Arginase-1促进前囊下白内障不同模型中晶状体上皮间质的转变,Cell Communication and Signaling

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Arginase-1促进前囊下白内障不同模型中晶状体上皮间质的转变
Cell Communication and Signaling ( IF 8.2 ) Pub Date : 2023-09-18 , DOI: 10.1186/s12964-023-01210-4
Qingyu Li _{1,

2} , Yuchuan Wang _{1,

2} , Luoluo Shi _{1,

2} , Qing Wang _{3,

4} , Guang Yang ₅ , Lin Deng _{1,

2} , Ye Tian _{1,

2} , Xia Hua ₆ , Xiaoyong Yuan _{1,

2}

Affiliation

精氨酸酶-1 (ARG1) 促进胶原蛋白合成和细胞增殖。ARG1在多种肿瘤细胞中高表达。本文研究了 ARG1 在上皮间质转化 (EMT) 相关白内障中的机制。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。用26号皮下注射针穿过中央角膜刺穿小鼠眼的右晶状体前囊。用 ARG1 靶向 (siARG1) 或阴性对照 siRNA (siNC) 转染人晶状体上皮细胞 (HLEC)。对于基因过表达，HLEC 用带有 ARG1 编码序列的质粒或空载体转染。添加含有或不添加转化生长因子β-2 (TGF-β2)的0.2%血清培养基6或24小时，分别检测mRNA或蛋白质。通过实时定量聚合酶链反应（RT-qPCR）、蛋白质印迹和免疫组织化学染色来测量相关基因的表达。使用 Transwell 测定和伤口愈合测定来确定细胞迁移。使用 Cell Counting Kit-8 测定、超氧化物测定试剂盒、NO 测定试剂盒和精氨酸酶活性试剂盒评估细胞增殖、超氧化物水平、一氧化氮 (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）、纤连蛋白和 Ki67 表达增加，而闭锁小带-1（ZO-1）表达减少。与空载体转染的 TGF-β2 处理后相比，过表达 ARG1 的 HLEC 中纤连蛋白和 I 型胶原 α1 链（胶原 1A1）表达增加，细胞迁移显着增加。这些效应可通过 ARG1 敲除来逆转。精氨酸酶相关途径在EMT中发挥重要作用。ARG1 过表达后，精氨酸酶相关途径酶的 mRNA 高表达。ARG1 敲除抑制了这些表达变化。Numidargistat (CB-1158) 二盐酸盐 (CB-1158) 是一种 ARG1 抑制剂，通过减少晶状体上皮细胞 (LEC) 的增殖和减少纤连蛋白、α-SMA、胶原蛋白 1A1 来抑制 TGF-β2 诱导的前囊下白内障 (ASC)和波形蛋白表达。与非前囊下白内障（non-ASC）患者相比，ASC患者ARG1、胶原1A1、波形蛋白、纤连蛋白和Ki67的表达显着升高。ARG1 可以调节 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。根据纤维化发生的部位，纤维化白内障可分为前囊下白内障或后囊膜混浊。纤维化白内障的机制尚不完全清楚。由创伤、炎症或辐射引起的纤维性混浊可在晶状体前囊下方积聚，导致前囊下白内障。后囊膜混浊是超声乳化人工晶状体植入术最常见的并发症之一，在年轻患者中发生率较高。我们首次证明 ARG1 可以调节纤维化白内障的 EMT。TGF-β2是LECs纤维化的主要原因。TGF-β2处理或小鼠晶状体囊损伤后，LECs中ARG1和纤连蛋白的表达增加。我们研究了 ARG1 调节纤维化白内障 EMT 的具体分子机制。由于 HLEC 中 ARG1 表达的敲低，精氨酸酶相关途径酶的 mRNA 表达降低。ARG1 过表达可逆转这些效应。此外，敲除 ARG1 会降低胶原蛋白 1A1、纤连蛋白和波形蛋白的表达。超氧化物水平；以及细胞迁移和 NO 水平升高。ARG1 过表达可逆转这些效应。用 CB-1158 对 ARG1 通路进行药理学阻断，可减少 LEC 的增殖，并减少小鼠晶状体中纤连蛋白、α-SMA、胶原蛋白 1A1 和波形蛋白的表达。我们认为ARG1通过直接激活精氨酸酶途径促进胶原蛋白1A1的产生，并通过减少NO产生和增加超氧化物水平导致晶状体纤维化，为预防和治疗纤维化白内障提供新机制。

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Arginase-1 promotes lens epithelial-to-mesenchymal transition in different models of anterior subcapsular cataract

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. Fibrotic cataracts can be classified as anterior subcapsular cataract or posterior capsular opacification depending on where fibrosis occurs. The mechanism of fibrotic cataracts is not fully understood. Fibrotic opacities induced by trauma, inflammation, or radiation can accumulate underneath the anterior lens capsule, causing anterior subcapsular cataract. Posterior capsular opacification is one of the most common complications of phacoemulsification with intraocular lens implantation, with a high incidence in young patients. We show for the first time that ARG1 can regulate EMT in fibrotic cataracts. TGF-β2 is the main cause of fibrosis in LECs. The expression of ARG1 and fibronectin in LECs increased after TGF-β2 treatment or mouse lens capsular injury. We investigated the specific molecular mechanisms by which ARG1 regulates EMT in fibrotic cataracts. The mRNA expression of enzymes of the arginase-related pathway was decreased due to knockdown of ARG1 expression in HLECs. These effects were reversed by ARG1 overexpression. Additionally, knockdown of ARG1 decreased collagen 1A1, fibronectin, and vimentin expression; superoxide levels; and cell migration and increased NO levels. These effects were reversed by ARG1 overexpression. Pharmacological blockade of the ARG1 pathway with CB-1158 reduced the proliferation of LECs and decreased fibronectin, α-SMA, collagen 1A1, and vimentin expression in mouse lenses. We believe that ARG1 promotes the production of collagen 1A1 by directly activating the arginase pathway and leads to lens fibrosis by reducing NO production and increasing superoxide levels, providing a new mechanism for the prevention and treatment of fibrotic cataracts.

更新日期：2023-09-18

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