Cytological and functional effect of complement 3a on Human Scleral Fibroblasts.

IF 1.6 4区医学 Q3 OPHTHALMOLOGY Cutaneous and Ocular Toxicology Pub Date : 2023-09-01 DOI:10.1080/15569527.2023.2226711

Kang Xiao, Ying Jie, Mingyue Luo, Qin Long

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Abstract

Purpose: The complement system is considered to play an important role in the progression of myopia, whereas the influence of complement activation on the human scleral fibroblasts (HSFs) remains unknown. Hence, the effect of complement 3a (C3a) on HSFs was investigated in this study.

Methods: HSFs were cultured with exogenous C3a at 0.1 μM for various periods following different measurement protocols, and cells without C3a treatment served as negative control (NC). Cell viability was investigated using the MTS assay after 3 days of C3a treatment. Cell proliferation was evaluated by the 5-Ethynyl-20-Deoxyuridine (EdU) assay following C3a stimulation for 24 hours. Apoptosis was assessed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining following C3a stimulation for 48 hours and the stained cells were analysed using flow cytometry. The levels of type I collagen and matrix metalloproteinase-2 (MMP-2) were analysed using ELISA following C3a stimulation for 36 and 60 hours. The level of CD59 were analysed using western blot following C3a stimulation for 60 hours.

Results: The MTS assay revealed that cell viability was attenuated by 13% and 8% after C3a for 2 and 3 days, respectively (P < 0.05). The EdU assay demonstrated a 9% decrease in proliferation rate for the C3a-treated cells after 24 hours (P < 0.05). The apoptosis analysis revealed an increased percentage of cells in early apoptosis (P = 0.02) and total apoptosis (P = 0.02) in the C3a-treated group. Compared with NC group, the level of MMP-2 was increased by 17.6% (P = 0.002), whereas the levels of type I collagen and CD59 were respectively decreased by 12.5% (P = 0.024) and 21.6% (P = 0.044) with C3a treatment for 60 hours.

Conclusions: These results indicated that C3a-induced complement activation is potentially involved in inducing myopic-associated scleral extracellular matrix remodelling via mediating the proliferation and function of HSFs.

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补体3a对人巩膜成纤维细胞的细胞学和功能影响。

目的:补体系统被认为在近视的进展中起重要作用，而补体激活对人巩膜成纤维细胞(hsf)的影响尚不清楚。因此，本研究探讨补体3a (C3a)对hsf的影响。方法:采用外源C3a (0.1 μM)培养hsf，并按不同的测量方案培养不同时期的hsf，不加C3a处理的细胞作为阴性对照(NC)。C3a处理3天后，采用MTS法检测细胞活力。C3a刺激24小时后，用5-乙基-20-脱氧尿苷(EdU)测定细胞增殖情况。C3a刺激48小时后，采用Annexin v -异硫氰酸荧光素(FITC)/碘化丙啶(PI)双染色法检测细胞凋亡，流式细胞术分析染色细胞凋亡情况。在C3a刺激36和60小时后，采用ELISA法分析I型胶原蛋白和基质金属蛋白酶-2 (MMP-2)水平。C3a刺激60小时后，用western blot分析CD59水平。结果:MTS检测显示，C3a处理组细胞活力下降13%，C3a处理组细胞凋亡率下降8% (P P P = 0.02)， C3a处理组细胞总凋亡率下降8% (P P P = 0.02)。与NC组相比，C3a治疗60 h后MMP-2水平升高17.6% (P = 0.002)， I型胶原和CD59水平分别下降12.5% (P = 0.024)和21.6% (P = 0.044)。结论:这些结果表明，c3a诱导的补体激活可能通过介导hsf的增殖和功能参与了近视相关巩膜细胞外基质的重塑。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Cutaneous and Ocular Toxicology 医学-毒理学

CiteScore

3.30

自引率

6.20%

发文量

审稿时长

1 months

期刊介绍： Cutaneous and Ocular Toxicology is an international, peer-reviewed journal that covers all types of harm to cutaneous and ocular systems. Areas of particular interest include pharmaceutical and medical products; consumer, personal care, and household products; and issues in environmental and occupational exposures. In addition to original research papers, reviews and short communications are invited, as well as concise, relevant, and critical reviews of topics of contemporary significance.