目的 为研究中华真地鳖血淋巴(Eupolyphaga sinensis Walker hemolymph, ESWH)对人红白血病(human erythroleukemia,HEL)细胞增殖及凋亡的影响并探究其机制。方法 采用不同质量浓度(10~60 mg·L-1)的ESWH体外作用于人红白血病细胞(HEL细胞)细胞,利用四甲基偶氮唑蓝(MTT)法测定细胞增殖活性;Hoechst 33258和Annexin V-FITC荧光染色观察细胞凋亡的形态学变化;Annexin/PI法检测HEL细胞凋亡率;流式细胞仪分析HEL细胞周期分布;荧光定量PCR(RT-qPCR)和Western blot技术检测HEL细胞中Bcl-2,Bax,caspase-3,caspase-8,Cyt C的mRNA和蛋白的表达。结果 ESWH能够抑制HEL细胞增殖,并呈一定的时间和浓度依赖关系;ESWH能够引起HEL细胞发生G2/M期阻滞,促进细胞凋亡;qRT-PCR和Western blot结果表明,ESWH可下调HEL细胞中Bcl-2的mRNA和蛋白的表达水平(P<0.01),上调Bax,Cyt C,cleaved caspase-3,cleaved caspase-8蛋白的表达水平(P<0.01)。结论 ESWH能抑制人红白血病HEL细胞增殖,并诱导其凋亡,其机制可能与激活线粒体凋亡通路有关。
Abstract
OBJECTIVE To explore the effect of Eupolyphaga sinensis Walker hemolymph (ESWH) on the proliferation and apoptosis of human erythroleukemia HEL cells. METHODS The cells were treated with different concentrations (10-60 mg·L-1) of ESWH in vitro. Cell viability was assessed by MTT assay, the morphological changes of apoptotic HEL cells were observed by Hoechst 33258 and Annexin V-FITC fluorescence staining, and the apoptosis rate was evaluated by Annexin/PI method. Furthermore, apoptosis and cell cycle distribution were analyzed with flow cytometry, and RT-qPCR and Western blot assay were employed to detect mRNA and protein expression levels of Bcl-2, Bax, caspase-3 and caspase-8, Cyt C. RESULTS ESWH inhibited HEL cells proliferation in a time and dose-dependent manner. ESWH could induce G2/M phase cell cycle arrest in HEL cells, promote cell apoptosis, down-regulate the mRNA and protein expression levels of Bcl-2 (P<0.01), and up-regulate the protein expression levels of Bax, Cyt C, cleaved caspase-3 and cleaved caspase-8 (P<0.01). CONCLUSION ESWH could inhibit proliferation and induce apoptosis of human erythroleukemia HEL cells, and the mechanism may be associated with activating the mitochondria-dependent pathway.
关键词
中华真地鳖 /
血淋巴 /
人红白血病细胞 /
凋亡 /
线粒体途径
{{custom_keyword}} /
Key words
Eupolyphaga sinensis Walker /
hemolymph /
HEL cells /
apoptosis /
mitochondrial pathway
{{custom_keyword}} /
中图分类号：
R932
{{custom_clc.code}}
({{custom_clc.text}})
{{custom_sec.title}}
{{custom_sec.title}}
{{custom_sec.content}}
参考文献
[1] ZHANG C X, TANG X D, CHENG J A. The utilization and industrialization of insect resources in China [J]. Entomol Res, 2010, 38(S1): S38-S47.
[2] DAI B, ZHAN Y, QI J, et al. Eupolyphaga sinensis Walker inhibits human chronic myeloid leukemia cell K562 growth by inducing G2/M phase cell cycle arrest and targeting EGFR signaling pathway and in S180 tumor-bearing mice [J]. Environ Toxicol Pharmacol, 2014, 37(3): 1177-1185.
[3] XU L, LAO L X, GE A, et al. Chinese herbal medicine for cancer pain [J]. Integr Cancer Ther, 2007, 6(3): 208-234.
[4] GE G F, YU C H, YU B, et al. Antitumor effects and chemical compositions of Eupolyphaga sinensis Walker ethanol extract [J]. J Ethnopharmacol, 2012, 141(1): 178-182.
[5] TANG Q F, DAI Y, XUE L L. Immunomodulatory effects of orally administered aqueous extract from Eupolyphaga sinensis Walker [J]. Afr J Biotechnol, 2010, 9(50): 8682-8686.
[6] DAI B, QI J, LIU R, et al. Eupolyphaga sinensis Walker demonstrates angiogenic activity and inhibits A549 cell growth by targeting the KDR signaling pathway [J]. Mol Med Rep, 2014, 10(3): 1590-1596.
[7] ZHAN Y Z, ZHANG H, LIU R, et al. Eupolyphaga sinensis Walker ethanol extract suppresses cell growth and invasion in human breast cancer cells [J]. Integr Cancer Ther, 2016, 15(1): 102-112.
[8] ZHANG Y M, ZHAN Y Z, ZHANG D D, et al. Eupolyphaga sinensis Walker displays inhibition on hepatocellular carcinoma through regulating cell growth and metastasis signaling [J]. Sci Rep, 2014, 4: 5518.
[9] BORSUK G, PTASZYN′SKA, OLSZEWSKI K, et al. A new method for quick and easy hemolymph collection from apidae adults [J]. PLoS One, 2017, 12(1): e0170487.
[10] BOULANGER N, BRUN R, EHRET-SABATIER L, et al. Immunopeptides in the defense reactions of Glossina morsitans to bacterial and Trypanosoma brucei brucei infections [J]. Insect Biochem Mol Biol, 2002, 32(4): 369-375.
[11] CHERNYSH S, KIM S I, BEKKER G, et al. Antiviral and antitumor peptides from insects [J]. Proc Natl Acad Sci USA, 2002, 99(20): 12628-12632.
[12] NAU K, LEWIS W D. Multiple myeloma: diagnosis and treatment [J]. Am Fam Physician, 2008, 78(7): 853-859.
[13] NAMBA T, MA Y H, INAGAKI K. Insect derived crude drugs in the chinese song dynasty [J]. J Ethnopharmacol, 1988, 24(2-3): 247-285.
[14] NOWAK V, PERSIJN D, RITTENSCHOBER D, et al. Review of food composition data for edible insects [J]. Food Chem, 2016, 193: 39-46.
[15] CAI H B, SUN X G, LIU Z F, et al. Effects of Dahuangzhechong pills on cytokines and mitogen activated protein kinase activation in rats with hepatic fibrosis [J]. J Ethnopharmacol, 2010, 132(1): 157-164.
[16] OTTO T, SICINSKI P. Cell cycle proteins as promising targets in cancer therapy [J]. Nat Rev Cancer, 2017, 17(2): 93-115.
[17] CHOWDHURY I, THARAKAN B, BHAT G K. Current concepts in apoptosis: the physiological suicide program revisited [J]. Cell Mol Biol Lett, 2006, 11(4): 506-525.
[18] GARTEL A L. Mechanisms of apoptosis induced by anticancer compounds in melanoma cells [J]. Curr Top Med Chem, 2012, 12(1): 50-52.
[19] CHEN Z, ZHU S W, MA L W, et al. Effect of trans-oleic acid on proliferation inhibition and apoptosis induction in H9c2 cardiomyocyte [J]. Chin Pharm J (中国药学杂志), 2020, 55(3): 199-205.
[20] STEVENS J M. Cytochrome C as an experimental model protein [J]. Metallomics, 2011, 3(4): 319-322.
[21] KIM P K, MAHIDHARA R, SEOL D W. The role of caspase-8 in resistance to cancer chemotherapy [J]. Drug Resist Updat, 2001, 4(5): 293-296.
{{custom_fnGroup.title_cn}}
脚注
{{custom_fn.content}}
基金
国家自然科学基金青年基金项目资助(81702914);农业昆虫与害虫防治专业研究生卓越人才计划项目资助(黔教研合ZYRC字[2013]010号)
{{custom_fund}}
PDF(4046 KB)
