[1]唐杨,刘川,康华利,等.褪黑素抑制氧化型低密度脂蛋白诱导血管平滑肌细胞增殖[J].第三军医大学学报,2021,43(17):1619-1626.
 TANG Yang,LIU Chuan,KANG Huali,et al.Melatonin inhibits ox-LDL-induced proliferation of vascular smooth muscle cells[J].J Third Mil Med Univ,2021,43(17):1619-1626.
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褪黑素抑制氧化型低密度脂蛋白诱导血管平滑肌细胞增殖(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
43卷
期数:
2021年第17期
页码:
1619-1626
栏目:
基础医学
出版日期:
2021-09-15

文章信息/Info

Title:
Melatonin inhibits ox-LDL-induced proliferation   of vascular smooth muscle cells
作者:
唐杨刘川康华利晋军
陆军军医大学(第三军医大学)第二附属医院心血管内科
Author(s):
TANG Yang LIU Chuan KANG Huali JIN Jun

Department of Cardiovascular Diseases, Second Affiliated Hospital, Army Medical University (Third Military Medical University), Chongqing, 400037, China

关键词:
动脉粥样硬化血管平滑肌细胞褪黑素氧化型低密度脂蛋白增殖
Keywords:
atherosclerosis vascular smooth muscle cells melatonin oxidized low-density lipoprotein proliferation
分类号:
R322.12; R329.28; R977.1
文献标志码:
A
摘要:

目的观察褪黑素(melatonin,Mel)对氧化型低密度脂蛋白(ox-LDL)诱导的血管平滑肌细胞(vascular smooth muscle cells,VSMCs)增殖功能的影响并探讨其可能的分子机制。方法①选用不同浓度(0、10、20、50、100 μg/mL)的ox-LDL诱导VSMCs 48 h后检测细胞增殖功能,观察各浓度ox-LDL对VSMCs功能的影响。②选择最佳浓度(50 μg/mL)的ox-LDL诱导VSMCs 0、6、12、24、48 h后检测细胞增殖功能,观察各时间点ox-LDL对VSMCs功能的影响。③50 μg/mL的ox-LDL诱导VSMCs 24 h后,再用1、1×103、1×105、1×106 nmol/L的Mel处理VSMCs 48 h,观察Mel作用不同时间对VSMCs增殖功能的影响。④使用ox-LDL联合Mel处理VSMCs。实验分4组:空白对照组(VSMCs正常培养不进行任何处理),Mel组(1×105 nmol/L Mel处理VSMCs 48 h),ox-LDL组(50 μg/mL ox-LDL诱导VSMCs 24 h),ox-LDL+ Mel组(使用50 μg/mL 的ox-LDL诱导VSMCs 24 h后,接着使用1×105 nmol/L的Mel处理VSMCs 48 h)。采用CCK-8法检测细胞增殖,流式细胞术检测细胞周期,免疫荧光染色和Western blot检测增殖标记物(PCNA)、α平滑肌肌动蛋白(α-SMA)、细胞周期素A(Cyclin A)、细胞周期抑制蛋白p21以及细胞外调节蛋白激酶1/2(ERK1/2)信号通路相关蛋白表达。结果与空白对照组比较,50 μg/mL ox-LDL处理VSMCs 24 h能显著诱导细胞增殖,经ox-LDL(50 μg/mL)诱导24 h后,S期与G1期细胞显著增加,G2期细胞显著减少,促进细胞迁移,上调PCNA、α-SMA、Cyclin A、p-ERK1/2蛋白表达,并下调p-p21蛋白水平,差异均具有统计学意义(P<0.01)。1、1×103、1×105、1×106 nmol/L Mel均可不同程度抑制 ox-LDL 诱导的 VSMCs增殖,与ox-LDL组比较,ox-LDL(50 μg/mL)处理VSMCs 24 h后接着使用Mel(1×105 nmol/L)处理48 h,可使G1 期与S期细胞显著减少,G2期细胞显著增加,降低PCNA、α-SMA、Cyclin A、p-ERK1/2蛋白表达,上调p-p21蛋白表达,差异均具有统计学意义(P<0.01)。结论Mel可显著抑制ox-LDL诱导的VSMCs的增殖,其作用机制可能与激活P21、阻断ERK1/2 信号通路相关。

Abstract:

ObjectiveTo explore the effect of melatonin (Mel) on the proliferation of vascular smooth muscle cells (VSMCs) induced by oxidized low-density lipoprotein (ox-LDL) and investigate the potential molecular mechanism. Methods① ox-LDL at different concentrations (0, 10, 20, 50 and 100 μg/mL) was used to treat VSMCs for 48 h, and cell proliferation was measured with CCK-8 assay. ② The effect of different time points (0, 6, 12, 24 and 48 h) on the cell viability was also detect after the VSMCs was treated with the optional concentration of ox-LDL (50 μg/mL). ③ After the VSMCs were induced by 50 μg/mL ox-LDL for 24 h, the cells were treated with Mel at 1, 1×103, 1×105 and 1×106 nmol/L for 48 h to determine the effect of Mel on cell viability. ④ The VSMCs were divided into 4 groups, that is, blank control group, Mel group (1×105 nmol/L Mel for 48 h), ox-LDL group (50 μg/mL ox-LDL for 24 h), and ox-LDL+Mel group (50 μg/mL ox-LDL for 24 h followed by 1×105 nmol/L Mel for 48 h). CCK-8 assay and flow cytometry were employed to detect cell proliferation and cell cycle, respectively. Immunofluorescence staining and Western blotting were adopted to determine the expression of cell proliferation markers, proliferating cell nuclear antigen (PCNA) and α-smooth muscle actin (α-SMA), cell cycle-related protein cyclin A (Cyclin A) and cyclin-dependent kinase inhibitor p21, as well as extracellular regulatory protein kinase 1/2 (ERK1/2). ResultsCompared with the blank control group, treatment of 50 μg/mL ox-LDL for 24 h in VSMCs significantly promoted cell proliferation, induced cell arrested at S and G1 phase, decreased the proportion of cells at G2 phase, improved cell migration, up-regulated the expression of PCNA, α-SMA, Cyclin A and p-ERK1/2, and down-regulated that of p-p21 protein (all P<0.01). Mel of 1, 1×103, 1×105 and 1×106 nmol/L inhibited the proliferative ability of VSMCs induced by ox-LDL treatment. Compared with the ox-LDL group, the ox-LDL+Mel group had significantly reduced proportion of G1 and S phase cells, enhanced proportion of G2 phase cells, decreased protein levels of PCNA, α-SMA, Cyclin A and p-ERK1/2, and increased level of p-p21 (P<0.01). ConclusionMel significantly inhibits the proliferation in VSMCs induced by ox-LDL, which might be related to activation of P21 and block of ERK1/2 signaling pathway.

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更新日期/Last Update: 2021-09-03