Filip Gabriela Adriana, Florea Adrian, Olteanu Diana, Clichici Simona, David Luminita, Moldovan Bianca, Cenariu Mihai, Scrobota Ioana, Potara Monica, Baldea Ioana
Department of Physiology, "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania.
Department of Cell and Molecular Biology, "Iuliu Hatieganu" University of Medicine and Pharmacy, Cluj-Napoca, Romania.
Mater Sci Eng C Mater Biol Appl. 2021 Apr;123:111974. doi: 10.1016/j.msec.2021.111974. Epub 2021 Feb 15.
The study aims to evaluate the impact of silver nanoparticles, phytosynthesized with polyphenols from Sambucus nigra L. (SN) fruit extract (AgSN), on dysplastic oral keratinocytes (DOK) and human gingival fibroblasts (HGF) in terms of cell viability and apoptosis. The morphology and ultrastructure of treated cells as well as the mechanisms involved in cell death induction were investigated in DOK cultures. The structure of AgSN was studied by using the appropriate analysis tools such as UV-Vis, transmission electron microscopy, Raman spectroscopy, dynamic light scattering (DLS) and zeta potential assessment. DOK and HGF were treated either with silver nanoparticles capped with Sambucus nigra L. extract or with SN extract. Untreated cells were used as controls. Viability was determined by MTS assay. Transmission electronic microscopy (TEM) was used to evaluate the intracellular localization of the nanoparticles at 4 and 24 h. Annexin V-FITC/propidium iodide staining and the expressions of p53, BAX, BCL2, NFkB, phosphorylated NFkB (pNFkB), pan AKT, pan phosphoAKT, LC3B and ɣHAX were evaluated to quantify the cell death. ELISA measurements of TNF-α and TRAIL was used for the study of the inflammatory response. Oxidative stress damage induced by nanoparticles was assessed by the malondialdehyde (MDA) level. Silver nanoparticles stimulated HGF proliferation and significantly diminished DOK viability at doses higher than 20 μg/ml. TEM analysis demonstrated the internalization of silver nanoparticles and showed ultrastructural changes of cells such as the appearance of vacuoles, autophagosomes, endosomes. AgSN inhibited the pro-survival molecules and regulators of apoptosis, diminished oxidative stress and inflammation and induced cell death through various mechanisms: necrosis, autophagy and DNA lesions. SN extract had antioxidant and anti-inflammatory effect and increased the DNA lesions and autophagy in DOK cells. Silver nanoparticles protected the normal cells and induced cell death in dysplastic cells by different mechanisms thus offering beneficial effects in the treatment of oral dysplasia.
本研究旨在评估由接骨木（SN）果实提取物中的多酚植物合成的银纳米颗粒（AgSN）对发育异常的口腔角质形成细胞（DOK）和人牙龈成纤维细胞（HGF）的细胞活力和凋亡的影响。在DOK培养物中研究了处理后细胞的形态和超微结构以及诱导细胞死亡的机制。使用适当的分析工具，如紫外可见光谱、透射电子显微镜、拉曼光谱、动态光散射（DLS）和zeta电位评估，研究了AgSN的结构。用接骨木提取物包覆的银纳米颗粒或SN提取物处理DOK和HGF。未处理的细胞用作对照。通过MTS测定法测定活力。透射电子显微镜（TEM）用于评估纳米颗粒在4小时和24小时时的细胞内定位。评估膜联蛋白V-FITC/碘化丙啶染色以及p53、BAX、BCL2、NFkB、磷酸化NFkB（pNFkB）、泛AKT、泛磷酸化AKT、LC3B和ɣHAX的表达以量化细胞死亡。通过ELISA测量TNF-α和TRAIL用于研究炎症反应。通过丙二醛（MDA）水平评估纳米颗粒诱导的氧化应激损伤。银纳米颗粒刺激HGF增殖，并在高于20μg/ml的剂量下显著降低DOK活力。TEM分析证明了银纳米颗粒的内化，并显示了细胞的超微结构变化，如空泡、自噬体、内体的出现。AgSN抑制促生存分子和凋亡调节因子，减少氧化应激和炎症，并通过各种机制诱导细胞死亡：坏死、自噬和DNA损伤。SN提取物具有抗氧化和抗炎作用，并增加了DOK细胞中的DNA损伤和自噬。银纳米颗粒通过不同机制保护正常细胞并诱导发育异常细胞死亡，从而在口腔发育异常的治疗中提供有益效果。
