王菊;李龙;曾家顺;
WANG Ju;LI Long;ZENG Jiashun;Department of Rheumatology Immunology,the Affiliated Hospital of Guizhou Medical University;

• 1:贵州医科大学附属医院风湿免疫科

摘要(Abstract)：

目的:观察桔梗皂苷D(PD)对人类风湿关节炎(RA)成纤维样滑膜细胞MH7A增殖和凋亡的影响及机制。方法:体外培养MH7A细胞,用不同浓度(5、10、15、20、25及30 mg/L) PD分别作用于MH7A细胞24 h,同时设置不含PDD的空白对照组,采用CCK-8实验检测PD对MH7A细胞增殖的影响;取有效增殖抑制的最低药物浓度作用于MH7A细胞,用Annexin V-FITC/PI双染色法流式细胞术检测PD对MH7A细胞凋亡的影响、PI单染法流式细胞术检测PD对MH7A细胞周期分布的影响、Western blotting法检测PD对MH7A细胞中Bax、Bcl-2和Caspase-9蛋白表达的影响。结果:与空白对照组比较,PD在5 mg/L浓度以上时,随着药物浓度的增加,MH7A细胞增殖率均降低、细胞凋亡率增加、G0/G1期细胞比例增加,差异有统计学意义(P <0.05);与空白对照组比较,PD药物各浓度组Caspase-9蛋白和Bax蛋白相对表达量升高,以10及15 mg/L组升高显著,差异有统计学意义(P <0.05或P <0.01); PD药物各浓度组细胞内Bcl-2蛋白相对表达量均显著下降,差异有统计学意义(P <0.05或P <0.01)。结论:PD能抑制MH7A细胞增殖和诱导其凋亡,推测其作用机制可能是将MH7A细胞阻滞在G0/G1期和调控线粒体凋亡途径相关蛋白的表达。
Objective: To observe the effect of platycodin D( PD) on proliferation and apoptosis of human rheumatoid arthritis( RA) fibroblast-like synovial MH7 A cells,and to explore its possible mechanism. Methods: Human MH7 A cells were cultured in vitro using different concentrations of PD( 5,10,15,20,25,and 30 mg/L) to function MH7 A cells for 24 h and set up the blank control group without PD at the same time,and then the effect on the proliferation of cells by CCK-8 experiment was detected. The MH7 A cells were functioned with PD with the best inhibitory effect on proliferation and without obvious cytotoxicity,and then the effect on cell apoptosis after PD action 24 h by Annexin V-FITC/PI double staining flow cytometry, and to detect the effect on cell cycle distribution after PD action 24 h by PI single staining flow cytometry,and to detect the effect on protein expression of Bax,Bcl-2,and Caspase-9 after PD action for 24 h by Western blotting. Results:Compared with the blank control group,the proliferation rate of MH7 A cells significantly decreased except 5 mg/L concentrations( P < 0. 01),and the cell apoptosis rate of MH7 A cells significantly increased( P < 0. 01),and the cell cycle distribution rate in G0/G1 phase of MH7 A cells significantly increased( P < 0. 01),and the protein expression of Bax,Caspase-9 increased,and the protein expression of Bcl-2 decreased. Conclusion: PD can obviously inhibit the proliferation of MH7 A cells and induce its apoptosis,and it is suggested that the possible mechanism of its action is to block MH7 A cells in G0/G1 phase of cell cycle and regulate the expression of related proteins in mitochondrial apoptotic pathways.

关键词(KeyWords)： 关节炎,类风湿;细胞增殖;细胞凋亡;桔梗皂苷D;MH7A细胞
arthritis,rheumatoid;cell proliferation;apoptosis;platycodin D(PD);MH7A cells

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81460254);; 贵州省科技计划项目[黔科合基础(2018)1137]

作者(Authors): 王菊;李龙;曾家顺;
WANG Ju;LI Long;ZENG Jiashun;Department of Rheumatology Immunology,the Affiliated Hospital of Guizhou Medical University;

DOI: 10.19367/j.cnki.2096-8388.2020.10.006

参考文献(References)：

• [1]MCINNES I B,SCHETT G. The pathogenesis of rheumatoid arthritis[J]. N Engl J Med,2011,365(23):2205-2219.
• [2]DANIEL A,TUHINA N,ALAN J S,et al. 2010 rheumatoid arthritis classification criteria:an American college of rheumatology/european league against rheumatism collaborative initiative[J]. Arthritis Rheum,2010,62:2569-2581.
• [3]SMOLEN J S,ALETAHA D,MCINNES I B. Rheumatoid arthritis[J]. Lancet,2016,388(10055):2023-2038.
• [4]THOMAS P,MüLLER L,RENATE E G,et al. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis[J]. Arthritis Research,2000,2(5):361-367.
• [5]NOSS E H,BRENNER M B. The role and therapeutic implications of fibroblast-like synoviocytes in inflammation and cartilage erosion in rheumatoid arthritis[J]. Immunological Reviews,2008,223:252-270.
• [6]邹葭霜，单进军，谢彤，等．桔梗皂苷D的研究进展[J]．中成药，2014,36(4):823-827.
• [7]MUHAMMAD K,AMARA M,HE Z,et al. Killing cancer with platycodin D through multiple mechanisms[J]. Journal of Cellular and Molecular Medicine,2016,20(3):389-402.
• [8]KWON O G,KU S K,AN H D,et al. The effects of Platycodin D,a saponin purified from Platycodi Radix,on collagen-induced DBA/1J mouse rheumatoid arthritis[J]. Evid Based Complement Alternat Med,2014,954508:1-16.
• [9] KEIJI M,AKIO M,HIROKAZU O. Establishment and characterization of a novel human rheumatoid fibroblastlike synoviocyte line,MH7A,immortalized with SV40 T antigen[J]. The Japanese Biochemical Society,1998,124(6):1153-1162.
• [10]刘健，章平衡，黄旦，等．中药及其单体对类风湿关节炎滑膜成纤维细胞增殖和凋亡的研究进展[J]．风湿病与关节炎，2018,7(11):66-70.
• [11]ZHANG Q,JIA L,MENG M Z,et al. Apoptosis induction of fibroblast-like synoviocytes is an important molecularmechanism for herbal medicine along with its active components in treating rheumatoid arthritis[J]. Biomolecules,2019,9(795):1-29.
• [12]BARTOK B,FIRESTEIN G S. Fibroblast-like synoviocytes:key effector cells in rheumatoid arthritis[J]. Immunol Rev,2010,233:233-255.
• [13]CHOI Y H,YOO D S,CHA M R,et al. Antiproliferative effects of saponins from the roots of Platycodon grandiflorum on cultured human tumor cells[J]. J Nat Prod,2010,73:1863-1867.
• [14]MICHAEL V V,ALISA E K. Cell Cycle Implications in the pathogenesis of rheumatoid arthritis[J]. Frontiers in Bioscience,2000,5:594-601.
• [15]PENTHALA N R,BOMMAGANI S. Heck products of parthenolide and melampomagnolide-B as anticancer modulators that modify cell cycle progression[J]. Eur J Med Chem,2014,85,517-525.
• [16]刘巧红，沈凌汛，滕云，等．类风湿关节炎滑膜细胞的增殖及细胞周期的研究[J]．临床内科杂志，2004,21(12):814-816.
• [17]XU C,SUN G,YUAN G,et al. Effects of platycodin D on proliferation,apoptosis and PI3K/Akt signal pathway of human glioma U251 cells[J]. Molecules,2014,19:21411-21423.
• [18]ZHOU R,LU Z,LIU K,et al. Platycodin D induces tumor growth arrest by activating FOXO3a expression in prostate cancer in vitro and in vivo[J]. Curr Cancer Drug Targets,2015,14:860-871.
• [19]HUA Q,XIAO Y D,YAN Z,et al. Platycodin D,a triterpenoid saponin from Platycodon grandiflorum,induces G2/M arrest and apoptosis in human hepatoma Hep G2cells by modulating the PI3K/Akt pathway[J]. Tumor Biol,2014,35:1267-1274.
• [20]STRASSER A,O’CONNOR L,DIXIT V M. Apoptosis signaling[J]. Annu Rev Biochem,2000,69:217-245.
• [21]刘雯，李守新．类风湿关节炎成纤维样滑膜细胞凋亡研究进展[J]．中华风湿病学杂志，2005,9(10):620-622.
• [22]SMITH M D,WEEDON H,PAPANGELIS V,et al. Apoptosis in the rheumatoid arthritis synovial membrane:modulation by disease-modifyinganti-rheumatic drug treatment[J]. Rheumatology(Oxford),2010,49(5):862-875.
• [23]ESTAQUIER J,VALLETTE F,VAYSSIERE J. The mitochondrial pathways of apoptosis[J]. Adv Exp Med Biol,2012,942:157-183.
• [24]CORY S,ADAMS J M. The Bcl-2 family:regulators of the cellular life or death switch[J]. Nat Rev Cancer,2002,2:647-656.
• [25]AOUACHERIA A,BAGHDIGUIAN S,LAMB H M,et al.Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins[J]. Neurochem Int,2017,109:141-161.

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