Loftus John P, Belknap James K, Black Samuel J
Department of Veterinary and Animal Sciences, University of Massachusetts, Paige Laboratory, Amherst, MA 01003, USA.
Vet Immunol Immunopathol. 2006 Oct 15;113(3-4):267-76. doi: 10.1016/j.vetimm.2006.05.010. Epub 2006 Jul 5.
We sought to determine whether a correlation exists between neutrophil infiltration and tissue matrix metalloproteinase-9 (MMP-9) content in digital laminae collected during the prodromal and acute phases of laminitis in horses treated with an aqueous black walnut heartwood extract (BWE). Hoof laminar tissue was obtained at the onset of leukopenia and at the onset of clinical signs of lameness from BWE-treated horses and at equivalent times from control horses. Thin sections of laminae were screened for neutrophils by immunohistochemistry with an anti-CD13 monoclonal antibody and extracts of the same tissues were screened for SDS-renaturable and native MMP-9 activities by denaturing and non-denaturing gelatin zymography. Samples were also screened for MMP-2 and MMP-9 gene expression by RT-qPCR. Control laminae were devoid of both MMP-9 and neutrophils, whereas neutrophils and SDS-renaturable MMP-9 activity were detected in laminae from BWE-treated horses and were strongly correlated at the acute stage of the disease at which time laminar MMP-9 gene expression was significantly (15-fold) elevated. In contrast, BWE-treatment did not significantly elevate MMP-2 gene or protein expression in the laminae. Interestingly, MMP-9 that was present in extracts of laminae from BWE-treated horses at both the prodromal and acute stages of the disease was mainly in the zymogen form, suggesting that the accumulation of the MMP did not contribute to pathology during these stages. However, elevated presence of the MMP-9 zymogen in the tissue would predispose it to catastrophic damage should conditions arise that cleave the regulatory propeptide domain.
我们试图确定，在接受黑胡桃心材水提取物（BWE）治疗的马患蹄叶炎前驱期和急性期采集的蹄叶中，中性粒细胞浸润与组织基质金属蛋白酶-9（MMP-9）含量之间是否存在相关性。在白细胞减少症发作时以及出现跛行临床症状时，从接受BWE治疗的马身上获取蹄叶组织，并在相同时间从对照马身上获取。通过用抗CD13单克隆抗体进行免疫组织化学筛选蹄叶薄片中的中性粒细胞，并用变性和非变性明胶酶谱法筛选相同组织提取物中的SDS可复性和天然MMP-9活性。还通过RT-qPCR筛选样品中的MMP-2和MMP-9基因表达。对照蹄叶中既没有MMP-9也没有中性粒细胞，而在接受BWE治疗的马的蹄叶中检测到了中性粒细胞和SDS可复性MMP-9活性，并且在疾病急性期两者密切相关，此时蹄叶MMP-9基因表达显著（15倍）升高。相比之下，BWE处理并未显著提高蹄叶中MMP-2基因或蛋白的表达。有趣的是，在疾病前驱期和急性期，接受BWE治疗的马的蹄叶提取物中存在的MMP-9主要以酶原形式存在，这表明在这些阶段MMP的积累并未导致病理变化。然而，如果出现切割调节性前肽结构域的情况，组织中MMP-9酶原的含量升高会使其易遭受灾难性损伤。
