ABSTRACT
AIM:To investigate the effect of tert-butylhydroquinone ( tBHQ) on the replicative senescence of bone marrow mesenchymal stem cells (BMSCs).METHODS: Late stage BMSCs were continuously treated with tBHQ at concentration of 30 μmol/L for 4 weeks and the cells were used for the following assays immediately .The proteasomal ac-tivity was determined by chemiluminescence method .The samples were subjected to CCK-8 assay and BrdU incorporation as well as flow cytometry analysis for analyzing the cell vitality and proliferation .Percentage of senescent cells was detected by senescence-associatedβ-galactosidase ( SA-β-Gal) staining.The expression of P53 was measured by Western blot .RE-SULTS:After the continuous treatment of tBHQ (30 μmol/L) for 4 weeks, the proteasomal activity of late stage BMSCs increased by 21.96%±1.98%(P<0.05).The cell vitality and survival were significantly increased with the increases in tBHQ doses till 40 μmol/L, and no cytotoxicity reaction with the increased dose of tBHQ till 120 μmol/L was observed . BrdU-positive cells, which represented the cell proliferation , were significantly increased (P<0.05).The proliferation in-dex was also significantly increased by flow cytometry analysis (P<0.05).The SA-β-Gal positive cells and the expression of P53 were decreased (P<0.05).CONCLUSION:tBHQ delays the proteasome dysfunction associated senescence pro-gress of BMSCs by increasing the proteasomal activity .
ABSTRACT
AIM:To investigate the effect of 18 alpha-glycyrrhetinic acid (18α-GA) on delaying the senescent progress and promoting the proliferation in late-passage bone marrow mesenchymal stem cells ( BMSCs ) .METHODS:Late-passage BMSCs were incubated with 2.0 mg /L 18α-GA or the same volume of DMSO for 30 d, and the cells were harvested to determine the proteasome activity .The expression of senescence-related proteins p53, p21 and p16 was detec-ted by senescence-associated β-galactosidase ( SA-β-Gal) staining and Western blot .The cell proliferation , the expression level of cell cycle-related proteins and cell cycle distribution of the cells were measured by CCK -8 assay, BrdU incorpora-tion, Western blot and flow cytometry.RESULTS:Compared with DMSO group, the proteasome activity in 18α-GA group increased significantly by about 0.2 times (P<0.01).SA-β-Gal-positive cells in 18α-GA group decreased, and cell stai-ning was lighter.The contents of p53 and p21 in 18α-GA group were decreased (P<0.05).The results of CCK-8 assay showed that the A value in 18α-GA group was 0.3 times higher than that in DMSO group (P<0.01).BrdU incorporation showed the increased proliferation in 18α-GA group compared with DMSO group ( P<0.05).The cells in G1 phase in 18α-GA group decreased significantly compared with DMSO group , while the cells in S phase increased significantly ( P<0.05).The expression level of cyclin D1 in 18α-GA group was 2.8 times higher than that in DMSO group (P<0.01), and the CDK4 level was 1.4 times higher than that in DMSO group (P<0.05).CONCLUSION:Activation of the pro-teasome activity by 18α-GA delays the aging process in the BMSCs and promotes the cell proliferation via up -regulation of the cell cycle-related proteins .