| 夏冰,洪滔.白杨黄素通过调控氧化应激反应保护脂多糖介导的炎症环境中人牙周膜干细胞的成骨分化能力[J].浙江中西医结合杂志,2021,31(10): |
| 白杨黄素通过调控氧化应激反应保护脂多糖介导的炎症环境中人牙周膜干细胞的成骨分化能力 |
| Effect of chrysin on osteogenic differentiation in human periodontal ligament stem cells with LPS-induced inflammatory condition |
| 投稿时间：2021-03-07 修订日期：2021-08-25 |
| DOI： |
| 中文关键词: 白杨黄素 hPDLSCs 成骨分化 LPS |
| 英文关键词:Chrysin hPDLSCs Osteogenic differentiation LPS |
| 基金项目:杭州市科技局项目 (20180533B76) |
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| 中文摘要: |
| 目的: 探讨白杨黄素对牙龈卟啉单胞菌(P.g)脂多糖(P.g LPS)介导的炎症环境中人牙周膜干细胞 (hPDLSCs)成骨分化能力的影响。方法：原代培养hPDLSCs, 采用流式细胞术鉴定后取第4代细胞进行实验。采用四唑盐(MTT)试验检测不同浓度P.g LPS (1, 10, 100 和1000 ng/mL)及白杨黄素 (0.1, 0.4, 1.6, 6.25, 25, 50, 100 μmol/L) 对hPDLSCs增殖能力的影响；P.g LPS与成骨诱导剂共培养24h, 48h, 72h和96h后，活性氧 (ROS)试剂盒检测hPDLSCs 的ROS含量, 实时荧光定量聚合酶链式反应 (RT-PCR)检测hPDLSCs锰超氧化物歧化酶(MnSOD), 铜锌超氧化物歧化酶(Cu/ZnSOD)和过氧化氢酶 (CAT) mRNA的表达; P.g LPS与成骨诱导剂共培养3天, 7天, 14天后, 碱性磷酸酶(ALP)试剂盒检测hPDLSCs 的ALP活性, RT-PCR法检测成骨相关转录因子(RUNX2), 锌指结构转录因子(OSX), 碱性磷酸酯酶(ALP) 和骨钙素(OCN) mRNA表达; 25 μmol/L 白杨黄素作用hPDLSCs后, 通过ROS试剂盒检测hPDLSCs 的ROS含量, 及实时PCR法检测抗氧化因子MnSOD、Cu/ZnSOD、CAT及成骨分化基因RUNX2、OSX、OCN和ALP的mRNA表达。结果：与对照组相比, MTT结果显示1000 ng/mL P.g LPS 作用hPDLSCs 72h [(0.51±0.03) 比 (0.68±0.02), 96h后(0.62±0.06) 比 (0.97±0.07), P均<0.05]均对细胞增殖活性受到显著抑制。并且25 μmol/L 的白杨黄素对hPDLSCs 增殖活性无抑制效应 [(99.8±1.02) 比 (100±1.02) %, P>0.05). 与常规成骨诱导液培养细胞相比, P.g LPS显著增加hPDLSCs的ROS含量至 (17.3±1.34) 比(3.12±1.21) ng/ml (P<0.01), 并显著降低抗氧化因子[Cu/ZnSOD, (0.89±0.24) 比 (2.84±0.27); CAT, (1.12±0.09) 比 (2.64±0.28), P均<0.05]和成骨分化基因表达 [ALP活性, (0.94±0.11) 比 (1.25±0.14); RUNX2, (1.42±0.13) 比 (1.97±0.16); OSX (1.97 ±0.16) 比 (2.68 ±0.19); OCN (1.23±0.11) 比 (2.56±0.17), P均<0.05]; 与P.g LPS+成骨诱导液组相比, 白杨黄素显著改善P.g LPS介导hPDLSCs的氧化状态及增强其成骨分化能力[RUNX2, (1.96±0.28) 比 (1.67±0.23); OSX (2.16±0.31) 比 (1.64±0.17), P均<0.05]。结论: 白杨黄素可能对P.g LPS介导的炎症环境中人牙周膜干细胞的成骨分化能力具有促进作用。 |
| 英文摘要: |
| ABSTRACT Objective To investigate the effects of chrysin on osteogenic differentiation under Porphyromonas gingivalis (P.g) lipopolysaccharide (P.g LPS)-induced inflammatory condition in human periodontal ligament stem cells (hPDLSCs). Method The primary cultured hPDLSCs were identified by flow cytometry, and the fourth generation cells were used for further studies. The MTT assay was employed to measure the effect of different concentrations of P.g LPS (1、10、100, and 1000 ng/mL) and chrysin (0.1, 0.4, 1.6, 6.25, 25, 50, 100 μmol/L) on the proliferation of hPDLSCs. The hPDLSCs were co-cultured with P.g LPS and an osteogenic inducer for 24h, 48h, 72h, and 96h, and the reactive oxygen species (ROS) kit was used to measure the ROS content of hPDLSCs. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the mRNA levels of manganese superoxide dismutase (MnSOD), Cu/Zn superoxide dismutase (Cu/ZnSOD), and Catalase (CAT); The hPDLSCs were co-cultured with P.g LPS and an osteogenic inducer for 3d, 7d, and 14d, and the alkaline phosphatase (ALP) activity of hPDLSCs was detected using an ALP kit, and then qRT-PCR was used to identify the RUNX family transcription factor 2 (RUNX2), Osterix (OSX), Alkaline phosphatase (ALP), and Osteocalcin (OCN) mRNA levels. With the chrysin (25 μmol/L) treatment, the concentration of ROS was detected using a ROS kit, and the mRNA levels of Anti-oxidant factors (MnSOD、Cu/ZnSOD、CAT) and osteogenic differentiation-related genes (RUNX2、OSX、OCN、ALP) were measured with RT-PCR. Results Compared with the control group, the result of MTT shown that 1000 ng/mL P.g LPS inhibited the proliferation under-treated for 72h [(0.51±0.03) 比 (0.68±0.02) and 96h (0.62±0.06) vs. (0.97±0.07), respectively; all P<0.05] in hPDLSCs. And, compared with the 0 μmol/L chrysin group, 25 μmol/L chrysin had no activity inhibition effect on hPDLSCs [(99.8±1.02) vs. (100±1.02) %, P>0.05). Besides, compared with the cultured cells with a conventional osteogenic inducer, the content of ROS was observably increased to (17.3±1.34) vs. (3.12±1.21) ng/ml (P<0.01), and the expressions of anti-oxidant factors [Cu/ZnSOD, (0.89±0.24) vs. (2.84±0.27); CAT, (1.12±0.09) vs. (2.64±0.28), all P<0.05] and osteogenic differentiation-related genes were significantly decreased by P.g LPS treatment [ALP activity, (0.94±0.11) vs. (1.25±0.14); RUNX2, (1.42±0.13) vs. (1.97±0.16); OSX (1.97 ±0.16) vs. (2.68 ±0.19); OCN (1.23±0.11) vs. (2.56±0.17), all P<0.05]. Compared with the P.g LPS+osteogenic inducer group, chrysin could improve the oxidation state by P.g LPS-mediated, and enhance the osteogenic differentiation of hPDLSCs [RUNX2, (1.96±0.28) vs. (1.67±0.23); OSX (2.16±0.31) vs. (1.64±0.17), all P<0.05]. Conclusion Chrysin might have a significant promoting effect on osteogenic differentiation in P.g LPS-induced hPDLSCs with inflammatory condition. |
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