Department of Microbial Biotechnology, School of Biology and Center of Excellence in Phylogeny of Living Organisms, College of Science, University of Tehran, Tehran, Iran.
Microbial Technology and Products Research Center, University of Tehran, Tehran, Iran.
J Appl Microbiol. 2018 Jan;124(1):254-266. doi: 10.1111/jam.13605. Epub 2017 Dec 18.
Vascular calcification (VC) is a significant pathological process in some life-threatening diseases. Several pathological mechanisms, including transdifferentiation of vascular smooth muscle cells to osteoblast-like cells and apoptosis are involved in VC. Compounds with an inhibitory effect on these processes are potentially efficient medications. In consideration of the multiple biological activities of Actinobacteria, this research was aimed at finding anti-VC metabolite-producing Actinobacteria.
After the isolation and identification of Actinobacteria, the effect of their fermentation broth extracts on the apoptosis rate was measured using various methods, for example, ethidium bromide/acridine orange staining, DNA laddering and diphenylamine assays. The effect of the most effective fermentation broth extract of Actinobacteria (FBEA) on the mRNA expression of runt-related transcription factor 2 (Runx2) and osteopontin (OPN) was examined. Finally, the most effective FBEA was fractionated and the chemical composition of anti-VC fractions was analysed using GC-MS. Various VC inhibition rates were observed in the tested FBEA (20 μg ml ; 17·9-60·15%). The inhibition of DNA fragmentation was 7-48%. The FBE with the greatest anticalcification activity belonged to Kribbella sp. UTMC 267 and, according to 16S rRNA analysis, Kribbella sancticallisti with a similarity of 98·53% is its nearest neighbour. The FBE of Kribbella sp. UTMC 267 reduced Runx2 mRNA expression by 2·95-fold and OPN mRNA expression by 28·57-fold, both of which are considered significant (P < 0·05). Finally, GC-MS analysis showed the existence of potent anti-oxidative and anti-inflammation agents in FBE of Kribbella sp. UTMC 267.
Actinobacterial metabolites can provide a new strategy for treating VC diseases by reducing the expression of osteogenic genes, the apoptosis rate and oxidative stress.
This study highlights the therapeutic potential of Kribbella sp. metabolites and Actinobacteria as a new natural source for drug discovery programs in the nonantibiotic bioactivity field.
血管钙化（VC）是一些危及生命的疾病中的一个重要病理过程。几种病理机制，包括血管平滑肌细胞向成骨样细胞的转化和细胞凋亡，都参与了 VC 的发生。具有这些过程抑制作用的化合物可能是有效的药物。鉴于放线菌具有多种生物学活性，本研究旨在寻找具有抗 VC 代谢产物产生能力的放线菌。
在分离和鉴定放线菌后，使用各种方法（例如溴化乙锭/吖啶橙染色、DNA 梯状电泳和二苯胺测定）测量其发酵液提取物对细胞凋亡率的影响。检测了放线菌发酵液提取物（FBEA）对 runt 相关转录因子 2（Runx2）和骨桥蛋白（OPN）mRNA 表达的影响。最后，对最有效的 FBEA 进行了分段，并使用 GC-MS 分析了抗 VC 馏分的化学成分。在测试的 FBEA（20μg/ml；17.9-60.15%）中观察到不同的 VC 抑制率。DNA 片段化的抑制率为 7-48%。具有最大抗钙化活性的 FBE 属于 Kribbella sp. UTMC 267，根据 16S rRNA 分析，与其相似度为 98.53%的 Kribbella sancticallisti 是其最近的亲缘关系。Kribbella sp. UTMC 267 的 FBE 使 Runx2 mRNA 表达降低了 2.95 倍，OPN mRNA 表达降低了 28.57 倍，这两者都被认为是显著的（P<0.05）。最后，GC-MS 分析表明，Kribbella sp. UTMC 267 的 FBE 中存在有效的抗氧化和抗炎剂。
放线菌代谢产物可以通过降低成骨基因的表达、细胞凋亡率和氧化应激来为治疗 VC 疾病提供新的策略。
本研究强调了 Kribbella sp. 代谢产物和放线菌作为非抗生素生物活性领域药物发现计划的新天然来源的治疗潜力。
