Department of Experimental Medicine, School of Medicine, University of Campania "Luigi Vanvitelli," Via L. De Crecchio, Naples, Italy.
Unit of Pharmacology, Department of Experimental Medicine, University of Campania "Luigi Vanvitelli", Naples, Italy.
PLoS One. 2020 Aug 6;15(8):e0236164. doi: 10.1371/journal.pone.0236164. eCollection 2020.
Hyaluronan (HA) is a nonsulfated glycosaminoglycan that has been widely used for biomedical applications. Here, we have analyzed the effect of HA on the rescue of primary cells under stress as well as its potential to recover muscle atrophy and validated the developed model in vitro using primary muscle cells derived from rats. The potentials of different HAs were elucidated through comparative analyses using pharmaceutical grade a) high (HHA) and b) low molecular weight (LHA) hyaluronans, c) hybrid cooperative complexes (HCC) of HA in three experimental set-ups. The cells were characterized based on the expression of myogenin, a muscle-specific biomarker, and the proliferation was analyzed using Time-Lapse Video Microscopy (TLVM). Cell viability in response to H2O2 challenge was evaluated by 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay, and the expression of the superoxide dismutase enzyme (SOD-2) was assessed by western blotting. Additionally, in order to establish an in vitro model of atrophy, muscle cells were treated with tumor necrosis factor-alpha (TNF-α), along with hyaluronans. The expression of Atrogin, MuRF-1, nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-kB), and Forkhead-box-(Fox)-O-3 (FoxO3a) was evaluated by western blotting to elucidate the molecular mechanism of atrophy. The results showed that HCC and HHA increased cell proliferation by 1.15 and 2.3 folds in comparison to un-treated cells (control), respectively. Moreover, both pre- and post-treatments of HAs restored the cell viability, and the SOD-2 expression was found to be reduced by 1.5 fold in HA-treated cells as compared to the stressed condition. Specifically in atrophic stressed cells, HCC revealed a noteworthy beneficial effect on the myogenic biomarkers indicating that it could be used as a promising platform for tissue regeneration with specific attention to muscle cell protection against stressful agents.
透明质酸（HA）是一种非硫酸化的氨基葡聚糖，已广泛应用于生物医学领域。在这里，我们分析了 HA 对压力下原代细胞的拯救作用及其恢复肌肉萎缩的潜力，并使用从大鼠中获得的原代肌肉细胞在体外验证了所开发的模型。通过使用制药级 a）高（HHA）和 b）低分子量（LHA）透明质酸、c）透明质酸的混合协同复合物（HCC）进行比较分析，阐明了不同 HA 的潜力。在三个实验方案中，基于肌肉特异性生物标志物肌球蛋白原的表达对细胞进行了表征，并使用延时视频显微镜（TLVM）分析了增殖情况。通过 3-[4,5-二甲基噻唑-2-基]-2,5-二苯基四氮唑溴化物（MTT）测定法评估细胞对 H2O2 挑战的存活率，并通过 Western 印迹法评估超氧化物歧化酶酶（SOD-2）的表达。此外，为了建立萎缩的体外模型，用肿瘤坏死因子-α（TNF-α）和透明质酸处理肌肉细胞。通过 Western 印迹法评估 Atrogin、MuRF-1、核因子 kappa-轻链增强子的激活 B 细胞（NF-kB）和 Forkhead-box-O-3（FoxO3a）的表达，以阐明萎缩的分子机制。结果表明，与未处理的细胞（对照）相比，HCC 和 HHA 分别使细胞增殖增加了 1.15 倍和 2.3 倍。此外，HA 的预处理和后处理均恢复了细胞活力，与应激条件相比，HA 处理的细胞中 SOD-2 的表达降低了 1.5 倍。特别是在萎缩应激细胞中，HCC 对肌生成生物标志物显示出显著的有益作用，表明它可用作组织再生的有前途的平台，特别注意保护肌肉细胞免受应激剂的影响。
