烟酰胺削弱罗格列酮诱导猪前体脂肪细胞凋亡的作用与分子机制

doi:10.3864/j.issn.0578-1752.2013.18.018

摘要/Abstract

摘要： 【目的】探讨烟酰胺（nicotinamide, NIC）和罗格列酮（rosiglitazone, RSG）对猪前体脂肪细胞凋亡的作用及分子机制，寻找通过凋亡方式调控脂肪细胞数目进而控制生脂的新途径。【方法】分别用含有 0、0.2、0.4、0.6、0.8 和 1.0 mmol•L-1 RSG的培养基处理猪前体脂肪细胞，在处理后的48 h，对不同处理组细胞进行Hoechst33258和Annexin-V-FITC/PI染色以观察细胞形态，流式细胞仪分析细胞凋亡情况；并收集细胞总蛋白，用Western blotting方法检测细胞凋亡关键基因的蛋白表达水平；基于RSG促凋亡的浓度梯度结果，选用1.0 mmol•L-1 RSG分别与含0、0.1、0.2和0.3 mmol•L-1 NIC的培养基共处理细胞，在处理后的48 h，染色并观察细胞形态，流式细胞仪分析细胞凋亡情况；并收集细胞总蛋白、细胞质蛋白和细胞核蛋白，检测细胞凋亡关键基因的蛋白水平。【结果】 RSG以浓度依赖方式诱导猪前体脂肪细胞凋亡，减少了细胞数目；Bcl-2和cleaved Caspase-3凋亡关键蛋白水平随RSG浓度增加而上升，Bax则下降；0.1（P＜0.05）、0.2（P＜0.01）和0.3 mmol•L-1 （P＜0.01）NIC显著削弱1 mmol•L-1 RSG诱导猪前体脂肪细胞凋亡，Bcl-2和cleaved Caspase-3水平显著被下调（P＜0.05），Bax则被上调（P＜0.05）；0.3 mmol•L-1 NIC明显有助于1 mmol•L-1 RSG处理的猪前体脂肪细胞中的Sirt1由细胞质向核转移，且抑制核中cleaved Caspase-3水平。【结论】NIC通过促进Sirt1由细胞质到核的转位以及下调核中cleaved Caspase-3水平削弱RSG诱导的猪前体脂肪细胞凋亡。

关键词: 猪 , 烟酰胺 , 罗格列酮 , 前体脂肪细胞 , 凋亡

Abstract: 【Objective】This study was performed to explore the effect and molecular mechanism of nicotinamide (NIC) and rosiglitazone (RSG) on apoptosis of porcine preadipocytes for finding a novel method to control adipogenesis through regulation of preadipocyte apoptosis.【Method】Cells were cultured in high glucose DMEM medium containing 0, 0.2, 0.4, 0.6, 0.8 and 1.0 mmol•L-1 RSG. After being treated for 48 h, cells were stained with Hoechst33258 and Annexin-V-FITC/PI and observed under microscope. Simultaneously, cells were harvested for analysis of flow cytometry and collection of total protein to detect protein levels of several genes related with apoptosis. Based on the above results, cells were further co-cultured in DMEM medium containing 0, 0.1, 0.2 and 0.3 mmol•L-1 NIN with 1 mmol•L-1 RSG. After being treated for 48 h, cells were stained and observed. Cells were harvested for analysis of flow cytometry and collection of total, cytoplasmic and nuclear proteins for Western blotting analysis.【Result】RSG induced preadipocyte apoptosis by a dose-dependent way and reduced cell number. The levels of Bcl-2 and cleaved Caspase-3 increased, whereas the level of Bax decreased in higher concentration of RSG. Moreover, 0.1（P＜0.05）、0.2（P＜0.01）and 0.3 mmol•L-1 （P＜0.01）NIC significantly attenuated RSG-induced apoptosis of preadipocytes. The levels of Bcl-2 and cleaved Caspase-3 were down-regulated, whereas the level of Bax was up-regulated in treatment with 0.3 mmol•L-1 NIN + 1 mmol•L-1 RSG. Compared with the control (1 mmol•L-1 RSG + 0 mmol•L-1 NIC), in treatment with 0.3 mmol•L-1 NIC + 1 mmol•L-1 RSG, Sirt1 was transfered from cytoplasm into nucleus and the level of cleaved Caspase-3 was reduced.【Conclusion】NIC attenuated RSG-induced apoptosis of porcine preadipocytes via cytoplasm/nucleus transposition of Sirt1 and down-regulating the level of nuclear cleaved Caspase-3.

Key words: pig , nicotinamide , rosiglitazone , preadipocyte , apoptosis

庞卫军, 许儒祥, 熊燕, 孙世铎, 杨公社. 烟酰胺削弱罗格列酮诱导猪前体脂肪细胞凋亡的作用与分子机制[J]. 中国农业科学, 2013, 46(18): 3878-3886.

PANG Wei-Jun, XU Ru-Xiang, XIONG Yan, SUN Shi-Duo, YANG Gong-She. Effects and Molecular Mechanism of Nicotinamide on Attenuating Apoptosis of Rosiglitazone-induced Porcine Preadipocytes[J]. Scientia Agricultura Sinica, 2013, 46(18): 3878-3886.

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