摘要
目的探讨安石榴苷对白细胞介素(IL)-1β诱导的软骨细胞凋亡的影响及其作用机制。方法采用噻唑蓝(MTT)法检测不同浓度安石榴苷(10、20、40、80、160 μmol/L)及不同组软骨细胞(对照组、IL-1β组、PUN+IL-1β组)活性的影响;流式细胞术检测各组细胞凋亡率;酶联免疫吸附试验(ELISA)法检测半胱氨酸天冬氨酸蛋白酶(Caspase)-3的活性;实时荧光定量聚合酶链反应(qRT-PCR)检测B细胞淋巴瘤/白血病-2相关X蛋白(bax)、B细胞淋巴瘤/白血病-2(bcl-2)、Caspase-3 mRNA表达;蛋白质印迹法(Western blot)检测磷脂酰肌醇3激酶(PI3K)/蛋白激酶B(Akt)信号通路相关蛋白的表达,多组间比较用单因素方差分析,两组间比较用t检验。结果 PUN+IL-1β组细胞活力[(54.40±4.72)%、(66.40±3.29)%、(82.00±4.53)%]高于IL-1β组[(42.20±3.77)%,t=25.759、45.179、40.497,P<0.05),表明PUN促进恢复细胞活力。IL-1β组凋亡率[(23.50±0.82)%]高于对照组[(4.42±0.57)%,t=64.117,P<0.05];PUN+IL-1β组细胞凋亡率[(16.90±0.81)%、(12.80±0.55)%、(7.43±0.62)%]低于IL-1β组(t=46.447、52.338、26.851,P<0.05)。IL-1β组Caspase-3活性[(183.80±7.19)%]高于对照组[(99.60±5.68)%,t=57.159,P<0.05];PUN+IL-1β组Caspase-3活性[(151.20±4.82)%、(130.60±5.55)%、(112.20±5.81)%]低于IL-1β组(t=70.193、52.620、43.218,P<0.05)。PUN+IL-1β组bax、Caspase-3 mRNA表达(2.27±0.08、1.81±0.05、1.40±0.04,2.39±0.10、1.84±0.09、1.39±0.05)低于IL-1β组(2.76±0.08、2.85±0.08),而bcl-2 mRNA表达(1.53±0.59、2.04±0.07、2.67±0.08)却明显增加(t=84.854、67.783、43.130,P<0.05)。PUN +IL-1β组p-PI3K(0.55±0.03、0.67±0.03、0.80±0.04)和p-Akt(0.62±0.04、0.75±0.03、0.85±0.03)蛋白质表达明显高于IL-1β组(0.45±0.04、0.48±0.03,t=48.833、56.706,P<0.05)。结论安石榴苷可通过激活PI3K/Akt通路来抑制IL-1β诱导的软骨细胞凋亡。
Objective To explore the effect of punicalagin on interleukin(IL)-1β-induced the apoptosis of chondrocytes and its related mechanisms.Methods Methyl thiazolyl tetrazolium(MTT)method was used to detect the effect of different concentrations of punicalagin on chondrocyte viability and cell viability in different groups.Flow cytometry was used to detect the apoptosis rate of each group.Enzyme linked immunosorbent assay(ELISA)method was conducted to detect the activity of cystein-aspartate protease(Caspase)-3.The expression of B cell lymphoma/leukemia-2 associated X protein(bax),B cell lymphoma/leukemia-2(bcl-2)and Caspase-3 mRNA was detected by quantitative real-time polymerase chain reaction(qRT-PCR).The protein level of phosphatidylinositol 3 kinase(PI3K)/protein kinase B(Akt)signaling pathway related proteins were detected by Western blotting.One-way analysis of variance was used for comparison between multiple groups,and t test was used for comparison between two groups.Results The cell viability of the PUN+IL-1βgroup[(54.40±4.72)%,(66.40±3.29)%,(82.00±4.53)%]was higher than the IL-1βgroup[(42.20±3.77)%,t=25.759,45.179,40.497,P<0.05],indicating that PUN promotes the restoration of cell viability.The apoptosis rate of IL-1βgroup[(23.50±0.82)%]was higher than the control group[(4.42±0.57)%,t=64.117,P<0.05];the apoptosis rate of PUN+IL-1βgroup[(16.90±0.81)%,(12.80±0.55)%,(7.43±0.62)%]was lower than the IL-1βgroup(t=46.447,52.338,26.851,P<0.05).The activity of Caspase-3 in the IL-1βgroup[(183.80±7.19)%]was higher than the control group(99.60±5.68)%,t=57.159,P<0.05);the activity of Caspase-3 in PUN+IL-1βgroup[(151.20±4.82)%,(130.60±5.55)%,(112.20±5.81)%]was lower than the IL-1βgroup(t=70.193,52.620,43.218,P<0.05).The expression of bax and Caspase-3 mRNA in the PUN+IL-1βgroup(2.27±0.08,1.81±0.05,1.40±0.04,2.39±0.10,1.84±0.09,1.39±0.05)was lower than that in the IL-1βgroup(2.76±0.08,2.85±0.08),while the expression of bcl-2 mRNA(1.53±0.59,2.04±0.07,2.67±0.08)increased significantly(t=
作者
刘飞飞
田青
宋瑞鹏
韩奇财
吴学建
Liu Feifei;Tian Qing;Song Ruipeng;Han Qicai;Wu Xuejian(Department of Orthopedics,the First Affiliated Hospital of Zhengzhou University,Zhengzhou 450052,China)
出处
《中华实验外科杂志》
CAS
北大核心
2021年第11期2104-2107,共4页
Chinese Journal of Experimental Surgery
