蛇床子素对L02细胞的毒性作用及其机制研究
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摘要
目的探究蛇床子素(osthole,Ost)对L02细胞的毒性损伤和作用机制,为中药蛇床子及其制剂的安全合理应用提供实验依据。方法以不同浓度Ost作用于L02细胞,MTT法检测细胞活性;乳酸脱氢酶(LDH)试剂盒测定细胞LDH释放率;Hoechst 33342染色法检测细胞核形态;Annexin V/PI双染法检测细胞凋亡;Western blot检测Bcl-2、Bax、pro-caspase-3、cleaved-caspase-3(p17)、p-Histon H3(Ser10)的表达。结果 L02细胞在Ost作用下活性下降,LDH释放率提高,且呈浓度依赖;Hoechst 33342染色荧光下可见细胞核皱缩碎裂;Annexin V/PI双染法结果表明凋亡率随浓度提高而上升。与对照组比较,50,100,200μmol·L~(-1)Ost作用24 h后,Bcl-2、pro-caspase-3、p-Histon H3(Ser10)表达水平降低,Bax、cleaved-caspase-3表达水平升高。结论 Ost对L02细胞有毒性损伤作用,呈一定的时间和浓度依赖性,可促进细胞凋亡,抑制细胞增殖。
OBJECTIVE To investigate the toxic effect of osthole(Ost) on L02 cells and explore its mechanism, which could provide an experimental evidence for the safe and rational application of Fructus cnidii and its preparation. METHODS Different concentrations of Ost were applied to L02 cells, MTT assay was used to detect cell activity; the release rate of lactate dehydrogenase(LDH) was analyzed using LDH kits; Hoechst 33342 staining was used to detect cell nucleus morphology; Annexin V/PI staining was used to detect apoptosis; Western blot was utilized to detect the expression of Bcl-2, Bax, pro-caspase-3, cleaved-caspase-3(p17) and p-Histon H3(Ser10). RESULTS The activity of L02 cells decreased under the action of Ost, while the release rate of LDH increased and appeared to be concentration dependent. The cell nucleus treated by Ost were shrunken and fragmented under Hoechst 33342 fluorescence staining. The results of double staining of Annexin V/PI showed that the apoptosis rate increased with the increase of Ost concentration. Compared with the control group, after Ost(50, 100, 200 μmol·L~(-1)) treatment for 24 h, the expression levels of Bcl-2, pro-caspase-3 and p-Histon H3(Ser10) decreased, and the expression levels of Bax and cleaved-caspase-3 increased. CONCLUSION Ost has toxic effects on L02 cells, which appeares a time and concentration dependent manner, as a result it will promote apoptosis and inhibit cell proliferation.
OBJECTIVE To investigate the toxic effect of osthole(Ost) on L02 cells and explore its mechanism, which could provide an experimental evidence for the safe and rational application of Fructus cnidii and its preparation. METHODS Different concentrations of Ost were applied to L02 cells, MTT assay was used to detect cell activity; the release rate of lactate dehydrogenase(LDH) was analyzed using LDH kits; Hoechst 33342 staining was used to detect cell nucleus morphology; Annexin V/PI staining was used to detect apoptosis; Western blot was utilized to detect the expression of Bcl-2, Bax, pro-caspase-3, cleaved-caspase-3(p17) and p-Histon H3(Ser10). RESULTS The activity of L02 cells decreased under the action of Ost, while the release rate of LDH increased and appeared to be concentration dependent. The cell nucleus treated by Ost were shrunken and fragmented under Hoechst 33342 fluorescence staining. The results of double staining of Annexin V/PI showed that the apoptosis rate increased with the increase of Ost concentration. Compared with the control group, after Ost(50, 100, 200 μmol·L~(-1)) treatment for 24 h, the expression levels of Bcl-2, pro-caspase-3 and p-Histon H3(Ser10) decreased, and the expression levels of Bax and cleaved-caspase-3 increased. CONCLUSION Ost has toxic effects on L02 cells, which appeares a time and concentration dependent manner, as a result it will promote apoptosis and inhibit cell proliferation.
引文
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[18]FAN X,XI H,ZHANG Z,et al.Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions[J].Acta Histochem,2017,119(3):198-204.
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[20]GARG A D,NOWIS D,GOLAB J,et al.Photodynamic therapy:illuminating the road from cell death towards anti-tumour immunity[J].Apoptosis,2010,15(9):1050-1071.
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[22]LI B,WAN Z,WANG H,et al.The role of histone H3phosphorylation at Ser10 in regulating proliferation and transformation of human nasopharyngeal carcinoma cells[J].Tumor(肿瘤),2014,34(10):894-902.
[23]LI B B,WAN Z,WANG H G,et al.The role of histone H3phosphorylation at Ser10 in regulating proliferation and transformation of human nasopharyngeal carcinoma cells[J].Tumor(肿瘤),2014,34(10):894-901.
[24]WANG B,MA W,XU X,et al.Phosphorylation of histone H3on Ser10 by auto-phosphorylated PAK1 is not essential for chromatin condensation and meiotic progression in porcine oocytes[J].J Anim Sci Biotechnol,2013,4(1):13.Doi:10.1186/2049-1891-4-13.
[2]GE J R,ZHENG H X,WAN X M.Expert consensus on the prevention and treatment for primary osteoporosis with traditional Chinese medicine[J].Chin J Osteoporosis(中国骨质疏松杂志),2015,21(9):1023-1028.
[3]张敏建,常德贵,贺占举,等.勃起功能障碍中西医结合诊疗指南(试行版)[J].中华男科学杂志,2016,22(8):751-757.
[4]QIN G,LI Y,PEI X,et al.Expert consensus of impotence treatment based on liver depression blood stasis and deficiency of kidney[J].Liaoning J Tradit Chin Med(辽宁中医杂志),2016,43(8):1622-1625.
[5]LIAO P C,CHIEN S C,HO C L,et al.Osthole regulates inflammatory mediator expression through modulating NF-κB,mitogen-activated protein kinases,protein kinase C,and reactive oxygen species[J].J Agric Food Chem,2010,58(19):10445-10451.
[6]YANG D,GU T,WANG T,et al.Effects of osthole on migration and invasion in breast cancer cells[J].Biosci Biotechnol Biochem,2010,74(7):1430-1434.
[7]ZHAO Y J,TANG D Z,CHENG S D,et al.Comparison study of the effect of different doses of osthole on OPG knockout mice and OVX rats[J].Chin J Osteoporosis(中国骨质疏松杂志),2015,21(2):147-151.
[8]ZHANG Z R,LEUNG W N,CHEUNG H Y,et al.Osthole:a review on its bioactivities,pharmacological properties,and potential as alternative medicine[J].Evid Based Complement Alternat Med,2015:919616.Doi:10.1155/2015/919616.
[9]WANG Q,ZHENG H,ZHOU Y,et al.Effect of osthole on the anti-breast cancer activity of TRAIL and its mechanism[J].Chin J Mod Appl Pharm(中国现代应用药学),2016,33(9):1141-1147.
[10]CHEN Y,WANG J,CHEN S,et al.Screening on toxicity of26 kinds of common orthopedic herbal medicine using Zebrafish model[J].J Nanjing Univ Tradit Chin Med(南京中医药大学学报),2016,32(5):465-469.
[11]华桦,赵军宁,邓治文.中药蛇床子的化学、药理毒理及临床应用研究进展[C].2010年全国中药学术研讨会论文集,2010.
[12]王亮,江涛,冯毅凡,等.代谢组学评价蛇床子提取物对大鼠毒理作用的初步研究[J].亚太传统医药,2010,6(10):20-22.
[13]华桦,赵军宁,鄢良春,等.蛇床子毒性效应谱及剂量-反应关系研究[J].中药药理与临床,2012,28(5):134-137.
[14]LI W N,XIAO G,LU D,et al.LD50 determination of osthole in mice[J].J Mod Med Health(现代医药卫生),2013,29(10):1444-1445.
[15]杨兴国,刘晓龙.蛇床子素对小鼠急性毒性的研究[J].中国畜牧兽医文摘,2013(8):196-198.
[16]WANG Q Q,ZHANG W L,YUAN X D,et al.Effect of mitochondria in apoptosis[J].Med Innov China(中国医学创新),2015(6):143-146.
[17]LIN C H,WU M R,LI C H,et al.Editor’s highlight:Periodic exposure to smartphone-mimic low-luminance blue light induces retina damage through Bcl-2/Bax-dependent apoptosis[J].Toxicol Sci,2017,157(1):196-210.
[18]FAN X,XI H,ZHANG Z,et al.Germ cell apoptosis and expression of Bcl-2 and Bax in porcine testis under normal and heat stress conditions[J].Acta Histochem,2017,119(3):198-204.
[19]LIU Z,DING Y,YE N,et al.Direct activation of Bax protein for cancer therapy[J].Med Res Rev,2015,36(2):313-341.
[20]GARG A D,NOWIS D,GOLAB J,et al.Photodynamic therapy:illuminating the road from cell death towards anti-tumour immunity[J].Apoptosis,2010,15(9):1050-1071.
[21]ZHONG M,YANG X F,DENG S P.Research progress in intrinsic and extrinsic mechanism of cell apoptosis[J].Pract J Clin Med(实用医院临床杂志),2014,11(2):170-174.
[22]LI B,WAN Z,WANG H,et al.The role of histone H3phosphorylation at Ser10 in regulating proliferation and transformation of human nasopharyngeal carcinoma cells[J].Tumor(肿瘤),2014,34(10):894-902.
[23]LI B B,WAN Z,WANG H G,et al.The role of histone H3phosphorylation at Ser10 in regulating proliferation and transformation of human nasopharyngeal carcinoma cells[J].Tumor(肿瘤),2014,34(10):894-901.
[24]WANG B,MA W,XU X,et al.Phosphorylation of histone H3on Ser10 by auto-phosphorylated PAK1 is not essential for chromatin condensation and meiotic progression in porcine oocytes[J].J Anim Sci Biotechnol,2013,4(1):13.Doi:10.1186/2049-1891-4-13.