Hesperetin inhibits PM(2.5)-induced apoptosis in H9c2 cells by attenuating oxidative stress and mitochondrial damage

Abstract

Objective: To investigate the effects of hesperetin on fine particulate matter (PM(2.5)) induced apoptosis in H9c2 cells and related mechanisms. Methods: 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. Results: (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)%, P<0.01), which was significantly reduced in H+PM(2.5) group ((34.80±2.21)%) (P=0.003 2 vs. PM(2.5) group, P<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)%, P<0.01), which was significantly reduced in H+PM(2.5) group ((35.08±3.90)%) (P=0.000 2 vs. PM(2.5) group, P<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)%, P<0.01), which was significantly decreased in H+PM(2.5) group ((13.41±2.89)%) (P<0.01 vs. PM(2.5) group, P=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%, P=0.000 9), which was significantly upregulated in H+PM(2.5) group ((92.95±6.82)%) (P=0.027 5 vs. PM(2.5) group, P=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%, P=0.000 2), which was significantly downregulated in H+PM(2.5) group ((200.45±4.31)%) (P=0.015 vs. PM(2.5) group, P<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%, P=0.002 7), which could be significantly downregulated in H+PM(2.5) group ((103.30±1.27)%) (P=0.01 vs. PM(2.5) group, P=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%, P<0.01), which was significantly downregulated in H+PM(2.5) group ((139.10±2.72)%) (P=0.001 6 vs. PM(2.5) group, P<0.01 vs. control group). Conclusions: 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.