芦荟苦素对非小细胞肺癌A549细胞增殖及凋亡的影响
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摘要
目的:研究芦荟苦素诱导人非小细胞肺癌(non-small cell lung cancer,NSCLC) A549细胞发生凋亡,从而抑制其增殖的作用机制。方法:取对数生长期的A549细胞,用细胞计数试剂(cell counting kit,CCK-8)检测考察不同浓度的芦荟苦素(0,2,4,8,16,32,64,128μmol·L~(-1))对A549细胞增殖的影响,5μmol·L~(-1)的顺铂作为阳性对照。用结晶紫染色测定芦荟苦素(0,16μmol·L~(-1))对A549细胞克隆形成的数目以及克隆形成的大小的作用;磷脂酰丝氨酸结合蛋白V/碘化丙啶(annexin V/propidium iodide,PI)凋亡试剂盒染色检测芦荟苦素对A549细胞凋亡的影响;赫斯特(Hoechst)染色检测细胞凋亡核固缩现象;蛋白免疫印迹法(Western blot)检测芦荟苦素对A549细胞中的B细胞淋巴瘤-特大型蛋白(B-cell lymphoma-extra large,Bclxl),B细胞淋巴瘤-特大型/B细胞淋巴瘤-2联合死亡促体(Bcl-xl/Bcl-2 associated death promoter,Bad),裂解半胱天冬酶-3[cleaved-Caspase3,cl-Caspase-3(Asp175)],半胱天冬酶-3(Caspase-3),裂解核糖聚合酶(cleaved-poly ADP-ribose polymerase,clPARP),核糖聚合酶(poly ADP-ribose polymerase,PARP)凋亡相关蛋白表达的影响。体内实验将5周龄裸鼠皮下接种2×106个A549细胞,并随机分为用药组和空白组,连续4周每天分别进行芦荟苦素或生理盐水腹腔注射,每周测量裸鼠肿瘤体积和小鼠体质量,并观察裸鼠的外观表现(精神、毛发、食欲、睡眠等)。结果:芦荟苦素呈浓度依赖性抑制A549细胞增殖及克隆形成的数量和大小(P <0. 05)。经芦荟苦素处理后,A549细胞凋亡的数量以及细胞发生核固缩的现象明显增加(P <0. 01),同时,芦荟苦素还明显下调了凋亡相关蛋白Bcl-xl的表达(P <0. 05),增加Bad蛋白表达(P <0. 01),同时还明显升高了cl-PARP(P <0. 01)和cl-Caspase-3(P <0. 05)表达水平。此外,在体内,芦荟苦素可明显缩小小鼠皮下肿瘤的体积,减轻肿瘤质量,并且小鼠外观表现优于空白组。结论:芦荟苦素可能通过诱导A549细胞凋亡从而达到抑制NSCLC的作用,并且用药安全。
Objective: To study the mechanism of aloesin in inducing apoptosis in human non-small cell lung cancer( NSCLC) A549 cells,so as to inhibit its proliferation. Method: A549 cells in logarithmic growth phase were collected,and cell counting kit-8( CCK-8) was used to detect the effect of different concentrations of aloesin( 2,4,8,16,32,64,128 μmol·L~(-1)) on the proliferation of A549. Effect of aloesin( 0,16 μmol·L~(-1))on the number of clones formed in A549 cells and the size of clone formation was determined by crystal violet staining. effect of aloesin on apoptosis of A549 cells was detected by annexin V/propidium iodide( PI) apoptosis kit staining. Hoechst staining was used to detect the phenomenon of apoptotic nuclear pyknosis. Western blot was used to detect aloesin's effect on death-related protein expressions of Bcl-xl/Bcl-2 associated death promoter( Bad), cleaved-Caspase-3, cl-Caspase-3( Asp175), Caspase-3, cleaved poly ADP-ribose polymerase( cl-PARP),poly ADP-ribose polymerase( PARP) in A549 cells. In vivo,5-week-old nude mice were subcutaneously inoculated with 2 × 106 A549 cells,and randomly divided into the medication group and the blank group. aloesin or normal saline was intraperitoneally injected for 4 weeks,and the tumor volume of nude mice was measured weekly. The body weight of the mice was observed,and the appearance of the nude mice was observed.Result: Aloesin inhibited the proliferation and cloning of A549 cells in a concentration-dependent manner( P <0. 05). After treatment with aloesin,the number of apoptosis and the phenomenon of nuclear pyknosis in A549 cells increased significantly( P < 0. 01). At the same time,aloesin significantly down-regulated the expression of apoptosis-related protein Bcl-xl( P < 0. 05),and increased the expression of Bad protein( P < 0. 01). The expression levels of cl-PARP( P < 0. 01) and cl-Caspase-3( P < 0. 05) were also significantly increased. In addition,in vivo,aloesin significantly shrank the volume of subcutaneous tumors in mice,reduced tumor weight,with a better appearance than that of the control group. Conclusion: Aloesin may inhibit the expression of NSCLC by inducing apoptosis of A549 cells,and is safe to use,with no inhibitory effect on the body weight of mice.
Objective: To study the mechanism of aloesin in inducing apoptosis in human non-small cell lung cancer( NSCLC) A549 cells,so as to inhibit its proliferation. Method: A549 cells in logarithmic growth phase were collected,and cell counting kit-8( CCK-8) was used to detect the effect of different concentrations of aloesin( 2,4,8,16,32,64,128 μmol·L~(-1)) on the proliferation of A549. Effect of aloesin( 0,16 μmol·L~(-1))on the number of clones formed in A549 cells and the size of clone formation was determined by crystal violet staining. effect of aloesin on apoptosis of A549 cells was detected by annexin V/propidium iodide( PI) apoptosis kit staining. Hoechst staining was used to detect the phenomenon of apoptotic nuclear pyknosis. Western blot was used to detect aloesin's effect on death-related protein expressions of Bcl-xl/Bcl-2 associated death promoter( Bad), cleaved-Caspase-3, cl-Caspase-3( Asp175), Caspase-3, cleaved poly ADP-ribose polymerase( cl-PARP),poly ADP-ribose polymerase( PARP) in A549 cells. In vivo,5-week-old nude mice were subcutaneously inoculated with 2 × 106 A549 cells,and randomly divided into the medication group and the blank group. aloesin or normal saline was intraperitoneally injected for 4 weeks,and the tumor volume of nude mice was measured weekly. The body weight of the mice was observed,and the appearance of the nude mice was observed.Result: Aloesin inhibited the proliferation and cloning of A549 cells in a concentration-dependent manner( P <0. 05). After treatment with aloesin,the number of apoptosis and the phenomenon of nuclear pyknosis in A549 cells increased significantly( P < 0. 01). At the same time,aloesin significantly down-regulated the expression of apoptosis-related protein Bcl-xl( P < 0. 05),and increased the expression of Bad protein( P < 0. 01). The expression levels of cl-PARP( P < 0. 01) and cl-Caspase-3( P < 0. 05) were also significantly increased. In addition,in vivo,aloesin significantly shrank the volume of subcutaneous tumors in mice,reduced tumor weight,with a better appearance than that of the control group. Conclusion: Aloesin may inhibit the expression of NSCLC by inducing apoptosis of A549 cells,and is safe to use,with no inhibitory effect on the body weight of mice.
引文
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[23] Wnk A,Andrzejewska E,Kobos J,et al. Molecular and immunohistochemical expression of apoptotic proteins Bax, Bcl-2 and Caspase-3 in infantile hemangioma tissues as an effect of propranolol treatment[J]. Immunol Lett, 2017, doi:10. 1016/j.imlet. 2017. 03. 005.
[24] YOU F L,LI Q,JIN G F,et al. Genistein protects against Aβ25-35 induced apoptosis of PC12 cells through JNK signaling and modulation of Bcl-2 family messengers[J]. BMC Neurosci,2017,doi:10. 1186/s12868-016-0329-9.
[2] Bray F, Ferlay J, Soerjomataram I. Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA Cancer J Clin,2018,doi:10. 3322/caac. 21492.
[3] Sean B K,Phil A C,Haval B,et al. Progress and prospects of early detection in lung cancer[J]. Open Biol,2017,doi:10. 1098/rsob. 170070.
[4]麦苏旦·艾山,再依奴热木·阿扎提.芦荟的认识与利用[J].世界最新医学信息文摘,2016,16(46):190-191.
[5]王晓辉,王伊林,靳小石.芦荟大黄素对肝癌Hep G2细胞生长、迁移及纤维状肌动蛋白的影响[J].中国实验方剂学杂志,2018,24(11):111-116.
[6]黎雅静,洪雪.芦荟大黄素对人肝癌Hep G2细胞端粒酶活性的影响及机制探讨[J].山东医药,2018,58(34):29-32.
[7]宋吉宁,董锦忠,陈文礼.芦荟大黄素对胃癌SGC-7901细胞株钙联蛋白/钙网蛋白循环及内质网应激凋亡的影响[J].中国临床研究,2018,31(6):725-729.
[8]兰海龙,王正辉,杨壮群,等.芦荟苦素对黑素细胞与角质形成细胞混合培养模型中黑素合成的影响[J].中国美容医学,2007,16(3):310-313.
[9] Hollinger J C,Angra K, Halder R M. Are natural ingredients effective in the management of hyperpigmentation? A systematic review[J]. J Clin Aesthet Dermatol,2018,11(2):28-37.
[10]杨月红.芦荟苦素的提取与分离纯化工艺研究[D].上海:华东理工大学,2012.
[11] Wahedi H M,Jeong M,Chae J K,et al. Aloesin from Aloe vera accelerates skin wound healing by modulating MAPK/Rho and Smad signaling pathways in vitro and in vivo[J]. Phytomedicine,2017,28(15):19-26.
[12] ZHANG L Q, LV R W, QU X D,et al. Aloesin suppresses cell growth and metastasis in ovarian cancer SKOV3 cells through the inhibition of the MAPK signaling pathway[J]. Anal Cell Pathol:Amst,2017,doi:10. 1155/2017/8158254.
[13]王立强,苗立成,吴迪,等.芦荟苦素对荷瘤小鼠的抑瘤率及对白血病小鼠生存期的影响[J].中国医院药学杂志,2003(2):15-17.
[14] Rotow J,Bivona T G. Understanding and targeting resistance mechanisms in NSCLC[J]. Nat Rev Cancer,2017,17(11):637-658.
[15] Rolfo C, Caglevic C, Santarpia M, et al.Immunotherapy in NSCLC:a promising and revolutionary weapon[J]. Adv Exp Med Biol,2017,doi:10. 1007/978-3-319-53156-4_5.
[16] LIN W F,LU J Y,CHENG B B,et al. Progress in research on the effects of traditional Chinese medicine on the tumor microenvironment[J]. J Integr Med,2017,15(4):282-287.
[17]朱元章,张贵彪,朱国福.中药复方抗肿瘤机制研究进展[J].中国实验方剂学杂志,2017,23(16):227-234.
[18] HOU L,ZHANG Y,YU B L,et al. Oocyte-G1promotes male germ cell apoptosis through activation of Caspase-3[J]. Gene,2018,doi:10. 1016/j. gene. 2018. 05. 099.
[19] Larsen B D, Srensen C S. The Caspase-activated DNase:apoptosis and beyond[J]. FEBS J,2017,284(8):1160-1170.
[20] Sergeeva T F,Shirmanova M V,Zlobovskaya O A.Relationship between intracellular p H, metabolic cofactors and Caspase-3 activation in cancer cells during apoptosis[J]. Biochim Biophys Acta Mol Cell Res,2017,1864(3):604-611.
[21] Paulsson J F,Schultz,S W,Khler M,et al. Real-time monitoring of apoptosis by Caspase-3-like protease induced FRET reduction triggered by amyloid aggregation[J]. Exp Diabetes Res, 2008, doi:10. 1155/2008/865850.
[22] Schenk R L,Strasser A,Dewson G. Bcl-2:long and winding path from discovery to therapeutic target[J].Biochem Biophys Res Commun,2017,482(3):459-469.
[23] Wnk A,Andrzejewska E,Kobos J,et al. Molecular and immunohistochemical expression of apoptotic proteins Bax, Bcl-2 and Caspase-3 in infantile hemangioma tissues as an effect of propranolol treatment[J]. Immunol Lett, 2017, doi:10. 1016/j.imlet. 2017. 03. 005.
[24] YOU F L,LI Q,JIN G F,et al. Genistein protects against Aβ25-35 induced apoptosis of PC12 cells through JNK signaling and modulation of Bcl-2 family messengers[J]. BMC Neurosci,2017,doi:10. 1186/s12868-016-0329-9.