Apigenin accelerates lipopolysaccharide induced apoptosis in mesenchymal stem cells through suppressing vitamin D receptor expression.
Apigenin accelerates lipopolysaccharide induced apoptosis in mesenchymal stem cells through suppressing vitamin D receptor expression.
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发表时间：
2011-11
影响因子：
6.1
通讯作者：
Huan-Tian Zhang;Z. Zha;Jia-hui Cao;Zu-jian Liang;Haoming Wu;M. He;X. Zang;Ping Yao;Jiaqing Zhang
中科院分区：
文献类型：
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作者：
Huan-Tian Zhang;Z. Zha;Jia-hui Cao;Zu-jian Liang;Haoming Wu;M. He;X. Zang;Ping Yao;Jiaqing Zhang
背景技术间充质干细胞（MSC）的移植被认为是组织工程的一种有前景的方法。然而，MSCs用于移植时，由于过度炎症等极其恶劣的微环境，会发生细胞凋亡。据报道，芹菜素 (API) 具有抗炎和抗氧化能力，可保护细胞免受炎症损伤和细胞死亡。在这里，我们研究了 API 对脂多糖 (LPS) 介导的 MSC 炎症和凋亡的调节作用，并进一步明确了其潜在机制。方法采用MTT法检测不同浓度API（0、5、10、20、40、80 μmol/L）作用24 h，LPS（0、0.5、5.0 μg/ml）作用6 h、24 h对MSCs活力的影响。在此基础上，在指定时间（6、12和24小时）用不同浓度的API（0-40 µmol/L）预处理MSC，然后暴露于5 µg/ml LPS 24小时。采用MTT、相差显微镜、膜联蛋白V/碘化丙啶(PI)双染流式细胞术(FCM)和Hoechst染色探讨API对5μg/ml LPS诱导MSCs 24小时的影响。此外，采用逆转录聚合酶链反应（RT-PCR）检测促炎因子包括环氧合酶-2（COX-2）、诱导型一氧化氮合酶（iNOS）、核因子-κB（NF-κB）、促凋亡基因caspase-3、Bad和抗凋亡基因Bcl-2的mRNA表达量。此外，利用AutoDock软件模拟API与维生素D受体（VDR）的对接分数。同时，使用蛋白质印迹和 RT-PCR 来研究 VDR 的蛋白和 mRNA 表达。结果用LPS 5μg/ml刺激MSCs 24小时作为过度炎症刺激诱导细胞凋亡的模型。 API（0～40μmol/L）对MSCs无毒作用；但除5 µmol/L浓度的API外，LPS诱导的MSCs在不同时间点均可降低COX-2、iNOS和NF-κB mRNA表达。相差显微镜、MTT、Hoechst染色和AnnexinV/PI双染FCM结果表明，随着API浓度的增加和给药时间的延长，MSCs发生明显的形态变化，细胞活力受到强烈抑制，同时与LPS单独组相比，LPS给药的MSCs凋亡加剧。此外，API以时间依赖性和浓度依赖性方式促进caspase-3、Bad mRNA表达并抑制Bcl-2 mRNA表达。进一步研究发现API的促凋亡作用与抑制VDR表达有关。结论原料药可抑制诱导炎症因子的表达，发挥较强的抗炎作用。然而，API不能保护LPS诱导的MSC凋亡，反而会放大凋亡。细胞凋亡与 Bad/Bcl-2 增加和 caspase-3 激活有关，这是通过抑制 VDR 表达介导的。
BACKGROUND Transplantation of mensenchymal stem cells (MSCs) has been proposed as a promising way for tissue engineering. However, the application of MSCs for transplantation will undergo apoptosis due to the extremely harsh microenvironment such as excessive inflammation. Apigenin (API) has been reported to protect cells against inflammatory damage and cell death by exhibiting anti-inflammatory and anti-oxidative capacity. Here we investigated the modulatory effects of API in lipopolysaccharide (LPS)-mediated inflammation and apoptosis of MSCs, and further defined the underlying mechanism. METHODS Effects of different concentrations of API (0, 5, 10, 20, 40 and 80 µmol/L) for 24 hours, and LPS (0, 0.5 and 5.0 µg/ml) for 6 hours and 24 hours on MSCs viability were assayed by MTT. Based on this, MSCs were pretreated with different concentrations of API (0 - 40 µmol/L) at the indicated times (6, 12 and 24 hours) followed by exposure to 5 µg/ml LPS for 24 hours. MTT, phase-contrast microscopy, annexinV/propidium iodide (PI) double stain flow cytometry (FCM) and Hoechst staining were applied to explore the effects of API on MSCs induced by 5 µg/ml LPS for 24 hours. In addition, reverse-transcription polymerase chain reaction (RT-PCR) was applied to detect the mRNA expression of pro-inflammatory factors including cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NF-κB), pro-apoptotic gene caspase-3, Bad, and anti-apoptotic gene Bcl-2. Moreover, AutoDock software was used to imitate the docking score of API and vitamin D receptor (VDR). In parallel, Western blotting and RT-PCR were used to investigate protein and mRNA expression of VDR. RESULTS MSCs stimulated with LPS 5 µg/ml for 24 hours was used as a model of apoptosis induced by over inflammatory stimulus. API (0 - 40 µmol/L) had non-toxic effect on MSCs; however, it could decrease mRNA expression of COX-2, iNOS and NF-κB at different time points in MSCs induced by LPS, except for API at the concentration of 5 µmol/L. RESULTS from phase-contrast microscopy, MTT, Hoechst staining and AnnexinV/PI double stain FCM demonstrated that with the increasing concentrations of API and extension of administrating time, significant morphological changes of MSCs occurred, viability of cells was strongly inhibited, and meanwhile, apoptosis of LPS-administrated MSCs was exacerbated, compared with LPS individual group. In addition, API promoted caspase-3, Bad mRNA expression and inhibited Bcl-2 mRNA expression in a time-dependent and concentration- dependent manner. Further study found that pro-apoptosis effect of API was related to suppress VDR expression. CONCLUSIONS API could inhibit the expression of inducible inflammatory factors, therefore exert the strong anti-inflammatory function. However, API could not protect MSC apoptosis induced by LPS but amplified the apoptosis. The apoptosis is related to Bad/Bcl-2 increasing and caspase-3 activation, which is mediated through suppressing VDR expression.