黄芪甲苷通过诱导M1型巨噬细胞极化发挥抗肿瘤作用的机制
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摘要
目的:探讨黄芪甲苷对巨噬细胞极化的影响及其可能的抗肿瘤免疫机制。方法:通过噻唑兰(methylthiazolyldiphenyl-tetrazolium bromide,MTT)比色法检测不同浓度黄芪甲苷作用不同时间,对巨噬细胞的细胞毒作用,以选取合适的黄芪甲苷作用浓度;将巨噬细胞与肿瘤细胞以1∶1进行共孵育,并用0. 1 mg·L~(-1)黄芪甲苷作用24 h,通过虫荧光素酶(luciferase,Luc)杀伤实验检测巨噬细胞对肿瘤细胞的杀伤效率;以0. 1 mg·L~(-1)黄芪甲苷作用巨噬细胞24 h,通过流式细胞术检测巨噬细胞CD16/32,CD206表达,通过实时荧光定量聚合酶链式反应(Real-time PCR)检测巨噬细胞诱导型一氧化氮合酶(inducible nitric oxide synthase,i NOS),精氨酸-1(Arginine-1,Arg-1)及细胞因子白细胞介素-1β(interleukin-1β,IL-1β),肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α),白细胞介素-12(interleukin-12,IL-12),白细胞介素-10(interleukin-10,IL-10),转化生长因子-β(transforming growth factor-β,TGF-β) mRNA表达,通过蛋白免疫印迹法(Western blot)检测巨噬细胞信号传导及转录激活因子1(Signal transducers and activators of transcription 1,STAT1),磷酸化信号传导及转录激活因子1(phosphorylation signal transducers and activators of transcription 1,p-STAT1)表达。结果:当黄芪甲苷质量浓度为0. 1 mg·L~(-1)时,对巨噬细胞无细胞毒作用。与空白组比较,Luc杀伤实验结果显示黄芪甲苷可以显著促进巨噬细胞介导的肿瘤杀伤; Real-time PCR结果显示黄芪甲苷可以诱导巨噬细胞表面M1型巨噬细胞标志CD16/32表达增高,促进M1型巨噬细胞特异性指标i NOS mRNA表达,同时诱导M1型巨噬细胞相关细胞因子IL-1β,TNF-α,IL-12 mRNA表达增高; Western blot结果显示黄芪甲苷可以促进巨噬细胞STAT1发生磷酸化。结论:黄芪甲苷可以通过促进巨噬细胞内STAT1发生磷酸化,进而诱导巨噬细胞向M1型巨噬细胞发生极化,并启动巨噬细胞相关的抗肿瘤免疫应答。
Objective: To investigate the effect of astragaloside on the macrophage polarization and the possible anti-tumor immunity mechanism of astragaloside. Method: The cytotoxic effect of different concentrations of astragaloside at different time points on macrophage was measured by methylthiazolyldiphenyl-tetrazolium bromide( MTT),in order to choose the suitable concentration of astragaloside,macrophages were co-cultured with tumor cells at the ratio 1 ∶ 1,and the effect of astragaloside on macrophage-mediated lysis of tumor cells was performed by biophotonic cytotoxicity assay after the mixed cells were effected with 0. 1 mg·L~(-1) astragaloside for 24 h.Macrophages were dealt with 0. 1 mg·L~(-1) astragaloside for 24 h,the expressions of CD16/32 and CD206 in macrophages were performed by flow cytometry,the mRNA expressions of macrophage inducible nitric oxide synthase( i NOS),Arginine-1( Arg-1),interleukin-1β( IL-1β),tumor necrosis factor-α( TNF-α),interleukin-12( IL-12),interleukin-10( IL-10) and transforming growth factor-β( TGF-β) were measured by Real-time PCR,the protein expressions of macrophage signal transducers and activators of transcription 1( STAT1) and phosphorylation signal transducers and activators of transcription 1( p-STAT1) were determined by Western blot.Result: Astragaloside had no effect on the viability of macrophages with 0. 1 mg·L~(-1). Compared with control group,astragaloside obviously enhanced the macrophage-mediated lysis of tumor cells according to the biophotonic cytotoxicity assay,induced the M1 macrophage marker CD16/32 expression according to flow cytometry,increased the mRNA expressions of i NOS,IL-1β,TNF-α and IL-12 according to the Real-time PCR,and promoted the phosphorylation of STAT1 in macrophages on the basis of Western blot. Conclusion: Astragaloside could induce M1 macrophage polarization by increasing the phosphorylation of STAT1,and initiate macrophage-related anti-tumor immunity response.
Objective: To investigate the effect of astragaloside on the macrophage polarization and the possible anti-tumor immunity mechanism of astragaloside. Method: The cytotoxic effect of different concentrations of astragaloside at different time points on macrophage was measured by methylthiazolyldiphenyl-tetrazolium bromide( MTT),in order to choose the suitable concentration of astragaloside,macrophages were co-cultured with tumor cells at the ratio 1 ∶ 1,and the effect of astragaloside on macrophage-mediated lysis of tumor cells was performed by biophotonic cytotoxicity assay after the mixed cells were effected with 0. 1 mg·L~(-1) astragaloside for 24 h.Macrophages were dealt with 0. 1 mg·L~(-1) astragaloside for 24 h,the expressions of CD16/32 and CD206 in macrophages were performed by flow cytometry,the mRNA expressions of macrophage inducible nitric oxide synthase( i NOS),Arginine-1( Arg-1),interleukin-1β( IL-1β),tumor necrosis factor-α( TNF-α),interleukin-12( IL-12),interleukin-10( IL-10) and transforming growth factor-β( TGF-β) were measured by Real-time PCR,the protein expressions of macrophage signal transducers and activators of transcription 1( STAT1) and phosphorylation signal transducers and activators of transcription 1( p-STAT1) were determined by Western blot.Result: Astragaloside had no effect on the viability of macrophages with 0. 1 mg·L~(-1). Compared with control group,astragaloside obviously enhanced the macrophage-mediated lysis of tumor cells according to the biophotonic cytotoxicity assay,induced the M1 macrophage marker CD16/32 expression according to flow cytometry,increased the mRNA expressions of i NOS,IL-1β,TNF-α and IL-12 according to the Real-time PCR,and promoted the phosphorylation of STAT1 in macrophages on the basis of Western blot. Conclusion: Astragaloside could induce M1 macrophage polarization by increasing the phosphorylation of STAT1,and initiate macrophage-related anti-tumor immunity response.
引文
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[2] Siegel R,Naishadham D,Jemal A. Cancer statistics,2013[J]. CA Cancer J Clin,2013,63(1):11-30.
[3] Albini A, Magnani E, Noonan D M. The tumor microenvironment:biology of a complex cellular and tissue society[J]. Q J Nucl Med Mol Imaging,2010,54(3):244-248.
[4] Schreiber R D, Old L J, Smyth M J. Cancer immunoediting:integrating immunity's roles in cancer suppression and promotion[J]. Science,2011,331(6024):1565-1570.
[5] Kerkar S P, Restifo N P. Cellular constituents of immune escape within the tumor microenvironment[J].Cancer Res,2012,72(13):3125-3130.
[6] YUAN R, LI S, GENG H, et al. Reversing the polarization of tumor-associated macrophages inhibits tumor matastasis[J]. Int Immunopharmacol,2017,49:30-37.
[7] LUO Y,WU J,ZHU X,et al. NK cell-dependent growth inhibition of Lewis lung cancer by Yu-Ping-Feng,an ancient Chinese herbal formula[J]. Med Inflamm,2016,doi:10. 1155/2016/3541283.
[8]陈卓,于卫江.注射用黄芪多糖对非小细胞肺癌放疗的作用及对机体免疫的影响[J].中国实验方剂学杂志,2013,19(6):309-313.
[9]刘苓霜.刘嘉湘辨治肺癌经验[J].中医文献杂志,2006,24(2):38-40.
[10]刘嘉湘,陈树森,郁仁存,等.肺癌证治[J].中医杂志,1986,27(3):4-7.
[11]桑国优,韦世秀,刘成军.黄芪抗肿瘤作用机制和临床应用研究进展[J].时珍国医国药,2008,19(12):3032-3034.
[12]杨苏钰,唐德才,曹子丰,等.黄芪甲苷配伍姜黄素对人卵巢癌HO-8910原位移植瘤转移的抑瘤作用[J].中国实验方剂学杂志,2017,23(6):155-160.
[13] Sica A, Mantovani A. Macrophage plasticity and polarization:in vivo veritas[J]. J Clin Invest,2012,122(3):787-795.
[14] Mosser D M,Edwards J P. Exploring the full spectrum of macrophage activation[J]. Nat Rev Immunol,2008,8(12):958-969.
[15] Lewis C E,Pollard J W. Distinct role of macrophages in different tumor microenvironments[J]. Cancer Res,2006,66(2):605-612.
[16] Mantovani A,Sozzani S,Locati M,et al. Macrophage polarization:tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes[J].Trends Immunol,2002,23(11):549-555.
[17] Goerdt S,Orfanos C E. Other functions,other genes:alternative activation of antigen-presenting cells[J].Immunity,1999,10(2):137-142.
[18] Mantovani A,Allavena P. The interaction of anticancer therapies with tumor-associated macrophage[J]. J Exp Med,2015,212(4):435-445.
[19] Mantovani A,Marchesi F,Malesci A,et al. Tumorassociated macrophages as treatment targets in oncology[J]. Nat Rev Clin Oncol,2017,14(7):399-416.
[20] Biswas S K, Allavena P, Mantovani A. Tumorassociated macrophage:functional diversity, clinical significance, and open questions[J]. Semin Immunopathol,2013,35(5):585-600.
[21] Hagemann T,Lawrence T,McNeish I,et al. Reeducating tumor-associated macrophages by targeting NF-kappaB[J]. J Exp Med, 2008, 205(6):1261-1268.
[22] KE S,Ziemiecki A,Wilks A F,et al. Polypeptide signaling to the nucleus through tyrosine phosphorylation of Jak and Stat proteins[J]. Nature, 1993, 366(6455):580-583.
[23] Darnell J E J, Kerr I M, Stark G R. Jak-STAT pathways and transcriptional activation in response to IFNs and other extracellular signaling proteins[J].Science,1994,264(5164):1415-1421.