HIPK2-p53信号通路在大鼠孕期砷暴露诱导子代孤独症样行为中的作用和机制研究

Genotoxicity caused by the exposure of environmental risk factors is one of the important pathogenesis of autism. HIPK2 is a key kinase that regulates the expression of a variety of genotoxic injuries-related transcription factors. Our previous animal study found that arsenic exposure during pregnancy induces autism-like behavior (ALB) of offspring, and the hypomethylation of HIPK2 in frontal cortex. Subsequent the clinical trials revealed that obviously increased expression of serum HIPK2 and its downstream regulatory transcription factors p53 was observed in children with autism, however, the role and mechanism of abnormal HIPK2-related signaling pathways in autism is still not clear. Therefore, we hypothesized that arsenic exposure during pregnancy could result in neuronal dysplasia of prefrontal cortex by HIPK2 hypomethylation and abnormal activation of p53, may contribute to the ALB in rat offspring. To this end, we will take advantage of the established model of arsenic exposure during pregnancy and in vitro arsenic exposure of frontal cortex neuron, using radionuclide labeling, flow cytometry, electrophysiological and RNA interference regulatory pathways to study the specific mechanism of ALB caused by abnormal regulation of HIPK2-p53 signaling pathway in arsenic exposure during pregnancy. The expected results will provide new strategies and targets for the prevention and treatment of autism.

环境危险因素暴露所致的遗传毒性损伤是孤独症重要发病机制之一。HIPK2是调控多种遗传毒性损伤相关转录因子表达的关键性激酶。本课题组前期动物实验发现大鼠孕期砷暴露可诱导子代孤独症样行为（autism-like behavior，ALB）、额叶皮层神经元HIPK2低甲基化；随后的临床实验检测孤独症患儿血清发现HIPK2及其下游调控的转录因子p53表达明显增加，但HIPK2相关通路的异常在孤独症中的作用和机制尚不清楚。因此，本项目提出孕期砷暴露可通过降低HIPK2甲基化异常活化p53致子代额叶皮层神经元发育障碍，从而诱导ALB的假说。为此，我们将利用已构建的大鼠孕期和额叶皮层神经元原代培养砷染毒模型，采用放射性核素标记、流式细胞检测、神经电生理、RNA干扰调节通路等手段，研究孕期砷暴露下HIPK2-p53信号通路调控异常所致ALB的具体作用机制。预期结果将为孤独症的防治提供新的策略和靶点。

研究背景和目的：砷是最常见的污染物之一。流行病学调查和动物实验研究已证实孕期砷暴露可以通过胎盘屏障，影响胎儿发育。然而，砷暴露与神经发育障碍性疾病孤独症谱系障碍（ASD）之间关系的研究在国内外尚处于起步阶段。通过构建孕期砷暴露模型，了解孕期砷暴露是否可致子代ASD样行为，探讨孕期砷暴露如何影响DNA甲基化以及这些表观遗传变化如何调节ASD的风险和/或严重程度。.研究内容和方法：建立并检测孕期砷暴露及丙戊酸暴露（VPA）ASD动物模型和正常对照组生长发育情况，并运用热板致痛法、倾斜板实验、社交及非社交实验、刻板行为检测、Momrris水迷宫等方法对模型大鼠进行行为学检测。对砷暴露组和对照组子代大鼠（n=6只/组，生后4-6周）的额叶皮层进行神经元甲基化检测。根据甲基化的检测结果，使用病例对照研究分析ASD患儿血清HIPK2和p53蛋白表达水平。进一步利用砷暴露动物模型探讨HIPK2-p53信号通路在大鼠孕期砷暴露诱导子代ASD样行为中的作用和机制。.结果：与对照组比较孕期砷暴露组大鼠睁眼时间、方向趋向能力、游泳协调能力、对伤害性刺激-痛觉的敏感性及学习和记忆能力无明显影响。砷暴露组和VPA-ASD模型组大鼠体重下降、社交能力下降及刻板行为明显增加，但VPA-ASD模型组尤为明显（Ps<0.05）。甲基化检测发现转录子上游2kb水平调控区域具有甲基化差异的基因共有51个，其中与神经发育有关的基因主要有：Pum2、HIPK2、Enpp6、DNM1、CPLx1、C1q14、Map3k13、Gys1 8个基因。病例对照研究表明ASD患儿血清中HIPK2和p53表达明显高于健康对照组儿童，且HIPK2与p53表达水具有明显的相关性。但血清HIPK2、p53水平与ASD症状严重程度无明显相关性。HIPK2和p53作为预测因子绘制ROC曲线，其曲线下面积AUC分别为0.825 (95% CI: 0.698-0.953, P<0.001) 和0.835 (95% CI: 0.703-0.966, P<0.001)。动物实验表明，孕期砷暴露及VPA-ASD模型额叶皮层神经元HIPK2-p53信号通路上调，子代大鼠额叶皮层凋亡增加。 .结论：额叶皮层甲基化异常从而上调HIPK2-P53通路导致神经元凋亡异常可能是砷暴露诱导子代ASD样行为的潜在机制，研究结果为ASD的防治提供新的策略和靶