Supplementary Material for: The Efficacy of Atelocollagen to Inhibit Fibrotic Proliferation in Tenon Tissue: in vitro study

Introduction: To evaluate the safety and efficacy of atelocollagen in preventing the fibrotic change of human tenon tissue induced by transforming growth factor β1 (TGFβ1) Methods: Primary cultured human Tenon’s fibroblasts (HTFs) were incubated with TGFβ1 alone, and with a various concentrations of atelocollagen respectively. Cell viability was measured by cell counting kit-8 (CCK8). 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 (RT-PCR), 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 HTFs 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. Results: The safety of atelocollagen in HTF was identified by CCK8 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. 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. Conclusion: Since atelocollagen gel effectively suppresses the proliferation of HTFs in TGFβ1 - induced transdifferentiation, it may be a potential therapeutic agent in glaucoma surgery.

引言：为评估去端肽胶原蛋白（atelocollagen）预防转化生长因子β1（transforming growth factor β1，TGFβ1）诱导的人腱组织纤维化改变的安全性与有效性。
方法：将原代培养的人Tenon囊成纤维细胞（human Tenon’s fibroblasts，HTFs）分别单独以TGFβ1孵育，或与不同浓度的去端肽胶原蛋白共同孵育。采用细胞计数试剂盒-8（cell counting kit-8，CCK8）检测细胞活力。通过定量反转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction，RT-PCR）、蛋白质印迹（western blot）及免疫荧光分析，检测α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、波形蛋白（vimentin）、纤连蛋白（fibronectin）、带状紧密连接脚手架蛋白（zonular occludens scaffolding protein，ZO-1）、细胞通信网络因子2（cellular communication network factor 2，CCN2）以及白细胞介素-6（interleukin-6，IL-6）的mRNA水平。此外，通过划痕愈合实验与胶原收缩实验，评估去端肽胶原蛋白对HTFs的抑制作用。为阐明去端肽胶原蛋白调控HTFs增殖的分子机制，采用蛋白质印迹检测磷酸化细胞外调节蛋白激酶（phospho-extracellular-signal-regulated kinases，pERK）/总细胞外调节蛋白激酶（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）以及pSmad3/tSmad3的蛋白表达比值。
结果：通过CCK8分析验证了去端肽胶原蛋白对HTFs的安全性。与仅经TGFβ1处理的细胞相比，经0.023%及0.046%去端肽胶原蛋白处理的HTFs中，α-SMA与vimentin的mRNA及蛋白表达水平均显著下调；经0.046%去端肽胶原蛋白处理的HTFs中ZO-1的表达水平则显著升高。经0.023%及0.046%去端肽胶原蛋白处理的HTFs中，纤连蛋白、CCN2及IL-6的蛋白表达水平均显著下调。α-SMA与ZO-1的免疫荧光观测结果与蛋白质印迹实验结果一致。在划痕愈合实验中，经0.005%去端肽胶原蛋白处理的HTFs的细胞迁移能力显著减弱。0.005%、0.011%及0.023%浓度的去端肽胶原蛋白均可在24h与48h时显著抑制TGFβ1诱导的胶原凝胶收缩。经TGFβ1处理的HTFs中pERK/tERK与pSmad3/tSmad3的蛋白表达比值升高，经0.023%及0.046%去端肽胶原蛋白处理后，该升高现象显著被抑制。
结论：鉴于去端肽胶原蛋白凝胶可有效抑制TGFβ1诱导的HTFs转分化过程中的细胞增殖，其有望成为青光眼手术的潜在治疗药物。