Xu Xiaogang, Liu Yanqing, Lan Menglong, Liu Fei, Xia Huimin, Zeng Jixiao
Department of Pediatric Surgery, Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangdong Provincial Clinical Research Center for Child Health, Guangzhou, China.
The First School of Clinical Medicine, Southern Medical University, Guangzhou, China.
Sci Prog. 2025 Apr-Jun;108(2):368504251336287. doi: 10.1177/00368504251336287. Epub 2025 Apr 29.
ObjectiveHirschsprung's disease (HSCR) is a rare congenital disorder attributed to the defects of enteric neural crest cells. We aim to identify characteristic phosphorylation proteins and preliminarily explore underlying related action mechanisms in HSCR.MethodsColon samples from HSCR patients underwent proteomic and phosphoproteomic sequencing to identify differentially expressed phosphoproteins (DEPPs) and proteins (DEPs). Interaction network construction and analysis of correlations with upstream phosphorylating kinases were employed to pinpoint core proteins. HSCR rat models were established through enema administration of Benzalkonium chloride and evaluated by measuring colon cross-sectional area, colon weight, AchE, and PGP9.5 levels. Histopathological damage was assessed via hematoxylin and eosin staining. Protein expression was analyzed using western blotting. Furthermore, the impact of SRC kinase in HSCR was investigated utilizing an SRC-specific inhibitor in HSCR rat models.ResultsA total of 5725 DEPPs were identified, with SRC kinase emerging as a key regulatory protein. In the HSCR rat model, SRC expression was elevated along with increased pCAV1 and FLNA levels. Notably, inhibition of SRC protein kinase activity by 1-(tert-butyl)-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d] pyrimidin-4-amine (PP2) led to reduced colon cross-sectional area and weight, an increase in the number of colonic ganglion cells, heightened AchE levels, enhanced PGP9.5 expression, and slight enlargement of the crypt, thereby alleviating HSCR symptoms in rats. Additionally, SRC kinase inhibition following PP2 treatment decreased the expression of pCAV1 and FLNA.ConclusionsInhibition of SRC kinase activity may potentially reduce CAV1/FLNA expression, ultimately alleviating the severity of HSCR in rats.
目的
先天性巨结肠（HSCR）是一种罕见的先天性疾病，归因于肠神经嵴细胞的缺陷。我们旨在鉴定特征性磷酸化蛋白，并初步探索HSCR潜在的相关作用机制。
方法
对HSCR患者的结肠样本进行蛋白质组学和磷酸蛋白质组学测序，以鉴定差异表达的磷酸化蛋白（DEPPs）和蛋白质（DEPs）。通过构建相互作用网络并分析与上游磷酸化激酶的相关性来确定核心蛋白。通过灌肠给予苯扎氯铵建立HSCR大鼠模型，并通过测量结肠横截面积、结肠重量、乙酰胆碱酯酶（AchE）和蛋白基因产物9.5（PGP9.5）水平进行评估。通过苏木精和伊红染色评估组织病理学损伤。使用蛋白质印迹法分析蛋白质表达。此外，在HSCR大鼠模型中利用SRC特异性抑制剂研究SRC激酶在HSCR中的作用。
结果
共鉴定出5725个DEPPs，SRC激酶成为关键调节蛋白。在HSCR大鼠模型中，SRC表达升高，同时pCAV1和丝状肌动蛋白（FLNA）水平增加。值得注意的是，1-（叔丁基）-3-（4-氯苯基）-1H-吡唑并[3,4-d]嘧啶-4-胺（PP2）抑制SRC蛋白激酶活性导致结肠横截面积和重量减小、结肠神经节细胞数量增加、AchE水平升高、PGP9.5表达增强以及隐窝轻度增大，从而减轻大鼠的HSCR症状。此外，PP2处理后抑制SRC激酶可降低pCAV1和FLNA的表达。
结论
抑制SRC激酶活性可能会降低CAV1/FLNA的表达，最终减轻大鼠HSCR的严重程度。
