Anti‑apoptotic effects of human placental hydrolysate against hepatocyte toxicity in vivo and in vitro

Abstract

Apoptosis and oxidative stress are essential for the pathogenesis of acute liver failure and fulminant hepatic failure. Human placental hydrolysate (hPH) has been reported to possess antioxidant and anti‑inflammatory properties. In the present study, the protective effects of hPH against D‑galactosamine (D‑GalN)‑ and lipopolysaccharide (LPS)‑induced hepatocyte apoptosis were investigated in vivo. In addition, the molecular mechanisms underlying the anti‑apoptotic activities of hPH against D‑GalN‑induced cell death in vitro were examined. Male Sprague‑Dawley rats were injected with D‑GaIN/LPS with or without the administration of hPH. Rats were sacrificed 24 h after D‑GaIN/LPS intraperitoneal injection, and the blood and liver samples were collected for future inflammation and hepatotoxicity analyses. Changes in cell viability, apoptosis protein expression, mitochondrial mass, mitochondrial membrane potential, reactive oxygen species generation, and the levels of proteins and mRNA associated with a protective mechanism were determined in HepG2 cells pretreated with hPH for 2 h prior to D‑GalN exposure. The findings suggested that hPH treatment effectively protected against D‑GalN/LPS‑induced hepatocyte apoptosis by reducing the levels of alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, interleukin‑6, and tumor necrosis factor‑α, and increasing the level of proliferating cell nuclear antigen. It was also found that hPH inhibited the apoptotic cell death induced by D‑GalN. hPH activated the expression of antioxidant enzymes, including superoxide dismutase, glutathione peroxidase, and catalase, which were further upregulated by the Kelch‑like ECH2‑associated protein 1‑p62‑nuclear factor‑erythroid 2‑related factor 2 pathway, a component of oxidative stress defense mechanisms. Furthermore, hPH markedly reduced cytosolic and mitochondrial reactive oxygen species and rescued mitochondrial loss and dysfunction through the reduction of damage‑regulated autophagy modulator, p53, and C/EBP homologous protein. Collectively, hPH exhibited a protective role in hepatocyte apoptosis by inhibiting oxidative stress and maintaining cell homeostasis. The underlying mechanisms may be associated with the inhibition of endoplasmic reticulum stress and minimization of the autophagy progress.

Figure 1

Protective effects of hPH treatment on D-GalN/LPS-induced acute liver failure. hPH (1.2, 2.4, or 3.6 ml/kg) was subcutaneously administered to rats three times at intervals of 24 h, followed by exposure to 700 mg/kg D-GalN and 15 µg/kg LPS (D-GalN/LPS). Livers from each experimental group were examined 24 h following D-GalN/LPS challenge. (A) Gross image of livers. Scale bar=1 cm (B) Animal weights (n=4-6) were measured just prior to sacrifice; with no statistical difference among groups. (C) Liver weights were measured following sacrifice, with no statistical difference among groups (n=4-6). (D) Survival rates of rats were observed for 24 h following D-GalN/LPS exposure (n=12). Serum (n=4-6) was collected from animals 24 h following exposure to D-GalN/LPS to determine levels of (E) AST, (F) ALT, (G) LDH, (H) IL-6, and (I) TNF-α. All data are presented as the mean ± standard error of the mean. *P<0.05 and ^**P<0.01, vs. D-GalN/LPS group. hPH, human placental hydrolysate; D-GalN, D-galactosamine; LPS, lipopolysaccharide; Cont, control; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.

Figure 2

Effects of hPH treatment on D-GalN/LPS-induced acute liver failure in tissue degeneration and apoptosis. (A) Histopathological staining. Upper panel (magnification, ×20), lower panel (magnification, ×40). Scale bar=100 µm. The arrows indicate apoptotic hepatocytes (black), a severely affected region of a lobule containing ballooned hepatocytes (red), and hepatocytes expanded by fat vacuoles (green). (B) Images showing PCNA staining. Scale bar=100 µm. (C) Apoptotic response to D-GalN/LPS in the liver tissue of rats was investigated using TUNEL staining. Scale bar=100 µm. (D) Stained sections were graded for histopathology using a four-point scale (0-3), with 0, 1, 2, and 3 representing no damage, mild damage, moderate damage, and severe damage, respectively. (E) PCNA-positive cells were significantly less numerous in the D-GalN/LPS-treated group than in the control group; the addition of hPH led to significant overexpression of PCNA in the liver compared with D-GalN/LPS treatment alone. (F) TUNEL-positive cells was higher in number in the group treated with D-GalN/LPS alone compared with the control group. However, apoptotic induction by D-GalN/LPS was further reduced in the groups treated with 1.2, 2.4, and 3.6 ml/kg of hPH. All data are presented as the mean ± standard error of the mean. ^*P<0.05 and ^**P<0.01, vs. D-GalN/LPS group. hPH, human placental hydrolysate; D-GalN, D-galactosamine; LPS, lipopolysaccharide; PCNA, proliferating cell nuclear antigen; Cont, control.

Figure 3

Effects of hPH treatment on D-GalN-induced apoptosis in HepG2 cells. (A) HepG2 cells were treated with 25, 50, or 100 mM D-GalN for 24 h. Treatment with 50 mM D-GalN yielded a cell viability of almost 50% compared with the vehicle control. The concentration of 50 mM D-GalN was used to determine the 50% inhibitory concentration. (B) HepG2 cells were treated with increasing concentrations of hPH or vehicle control for 24 h. HepG2 cells were treated with hPH (5%) for 2 h prior to D-GalN stimulation (50 mM). After 24 h, the hepatoprotective effects of hPH were assessed with the (C) Cell Counting Kit-8 assay and (D) LDH release test for cell viability, and with an (E) inverted phase-contrast microscope for morphological changes (scale bar=50 µm). HepG2 cells were stimulated with D-GalN (50 mM, 24 h) in the presence or absence of 5% hPH. (F) Cells were subjected to fluorescence microscopy (PI stain only) and (G) Annexin V/PI staining analyzed by a microplate (scale bar=50 µm). (H) DNA fragmentation (red arrow) and nuclear condensation (white arrow) were detected by DAPI staining under each condition (scale bar=5 µm). For western blot analysis, cell lysates were collected and subjected to sodium dodecyl sulfate-PAGE, followed by immunoblot analysis using anti-BCL2, anti-BAX and anti-PARP antibodies. Anti-GAPDH was used as a loading control. (I) Representative images and (J) densitometry. All data are presented as the mean ± standard error of the mean. ^**P<0.01, vs. D-GalN group. hPH, human placental hydrolysate; D-GalN, D-galactosamine; Cont, control; LDH, lactate dehydrogenase; BCL2, B-cell lymphoma 2; BAX, Bcl-2-associated X protein; PARP, poly (ADP) ribose polymerase; PI, propidium iodide.

Figure 4

D-GalN-induced mitochondrial dysfunction and ROS overgeneration in HepG2 cells are improved by hPH treatment. HepG2 cells were stimulated with D-GalN (50 mM, 24 h) in the presence or absence of 5% hPH. (A) HepG2 cells post-hPH treatment were immunostained with anti-Tomm20 antibody, followed by the addition of Cy3-conjugated secondary antibody. Nuclei were identified using DAPI staining (scale bar=5 µm). (B) MitoTracker fluorescence signals for mitochondrial mass were measured. (C) ΔΨm was measured under the indicated conditions using the ΔΨm-sensitive fluorochrome MitoTracker Red CMXRos. (D) Cells were stained with 2′,7′-dichlorofluorescin diacetate for 30 min to measure intracellular hydrogen peroxide. (E) Cells were stained with MitoSOX for 30 min to measure mitochondrial ROS. Bar graphs show relative analysis. All data are presented as the mean ± standard error of the mean. ^**P<0.01, vs. D-GalN group. hPH, human placental hydrolysate; D-GalN, D-galactosamine; Con, control; MMP/ΔΨm, mitochondrial membrane potential; ROS, reactive oxygen species.

Figure 5

Upregulation of antioxidant enzymes and regulation of Keap1-Nrf2 in hPH-treated HepG2 cells. (A) Lysates from hPH-induced HepG2 cells were immunoblotted with anti-SOD-1, anti-SOD-2, anti-GPx, or anti-catalase antibodies. Representative images (left penal), densitometry results (right panel). All data are presented as the mean ± standard error of the mean. ^*P<0.05, ^**P<0.01 vs. 0 h time point. (B) Following treatment with hPH, the expression levels of p-p62, p62, Keap1, and HO-1 were determined by western blot analysis. Representative images (left penal), densitometry results (right panel). All data are presented as the mean ± standard error of the mean. ^*P<0.05, ^**P<0.01, vs. 0 h time point. (C) Nuclear localization of Nrf2 in hPH-treated HepG2 cells compared with untreated HepG2 cells. Lysates from hPH-treated HepG2 cells were immunoblotted with anti-Nrf2, anti-Lamin B1, or anti-GAPDH antibodies. Representative images (left penal), densitometry results (right panel). All data are presented as the mean ± standard error of the mean. ^*P<0.05, ^**P<0.01, vs. 0 h time point. (D) HepG2 cells post-hPH treatment were immunostained with anti-Nrf2 antibody. Nuclei were identified using DAPI staining (scale bar=20 µm). hPH, human placental hydrolysate; SOD, superoxide dismutase; GPx, glutathione peroxidase; Keap1, Kelch-like ECH2-associated protein 1; HO-1, heme oxygenase-1; p-p62; Nrf2, nuclear factor-E2-related factor 2.

Figure 6

hPH treatment results in altered autophagy regulation in HepG2 cells following D-GalN stimulation. (A) HepG2 cells treated with hPH exhibit minimized autophagy flux. To detect autophagosomes, cells were immunostained with anti-LC3 antibody. Nuclei were identified using DAPI staining (scale bar=10 µm). (B) Cell lysates were immunoblotted with anti-LC3 I/II, anti-DRAM, anti-CHOP, anti-p53, or anti-GAPDH antibodies. Representative western blot images (left panel) and densitometry results (right panel). All data are presented as the mean ± standard error of the mean. ^**P<0.01, vs. D-GalN group. (C) Transcript levels of ER stress- and autophagy-related genes were determined by reverse transcription-quantitative polymerase chain reaction analysis. All data are presented as the mean ± standard error of the mean. ^**P<0.01, vs. D-GalN group. hPH, human placental hydrolysate; D-GalN, D-galactosamine; Cont, control; LC3, microtubule-associated protein 1A/1B-light chain 3; DRAM, damage-regulated autophagy modulator; CHOP, C/EBP homologous protein; ATF, activating transcription factor; BECN1; beclin 1; LAMP1, lysosomal-associated membrane protein 1.