FGF2 Regulates Hypoxia-Induced Proliferation and Collagen Metabolism of Scleral Fibroblasts Through the PERK/EIF2α/ATF4 Signaling Pathway
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摘要:
目的 研究FGF2对缺氧诱导的巩膜成纤维细胞（scleral fibroblasts，SF）增殖和胶原的影响并探讨其可能调节的下游信号通路。 方法 用5% O2刺激SF 24 h诱导近视SF模型，RT-qPCR检测FGF2 mRNA表达，Western blot检测FGF2蛋白表达。细胞计数试剂盒8（Cell Count Kit-8，CCK-8）、流式细胞术和Western blot分别检测细胞增殖活力、细胞凋亡和胶原代谢相关蛋白collagen Ⅰ、MMP2以及通路蛋白PERK、p-PERK、EIF2α、EIF2α、ATF4表达。 结果 缺氧刺激可促进FGF2 mRNA和蛋白表达升高（P < 0.01）并激活PERK/EIF2α/ATF4通路（P < 0.001），抑制SF细胞增殖（P < 0.001）和collagen Ⅰ表达（P < 0.001），诱导MMP2表达（P < 0.001）及细胞凋亡（P < 0.001）。敲降FGF2或经PERK抑制剂GSK2606414处理可逆转缺氧对SF细胞的作用，促进细胞增殖活力（P < 0.001）和collagen Ⅰ表达（P < 0.01），减少细胞凋亡（P < 0.01）。 结论 FGF2通过调节PERK/EIF2α/ATF4通路的激活，影响缺氧诱导的SF增殖及胶原代谢。 Abstract:Objectives To investigate the effects of FGF2 on the proliferation and collagen production of hypoxia-induced scleral fibroblasts （SF） and explore the downstream signaling pathways it regulates. Methods 5% O2 was used to stimulate the SF to induce myopia SF model for 24 hours. RT-qPCR was used to detect FGF2 mRNA expression, and Western blot analysis was used to check FGF2 protein expression. The Cell Counting Kit-8 （CCK-8）, flow cytometry, and Western blot were used to assess cell proliferation vitality, cell apoptosis, and the expression of collagen metabolism-related proteins collagen I, MMP2, and pathway proteins PERK, p-PERK, EIF2α, EIF2α, and ATF4. Results Hypoxia increased FGF2 mRNA and protein expression （P < 0.01） , activated the PERK/EIF2α/ATF4 pathway （P < 0.001）, inhibited SF cell proliferation （P < 0.001） and collagen I expression （P < 0.001）, while induced MMP2 expression （P < 0.001） and apoptosis （P < 0.001）. Knocking down FGF2 or treating with PERK inhibitor GSK2606414 reversed the effect of hypoxia on SF cells, increased cell proliferation （P < 0.001） and collagen Ⅰ expression （P < 0.01）, and suppressed cell apoptosis （P < 0.01）. Mechanism study revealed that FGF2 knockdown dampened the activation of PERK/EIF2α/ATF4 pathway. Conclusion FGF2 affects hypoxia-induced SF proliferation and collagen metabolism by regulating the activation of PERK/EIF2α/ATF4 signaling pathway. -
Key words:
- Scleral fibroblasts /
- Hypoxia /
- FGF2 /
- Proliferation /
- Collagen metabolism
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图 1 FGF2在缺氧诱导的SF中高表达
A：缺氧诱导的SF中FGF2 mRNA相对表达；B：缺氧诱导的SF中FGF2蛋白表达电泳图。C：FGF2蛋白表达统计学分析。**P < 0.01。Ctrl：正常对照组，Hypoxia：缺氧组。
Figure 1. Overexpression of FGF2 in hypoxia-induced SF
图 2 FGF2调控缺氧诱导的SF增殖和胶原代谢
A：转染si-FGF2的SF中FGF2蛋白相对表达水平；B：细胞增殖活力；C～D：细胞凋亡率；E：collagen Ⅰ和MMP2蛋白表达电泳图；F：collagen Ⅰ和MMP2蛋白表达统计学分析；G：PERK、p-PERK、EIF2α、p-EIF2α、ATF4蛋白表达电泳图；H：p-PERK/PERK蛋白表达统计学分析；I：p-EIF2α/EIF2α蛋白表达统计学分析；J：ATF4蛋白表达统计学分析。*P < 0.05，**P < 0.01，***P < 0.001。Ctrl：正常对照组，Hypoxia：缺氧组。
Figure 2. FGF2 regulates proliferation and collagen metabolism in hypoxia-induced SF
图 3 PERK/EIF2α/ATF4通路调控缺氧诱导的SF增殖及胶原代谢
A：PERK、p-PERK、EIF2α、p-EIF2α、ATF4蛋白的蛋白表达电泳图；B：p-PERK/PERK蛋白表达统计学分析；C：p-EIF2α/EIF2α蛋白表达统计学分析；D：ATF4蛋白表达统计学分析；E：细胞增殖活力；F：细胞凋亡率统计；G：细胞凋亡图；H：collagen Ⅰ和MMP2蛋白表达电泳图：I：collagen Ⅰ蛋白表达统计学分析；J：MMP2蛋白表达统计学分析。*P < 0.05，**P < 0.01，***P < 0.001。Ctrl：正常对照组，Hypoxia：缺氧组。
Figure 3. PERK/EIF2α/ATF4 pathway regulates proliferation and collagen metabolism in hypoxia-induced SF
表 1 引物序列
Table 1. Primer sequences
目的基因 引物序列
（F: Forward，R: Reverse，5’-3’）FGF2 F: GTCAAACTACAACTCCAAGCAG R: GAAACACTCTTCTGTAACACACTT GAPDH F: GTGGAGTCATACTGGAACATGTAG R: AATGGTGAAGGTCGGTGTG 
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