Department of Ophthalmology, Pusan National University School of Medicine, Yangsan, Republic of Korea.
Research Institute for Convergence of Biomedical Science and Technology, Pusan National University Yangsan Hospital, Yangsan, Republic of Korea.
Ophthalmic Res. 2023;66(1):86-98. doi: 10.1159/000525762. Epub 2022 Aug 11.
The aim of the study was to evaluate the safety and efficacy of atelocollagen in preventing the fibrotic change of human tenon tissue induced by transforming growth factor β1 (TGFβ1).
Primary cultured human Tenon's fibroblasts (HTFs) were incubated with TGFβ1 alone and with various concentrations of atelocollagen, respectively. Cell viability was measured by Cell Counting Kit-8 (CCK-8). The mRNA levels of α-smooth muscle actin (α-SMA), vimentin, fibronectin, zonular occludens scaffolding protein (ZO-1), cellular communication network factor 2 (CCN2), and interleukin 6 (IL-6) were measured by quantitative reverse transcription polymerase chain reaction, Western blot, and immunofluorescence analysis. Wound healing assay and collagen contraction assay were additionally evaluated for identifying the inhibitory effect of atelocollagen in HTFs. To elucidate the mechanism by which atelocollagen affects HTF proliferation, the phospho-extracellular-signal-regulated kinases (pERK)/total-extracellular-signal-regulated kinases (tERK), phospho-focal adhesion kinase (pFAK)/total-focal adhesion kinase (tFAK), and pSmad3/tSmad3 protein expression ratios were measured by Western blot.
The safety of atelocollagen in HTF was identified by CCK-8 analysis. The expression of α-SMA and vimentin in HTFs treated with 0.023% and 0.046% atelocollagen significantly decreased at both mRNA and protein levels, while that of ZO-1 in 0.046% atelocollagen increased compared with TGFβ1-treated cells. The protein expression of fibronectin, CCN2, and IL-6 in HTFs treated with 0.023% and 0.046% atelocollagen significantly decreased. The immunofluorescence microscopy of α-SMA and ZO-1 showed results similar to those of the Western blot. In the wound-scratch assays, cell migration was significantly attenuated in HTFs treated with 0.005% atelocollagen. Atelocollagen at 0.005, 0.011, and 0.023% significantly inhibited the gel contraction induced by TGFβ1 at both 24 h and 48 h. The increase in pERK/tERK and pSmad3/tSmad3 protein expression ratios in TGFβ1-treated HTFs significantly decreased after treatment with 0.023 and 0.046% atelocollagen.
Since atelocollagen gel effectively suppresses the proliferation of HTFs in TGFβ1-induced transdifferentiation, it may be a potential therapeutic agent in glaucoma surgery.
本研究旨在评估去端肽胶原（atelocollagen）在预防转化生长因子 β1（TGFβ1）诱导的人眼Tenon 组织纤维化改变中的安全性和有效性。
原代培养的人眼Tenon 成纤维细胞（HTFs）分别用 TGFβ1 单独和不同浓度的去端肽胶原孵育。通过细胞计数试剂盒-8（CCK-8）测量细胞活力。通过定量逆转录聚合酶链反应、Western blot 和免疫荧光分析测量α-平滑肌肌动蛋白（α-SMA）、波形蛋白、纤维连接蛋白、连接蛋白 2（zonular occludens scaffolding protein，ZO-1）、细胞通讯网络因子 2（cellular communication network factor 2，CCN2）和白细胞介素 6（interleukin 6，IL-6）的 mRNA 水平。另外，通过划痕愈合试验和胶原收缩试验来评估去端肽胶原对 HTFs 的抑制作用。为了阐明去端肽胶原影响 HTF 增殖的机制，通过 Western blot 测量磷酸化细胞外信号调节激酶（phospho-extracellular-signal-regulated kinases，pERK）/总细胞外信号调节激酶（total-extracellular-signal-regulated kinases，tERK）、磷酸化黏着斑激酶（phospho-focal adhesion kinase，pFAK）/总黏着斑激酶（total-focal adhesion kinase，tFAK）和 pSmad3/tSmad3 蛋白表达比值。
通过 CCK-8 分析鉴定了去端肽胶原在 HTF 中的安全性。用 0.023%和 0.046%去端肽胶原处理的 HTFs 中，α-SMA 和波形蛋白的 mRNA 和蛋白水平表达明显降低，而 0.046%去端肽胶原处理的细胞中 ZO-1 的蛋白表达增加。用 0.023%和 0.046%去端肽胶原处理的 HTFs 中，纤维连接蛋白、CCN2 和 IL-6 的蛋白表达明显降低。α-SMA 和 ZO-1 的免疫荧光显微镜观察结果与 Western blot 结果相似。在划痕试验中，用 0.005%去端肽胶原处理的细胞迁移明显受到抑制。0.005%、0.011%和 0.023%的去端肽胶原在 24 h 和 48 h 时显著抑制 TGFβ1 诱导的凝胶收缩。用 0.023%和 0.046%去端肽胶原处理后，TGFβ1 处理的 HTFs 中 pERK/tERK 和 pSmad3/tSmad3 蛋白表达比值的增加明显降低。
由于去端肽胶原凝胶能有效抑制 TGFβ1 诱导的转分化 HTFs 的增殖，因此它可能是青光眼手术的一种潜在治疗剂。
