巴戟天含药血清对人骨肉瘤MG63细胞凋亡及JNK信号通路的影响
|
摘要
目的观察巴戟天含药血清对人骨肉瘤MG63细胞凋亡的影响,并探讨其潜在机制。方法取4周龄SD大鼠12只,随机分4组,分别给予低、中、高三种浓度巴戟天溶液(0. 54、1. 08、2. 16 g/kg)及蒸馏水灌胃,分离血清。体外培养人骨肉瘤MG63细胞,采用不同浓度的巴戟天含药血清干预,以空白血清为对照。采用MTT法检测巴戟天含药血清干预MG63细胞24、48 h的增殖率,流式细胞术检测凋亡率,Hoechst 33258荧光染色法检测细胞凋亡形态,Western-blotting法检测JNK信号通路相关蛋白JNK、p-JNK、Bad、p-Bad、Cleaved caspase-3的表达情况。结果中、高浓度的巴戟天含药血清能有效抑制MG63细胞增殖(P <0. 01或P <0. 05),但干预48 h对比24 h并无显著差异;同时,高浓度组MG63细胞较对照组凋亡率显著增加(P <0. 01),细胞分布散乱,胞核固缩,细胞透亮,呈现典型的凋亡形态学特征;高浓度的巴戟天含药血清能显著增加MG63细胞JNK总蛋白及磷酸化p-JNK蛋白的表达(P <0. 01或P <0. 05),上调p-Bad和Cleaved caspase-3蛋白表达(P <0. 01),但对Bad总蛋白表达无明显影响。结论巴戟天含药血清能促进人骨肉瘤MG63细胞凋亡,其作用机制与激活JNK信号通路有关。
Objective To investigate the effects of Morinda officinalis sera on apoptosis of human osteosarcoma MG63 cells and to elucidate its underlying mechanism. Methods 12 SD rats aged 4 weeks were randomly divided into 4 groups. The rats were given low,medium and high concentrations of Morinda officinalis(0. 54,1. 08,2. 16 g/kg) and distilled water to separate the serum. Human osteosarcoma MG63 cells were cultured in vitro,and different concentrations of Morinda officinalis medicated serum were used to intervene,and blank serum was used as a control. MTT assay was used to detect the proliferation rate of MG63 cells in MG63 cells at 24 and 48 h. The apoptosis rate was detected by flow cytometry and apoptosis morphology was detected by Hoechst 33258 fluorescent staining method. The JNK signaling pathway was detected by Western-blotting method. Expression of related proteins JNK,p-JNK,Bad,p-Bad,and Cleaved caspase-3 were detected. Results Medium and high concentrations of Morinda officinalis sera could effectively inhibit the proliferation of MG63 cells( P < 0. 01 or P <0. 05),but there was no significant difference between 48 h and 24 h after intervention. Meanwhile,MG63 cells in the high concentration group were compared with the control group. The mortality rate increased significantly(P < 0. 01),the cell distribution was scattered,the nucleus was pyknosis,and the cells were translucent,showing typical apoptotic morphological features. Western-blotting results showed that high concentration of Morinda officinalis sera could significantly increase the expression of JNK total protein and phosphorylated p-JNK protein in MG63 cells( P < 0. 01 or P < 0. 05),and up-regulate p-Bad and Cleaved caspase-3 protein expression( P <0. 01),but no significant effect on total protein expression of Bad. Conclusion Morinda sinensis-containing serum can promote apoptosis of human osteosarcoma MG63 cells,and its mechanism is related to activation of JNK signaling pathway.
Objective To investigate the effects of Morinda officinalis sera on apoptosis of human osteosarcoma MG63 cells and to elucidate its underlying mechanism. Methods 12 SD rats aged 4 weeks were randomly divided into 4 groups. The rats were given low,medium and high concentrations of Morinda officinalis(0. 54,1. 08,2. 16 g/kg) and distilled water to separate the serum. Human osteosarcoma MG63 cells were cultured in vitro,and different concentrations of Morinda officinalis medicated serum were used to intervene,and blank serum was used as a control. MTT assay was used to detect the proliferation rate of MG63 cells in MG63 cells at 24 and 48 h. The apoptosis rate was detected by flow cytometry and apoptosis morphology was detected by Hoechst 33258 fluorescent staining method. The JNK signaling pathway was detected by Western-blotting method. Expression of related proteins JNK,p-JNK,Bad,p-Bad,and Cleaved caspase-3 were detected. Results Medium and high concentrations of Morinda officinalis sera could effectively inhibit the proliferation of MG63 cells( P < 0. 01 or P <0. 05),but there was no significant difference between 48 h and 24 h after intervention. Meanwhile,MG63 cells in the high concentration group were compared with the control group. The mortality rate increased significantly(P < 0. 01),the cell distribution was scattered,the nucleus was pyknosis,and the cells were translucent,showing typical apoptotic morphological features. Western-blotting results showed that high concentration of Morinda officinalis sera could significantly increase the expression of JNK total protein and phosphorylated p-JNK protein in MG63 cells( P < 0. 01 or P < 0. 05),and up-regulate p-Bad and Cleaved caspase-3 protein expression( P <0. 01),but no significant effect on total protein expression of Bad. Conclusion Morinda sinensis-containing serum can promote apoptosis of human osteosarcoma MG63 cells,and its mechanism is related to activation of JNK signaling pathway.
引文
[1]Li Y,Zhou Q,Hu Z,et al. 5-aminolevulinic acid-based sonodynamic therapy induces the apoptosis of osteosarcoma in mice[J]. Plos One,2015,10(7):e0132074.
[2]Zhou Z,Li Y,Yan X,et al. Does rarity mean imparity? Biological characteristics of osteosarcoma cells originating from the spine[J]. J Cancer Res Clin Oncol,2017,143(10):1959-1969.
[3]黄慧,陈健,何剑全,等.巴戟天含药血清对成骨-破骨细胞共育体系OPGmRNA、RANKLmRNA表达的影响[J].中国骨质疏松杂志,2015,21(1):67-71.
[4]傅应亚,黎友伦. JNK信号通路与细胞凋亡[J].重庆医科大学学报,2014,39(3):281-283.
[5]杨全会,许荣焜.细胞凋亡与肿瘤[J].生理科学进展,2006,37(4):373-377.
[6]李金贵,刘川,万华,等.白藜芦醇诱导前列腺癌细胞凋亡的实验研究[J].遵义医学院学报,2018,41(3):272-276.
[7]Barichievy S,Naidoo J,BoulléM,et al. Viral apoptosis evasion via the MAPK pathway by use of a host long noncoding RNA[J]. Front Cell Infect Microbiol,2018,8(3):263-269.
[8] Bommareddy A,Crisamore K,Fillman S,et al. Survivin down-regulation byα-Santalol is not mediated through PI3K-AKT pathway in human breast cancer cells[J].Anticancer Res,2015,35(10):5353-5357.
[9]Qin W,Liu P,Zhang R,et al. JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells[J]. Connect Tissue Res,2014,55(3):217-224.
[10]王晓天,刘晓梅,尤红娟,等. JNK磷酸化Bad在脑缺血神经元凋亡中的作用[J].中国现代医生,2014,52(31):1-3.
[11]Naveen Kumar S K,Hemshekhar M,Sundaram M S. Cell-free methemoglobin drives platelets to apoptosis via mitochondrial ROS-mediated activation of JNK and p38MAP kinase[J]. Biochem Biophys Res Commun,2017,491(1):183-191.
[12]Wang X T,Pei D S,Xu J,et al. Opposing effects of Bad phosphorylation at two distinct sites by Akt1 and JNK1/2on ischemic brain injury[J]. Cell Signal,2007,19(9):1844-1856.
[2]Zhou Z,Li Y,Yan X,et al. Does rarity mean imparity? Biological characteristics of osteosarcoma cells originating from the spine[J]. J Cancer Res Clin Oncol,2017,143(10):1959-1969.
[3]黄慧,陈健,何剑全,等.巴戟天含药血清对成骨-破骨细胞共育体系OPGmRNA、RANKLmRNA表达的影响[J].中国骨质疏松杂志,2015,21(1):67-71.
[4]傅应亚,黎友伦. JNK信号通路与细胞凋亡[J].重庆医科大学学报,2014,39(3):281-283.
[5]杨全会,许荣焜.细胞凋亡与肿瘤[J].生理科学进展,2006,37(4):373-377.
[6]李金贵,刘川,万华,等.白藜芦醇诱导前列腺癌细胞凋亡的实验研究[J].遵义医学院学报,2018,41(3):272-276.
[7]Barichievy S,Naidoo J,BoulléM,et al. Viral apoptosis evasion via the MAPK pathway by use of a host long noncoding RNA[J]. Front Cell Infect Microbiol,2018,8(3):263-269.
[8] Bommareddy A,Crisamore K,Fillman S,et al. Survivin down-regulation byα-Santalol is not mediated through PI3K-AKT pathway in human breast cancer cells[J].Anticancer Res,2015,35(10):5353-5357.
[9]Qin W,Liu P,Zhang R,et al. JNK MAPK is involved in BMP-2-induced odontoblastic differentiation of human dental pulp cells[J]. Connect Tissue Res,2014,55(3):217-224.
[10]王晓天,刘晓梅,尤红娟,等. JNK磷酸化Bad在脑缺血神经元凋亡中的作用[J].中国现代医生,2014,52(31):1-3.
[11]Naveen Kumar S K,Hemshekhar M,Sundaram M S. Cell-free methemoglobin drives platelets to apoptosis via mitochondrial ROS-mediated activation of JNK and p38MAP kinase[J]. Biochem Biophys Res Commun,2017,491(1):183-191.
[12]Wang X T,Pei D S,Xu J,et al. Opposing effects of Bad phosphorylation at two distinct sites by Akt1 and JNK1/2on ischemic brain injury[J]. Cell Signal,2007,19(9):1844-1856.