p38介导β-羟丁酸对原代培养犊牛肝细胞凋亡的影响

本试验旨在探讨β-羟丁酸（BHBA）对体外原代培养犊牛肝细胞凋亡的影响以及p38MAPK在其中的调控作用。选择培养72h的肝细胞，添加不同浓度的BHBA（0、0．6、1．2、2．4mmol／L），培养9h，每个浓度3个重复。另外选取细胞，p38MAPK抑制剂SB203580（10pmol／L）预处理1h后，添加BHBA，使其终浓度为2．4retool／L；PI／An—nexinV—FITC双染，运用荧光显微镜观察肝细胞凋亡情况；ELISA法检测p38的活性；实时荧光定量PCR方法检测p38、Caspase-3、Caspase9、bcl2基因的mRNA表达水平。结果表明，与对照组相比，1．2和2．4mmol／I—BHBA组的肝细胞凋亡明显增加；p38酶活性明显升高；p38、caspase-3和caspase-9基因mRNA表达水平均显著增加（P〈0．01）。bcl2基因mRNA表达水平显著降低（P〈0．05）；添加p38抑制剂后，caspase-3和caspase-9基因mRNA表达水平显著降低（P〈0．01），bcl-2基因mRNA表达水平显著增加（P〈O．05）。高浓度的BHBA可以诱导体外原代培养犊牛肝细胞凋亡，p38在BHBA诱导的细胞凋亡中发挥重要作用。

B-hydroxybutyrate induce the apoptosis of primary cultured bovine hepatocytes mediated by p38

The objective of this study was to investigate the effect of β-hydroxybutyrate （BHBA） on the apoptosis of primary cultured bovine bepatocytes and the important role of p38 in the BHBA- induced apoptosis. After hepatocytes were cultured for 72 h,different concentrations of BHBA （0, 0.6,1.2,2.4 mmol/L） were added with three replications in one group and cultured for another 9 h. Hepatocytes were pretreated with p38 inhibitor SB203580 （10 μmol/L） for 1 h,then BHBA were added in the final concentration of 2.4 mmol/L. Apoptosis of hepatocytes was observed by fluorescence microscopy after stained with PI/Annexin V-FITC. The enzyme activity of p38 was detected by available p38 activity kits at the same time. The mRNA expression levels of p38 and apoptotic related genes including caspase-3, caspase-9 and bcl-2 were detected by using real-time fluorescence quantitative RT-PCR（qRT-PCR）. The results showed that the apoptotic cells in high concentrations of BHBA （1.2,2.4 mmol/L） were significantly increased compared with the con- trol group. P38 enzyme activity was gradually increased in a concentration dependent manner,with significant difference in the high concentration of BHBA groups （1.2 and 2.4 mmol/L） （P〈0.05 or P〈0.01）. The gene expression levels of p38, caspase-3 and caspase-9 significantly increased in high concentrations of BHBA （1.2 and 2.4 mmol/L） groups compared with the control group （P〈0.05 or P〈0.01） ,while the bcl-2 gene expression levels was significantly lower than control group （P〈0.05）. Compared with the high BHBA concentration group （2.4 mmol/L） ,caspase-3 and caspase 9 gene expression levels were significantly lower（P〈0.01） in the group pretreated p38 inhibitor. while the bcl-2 gene expression levels increased significantly （P〈0.05）. In summary,high concen tration of BHBA can induce the apoptosis of bovine hepatocytes cultured in vitro ,and p38 signa ling pathway plays an important role in the hepatocyte apoptosis induced by BHBA.