Zhou Xiaoyan, Sun Yan, Yang Guoshuai
Department of Hemodialysis, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Haikou, 570208, China.
Department of Neurology, Haikou Affiliated Hospital of Central South University Xiangya School of Medicine, Haikou, 570208, China.
In Vitro Cell Dev Biol Anim. 2025 Jul 9. doi: 10.1007/s11626-025-01057-8.
5-Methoxytryptophan (5-MTP), a candidate biomarker for chronic kidney disease (CKD), has an undefined role in cerebrovascular pathophysiology. To investigate this, we employed a folic acid (FA)-induced CKD to simulate cerebrovascular complications in vivo. Additionally, in vitro models of cerebral ischemia and cerebrovascular endothelial cell injury were established. 5-MTP was administered to rats and cells, along with nuclear factor-κB (NF-κB) expression. The pathological characteristics of kidney and brain tissue were observed by histological staining. Cell proliferation was assessed using the Cell Counting Kit 8, while tube formation and migration were examined using tube formation and wound healing assays. Cell apoptosis was detected using both TdT-mediated dUTP-biotin nick end labeling and flow cytometry. Levels of renal injury markers, blood biomarkers of cerebrovascular disease, and inflammatory cytokines were measured using biochemical assays. Quantitative real-time PCR and Western blot were used to detect the mRNA and protein expression, respectively. Key findings revealed that FA successfully induced CKD in rats, which subsequently exacerbated cerebrovascular dysfunction. 5-MTP reduced the levels of proteinuria, N-acetyl-beta-D-glucosaminidase, nephrin, endothelin-1, von Willebrand factor, and thrombomodulin; improved the degree of renal fibrosis and structural damage to the brain tissue; and inhibited cell apoptosis in rats. In vitro, 5-MTP promoted cell proliferation, tube formation, migration, and the upregulation of B-cell lymphoma-2 and caspase-3 expression. This treatment also led to an increase in interleukin (IL)-10 levels while suppressing cell apoptosis, Bcl-2-associated X protein (Bax), and cleaved caspase-3 expression. Furthermore, it reduced the IL-6 and tumor necrosis factor-alpha levels. NF-κB overexpression reversed the effects of 5-MTP in vitro and in vivo. Our results demonstrate that 5-MTP ameliorated CKD-induced cerebrovascular injury through the NF-κB pathway, indicating its potential as an innovative and efficacious therapeutic target for CKD-induced cerebrovascular dysfunction.
5-甲氧基色氨酸（5-MTP）作为慢性肾脏病（CKD）的一种候选生物标志物，在脑血管病理生理学中的作用尚不明确。为了对此进行研究，我们采用叶酸（FA）诱导的CKD模型在体内模拟脑血管并发症。此外，还建立了脑缺血和脑血管内皮细胞损伤的体外模型。将5-MTP给予大鼠和细胞，并检测核因子κB（NF-κB）的表达。通过组织学染色观察肾脏和脑组织的病理特征。使用细胞计数试剂盒8评估细胞增殖，通过管腔形成和伤口愈合试验检测管腔形成和迁移情况。使用末端脱氧核苷酸转移酶介导的dUTP生物素缺口末端标记法和流式细胞术检测细胞凋亡。采用生化检测方法测定肾损伤标志物、脑血管疾病血液生物标志物和炎性细胞因子的水平。分别使用定量实时聚合酶链反应和蛋白质印迹法检测mRNA和蛋白质表达。主要研究结果显示，FA成功诱导大鼠发生CKD，随后加重了脑血管功能障碍。5-MTP降低了蛋白尿、N-乙酰-β-D-氨基葡萄糖苷酶、nephrin、内皮素-1、血管性血友病因子和血栓调节蛋白的水平；改善了肾纤维化程度和脑组织的结构损伤；并抑制了大鼠细胞凋亡。在体外，5-MTP促进细胞增殖、管腔形成、迁移以及B细胞淋巴瘤-2和半胱天冬酶-3表达上调。该处理还导致白细胞介素（IL）-10水平升高，同时抑制细胞凋亡、Bcl-2相关X蛋白（Bax）和裂解的半胱天冬酶-3表达。此外，它降低了IL-6和肿瘤坏死因子-α水平。NF-κB过表达在体外和体内均逆转了5-MTP的作用。我们的结果表明，5-MTP通过NF-κB途径改善了CKD诱导的脑血管损伤，表明其作为CKD诱导的脑血管功能障碍的一种创新且有效的治疗靶点的潜力。
