Department of Comparative Biosciences, College of Veterinary Medicine, University of Illinois Urbana-Champaign, Urbana, IL, USA.
Department of Obstetrics and Gynecology, College of Medicine, University of Florida, Gainesville, FL, USA.
Vet Ophthalmol. 2021 May;24(3):279-287. doi: 10.1111/vop.12888. Epub 2021 Apr 9.
Matrix metalloproteinases (MMPs)-2 and -9 are present in corneal ulcers, and an imbalance between MMPs and tissue inhibitors of metalloproteinases (TIMPs) leads to further corneal degradation. Amniotic membrane homogenate (AMH) has proteolytic properties beneficial for corneal healing, but it is unknown whether AMH possesses TIMPs or effectively inhibits MMP-2 and MMP-9 activity.
To determine if bovine and equine AMH reduce in vitro MMP-2 and MMP-9 activities associated with the presence of TIMPs.
Undiluted and diluted twofold series (0-fold to 16-fold dilutions) of equine amniotic membrane homogenates (EAMH, n = 8) and bovine amniotic membrane homogenates (BAMH, n = 8) were subjected to fluorescence resonance energy transfer, and the fluorescence emitted was recorded over time. Average fluorescence was calculated versus recombinant concentration. Enzyme-linked immunosorbent assays for TIMPs 1-4 were applied to quantify TIMPs in the samples.
AMH from both species were able to inhibit MMP-2 and MMP-9 activities in vitro, and the inhibition efficacy decreased gradually with dilution. BAMH was significantly more effective than EAMH at inhibiting MMP-2 and MMP-9 in vitro. TIMPs -2 and -3 were present in EAMH and BAMH. TIMP-1 was detected only in BAMH, and TIMP-4 was not detected in any samples.
Both EAMH and BAMH directly inhibited MMP-2 and MMP-9 in vitro without dilution, and BAMH showed better inhibition of MMP-2 and MMP-9 before and after dilution compared to EAMH.
基质金属蛋白酶（MMPs）-2 和 -9 存在于角膜溃疡中，MMPs 和金属蛋白酶组织抑制剂（TIMPs）之间的失衡导致进一步的角膜降解。羊膜匀浆（AMH）具有有益于角膜愈合的蛋白水解特性，但尚不清楚 AMH 是否具有 TIMPs 或是否能有效抑制 MMP-2 和 MMP-9 的活性。
确定牛和马 AMH 是否能降低与 TIMPs 存在相关的体外 MMP-2 和 MMP-9 活性。
未经稀释和以两倍系列稀释（0 倍至 16 倍稀释）的马羊膜匀浆（EAMH，n=8）和牛羊膜匀浆（BAMH，n=8）接受荧光共振能量转移，记录随时间发射的荧光。平均荧光强度与重组浓度相对应进行计算。酶联免疫吸附试验（ELISA）用于检测样品中的 TIMPs 1-4。
两种来源的 AMH 均能在体外抑制 MMP-2 和 MMP-9 的活性，抑制效果随稀释度逐渐降低。BAMH 比 EAMH 在体外抑制 MMP-2 和 MMP-9 的效果更显著。EAMH 和 BAMH 中均存在 TIMP-2 和 TIMP-3。TIMP-1 仅存在于 BAMH 中，而任何样本中均未检测到 TIMP-4。
EAMH 和 BAMH 均可在未经稀释的情况下直接抑制 MMP-2 和 MMP-9 的体外活性，且 BAMH 在稀释前后对 MMP-2 和 MMP-9 的抑制作用均优于 EAMH。
