Phenylephrine effects on the morphology of osteoblasts and expression of nicotinamide phosphoribosyltransferase under oxidative stress

doi:10.3969/j.issn.2095-4344.0814

Abstract

Abstract:

BACKGROUND: Phenylephrine has been proved to exert a protective effect on radiant-induced salivary gland and epithelial cell injuries, but its effect on hydrogen peroxide (H2O2)-induced oxidative stress in osteoblasts are not fully understood.
OBJECTIVE: To explore the effect of phenylephrine on H2O2-induced oxidative stress in osteoblasts, and to explore the mechanism underlying the regulation by the expression level of nicotinamide phosphoribosyltransferase (Nampt).
METHODS: Primary osteoblasts were cultured and randomly divided into four groups: blank control group, H2O2 group, phenylephrine group, and combination group (0.5 hour pretreatment of 1×10-5 mol/L phenylephrine, and then given 300 μmol/L H2O2). The morphology of osteoblasts was observed at different time points. Osteoblasts were collected after 24-hour culture, and total RNA and protein were then extracted to detect the mRNA and protein expression levels of Nampt by RT-PCR and western blot assay, respectively.
RESULTS AND CONCLUSION: Compared with the blank control group, reduced osteoblasts and evident cell shrinks were observed in the H2O2 group, while the number of osteoblasts significantly increased in the combined group compared with the H2O2 group at 12, 24 and 48 hours of culture. RT-PCR results showed that the mRNA level of Nampt in the H2O2 group was reduced by 31.23% of that in the blank control group, while the mRNA level of Nampt in the combination group was dramatically increased by 206.20% of that in the H2O2 group at 24 hours of culture (both P < 0.05). Furthermore, western blot assay findings revealed that the protein level of Nampt in the H2O2 group was reduced by 67.98% of that in the blank control group, while the protein level of Nampt in the combination group was increased by 152.25% of that in the H2O2 group at 24 hours of culture (both P < 0.05). Our results indicate that phenylephrine can alleviate the shrink and atrophy of osteoblasts caused by H2O2, thereby exerting protective effect by up-regulating the mRNA and protein levels of Nampt that may be a regulatory gene.

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: Osteoblasts, Cell Proliferation, Cell Differentiation, Oxidative Stress, Transaminases, Tissue Engineering

CLC Number:

R318

Zhang Ju, Han Li-chi, Wang Yi-fei, Yan Xiao-xu. Phenylephrine effects on the morphology of osteoblasts and expression of nicotinamide phosphoribosyltransferase under oxidative stress[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(8): 1161-1166.

Figures/Tables 1

2.1 大鼠乳鼠成骨细胞形态原代组织块法培养成骨细胞，细胞从组织块边缘长出，5 d后于倒置显微镜下可见，细胞呈三角形、梭形、其他不规则形等多种形态，细胞周围有长短不一的突起相互连接，胞浆丰富、胞核清晰可见(图1A)。传代后细胞生长迅速，5-7 d后进入指数生长期，细胞呈集落式生长，细胞间呈铺路石状汇合，细胞密集处可见复层式生长，此时细胞形态不明显、分界较模糊(图1B)。 2.2 成骨细胞的增殖情况不同时间点细胞增殖率升高与降低趋势基本一致，H2O2浓度从0 μmol/L分别到 50 μmol/L或100 μmol/L，细胞的增殖率有所增加，之后随着浓度增加至200，300及400 μmol/L细胞增殖率降低(表1)。各组细胞增殖率升高与0 μmol/L比较差异不具有显著性意义；不同时间点H2O2浓度超过100 μmol/L时，成骨细胞增殖率降低，其中在24 h和48 h时间点，300，400 μmol/L增殖率降低明显，其细胞增殖率降低与0 μmol/L比较差异有显著性意义。为有效模拟氧化损伤，应用终浓度为 300 μmol/L的H2O2作为成骨细胞氧化损伤浓度。 2.3 H2O2氧化应激后大鼠成骨细胞形态于倒置显微镜下观察可见：苯肾上腺素组较空白对照组细胞密度和形态未见明显变化；单纯H2O2处理组细胞正常连接消失、细胞皱缩、呈散在分布。细胞数量减少，随时间增加变化更加明显；苯肾上腺素+H2O2组在处理12，24和48 h后细胞空泡状改变明显减少，细胞略有皱缩，较单纯H2O2处理组轻微(图2)。 2.4 大鼠成骨细胞Nampt mRNA表达水平苯肾上腺素预处理及H2O2氧化应激处理24 h后，单纯H2O2处理组Nampt mRNA表达较空白对照组减少31.23%(P < 0.05)，苯肾上腺素+H2O2组Nampt mRNA表达较空白对照组减少52.51%(P < 0.01)，但较单纯H2O2处理组增加206.20% (P < 0.05) (图3)。结果表明，苯肾上腺素预处理可促进氧化应激损伤后大鼠成骨细胞Nampt mRNA的表达。 2.5 大鼠成骨细胞Nampt蛋白表达水平苯肾上腺素预处理及H2O2氧化应激处理24 h后，单纯H2O2处理组Nampt蛋白表达较空白对照组减少67.98%(P < 0.05)；苯肾上腺素+H2O2组Nampt蛋白表达较空白对照组减少23.81% (P < 0.05)，但苯肾上腺素+H2O2组Nampt蛋白表达较单纯H2O2处理组增加152.25%(P < 0.05) (图4)。该结果显示，苯肾上腺素预处理氧化应激的成骨细胞可明显上调其Nampt蛋白的表达。"

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