Cao J, Lyu J Y, Zhang M S, Shi R Z, Feng Q A
Department of Intensive Care Unit, First Hospital of Shanxi Medical University, Taiyuan 030001, China.
Zhonghua Xin Xue Guan Bing Za Zhi. 2018 May 24;46(5):382-389. doi: 10.3760/cma.j.issn.0253-3758.2018.05.011.
To investigate the effects of hesperetin on fine particulate matter (PM(2.5)) induced apoptosis in H9c2 cells and related mechanisms. H9c2 cells were divided into 4 groups: control group (cells were cultured without intervention), PM(2.5) group (cells were treated with 800 µg/ml PM(2.5)), hesperetin group (H group, cells were treated by 40 µmol/L hesperetin for 1 h at 37 ℃), and hesperetin+PM(2.5) group (H+PM(2.5) group, cells were pretreated with hesperetin before PM(2.5) treatment). Cells were cultured for corresponding interval. Apoptotic cells were detected by Annexin Ⅴ-FITC/PI apoptosis detection kit and Hoechst staining. The intracellular reactive oxygen species (ROS) levels were measured by DCFH-DA Fluorescence Probe and mitochondrial membrane potential (MMP) was detected with JC-1 staining, respectively in these groups. Apoptotic related protein and phosphorylated MAPK expression levels were determined by Western blot. (1) Flow cytometry results showed that the apoptosis rate of PM(2.5) group ((48.94±3.20)%) was significantly higher than that of control group ((8.13±1.40)%, <0.01), which was significantly reduced in H+PM(2.5) group ((34.80±2.21)%) (0.003 2 vs. PM(2.5) group, <0.01 vs. control group). The number of Hoechst 33258 positive apoptotic cells was distinctly less in H+PM(2.5) group than in PM(2.5) group. (2) The ROS levels was significantly higher in PM(2.5) group ((49.69±5.05)%) than in control group (10.57±1.33)%, <0.01), which was significantly reduced in H+PM(2.5) group ((35.08±3.90)%) (0.000 2 vs. PM(2.5) group, <0.01 vs. control group). (3) Green fluorescence indicating the JC-1 monomer form, which represented MMP loss of H9c2 cells, was significantly higher in PM(2.5) group ((20.28±4.69)%) than in control group ((10.50±2.72)%, <0.01), which was significantly decreased in H+PM(2.5) group ((13.41±2.89)%) (<0.01 vs. PM(2.5) group, 0.029 4 vs. control group). (4) The expression levels of Bcl-2 protein of H9c2 cells was lower in PM(2.5) group ((76.94±4.52)%) than in control group (100%, 0.000 9), which was significantly upregulated in H+PM(2.5) group ((92.95±6.82)%) (0.027 5 vs. PM(2.5) group, 0.15 vs. control group). The expression levels of cleaved caspase-3 protein of H9c2 cells was significantly higher in PM(2).5 group ((243.98±17.94)%) than in control group (100%, 0.000 2), which was significantly downregulated in H+PM(2.5) group ((200.45±4.31)%) (0.015 vs. PM(2.5) group, <0.01 vs. control group). (5) The expression levels of phosphorylated p38 MAPK protein of H9c2 cells was higher in PM(2.5) group ((118.90±4.78)%) than in control group(100%, 0.002 7), which could be significantly downregulated in H+PM(2.5) group ((103.30±1.27)%) (0.01 vs. PM(2.5) group, 0.05 vs. control group). The expression levels of phosphorylated ERK protein of H9c2 cells was higher in PM(2.5) group ((163.50±4.98)%) than in control group (100%, <0.01), which was significantly downregulated in H+PM(2.5) group ((139.10±2.72)%) (0.001 6 vs. PM(2.5) group, <0.01 vs. control group). Hesperetin protects H9c2 cells from PM(2.5) stimulation through reducing oxidative stress and protecting mitochondrial function, regulating the expression of apoptotic associated proteins as well as MAPK signal pathway, thus inhibiting H9c2 cells apoptosis.
探讨橙皮素对细颗粒物（PM(2.5)）诱导的H9c2细胞凋亡的影响及其相关机制。将H9c2细胞分为4组：对照组（细胞无干预培养）、PM(2.5)组（细胞用800 µg/ml PM(2.5)处理）、橙皮素组（H组，细胞在37℃用40 µmol/L橙皮素处理1小时）和橙皮素+PM(2.5)组（H+PM(2.5)组，细胞在PM(2.5)处理前用橙皮素预处理）。细胞培养相应时间。采用AnnexinⅤ-FITC/PI凋亡检测试剂盒和Hoechst染色检测凋亡细胞。分别用DCFH-DA荧光探针检测细胞内活性氧（ROS）水平，用JC-1染色检测线粒体膜电位（MMP）。采用蛋白质免疫印迹法检测凋亡相关蛋白和磷酸化MAPK的表达水平。（1）流式细胞术结果显示，PM(2.5)组凋亡率（(48.94±3.20)%）显著高于对照组（(8.13±1.40)%, <0.01），H+PM(2.5)组凋亡率（(34.80±2.21)%）显著降低（与PM(2.5)组比较，P = 0.003 2；与对照组比较，<0.01）。H+PM(2.5)组Hoechst 33258阳性凋亡细胞数明显少于PM(2.5)组。（2）PM(2.5)组ROS水平（(49.69±5.05)%）显著高于对照组（10.57±1.33)%, <0.01），H+PM(2.5)组ROS水平（(35.08±3.90)%）显著降低（与PM(2.5)组比较，P = 0.000 2；与对照组比较，<0.01）。（3）代表H9c2细胞MMP丧失的绿色荧光（即JC-1单体形式）在PM(2.5)组（(20.28±4.69)%）显著高于对照组（(10.50±2.72)%, <0.01），H+PM(2.5)组绿色荧光显著降低（与PM(2.5)组比较，<0.01；与对照组比较，P = 0.029 4）。（4）PM(2.5)组H9c2细胞Bcl-2蛋白表达水平（(76.94±4.52)%）低于对照组（100%，P = 0.000 9），H+PM(2.5)组Bcl-2蛋白表达水平显著上调（(92.95±6.82)%）（与PM(2.5)组比较，P = 0.027 5；与对照组比较，P = 0.15）。PM(2).5组H9c2细胞裂解型caspase-3蛋白表达水平（(243.98±17.94)%）显著高于对照组（100%，P = 0.000 2），H+PM(2.5)组裂解型caspase-3蛋白表达水平显著下调（(200.45±4.31)%）（与PM(2.5)组比较，P = 0.015；与对照组比较
