Department of Cataract, Tianjin Eye Hospital, Tianjin, China.
Tianjin Key Lab of Ophthalmology and Visual Science, Tianjin, China.
Cell Commun Signal. 2023 Sep 18;21(1):236. doi: 10.1186/s12964-023-01210-4.
Arginase-1 (ARG1) promotes collagen synthesis and cell proliferation. ARG1 is highly expressed in various tumour cells. The mechanisms of ARG1 in epithelial-to-mesenchymal transition (EMT)-associated cataracts were studied herein.
C57BL/6 mice, a human lens epithelial cell line (HLEC-SRA01/04), and human lens capsule samples were used in this study. The right lens anterior capsule of the mouse eye was punctured through the central cornea with a 26-gauge hypodermic needle. Human lens epithelial cells (HLECs) were transfected with ARG1-targeted (siARG1) or negative control siRNA (siNC). For gene overexpression, HLECs were transfected with a plasmid bearing the ARG1 coding sequence or an empty vector. Medium containing 0.2% serum with or without transforming growth factor beta-2 (TGF-β2) was added for 6 or 24 h to detect mRNA or protein, respectively. The expression of related genes was measured by quantitative real-time polymerase chain reaction (RT-qPCR), western blotting, and immunohistochemical staining. Transwell assays and wound healing assays were used to determine cell migration. Cell proliferation, superoxide levels, nitric oxide (NO) levels, and arginase activity were estimated using Cell Counting Kit-8 assays, a superoxide assay kit, an NO assay kit, and an arginase activity kit.
ARG1, alpha-smooth muscle actin (α-SMA), fibronectin, and Ki67 expression increased after lens capsular injury, while zonula occludens-1 (ZO-1) expression decreased. Fibronectin and collagen type I alpha1 chain (collagen 1A1) expression increased, and cell migration increased significantly in ARG1-overexpressing HLECs compared with those transfected with an empty vector after TGF-β2 treatment. These effects were reversed by ARG1 knockdown. The arginase-related pathway plays an important role in EMT. mRNAs of enzymes of the arginase-related pathway were highly expressed after ARG1 overexpression. ARG1 knockdown suppressed these expression changes. Numidargistat (CB-1158) dihydrochloride (CB-1158), an ARG1 inhibitor, suppressed TGF-β2-induced anterior subcapsular cataract (ASC) by reducing the proliferation of lens epithelial cells (LECs) and decreasing fibronectin, α-SMA, collagen 1A1, and vimentin expression. Compared with that in nonanterior subcapsular cataract (non-ASC) patients, the expression of ARG1, collagen 1A1, vimentin, fibronectin, and Ki67 was markedly increased in ASC patients.
ARG1 can regulate EMT in EMT-associated cataracts. Based on the pathogenesis of ASC, these findings are expected to provide new therapeutic strategies for patients.
精氨酸酶 1（ARG1）促进胶原蛋白合成和细胞增殖。ARG1 在各种肿瘤细胞中高度表达。本研究旨在探讨 ARG1 在与上皮间质转化（EMT）相关的白内障中的作用机制。
本研究使用 C57BL/6 小鼠、人晶状体上皮细胞系（HLEC-SRA01/04）和人晶状体囊样本。通过中央角膜用 26 号皮下注射针穿刺小鼠眼球的右晶状体前囊。用靶向 ARG1 的（siARG1）或阴性对照 siRNA（siNC）转染人晶状体上皮细胞（HLECs）。为了基因过表达，用携带 ARG1 编码序列的质粒或空载体转染 HLECs。分别用含 0.2%血清的培养基加入或不加入转化生长因子β2（TGF-β2）孵育 6 或 24 h，以检测 mRNA 或蛋白。通过定量实时聚合酶链反应（RT-qPCR）、western blot 和免疫组织化学染色测量相关基因的表达。使用 Transwell 测定和划痕愈合测定分别测定细胞迁移。通过细胞计数试剂盒-8 测定法、超氧化物测定试剂盒、一氧化氮（NO）测定试剂盒和精氨酸酶活性试剂盒分别评估细胞增殖、超氧化物水平、NO 水平和精氨酸酶活性。
晶状体囊损伤后，ARG1、α-平滑肌肌动蛋白（α-SMA）、纤维连接蛋白和 Ki67 的表达增加，而封闭蛋白-1（ZO-1）的表达减少。与转染空载体的细胞相比，转染 ARG1 过表达质粒的细胞在 TGF-β2 处理后，纤维连接蛋白和 I 型胶原蛋白α1 链（胶原 1A1）的表达增加，细胞迁移明显增加。ARG1 敲低可逆转这些变化。精氨酸酶相关途径在 EMT 中发挥重要作用。ARG1 过表达后，精氨酸酶相关途径的酶的 mRNA 表达水平升高。ARG1 敲低抑制了这些表达变化。ARG1 抑制剂 Numidargistat（CB-1158）二盐酸盐（CB-1158）通过减少晶状体上皮细胞（LECs）的增殖和降低纤维连接蛋白、α-SMA、胶原 1A1 和波形蛋白的表达，抑制 TGF-β2 诱导的前囊下白内障（ASC）。与非前囊下白内障（非 ASC）患者相比，ASC 患者的 ARG1、胶原 1A1、波形蛋白、纤维连接蛋白和 Ki67 的表达明显增加。
ARG1 可调节 EMT 相关白内障中的 EMT。基于 ASC 的发病机制，这些发现有望为患者提供新的治疗策略。
