Department of Laboratory Medicine and Sichuan Provincial Key Laboratory for Human Disease Gene Study, Sichuan Provincial People's Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, China.
Department of Clinical Laboratory, Sinopharm Dongfeng General Hospital, Hubei University of Medicine, Shiyan, Hubei 442008 China.
Int Immunopharmacol. 2024 Oct 25;140:112827. doi: 10.1016/j.intimp.2024.112827. Epub 2024 Aug 8.
Hyperhomocysteine has been recognized as an independent risk factor of multiple diseases, including several eye diseases. In this study, we aim to investigate whether increased homocysteine (Hcy) is related to cataracts, and to explore whether dysregulation of mTOR-mediated autophagy and connexin expression are underlying mechanisms.
We first developed a method of liquid chromatography tandem mass spectrometry to accurately measure serum concentrations of Hcy in 287 cataract patients and 334 healthy controls. Next, we treated human lens epithelial (HLC-B3) cells with Hcy at different concentrations and durations, and then analyzed expression of autophagy-related markers and connexins, as well as phosphorylated mTOR (p-mTOR) in these cells by Western blotting. Formation of autophagic vacuoles and intracellular Ca in the Hcy-treated cells were observed by fluorescence microscopy. Further, we performed a rescue experiment in the Hcy-treated HLC-B3 cells by pre-incubation with rapamycin, an mTOR inhibitor.
The serum levels of Hcy in patients with cataracts were significantly increased compared to those in healthy controls. In cultured HLC-B3 cells, expression of autophagy related markers (LC3B and Beclin1) and connexins (Cx43 and Cx50) was inhibited by Hcy treatment in a dose- and duration-dependent manner. Accumulation of Ca in the Hcy-treated lens epithelial cells was observed as a consequence of reduced connexin expression. Meanwhile, expression of p-mTOR increased, representing up-regulation of the mTOR pathway. Importantly, inhibition of autophagy and connexin expression due to hyperhomocysteine was rescued via mTOR suppression by pretreatment with rapamycin in HLC-B3 cells.
Our results demonstrate that hyperhomocysteine might promote cataract development through two mTOR-mediated pathways in the lens epithelial cells: 1) dysregulation of autophagy and 2) accumulation of intracellular calcium via decreased connexin expression.
高同型半胱氨酸血症已被认为是多种疾病的独立危险因素，包括几种眼部疾病。在本研究中，我们旨在研究同型半胱氨酸（Hcy）升高是否与白内障有关，并探讨 mTOR 介导的自噬和连接蛋白表达失调是否是潜在的机制。
我们首先开发了一种液相色谱串联质谱法，以准确测量 287 例白内障患者和 334 例健康对照者血清 Hcy 浓度。接下来，我们用不同浓度和时间的 Hcy 处理人晶状体上皮（HLC-B3）细胞，然后用 Western blot 分析这些细胞中自噬相关标志物和连接蛋白以及磷酸化 mTOR（p-mTOR）的表达。用荧光显微镜观察 Hcy 处理细胞中自噬小体的形成和细胞内 Ca2+。进一步，我们在 Hcy 处理的 HLC-B3 细胞中通过预先用 rapamycin（mTOR 抑制剂）孵育进行挽救实验。
与健康对照组相比，白内障患者血清 Hcy 水平明显升高。在培养的 HLC-B3 细胞中，自噬相关标志物（LC3B 和 Beclin1）和连接蛋白（Cx43 和 Cx50）的表达在 Hcy 处理时呈剂量和时间依赖性抑制。由于连接蛋白表达减少，Hcy 处理的晶状体上皮细胞中 Ca2+积累。同时，mTOR 通路被上调，表现为 p-mTOR 表达增加。重要的是，在 HLC-B3 细胞中，通过 rapamycin 预处理抑制 mTOR 可挽救由于高同型半胱氨酸血症引起的自噬和连接蛋白表达抑制。
我们的结果表明，高同型半胱氨酸血症可能通过两条 mTOR 介导的途径促进晶状体上皮细胞白内障的发生：1）自噬失调；2）通过连接蛋白表达减少导致细胞内钙积累。
