目的：尿道下裂是男性常见的先天性疾病，通常伴有多系统异常，尤其重度尿道下裂常伴有各种严重并发症。国外有研究表明，患有重度尿道下裂的儿童在成年后容易患高血压、动脉粥样硬化和其他心血管疾病，早期预防尿道下裂及其并发症是必要的。然而，国内尚缺乏针对尿道下裂患儿心血管功能的研究，并且以往关于尿道下裂的发病机制及其可能的并发症的研究报道较少，尿道下裂的具体发病机制尚不清楚。本研究拟通过蛋白质组学方法对尿道下裂差异表达蛋白进行研究，并对尿道下裂患儿的临床资料进行回顾性分析，旨在为研究尿道下裂的发病机制和预后提供进一步的理论基础。

方法：

选取在2021年至2023年间于首都儿科研究所附属儿童医院泌尿外科行手术治疗的尿道下裂患儿作为研究对象，分别纳入44例轻度，65例中度和43例重度尿道下裂患儿，另外为三组病例各选取50名年龄匹配的健康患儿作为对照，采集所有研究对象血清学结果及临床资料进行数据分析。

选取在2022年4月至2022年9月间于首都儿科研究所附属儿童医院泌尿外科行手术治疗的尿道下裂患儿作为研究对象，分别纳入9例轻度，6例中度，5例重度尿道下裂患儿，另外同时选取5名于本院泌尿外科行包皮环切术的健康患儿作为对照，采集所有研究对象的包皮组织。

基于定量蛋白质组学方法，鉴定并筛选轻度、中度、重度尿道下裂和对照组患儿包皮组织中的DEPs，对DEPs进行PPI分析，并对尿道下裂患儿包皮组织中显著上调的差异表达蛋白进行验证。

应用现代生物信息学分析方法，对DEPs进行GO功能富集分析、KEGG通路富集分析，然后，对尿道下裂致病机制及其可能的并发症进行探讨。

结果：1.与对照组相比，我们发现sSBP、总胆固醇和低密度脂蛋白在轻、中、重度尿道下裂患儿中均显著增高。

2. 轻度尿道下裂患儿包皮组织差异表达蛋白以上调为主，中、重度尿道下裂患儿包皮组织差异表达蛋白以下调为主。在尿道下裂患儿中，AGT、FGG、FGB和SERPINA1蛋白表达水平显著升高(AGT**、FGG*、FGB*、SERPINA1*(*P<0.05,** P<0.01））。轻度尿道下裂组AGT蛋白水平略有升高(p=0.11)，但差异无统计学意义,中度、重度尿道下裂组患者均显著升高(中度：p<0.05;重度: p<0.01)。中度尿道下裂组AGT mRNA表达水平显著升高(p<0.05)，此外，轻、中度尿道下裂组APOC3 mRNA表达水平均显著升高(p<0.01)。IHC分析也显示，中度尿道下裂包皮组织中AGT蛋白水平上调(p<0.001)。

3. 在生物过程中，DEPs主要富集于正向调节胆固醇酯化、调节胆固醇酯化、蛋白-脂复合物重塑、血浆脂蛋白颗粒重塑、血浆脂蛋白颗粒组织、蛋白-脂复合物亚基组织。在细胞成分方面，大多数DEPs主要位于细胞外外泌体和膜结合囊泡中。在分子功能上，DEPs主要与2型血管紧张素受体结合、1型血管紧张素受体结合、血管紧张素受体结合等相关功能相关。在KEGG富集途径中，我们发现肾素-血管紧张素系统、胆固醇代谢和皮质醇合成和分泌是重要的途径。这些DEPs的GO注释和富集分析表明，尿道下裂患儿容易发生脂质代谢紊乱和心血管疾病。

结论： 本研究提示随着尿道下裂患儿容易发生脂质代谢紊乱和心血管疾病。我们的研究结果为尿道下裂患儿早期监测血脂和血压以预防心血管疾病提供了理论依据。

purposes: Hypospadias is a common congenital disease in men,and is often accompanied by multiple system abnormalities,severe hypospadias is often accompanied by various serious complications.Studies have shown that children with severe hypospadias are prone to hypertension, atherosclerosis and other CVDs in adulthood. Therefore, early prevention of hypospadias and its complications is necessary. However, no studies have focused on cardiovascular function in children with hypospadias in China. Few previous studies have reported on the mechanism of hypospadias and its possible complications,and the specific pathogenesis of hypospadias is still unclear.In this study, the differential expression of hypospadias protein was studied by proteomic methods, and clinical data of children with hypospadias were retrospectively analyzed, aiming to provide further theoretical basis for the study of the pathogenesis and prognosis of hypospadias.

Methods:

Children with hypospadias who underwent surgery in the Department of Urology, Children's Hospital Affiliated to Capital Institute of Pediatrics from 2021 to 2023 were selected as the study objects. We included 44 children with mild hypospadias, 65 with moderate hypospadias and 43 with severe hypospadias. Meanwhile, we selected 50 age-matched controls for each of the three groups of cases: mild, moderate, and severe hypospadias.Serological indicators and clinical data of all subjects were collected for data analysis.

Children with hypospadias who underwent surgery in the Department of Urology, Children's Hospital Affiliated to Capital Institute of Pediatrics from April , 2022 to September , 2022 were selected as the study objects. We included 9 children with mild hypospadias, 6 with moderate hypospadias, 5 with severe hypospadias.In addition,5 healthy children who underwent circumcision in the Department of Urology in our hospital were selected as controls, and the foreskin tissue of all subjects were collected.

Based on quantitative proteomics, the DEPs in the foreskin tissue of children with mild, moderate and severe hypospadias and the control group were identified and screened. PPI analysis was performed on the DEPs. The significantly upregulated differentially expressed proteins in the prepuse tissues of children with hypospadias were verified.

Modern bioinformatics analysis methods were applied to conduct GO functional enrichment analysis and KEGG pathway enrichment analysis of DEPs, and then we explore the pathogenic mechanism of hypospadias and its possible complications.

Results:

 We found that sSBP,TC and LDL were significantly increased in children with mild, moderate and severe hypospadias, compared with control group.

DEPs in foreskin tissue in children with mild hypospadias were mainly upregulated, and DEPs in preputial tissue in children with moderate or severe hypospadias were mainly downregulated.the relative expression levels of AGT,FGG,FGB and SERPINA1 proteins were significantly increased in children with hypospadias(AGT**、FGG*、FGB*、SERPINA1*(*P < 0.05,** P < 0.01））. The level of AGT protein was slightly increased in the mild hypospadias group(p=0.11), but there was no statistical significance. It was significantly increased in moderate and severe hypospadias groups (moderate: p < 0.05; severe: p < 0.01). Similarly, AGT mRNA level was increased in the foreskin tissue of children in the mild hypospadias group (p=0.11), but this was not significant. AGT mRNA level was significantly increased in the moderate hypospadias group (p < 0.05), which was consistent with the results of western blotting. Moreover, APOC3 mRNA level was also significantly increased in children with mild and moderate hypospadias(p<0.01).The IHC analysis also showed that the level of AGT protein was up-regulated in the foreskin tissue of moderate hypospadias (p< 0.001).

With regard to biological processes, DEPs were mainly enriched  in positive regulation of cholesterol esterification, regulation of cholesterol  esterification,protein-lipid complex remodeling,plasma lipoprotein particle remodeling, plasma lipoprotein particle organization, protein-lipid complex subunit organization(Fig.4a).Other non-top 20  enrichments included the regulation of plasma lipoprotein particle levels (p=0.0007), regulation of lipase activity (p=0.0009) and renin-angiotensin regulation of aldosterone production (p=0.0018). With regard to cell components, most of the DEPs were mainly located in extracellular exosomes and membrane-bound vesicles. With regard to molecular function, the DEPs were mainly associated with type 2 angiotensin receptor binding, type 1 angiotensin receptor binding, angiotensin receptor binding and other related functions.In the KEGG enrichment pathway,we found that the significant pathways were the renin-angiotensin system(p=0.0172),cholesterol metabolism(p= 0.0384),and cortisol synthesis and secretion(p=0.0468).These results are highly consistent with our previous epidemiological findings,which proves that children with hypospadias do have the risk of cardiovascular and cerebrovascular diseases.

Conclusion:  This study suggests that children with hypospadias are prone to lipid metabolism disorders and cardiovascular diseases. Our results provide a theoretical basis for early monitoring of blood lipids and blood pressure in children with hypospadias to prevent cardiovascular diseases.