Thirty male adult Sprague-Dawley rats, weighing 300±20 g, ~8 weeks of age were obtained from the Centers for Disease Control and Prevention of Wuhan (Wuhan, China), and were maintained in the Animal Experimental Center of Zhongnan Hospital, Wuhan University (Wuhan China). Prior to the experiments, standard chow and water were given to the rats ad libitum, and they were housed at 20–25°C and 50–70% humidity with a 12 h light/dark cycle. All the rats were randomized into three groups equally: Sham-operation group (S group), BD group, and 4-phenylbutyric acid group (4-PBA group), with 10 rats in each group. All animals were fed in the Animal Experiment Center of Zhongnan Hospital of Wuhan University 7 days before the experimental operation. All experiment operations were performed in accordance with the Experimental Animal Regulations of the People's Republic of China and the Guide for the Care and Use of Laboratory Animals (13). Prior to the establishment of animal model all animals were fasted overnight but had free access to water.

The BD model was established by intracranial inflating (14). All animals were anesthetized with pentobarbital solution (50 mg/kg) via an intraperitoneal injection; atropine solution (0.03 mg/kg) was used to decrease secretion of glands. Following anesthesia, rats were fixed on a homeothermic warming blanket linking to an Intelligent Temperature Controller (Taimeng Biological Technology Co., Ltd.) with the temperature probe placed into the anus to maintain temperature at ~38±1°C. Skin of calva was prepared. The scalp and the epicranial muscles and periosteum were excised. A 2-mm diameter hole was drilled on the skull at the intersection of sagittal line and the midpoint of two eyes. A 3F Fogarty catheter balloon (Fogarty Arterial Embolectomy Catheter, 3F, Edwards Lifesciences Co.) was put into the epidural space completely through the burr hole. Then, the catheter was gradually inflated to a final volume of ~2,000 ml. The femoral artery was catheterized to monitor blood pressure. The femoral vein was catheterized to prepare for fluid administration. An electrocardiogram and electroencephalogram were monitored using the Biological Function Date Acquisition and Monitoring System (Taimeng Biological Technology Co., Ltd.); tracheal intubation was also performed. The criterions of BD were referred to China Adult Brain Death Determination Technology Specifications and USA Adult Brain Death Diagnostics Guide (15). Rats of the BD group had underwent BD which was maintained for 6 h. Sham-operation rats were anesthetized, skull-drilled and intracranial catheterized but without inflating; anesthesia was maintained for 6 h. 4-PBA was administered 300 mg/kg via an intraperitoneal injection when the BD model was established; liver specimens were harvested 6 h after BD.

After 6 h of BD, the peritoneal cavity was opened. The falciform ligament and bilateral deltoid ligament were moved to carefully expose the liver. The liver was gently moved with a sterile swab and then placed on a piece of sterile gauze. The color and appearance of the liver was analyzed under a fine-light view.

Following reperfusion, about 0.25 g liver samples were fixed with 10% buffered formalin for 24 h at room temperature (pH=7.2; cat. no. G2161; Beijing Solarbio Science & Technology, Co., Ltd.), then put into 75, 80, 85, 90, 95 and 100% alcohol for 1 h respectively, so as to fully dehydrate the tissues. Following dehydration, the tissues were soaked in 50% alcohol + 50% xylene solution for 30 min, and then soaked in 100% xylene until they became transparent. The transparent samples were soaked in liquid paraffin at 60°C twice (1 h each) until the paraffin had completely infiltrated. The samples after paraffin immersion were embedded in paraffin wax and cut into 4-µm sections for histological analysis via hematoxylin-eosin staining (H&E; hematoxylin staining for 5–15 min and eosin staining for 1–3 min; all performed at room temperature). Sections were analyzed under a confocal microscope (magnification, ×200; Nikon A1R/A1; Nikon Corporation) and images were obtained. A total of 6 fields of view per section were randomly selected for the assessment of liver damage. Numerical assessment of liver damage was conducted according to the histological criteria for assessment of liver damage.

Protein expression of glucose-regulated protein 78 (Grp78), ATF6, PP2A and phosphorylated PP2A (PP2A Y307) were examined by western blotting. Frozen liver tissues were cut into pieces and solubilized in radioimmunoprecipitation assay buffer (cat. no. G2002; Wuhan Servicebio Technology Co., Ltd.) with phenylmethanesulfonyl fluoride 10 µl/ml. The turbid liquid was centrifuged for 15 min at 12,000 × g at 4°C. Protein concentration of the supernatant was determined by the bicinchoninic acid kit (cat. no. G2026; Wuhan Servicebio Technology Co., Ltd.). Then, the extracted protein was mixed with 5X loading buffer containing bromophenol blue as an indicator, which was then heated to 100°C for 10 min. 10% SDS-PAGE was performed with equal protein quantity each group using a constant voltage of 80 V in spacer gel and 120 V in separation gel. After electrophoresis, proteins were transferred to a polyvinylidenedifluoride filter membrane with a constant electric current of 278 mA in a mixture of ice and water. The membrane was blocked in 5% nonfat milk for 1 h at room temperature and incubated overnight at 4°C with anti-Grp78 (1:1,000, bs-1219R, Beijing Biosynthesis Biotechnology Co., Ltd., China), anti-ATF6 (1:1,000, bs-1634R, Beijing Biosynthesis Biotechnology Co., Ltd., China), anti-total PP2A (1:1,000, ab32104, Abcam), anti-PP2A Y307 (1:1,000, AF1756, Beyotime Institute of Biotechnology) and anti-β-actin (1:3,000, AF0003, Beyotime Institute of Biotechnology). Following washing with TBS-T, the membranes were then incubated for 2 h in anti-rabbit IgG (1:3,000, GB23303, Wuhan Goodbio Technology Co. Ltd.) at 37°C. Finally, the bands were visualized using an enhanced chemiluminescence reagent (cat. no. G2020; Wuhan Servicebio Co., Ltd.). Then, the bands were scanned and analyzed with Image-Pro Plus v6.0 software (Media Cybernetics, Inc.).

The activation of PP2A was detected with a quantitative detection kit (GMS50042.3, Genmed Scientific Inc.) by reactive colorimetry. Washing buffer (3 ml) was added to 500 mg liver tissue of each group; following washing they were placed in liquid nitrogen overnight. The next day 500 µl lysate buffer was added to each group and fully lysed with vortex oscillation. Then all the lysate were incubated for 30 min at 4°C and centrifuged for 5 min at 16,000 × g. Protein concentration of each group was detected by the bicinchoninic acid kit (cat. no. G2026; Wuhan Servicebio Technology Co., Ltd.) for later use. According to the protocol, the standard curves were drawn with negative and standard solutions of different concentrations. Then, 20 µl protein lysates of each group were taken to measure the background phosphorus concentration, total active phosphorus concentration and non-specific active phosphorus concentration by spectrophotometer (Shanghai CHOIF Co., Ltd.) and the specific phosphorus concentration of each sample was calculated according to the manufacturer's protocol.

Cell apoptosis of each group was detected by TUNEL assay. Detection was performed with the One Step TUNEL Apoptosis Assay Kit (Beyotime Institute of Biotechnology); 4-µm paraffin sections were deparaffinized in xylene (Sinopharm Chemical Reagent Co., Ltd.) three times (10 min each) and rehydrated in alcohol (anhydrous, 95, 80, 75%) and double distilled water successively, 5 min each. Then, the sections were treated with proteinase K for 20 min at room temperature and subsequently incubated at room temperature with a mixture of fluorescent labeling solution and TdT enzyme for 1 h in a the dark. After washing in PBS and drying, sections were incubated with DNase I for 10 min in the dark at room temperature. Then the sections were gently shaken to dry, DAPI staining solution was added to the tissue, incubated in dark for 10 min at room temperature. Finally the sections were washed with PBS for 3 times, 5 min each time, and sealed with anti-fluorescence quenching agent. The fluorescein isothiocyanate-labeled TUNEL-positive cells were imaged using fluorescent microscopy with 488 nm excitation and 530 nm emission wavelengths, and DAPI with 364 nm excitation and 454 nm emission wavelengths. The cells labeled with green fluorescence were deemed apoptotic cells.

Statistical analysis was performed with SPSS for windows version 17.0 (SPSS, Inc.). Continuous variables were presented as the mean ± standard deviation unless explicitly noted otherwise. Statistical significance between groups was tested by one-way ANOVA with Tukey's post-hoc test. P<0.05 was considered to indicate a statistically significant difference.

As presented in Fig. 1, variations in the macroscopic appearance of livers were observed. Livers of the S group were normally red, with a clear edge and smooth surface. BD livers were aubergine in color and the edge of liver was blunt with notable edema. 4-PBA livers had a more pronounced shape and were brighter in color than the BD group, but less so than the S group.

Hematoxylin-eosin staining was performed using the liver sections of the three groups, which were then observed under an optical microscope; representative images of analysis were presented in Fig. 2. In the S group, livers were histologically normal, as depicted by an orderly matrix of the hepatic sinusoid and an integrated hepatic lobule. In the BD group the hepatic sinusoid was misaligned; the hepatic lobule was malformed, while central venous endothelial injury, swelled liver cells and apoptosis were observed. Treatment with 4-PBA lessened the injury of to the hepatic lobule associated with BD.

The extent of liver cell apoptosis was presented in Fig. 3. Liver specimens after TUNEL straining were observed with an inverted fluorescence microscope. Green fluorescence was positive a result of the TUNEL assay, which indicated cell death, while blue fluorescence indicated positive DAPI staining for the total number of cells. The results showed that the apoptosis of the S group was only 10±1%, which is significantly lower than the BD (80±2%, P<0.05) and 4-PBA (68±2%, P<0.05) groups, while differences between the 4-PBA and BD groups were statistically significant (P<0.05; Table I).

Activation of PP2A was detected via colorimetry. As shown in Fig. 4, compared with the S group (1.898±0.337 nmolP/mg/min), the PP2A activity of the BD (4.008±0.566 nmolP/mg/min, P=0.005) and 4-PBA (2.874±0.241 nmolP/mg/min, P=0.015) groups were significantly increased. In addition, PP2A activation in the BD group was significantly higher than in the 4-PBA group (P=0.033).

The expression of Grp78 and ATF6 was detected by western blotting to assess the activation of ERS (Fig. 5). The results showed that the expression of Grp78 was significantly increased in the BD (2.792±0.151, P<0.001) and 4-PBA (2.011±0.174, P=0.003) group compared with the S group (0.881±0.014). Consistent with the increase Grp78 expression, ATF6 expression was also increased significantly in the BD (1.287±0.116, P<0.01) and 4-PBA (0.961±0.108, P=0.014) groups than the S group (0.692±0.155). The expression of Grp78 and ATF6 after 4-PBAtreatment were significantly lower than in the BD group (P=0.015 and 0.003, respectively).

Total expression of total PP2A and phosphorylated PP2A (PP2A Y307) were detected by western blotting. The results indicated no significant differences in total PP2A between the S and BD (2.096±0.101 vs. 2.197±0.109, P=0.586; Fig. 6). The expression of PP2A Y307 was significantly decreased after BD than in the S group (0.467±0.066 vs. 3.780±0.473, P=0.002), indicating that BD suppressed the phosphorylation of PP2A, which promoted the activation of PP2A. In addition, the expression of PP2A Y307 of the 4-PBA (1.632±0.222) group was significantly higher than the BD group (P=0.007) but lower than the S group (P=0.015; Fig. 6).