ABSTRACT
Objective@#Investigate the effects of inducible ppp2r1a knockout on main physiological function in adult mice and study the mechanism.@*Methods@#Ppp2r1aflox/flox mice and CAGG-CreER mice were hybridized to obtain 20 CAGG-CreER ppp2r1aflox/flox and 20 mice in homozygous group. Two groups of mice were divided into 4 groups respectively, finally we got 8 groups with 5 mice in each group. Tamoxifen was injected intraperitoneally to acquire inducible ppp2r1a knockout mice. The knockout efficiency of PP2A Aα in vital organs was measured by Western blot. At 0, 2, 4 and 6 days after injection, we measured body weight, histopathological change, peripheral blood cell counts and blood biochemical. Real-time PCR was performed to measure expression of liver glucolipid metabolism genes.@*Results@#After tamoxifen injection for 6 days, the knockout efficiency of PP2A Aα in vital organs was 35%, 12%, 15%, 60%, 69% and 72%, respectively in heart, liver, spleen, lung, kidney and brain. After tamoxifen injection for 6 days, the weight of homozygous mice was lower than that of wild type mice, with values of (17.42±1.76) g and (21.69±1.82) g, respectively (P<0.05). Moreover, the activity level, abdominal and renal fat were significantly decreased in homozygous mice. Homozygous mice survived no more than 7 days. Compared with wild type mice, the organ coefficient of spleen of homozygous mice was decreased at the 6th day, with values of (0.59±0.10)% and (0.36±0.05)% respectively (P<0.05). Obvious spleen atrophy and marked decrease of nucleated cells were showed by performing HE staining. Tunel staining revealed increased apoptosis ratio of splenic lymphocytes in homozygous mice. The levels of alanine aminotransferase (ALT) and aspartate transaminase (AST) of homozygous mice were higher than wild type mice (P<0.05). The values of ALT and AST in homozygous mice were (153.68±62.80) U/L and (193.2±44.28) U/L. The corresponding values in wild type mice were (41.02±12.91) U/L and (69.40±9.55) U/L. The above results indicated that ppp2r1a knockout caused liver damage. Blood sugar level of homozygous mice was lower than in wild type mice (P<0.05), with values of (4.20±1.99) mmol/L and (8.88±0.65) mmol/L respectively. Plasma total cholesterol (TC), high density lipoprotein (HDL) and β-hydroxybutyric acid (β-HB) level of homozygous mice were higher than those of wild type mice (P<0.05). The values of TC, HDL and β-HB in homozygous mice were (3.12±0.39), (1.53±0.38) and (2.49±0.89) mmol/L. The corresponding values in wild type mice were (1.69±0.92), (0.78±0.50) and (0.45±0.30) mmol/L respectively. The above results indicated that ppp2r1a loss interfered glucose and cholesterol metabolism. In addition, we also found that the white blood cell count (WBC) and lymphocyte count (LYM) of homozygous mice were lower than in wild type mice (P<0.05). The values of WBC and LYM in homozygous mice were (1.88±0.89)×109/L and (0.92±0.37)×109/L respectively. The corresponding values in wild type mice were (3.91±0.80)×109/L and (2.74±0.52)×109/L respectively. The mRNA levels of glucose-6-phosphatase (G6P) and phosphoenolpyruvate carboxykinase (PEPCK) of homozygous were lower than wild type mice (P<0.05). The fold change of G6P and PEPCK in homozygous mice was 0.46±0.11 and 0.72±0.07 respectively. The corresponding fold change in wild type mice was 1.02±0.07 and 1.02±0.06 respectively.@*Conclusion@#Whole body ppp2r1a is essential for the survival of adult mice, due to the important role in maintaining the metabolism of glucose and cholesterol of liver.
ABSTRACT
Objective@#To investigate the effect of polycyclic aromatic hydrocarbons (PAHs) exposure on the level of histone H3Ser10 phosphorylation (p-H3S10) and DNA damage degree in peripheral blood lymphocyte (PBLCs).@*Method@#75 coke oven workers from Benxi steel plant in Liaoning Province of China (PAHs-exposed group) and local 50 hot rolling workers (control group) were recruited in this study with age, working years, labor intensity and high temperature for matching factors using cluster sampling method in 2014. HPLC-fluorescence was performed to determine the level of urinary 1-hydroxypyrene (1-OHP), DNA damage and specific histone modification were measured in PBLCs of the subjects through comet assay and ELISA assay, respectively. Linear regression model analysis was used to analyze the differences among PAHs exposure, DNA damage and p-H3S10 level in two groups. The Mediation analysis was used to analyze the regulated relationships between urinary 1-OHP, DNA damage and histone modification through the bootstrap method.@*Results@#Age of the control and the exposed group were (45.32±8.32) and (43.87±5.67) years old (P=0.284). The concentration of urinary 1-OHP, OTM value, Tail DNA% and p-H3S10 level in exposure group were higher than that in control group, while the M (P5-P95) of p-H3S10 levels in control and exposed group were 2.21 (0.68-4.71), 4.54 (1.85-23.91) (P<0.001). The degree p-H3S10 level was increased after the subgroups which were (2.59±1.19)%, (3.24±2.81)%, (5.55±3.25)%, (8.77±7.84)%, respectively, divided by quantitated 1-OHP concentration as P0-P25, P26-P50, P51-P75 and P76-P100 (P<0.001). We also found the correlations between urinary 1-OHP and p-H3S10 level or OTM value or Tail DNA%, β (95%CI) were 0.264 (0.167-0.360), 0.500 (0.299-0.702), and 0.510 (0.384-0.671), respectively (P<0.001). Similar result was also observed between p-H3S10 level and OTM value or Tail DNA%, β (95%CI) were 0.149 (0.073-0.226) and 0.220 (0.132-0.308) (P<0.001). Moreover, the mediation effect value of DNA damage on PAHs induced p-H3S10 alteration was 0.054(P=0.040).@*Conclusion@#The results suggested that PAHs exposure could induce DNA damage and an increase in histone H3Ser10 phosphorylation in PBLCs. Particularly, the alteration of H3S10 phosphorylation may play an important role in regulating cell DNA damage repair.