Huang Denghao, Lei Jie, Li Xingrui, Jiang Zhonghao, Luo Maoxuan, Xiao Yao
Luzhou Key Laboratory of Orofacial Reconstruction and Regeneration, The Affiliated Stomatology Hospital of Southwest Medical University & Institute of Stomatology, Southwest Medical University, Luzhou, China.
Cell Biology Technology Platform, Public Experiment Technology Center of Southwest Medical University, Luzhou, China.
Stem Cells Int. 2022 Sep 20;2022:9806887. doi: 10.1155/2022/9806887. eCollection 2022.
Angiogenic tissue engineering is a vital problem waiting to be settled for periodontal regeneration. Erythropoietin, a multieffect cytokine, has been reported as a protective factor for cell fate. According to our previous study, erythropoietin has a significantly angiogenic effect on periodontal ligament stem cells. To further explore its potential effects and mechanism, we studied biological behaviors of periodontal ligament stem cells under inflammatory microenvironment induced by different concentrations (0, 10, 20, 50, and 100 ng/mL) of tumor necrosis factor- (TNF-) and examined how different concentrations (0, 5, 10, 20, and 50 IU/mL) of erythropoietin changed biological behaviors of periodontal ligament stem cells.
Cell Counting Kit-8 was used for cell proliferation assay. Annexin V-PI-FITC was used for cell apoptosis through flow cytometry. Matrigel plug was adopted to measure the angiogenic capacity . RNA sequencing was used to detect the downstream signaling pathway. Quantitative real-time polymerase chain reaction was conducted to examine mRNA expression level. Western blot and immunofluorescence were applied to testify the protein expression level.
Periodontal ligament stem cells upregulated apoptosis and suppressed autophagy and angiogenesis under inflammatory microenvironment. Erythropoietin could activate autophagy to rescue apoptosis and angiogenesis levels of periodontal ligament stem cells through the Akt/Erk1/2/BAD signaling pathway under inflammatory microenvironment.
Erythropoietin could protect periodontal ligament stem cells from inflammatory microenvironment, which provided a novel theory for periodontal regeneration.
血管生成性组织工程是牙周再生亟待解决的关键问题。促红细胞生成素是一种具有多种效应的细胞因子,已被报道为细胞命运的保护因子。根据我们之前的研究,促红细胞生成素对牙周膜干细胞具有显著的血管生成作用。为了进一步探究其潜在作用及机制,我们研究了不同浓度(0、10、20、50和100 ng/mL)肿瘤坏死因子-(TNF-)诱导的炎症微环境下牙周膜干细胞的生物学行为,并检测了不同浓度(0、5、10、20和50 IU/mL)促红细胞生成素如何改变牙周膜干细胞的生物学行为。
使用细胞计数试剂盒-8进行细胞增殖检测。采用膜联蛋白V-碘化丙啶-异硫氰酸荧光素通过流式细胞术检测细胞凋亡。采用基质胶栓检测血管生成能力。利用RNA测序检测下游信号通路。进行定量实时聚合酶链反应检测mRNA表达水平。应用蛋白质免疫印迹和免疫荧光法验证蛋白质表达水平。
在炎症微环境下,牙周膜干细胞的凋亡上调,自噬和血管生成受到抑制。在炎症微环境下,促红细胞生成素可通过Akt/Erk1/2/BAD信号通路激活自噬,以挽救牙周膜干细胞的凋亡和血管生成水平。
促红细胞生成素可保护牙周膜干细胞免受炎症微环境的影响,这为牙周再生提供了一种新理论。