乳鼠共144隻,每箇時間點8隻.頸靜脈註射不同劑量膽紅素(15 mg/kg、30 mg/kg和50 mg/kg)建立新生SD大鼠高膽紅素血癥模型,1h時腹腔註入LPS 1 mg/kg.在註入膽紅素後2h、5h處死大鼠,取脾髒,採用免疫組織化學法檢測脾p-p38MAPK蛋白的錶達,TUNEL法檢測脾細胞凋亡情況.結果 1.膽紅素對脾p-p38MAPK蛋白錶達的影響:低、中水平範圍的膽紅素可抑製p-p38MAPK的錶達(Ⅳa組2h、5h的q值分彆為20.93、10.37,P均＜0.01;Ⅳb組2h、5h的q值分彆為79.97、14.79,P均＜0.01),水平越高,抑製作用越明顯,持續時間越長;高水平範圍的膽紅素激活p-p38MAPK錶達(Ⅳc組2h、5h、24 h的q值分彆為32.55、19.23、27.72,P均＜0.01).2.膽紅素對脾細胞凋亡的影響:LPS單獨作用使脾細胞凋亡增加(q=54.62,P＜0.01),低水平範圍膽紅素對脾細胞凋亡無明顯影響(q=43.92,P＞0.05);低、中水平範圍的膽紅素與LPS共同作用可減少脾細胞凋亡(qⅣa=4.48,P＜0.01;qⅣb =2.07,P ＜0.05);高水平範圍的膽紅素可增加細胞凋亡(q =5.08,P＜0.01).結論 低、中水平膽紅素可抑製p-p38MAPK錶達的趨勢,高水平則促進p-p38MAPK錶達,且隨著水平的升高,作用時間延長.高水平膽紅素對脾細胞有毒性作用,細胞凋亡增多.提示膽紅素對免疫細胞的影響可能與調節TLRs信號通路中p38MAPK的燐痠化及誘導細胞的凋亡有關.
목적 건립신생대서고담홍소혈증모형,탐토정맥주입담홍소대비린산화p38사렬원활화단백격매(p38MAPK)(p-p38 MAPK)단백표체화세포조망적영향.방법 선용청길급7～8d신생SD대서,자웅불한,체질량12.0～15.0g,수궤추첨법분위공백대조조(Ⅰ조)、지다당(LPS)대조조(Ⅱ조)、15 mg/kg담홍소대조조(Ⅲ조)、15 mg/kg담홍소+LPS조(Ⅳa조)、30 mg/kg담홍소+LPS조(Ⅳb조)화50 mg/kg담홍소+LPS조(Ⅳc조),공6조,매조설치2h、5h、24h3개시간점,재실험과정중200지유서유부분사망,최종납입통계수거적유서공144지,매개시간점8지.경정맥주사불동제량담홍소(15 mg/kg、30 mg/kg화50 mg/kg)건립신생SD대서고담홍소혈증모형,1h시복강주입LPS 1 mg/kg.재주입담홍소후2h、5h처사대서,취비장,채용면역조직화학법검측비p-p38MAPK단백적표체,TUNEL법검측비세포조망정황.결과 1.담홍소대비p-p38MAPK단백표체적영향:저、중수평범위적담홍소가억제p-p38MAPK적표체(Ⅳa조2h、5h적q치분별위20.93、10.37,P균＜0.01;Ⅳb조2h、5h적q치분별위79.97、14.79,P균＜0.01),수평월고,억제작용월명현,지속시간월장;고수평범위적담홍소격활p-p38MAPK표체(Ⅳc조2h、5h、24 h적q치분별위32.55、19.23、27.72,P균＜0.01).2.담홍소대비세포조망적영향:LPS단독작용사비세포조망증가(q=54.62,P＜0.01),저수평범위담홍소대비세포조망무명현영향(q=43.92,P＞0.05);저、중수평범위적담홍소여LPS공동작용가감소비세포조망(qⅣa=4.48,P＜0.01;qⅣb =2.07,P ＜0.05);고수평범위적담홍소가증가세포조망(q =5.08,P＜0.01).결론 저、중수평담홍소가억제p-p38MAPK표체적추세,고수평칙촉진p-p38MAPK표체,차수착수평적승고,작용시간연장.고수평담홍소대비세포유독성작용,세포조망증다.제시담홍소대면역세포적영향가능여조절TLRs신호통로중p38MAPK적린산화급유도세포적조망유관.
Objective To explore the effects of bilirubin on myeloid differentiation factor phospho-p38 mitogen-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,tinally,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 bilirubin (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:bilirubin 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 ;qⅣ b =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 bilirubin (q =43.92,P ＞ 0.05).Low-mid concentration of bilirubin 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 bilirubin with LPS increased the AI of splenocytes (q =5.08,P ＜ 0.01).Conclusions Bilirubin in low-mid concentrations of range could inhibit the 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.
