Department of Ophthalmology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China.
Department of Ophthalmology, The First Affiliated Hospital of Guangxi Medical University, Nanning, China,
Ophthalmic Res. 2020;63(3):284-294. doi: 10.1159/000503724. Epub 2020 Feb 25.
Transient receptor potential canonical (TRPC) channels are involved in neovascularization repairing after vascular injury in many tissues. However, whether TRPCs play a regulatory role in the development of diabetic retinopathy (DR) has rarely been reported. In the present study, we selected TRPC1, 3, and 6 to determine their roles and mechanism in human retina vascular endothelial cells (HREC) under high glucose (HG) conditions.
HRECs were cultured in vitro under HG, hyper osmosis, and normal conditions. The expression of TRPC1, 3, and 6 in the cells at 24 and 48 h were detected by RT-polymerase chain reaction (PCR), Western blot and cell immunohistochemistry (IHC); In various concentrations, SKF96365 acted on HG cultured HRECs, the expression of vascular endothelial growth factor (VEGF) were detected by the same methods above; and the CCK-8, Transwell, cell scratch assay, and Matrigel assay were used to assess cell proliferation, migration, and lumen formation.
The RT-PCR, Western blot, and IHC results showed that TRPC1 expression was increased, and TRPC6 mRNA expression was increased under high-glucose conditions. SKF96365 acted on HG cultured HRECs that VEGF expression was significantly decreased. The CCK-8 assay, Transwell assay, cell scratch assay, and Matrigel assay showed that cell proliferation, migration, and lumen formation were downregulated by SKF96365.
HG can induce increased expression of TRPC1 and 6 in HRECs. Inhibition of the TRPC pathway not only can decrease VEGF expression but also can prevent proliferation, migration, and lumen formation of HRECs induced by HG. Inhibition of TRPC channels is expected to become a drug target for DR.
瞬时受体电位经典型（TRPC）通道参与多种组织血管损伤后的新生血管修复。然而，TRPC 通道是否在糖尿病视网膜病变（DR）的发生发展中起调节作用鲜有报道。本研究选择 TRPC1、3 和 6，以确定其在高糖（HG）条件下人视网膜血管内皮细胞（HREC）中的作用及机制。
体外培养 HREC，在 HG、高渗和正常条件下培养。采用 RT-PCR、Western blot 和细胞免疫组化（IHC）检测细胞在 24 和 48 h 时 TRPC1、3 和 6 的表达；采用不同浓度的 SKF96365 作用于 HG 培养的 HRECs，采用上述相同方法检测血管内皮生长因子（VEGF）的表达；采用 CCK-8 法、Transwell 法、细胞划痕实验和 Matrigel 实验评估细胞增殖、迁移和管腔形成。
RT-PCR、Western blot 和 IHC 结果显示，高糖条件下 TRPC1 表达增加，TRPC6 mRNA 表达增加。SKF96365 作用于 HG 培养的 HRECs 后，VEGF 表达明显下降。CCK-8 实验、Transwell 实验、细胞划痕实验和 Matrigel 实验表明，SKF96365 下调细胞增殖、迁移和管腔形成。
HG 可诱导 HRECs 中 TRPC1 和 6 的表达增加。TRPC 通路的抑制不仅可以降低 VEGF 的表达，还可以阻止 HG 诱导的 HRECs 的增殖、迁移和管腔形成。抑制 TRPC 通道有望成为 DR 的药物靶点。
