[Effects and mechanism of pyrroloquinoline quinine on mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells under oxidative stress].

ABSTRACT

Objective: To observe the effects of pyrroloquinoline quinine (PQQ) on the mitochondrial function and cell survival of rat bone marrow mesenchymal stem cells (BMSCs) under oxidative stress, and to explore its mechanism. Methods: BMSCs of rats were cultured in vitro with Dulbecco's minimum essential medium/F12 medium containing fetal bovine serum in the volume fraction of 10% (hereinafter referred to as normal medium). The rat BMSCs of third to fifth passages in logarithmic growth phase were selected for the following experiments. (1) The cells were divided into normal control group, normal control+ PQQ group, hydrogen peroxide (H(2)O(2)) alone group, and H(2)O(2)+ PQQ group. The cells in normal control group were cultured in normal medium for 24 hours; the cells in normal control+ PQQ group were cultured in normal medium containing 100 µmol/L PQQ for 24 hours; the cells in H(2)O(2) alone group were cultured in normal medium containing 200 µmol/L H(2)O(2) for 24 hours; the cells in H(2)O(2)+ PQQ group were pre-incubated with normal medium containing 100 µmol/L PQQ for 2 hours, and then with H(2)O(2) added to the concentration of 200 µmol/L and cultured for 24 hours. The cell morphology of each group was observed under the inverted phase contrast microscope, and the cell survival rate was detected by cell count kit 8 method. (2) Five batches of cells were collected, and the cells of each batch were divided into normal control group, H(2)O(2) alone group, and H(2)O(2)+ PQQ group. The cells in each group received the same treatment as that in the corresponding group of experiment (1). After 24 hours of culture, one batch of cells was collected for apoptosis detection by flow cytometry, and the apoptosis rate was calculated. One batch of cells was subjected to mitochondrial membrane potential assay and JC-1 fluorescent staining observation using the JC-1 mitochondrial membrane potential detection kit and the inverted phase contrast fluorescence microscope, respectively. One batch of cells was collected for mitochondrial morphology observation under the transmission electron microscope. One batch of cells was subjected to catalase (CAT) and superoxide dismutase (SOD) activity assay by CAT activity assay kit and SOD activity assay kit, respectively. One batch of cells was subjected to Western blotting for determination of protein level of Epac1, adenine monophosphate activated protein kinase (AMPK), phosphorylated AMPK, cysteinyl aspartate-specific proteinase 3 (caspase-3), and cleaved caspase-3, and the phosphorylation level of AMPK and cleaved caspase-3/caspase-3 ratio were calculated. Six replicates were measured in each group for each index except for morphological observation. Data were statistically analyzed with one-way analysis of variance and independent sample equal variance t test. Results: (1) After 24 hours of culture, compared with those in normal control group (the cell survival rate was set to 100.0%), there was an increase in cell vacuole and a decrease in cell number in H(2)O(2) alone group, and the cell survival rate was significantly reduced to (74.3±2.9)% (t=6.39, P<0.01). Compared with those in H(2)O(2) alone group, the cell morphology of H(2)O(2)+ PQQ group was significantly improved, and the cell survival rate was significantly increased to (116.9±4.2)% (t=6.92, P<0.01); the cell survival rate in normal control+ PQQ group was (101.2±1.1)%, close to that of control group (t=1.06, P>0.05). (2) After 24 hours of culture, compared with (13.6±1.0)% in normal control group, the apoptosis rate of cells in H(2)O(2) alone group was significantly increased to (37.1±2.0)% (t=10.57, P<0.01). Compared with that in H(2)O(2) alone group, the apoptosis rate of cells in H(2)O(2)+ PQQ group was significantly declined to (17.0±0.7)% (t=9.49, P<0.01). (3) After 24 hours of culture, compared with those in normal control group, the mitochondrial membrane potential of cells in H(2)O(2) alone group was depolarized, the JC-1 fluorescent dye mainly existed in the cytoplasm in the form of monomer, which emitted green fluorescence, and a significant decrease in mitochondrial membrane potential was shown (t=4.18, P<0.01). Compared with those in H(2)O(2) alone group, the mitochondrial membrane potential of cells in H(2)O(2)+ PQQ group was increased to normal level (t=4.43, P<0.01), and the JC-1 fluorescent dye accumulated in mitochondria following the polarized mitochondrial membrane potential and emitted red fluorescence. (4) After 24 hours of culture, compared with that in normal control group, the mitochondrial structure of cells in H(2)O(2) alone group was disordered, with disappeared mitochondrial cristae and decreased mitochondrial matrix density. Compared with that in H(2)O(2) alone group, the mitochondrial structure of cells in H(2)O(2)+ PQQ group was regular and intact, with clearly visible mitochondrial cristae and increased mitochondrial matrix density. (5) After 24 hours of culture, compared with those in normal control group, the CAT activity of cells in H(2)O(2) alone group was significantly increased (t=4.54, P<0.05), and the SOD activity was significantly decreased (t=3.93, P<0.05). Compared with those in H(2)O(2) alone group, the CAT activity of cells in H(2)O(2)+ PQQ group was obviously increased (t=8.65, P<0.01), while there was no significant change in the SOD activity (t=0.72, P>0.05). (6) After 24 hours of culture, compared with those in normal control group, the protein expression of Epac1 of cells in H(2)O(2) alone group was significantly decreased (t=4.67, P<0.01), while the AMPK phosphorylation level and the cleaved caspase-3/caspase-3 ratio were significantly increased (t=7.88, 3.62, P<0.01). Compared with those in H(2)O(2) alone group, the protein expression of Epac1 and the AMPK phosphorylation level of cells in H(2)O(2)+ PQQ group were both significantly increased (t=4.34, 16.37, P<0.01), while the cleaved caspase-3/caspase-3 ratio was significantly declined (t=3.17, P<0.05). Conclusions: Pretreatment with PQQ can improve the mitochondrial function, reduce cell apoptosis rate, and enhance cell survival rate of rat BMSCs under oxidative stress, which may be related to the up-regulation of Epac1 protein expression, activation of AMPK signaling pathway, and down-regulation of cleaved caspase-3 protein level.