GHD矮小儿童血清IGF-1水平与血铅的相关性研究及其机制探讨

Correlation between serum IGF-1 and blood lead level in short stature children with growth hormone deficiency

目的通过生长激素缺乏症（GHD）儿童矮小发生与体内胰岛素样生长因子1（IGF-1）水平、血铅含量之间的相关性研究，以及铅暴露大鼠模型IGF-1相关信号分子的改变，为阐明铅诱导儿童矮小的发生及其致病机制研究提供科学依据，为铅暴露致儿童矮小的监测、防治提供可行途径。方法采用临床病例对照研究。自2011年6月至2013年3月依据相应诊断标准纳入880例西京医院儿科生长发育门诊就诊矮小患儿，通过生长激素（GH）激发试验GH峰值将患儿分为GHD和特发性矮小（ISS）组，检测并比较不同组别、性别患儿血清IGF-1和血铅水平；醋酸铅饮水法建立生长发育期大鼠铅中毒模型，利用原子吸收分光光度法测定血铅含量确定建模成功；利用蛋白印迹（Western blot）分析确证染铅大鼠脑组织IGF-1相关信号通路分子促分裂原活化蛋白激酶（MAPK）、磷脂酰肌醇-3-激酶／蛋白激酶B（P13K／AKT）的变化。采用均值t检验和非参数Mann—Whitney检验等方法进行统计学分析。结果880例矮小患儿中，GHD患儿362例（41．14％）。GHD组患儿IGF-1水平（20．02±11．24）μmo]／L（女），（20．74±13．39）μmol／L（男）均低于ISS组患儿IGF-1水平（46．58±27．00）μmol／L（女）、（35．91±20．05）μmol／L（男），z分别为10．45和9．98，P均〈0．01。GHD组血铅水平（0．49±0．18）μmol／L（女），（0．46±0．18）μmol／L（男）高于ISS组血铅（0．32±0．11）μmol／L（女），（0．34±0．13）μmol／L（男），t分别为-10．91和-9．056，P均〈0．01。铅暴露后大鼠血铅含量逐渐增加，染铅6周时趋于稳定。铅暴露导致信号通路MAPK、AKT中信号分子细胞外信号调节激酶（ERK1／2）、c-Jun氨基末端激酶（JNK）、p38丝裂原活化蛋白激酶（p38）、AKT473、AKT308磷酸化较总酶的蛋白相对表达量增加显著，AKT308／t—AKT组中x,1N与300ppm（1ppm醋酸铅=2．64×10μmol／L组蛋白相对表达量分别为1．320±0．071，2．960±0．552，F=19．360，P〈0．01；AKT473／t—AKT组中对照与300ppm组蛋白相对表达量分别为0．3114-0．038，1．018±0．282，F=16．101，P〈0．01；p-ERK／t-ERK组中对照与300ppm组蛋白相对表达量分别为1．173±0．109，6．4384-0．748，F=72．054，P〈0．01；p-JNK／t-JNK组中对照与300ppm组蛋白相对表达量分别为1．249±0．129，4．869±0．907，F=35．528，P〈0．01；P—P38／t-P38组中对照与300ppm组蛋白相对表达量分别为0．083±0．022，0．500±0．038，F=57．29，P〈0．01。结论GHD矮小儿童IGF-1水平减低可能与血铅水平升高相关，铅可能通过增加MAPK和AKT等IGF-1相关信号通路分子的磷酸化从而影响IGF-1介导的GH促生长作用，进而导致儿童GHD及矮小的发生。

Objective To investigate the correlation between serum insulin-like growth factor-1 （IGF-1） and blood lead levels in short stature children with growth hormone deficiency （GHD） , and study the changes of IGF-1 signal molecules in lead exposed rats, providing evidence for clarifying the pathogenesis of lead induced short stature in children. Methods Totally 800 short stature children were recruited in the clinical case-control study during June 2011 to March 2013 and were divided into GHD group or idiopathic short stature （ISS） group according to the their GH peak in growth hormone stimulation test. The serum IGF-1 levels and blood lead levels were determined. A lead poisoning model in rats was established and Western blot assay was employed to detect the phosphorylation of signaling molecules （ MAPK and PI3K/ AKT） related to IGF-1 signaling pathway. The average independent samples T-test and non-parametric Mann-Whitney test were used for statistical analysis. Results GHD children accounted for 41.14% （362 cases） of the 880. short stature cases. Serum IGF-1 levels in GHD group were （20. 02 ±1. 24） μmol/L in female and （20. 74 ±13.39 ） μmol/L in male, which were significantly lower than the ISS Group with （46. 58 ±27.00 ） μmol/L in female and （35.91± 20.05 ）μmol/L in male （t = 10.45, 9.98 respectively, both P 〈0. 01. ） However, the blood lead levels of GHD group [ （0. 49 ±0. 18）μ mol/L in female, （0. 46 ±0. 18 μmol/L）] in male were significantly higher than those in ISS group [ （0. 32 ±0. 11 ） μmol/L in female,（0. 34±0. 13 μmol/L in male]. All had a statistically significant difference （t = - 10. 91 and - 9. 056, both P 〈 0. 01 ）. Atomic absorption speetrophotometry showed the blood lead level in rats treated with lead containing water for 6 weeks significantly increased when compared with control group. Western blot assay confirmed that the protein expression of phosphorylated ERK1/2, JNK, p38, AKT473 and AKT308 increased significantly than the total enzyme in lead exposure rats. In AKT308/t-AKT group, the protein expression levels in the control group and 300 ppm（ 1 ppm =2. 64 × 10 -6mol/L） group were 1. 320 ±0. 071 and 2. 960±9. 552（F = 19. 360 ,P 〈0.01 ）. In AKT473/t-AKT group, the protein expression levels in the control group and 300 ppm group were 0. 311 ± 0. 038 and 1. 018± 0. 282, respectively（ F = 16. 101, P 〈 0.01 ） . In p-ERK/t-ERK group, the protein expression levels in the control group and 300 ppm group were 1. 173±0. 109 and 6. 438 ±0. 748（F =72. 054, P 〈0.01 ）. In p-JNK/t-JNK group, the protein expression levels in the control group and 300 ppm group were 1. 249 ±0. 129 and 4. 869 ±0. 907 （ F = 35. 528, P 〈 0. 01 ）. In p-P38/t-P38 group, the protein expression levels in the control group and 300 ppm group were 0. 083 ±0. 022 and 0. 500 ± 0. 038 （ F = 57. 29, P 〈 0. 01 ）. Conclusions The study suggests that reduction in IGF-1 in children with GHD is associated with an increased blood lead level. Lead possibly affects the IGF-l-mediated growth-promoting effect of GH by increasing the phosphorylation of molecules involved in MAPK, AKT and other IGF-l-related signaling pathways, eventually leading to the occurrence of child GHD and short stature.