Department of Endocrinology, the Second Affiliated Hospital, Kunming Medical University, Kunming, Yunnan, China.
Department of Epidemiology and Biostatistics, School of Public Health, Kunming Medical University, Kunming, Yunnan, China.
PeerJ. 2024 Aug 14;12:e17676. doi: 10.7717/peerj.17676. eCollection 2024.
The incidence of diabetes-associated cognitive dysfunction (DACD) is increasing; however, few clinical intervention measures are available for the prevention and treatment of this disease. Research has shown that proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, particularly SBC-115076, have a protective effect against various neurodegenerative diseases. However, their role in DACD remains unknown. In this study, we aimed to explore the impact of PCSK9 inhibitors on DACD.
Male Sprague-Dawley (SD) rats were used to establish an animal model of type 2 diabetes mellitus (T2DM). The rats were randomly divided into three groups: the Control group (Control, healthy rats, = 8), the Model group (Model, rats with T2DM, = 8), and the PCSK9 inhibitor-treated group (Treat, T2DM rats treated with PCSK9 inhibitors, = 8). To assess the spatial learning and memory of the rats in each group, the Morris water maze (MWM) test was conducted. Hematoxylin-eosin staining and Nissl staining procedures were performed to assess the structural characteristics and functional status of the neurons of rats from each group. Transmission electron microscopy was used to examine the morphology and structure of the hippocampal neurons. Determine serum PCSK9 and lipid metabolism indicators in each group of rats. Use qRT-PCR to detect the expression levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) in the hippocampal tissues of each group of rats. Western blot was used to detect the expression of PCSK9 and low-density lipoprotein receptor (LDLR) in the hippocampal tissues of rats. In addition, a 4D label-free quantitative proteomics approach was used to analyse protein expression in rat hippocampal tissues. The expression of selected proteins in hippocampal tissues was verified by parallel reaction monitoring (PRM) and immunohistochemistry (IHC).
The results showed that the PCSK9 inhibitor alleviated cognitive dysfunction in T2DM rats. PCSK9 inhibitors can reduce PCSK9, total cholesterol (TC), and low-density lipoprotein (LDL) levels in the serum of T2DM rats. Meanwhile, it was found that PCSK9 inhibitors can reduce the expression of PCSK9, IL-1β, IL-6, and TNF-α in the hippocampal tissues of T2DM rats, while increasing the expression of LDLR. Thirteen potential target proteins for the action of PCSK9 inhibitors on DACD rats were identified. PRM and IHC revealed that PCSK9 inhibitors effectively counteracted the downregulation of transthyretin in DACD rats.
This study uncovered the target proteins and specific mechanisms of PCSK9 inhibitors in DACD, providing an experimental basis for the clinical application of PCSK9 inhibitors for the potential treatment of DACD.
糖尿病相关认知功能障碍（DACD）的发病率正在上升；然而，针对这种疾病的预防和治疗措施却很少。研究表明，前蛋白转化酶枯草溶菌素/柯萨奇蛋白酶 9（PCSK9）抑制剂，特别是 SBC-115076，对各种神经退行性疾病具有保护作用。然而，其在 DACD 中的作用尚不清楚。本研究旨在探讨 PCSK9 抑制剂对 DACD 的影响。
雄性 Sprague-Dawley（SD）大鼠用于建立 2 型糖尿病（T2DM）动物模型。大鼠随机分为三组：对照组（Control，健康大鼠，n = 8）、模型组（Model，T2DM 大鼠，n = 8）和 PCSK9 抑制剂治疗组（Treat，T2DM 大鼠用 PCSK9 抑制剂治疗，n = 8）。为了评估各组大鼠的空间学习和记忆能力，进行了 Morris 水迷宫（MWM）测试。进行苏木精-伊红（H&E）染色和尼氏染色程序，以评估各组大鼠神经元的结构特征和功能状态。使用透射电子显微镜观察海马神经元的形态和结构。测定各组大鼠血清 PCSK9 和脂质代谢指标。使用 qRT-PCR 检测各组大鼠海马组织中白细胞介素（IL）-1β、IL-6 和肿瘤坏死因子-α（TNF-α）的表达水平。使用 Western blot 检测各组大鼠海马组织中 PCSK9 和低密度脂蛋白受体（LDLR）的表达。此外，采用 4D 无标记定量蛋白质组学方法分析大鼠海马组织中的蛋白质表达。通过平行反应监测（PRM）和免疫组织化学（IHC）验证海马组织中选定蛋白的表达。
结果表明，PCSK9 抑制剂减轻了 T2DM 大鼠的认知功能障碍。PCSK9 抑制剂可降低 T2DM 大鼠血清中的 PCSK9、总胆固醇（TC）和低密度脂蛋白（LDL）水平。同时，发现 PCSK9 抑制剂可降低 T2DM 大鼠海马组织中 PCSK9、IL-1β、IL-6 和 TNF-α的表达，同时增加 LDLR 的表达。鉴定出 13 种潜在的 PCSK9 抑制剂作用于 DACD 大鼠的靶蛋白。PRM 和 IHC 显示 PCSK9 抑制剂可有效逆转 DACD 大鼠转甲状腺素的下调。
本研究揭示了 PCSK9 抑制剂在 DACD 中的作用靶点和具体机制，为 PCSK9 抑制剂在 DACD 的潜在治疗中的临床应用提供了实验依据。
