目的 研究反式油酸(9t-C18:1)对H9c2心肌细胞增殖抑制及诱导凋亡作用并探寻其可能的机制。方法 体外培养H9c2大鼠心肌细胞,9t-C18:1高、中、低剂量组及阴性对照组(NC)分别作用于随机分组的H9c2细胞。采用CCK-8法检测细胞增殖能力;AO-EB染色后观察细胞形态学变化;流式细胞仪检测细胞内活性氧(ROS)及细胞凋亡情况;实时定量PCR(QRT-PCR)法检测Bcl-2、Bax基因表达,免疫荧光染色后流式细胞仪检测Bcl-2、Bax蛋白表达。结果 荧光显微镜下可见,9t-C18:1作用后的H9c2细胞出现典型的凋亡形态学特征;与NC组比较,随着9t-C18:1剂量的增加细胞增殖抑制作用明显,ROS、细胞凋亡率显著升高,Bcl-2基因及蛋白表达下调,Bax基因及蛋白表达上调(P<0.05,P<0.01)。结论 9t-C18:1能抑制H9c2细胞增殖并诱导其凋亡,其机制可能与促进细胞线粒体凋亡通路有关。
Abstract
OBJECTIVE To investigate the effects and possible mechanism of trans-oleic acid (9t-C18:1) on proliferation inhibition and induction apoptosis in H9c2 cardiomyocyte. METHODS H9c2 rat cardiomyocytes were cultured in vitro. High, medium and low (600, 300, 150 μmol·L-1) dose of 9t-C18:1 groups and the negative control (NC) group were administered to H9c2 cardiomyocytes. The effect of 9t-C18:1 on cell proliferation was tested using cell counting kit-8 (CCK-8) assay. Morphological changes of cells were observed by AO-EB staining. Intracellular reactive oxygen species (ROS) and apoptosis were detected by flow cytometry. The expression of Bcl-2 and Bax genes was detected by quantitative real time- polymerase chain reaction (QRT-PCR). The expression of Bcl-2 and Bax protein was determined by flow cytometry after immunofluorescence staining. RESULTS The typical morphological characteristics of apoptosis were observed by fluorescence microscope. The result of CCK-8 assay indicated that 9t-C18:1 have an certainly inhibitory effect on the proliferation of H9c2 cells. ROS level and apoptosis rate were significantly increased. Bcl-2 gene and protein expression were down-regulated, and Bax gene and protein expression were up-regulated, compared with NC group(P<0.05, P<0.01). CONCLUSION 9t-C18:1 can inhibit the proliferation and induce the apoptosis of H9c2 cardiomyocyte, and its mechanism may be related to promoting the mitochondrial apoptotic pathway.
关键词
反式油酸(9t-C18:1) /
H9c2心肌细胞 /
增殖抑制 /
细胞凋亡
{{custom_keyword}} /
Key words
trans-oleic acid (9t-C18:1) /
H9c2 cardiomyocyte /
proliferation inhibition /
apoptosis
{{custom_keyword}} /
中图分类号：
R965
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] SHEN J F, ZHANG Z Y. Safety of trans fatty acids and recent research progress [J]. J Chin Cere Oils Assoc(中国粮油学报), 2005, 20(4):88-90.
[2] COUNIL E, JULIEN P, LAMARCHEB, et al. Association between trans-fatty acids in erythrocytes and pro-atherogenic lipid profiles among Canadian Inuit of Nunavik possible influences of sex and age [J]. Br J Nutr, 2009, 102(5):766-776.
[3] LI X P. Effect of trans fatty acid can proliferation and function of human umbilical vein smooth muscle cells [D]. Nanchang: Nanchang University,2013.
[4] ROZENN N L, IRENA B K, TRIVELLORE E R. Cell membrane trans fatty acids and the risk of primary cardic arrest [J]. Circulation, 2002, 105:697-701.
[5] MOZAFFARIAN D, ARO A, WILLETT W C. Health effects of trans-fatty acids: experimental and observational evidence [J]. Eur J Clin Nutr, 2009, 63(suppl 2):5-21.
[6] OOMEN C M, OCKE M C, FESKENS E J, et al. Association between trans fatty acids intake and 10-year risk of coronary heart disease in the Zutphen Elderly Study a prospective population-based study [J]. Lancet, 2001, 357(9258):746-751.
[7] DOWNAR D Z, KOSMIDER A, NARUSZEWICZ M. Trans fatty acids induce apoptosis in human endothelial cells [J]. Physiol Pharmacol, 2005, 56(4):611-625.
[8] KONDOH Y, KAWADA T, URADE R. Activation of caspase-3 in HepG2 cells by elaidic acid (t18:1) [J]. Biochim Biophys Acta, 2007, 1771(4):500-505.
[9] BAINES C P, MOLKENTIN J D. Stress signaling pathways that modulate cardiac myocyte apoptosis [J]. J Mol Cell Cardiol, 2005, 38(1):47-62.
[10] WANG L W, LIU F Z, LU J K, et al. Effect of flavonols on caspase-3, Bcl-2 and Bax expression in cardiomyocytes apoptosis [J]. Chin Pharm J(中国药学杂志), 2007, 42(7):501-504.
[11] KIMES B W, BRANDT B L. Properties of clonal muscle cell line from rat heart [J]. Exp Cell Res, 1976, 98(2):367-381.
[12] JOSE CORBALAN J, VATNER D E, VATNE R S F. Myocardial apoptosis in heart disease: does the emperor have clothes? [J]. Basic Res Cardiol, 2016, 111:31.
[13] KUZMICIC J, DEL CAMPO A, LPEZ-CRISOSTO C, et al. Mitochondrial dynamics: a potential new therapeutic target for heart failure [J]. Rev Esp Cardiol, 2011, 64 (10):916-923.
[14] BOTT-FLGEL L, WEIG H J, UHLEIN H, et al. Quantitative analysis of apoptotic markers in human end-stage heart failure [J]. Eur J Heart Fail, 2008, 10(2):129-132.
[15] TSIPIS A, ATHANASSIADOU A M, ATHANASSIADOU P, et al. Apoptosis-related factors p53, bcl-2 and the defects of force transmission in dilated cardiomyopathy [J]. Pathol Res Pract, 2010, 206(9):625-630.
[16] FENG W S, YANG F F, ZHANG L, et al. Protective effects of aqueous extract from descurainia sophia on doxorubicin-induced cardiomyocyte injuryvia suppressing apoptosis and oxidative stress [J]. Chin Pharm J(中国药学杂志), 2018, 53(23):1999-2007.
[17] BI L, YAN X, CHEN W, et al. Antihepatocellular carcinoma potential of tetramethylpyrazine induces cell cycle modulation and mitochondrial-dependent apoptosis: regulation of p53 signaling pathway in Hep G2 cells in vitro [J]. Integr Cancer Ther, 2016, 15(2):226-236.
[18] MA L W, ZHOU L, LI S L, et al. Effect of 12-deoxyphorbol-13-palmitate on proliferation inhibition and apoptosis induction of HL60 cells [J]. Chin Pharm J(中国药学杂志),2018,53 (1):30-34.
[19] SONG R S, XIAO X H, LIU Z L, et al. Effects of a monomer purified from paris polyphylla (PP-22) on proliferation and apoptosis of human gastric carcinoma MGC803 cells [J]. Chin Pharm J(中国药学杂志), 2015, 50(18):1600-1606.
[20] LIU H C, ZHANG Y, ZHANG S, et al. Correlation research on the protein expression (p75 NTR, bax, bcl-2 and caspase-3) and cortical neuron apoptosis following mechanical injury in rat [J]. Eur Rev Med Pharmacol Sci, 2015, 19(18):3459-3467.
[21] WANG S J, GUO X, ZUO H, et al. Chondrocyte apoptosis and expression of Bcl-2, Bax, Fas, and iNOS in articular cartilage in patients with Kashin-Beck disease [J]. J Rheumatol, 2006, 33(3):615-619.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
齐齐哈尔医学院院内基金资助(QY2016CX-03)
{{custom_fund}}
PDF(4155 KB)
