To investigate the role of PTTG1 gene in ethanol-induced acute injury of LO2 hepatocytes.

LO2 hepatocytes were treated with ethanol solution of different concentrations 0, 100, 200, 400, 800, 1 600 mmol/L for 0, 3, 6, 12, 24 and 48 h, respectively. Cell morphology was observed under microscope to explore the optimal parameters for the establishment of ethanol-induced injury models. Normal human LO2 hepatocyte cell line was infected with fluorescent lentivirus vector carrying PTTG1 shRNA. The stable transfected cell lines with PTTG1 knock-down were screened by Purimycin and assigned in the PTTG1/ KD group. The cell lines with empty vector were allocated into the PTTG1/vector group. According to the explored parameters, the cell lines were treated with 400 mmol/L ethanol for 24 h. The morphological changes and severity of injury were observed by HE staining. The expression levels of PTTG1 and apoptosis-related protein of cleaved-caspase3 were detected by Western blot. The expression levels of PTTG1 among multiple groups were statistically analyzed by One-way ANOVA and LSD-t test and the comparison between two groups was conducted using t test.

Under the microscope in preliminary trial, significant morphological changes of acute injury were observed after exposure to 400 mmol/L ethanol for 24 h. Hence, the experimental parameters of the model were determined. The expression of PTTG1 was up-regulated with the increase of ethanol concentration and treatment time, and reached the highest level when using 400 mmol/L ethanol for 24 h (LSD-t=6.90, 4.14; P<0.05). HE staining demonstrated that the number of apoptotic cells in PTTG1/KD group was significantly higher than that in PTTG1/vector group. Western blot showed that the expression of PTTG1 in PTTG1/KD group was 0.22±0.11, significantly lower than 1.09±0.11 in PTTG1/vector group (t=-11.60, P<0.05). However, the expression of cleaved-caspase3 in PTTG1/KD group was 0.05±0.01, significantly higher than 0.03 in PTTG1/vector group (t=8.20, P<0.05).

Ethanol can cause changes of expression of PTTG1 gene in LO2 hepatocytes. PTTG1 gene can alleviate the ethanol-induced apoptosis of hepatocytes, which probably plays a role of anti-apoptosis in acute ethanol-induced liver injury.