Histological observations
Mouse livers treated with DCE-2.5 showed hypertrophic hepatocytes containing more eosinophilic cytoplasms than non-treated controls. Most hepatic cords were thickened with hypertrophic hepatocytes, but their sinusoidal spaces were well preserved. Many hepatocytes showed increased heterochromatic nuclei, but no necrotic hepatocytes were observed during the histological observation (Fig. 1b). Mouse livers treated with DCE-5 consistently showed hypertrophic hepatocytes and narrow sinusoidal spaces (Fig. 1c), while those treated with DCE-10 showed shrunken hepatocytes and had larger sinusoidal spaces than non-treated controls (Fig. 1d).
Immunohistochemical observation
Representative antibodies of GST-1, PARP, MMP-9, and HGF-1 were applied to mouse liver sections. Hepatocyte regeneration and expressions of protective proteins, HGF-1 and GST-1, were dose-dependently increased in mouse livers treated with DCE-2.5, DCE-5, or DCE-10. HGF-1 was strongly expressed at hepatocyte membranes in the DCE-5 group, while GST-1 was strongly positive in hepatocyte cytoplasms in the DCE-10 group (Fig. 2A1–A4, B1–B4). Mouse livers treated with DCE-2.5, DCE-5, or DCE-10 were positive for PARP and MMP-9 (cellular apoptosis and scavenging proteins, respectively). Both PARP and MMP-9 were strongly positive in the DCE-2.5 group, and consistently positive in the DCE-5 and DCE-10 groups (Fig. 2C1–C4, D1–D4).
Effects of DCE on the expressions of proliferation-related proteins in mouse livers
Mouse livers treated with DCE-2.5 or DCE-5 showed higher expressions of proliferation-related proteins (PLK4 (107.5%) and MPM2, (105.6%)) but lower p14 expression (94.7%) than non-treated controls. The expression levels of other proliferation-related proteins, PCNA, Ki-67, CDK4, cyclin D2, p16, p21, p27, and lamin A/C like those of the control housekeeping proteins (β-actin, α-tubulin, and glyceraldehyde-3-phosphate dehydrogenase, GAPDH) changed by less than ± 5% in response to DCE (Fig. 3A1). However, PCNA, Ki-67, CDK4, cyclin D2, and lamin A/C were upregulated; and p16, p21, and p27 were downregulated dose-dependently by DCE (Fig. 3A2). These results suggested that DCE-2.5 and DCE-5 slightly enhanced the proliferation of murine hepatocytes.
Effects of DCE on the expressions of cMyc/MAX/MAD network proteins in mouse livers
Mouse livers treated with DCE showed expression changes of cMyc, MAX, and MAD of < ± 5% as was observed for control housekeeping proteins (Fig. 3B1, B2), indicating DCE did not activate cMyc/MAX signaling in murine hepatocytes.
Effects of DCE on the expressions of p53/Rb/E2F signaling proteins in mouse livers
DCE-2.5 or DCE-5 altered the expressions of p53, MDM2, Rb-1, E2F-1, and CDK4 by < ± 5%, which was similar to those observed for control housekeeping proteins, but DCE-10 increased the expression of CDK4 (105.4%) and reduced the expression of Rb-1 (94%) as compared with non-treated control livers (Fig. 3C1, C2). These results suggest that DCE-2.5 and DCE-5 did not activate p53/Rb/E2F signaling in murine hepatocytes.
Effects of DCE on the expression of Wnt1/β-catenin signaling proteins in mouse livers
Mouse livers treated with DCE showed expression changes in Wnt1, β-catenin, APC, and TCF-1 of < ± 5%, which was similar to that observed for control housekeeping proteins, but had reduced snail levels (93.9%) (Fig. 3D1, D2). These results suggested DCE did not activate Wnt/β-catenin signaling in murine hepatocytes.
Effects of DCE on the expressions of epigenetic modification-related proteins in mouse livers
Mouse livers treated with DCE showed DMAP1, KDM4D, and MBD4 level changes of < ± 5%, as was observed for control housekeeping proteins, but the expressions of histone H1, HDAC10, and DNMT1 reduced to 91%, 93.8%, and 89%, respectively, after treatment with DCE-5 or DCE-10 (Fig. 4A1, A2). These results suggested DCE slightly activated epigenetic modification by downregulating histone H1, HDAC10, and DNMT1.
Effects of DCE on the expressions of protein translation-related proteins in mouse livers
Mouse livers treated with DCE showed expression changes of DHS, eIF5A-1, eIF5A-2, and eIF2AK3 of < ± 5%, like control housekeeping proteins, but showed slight decreases in the levels of DOHH (91.6%) after treatment with DCE-5 or DCE-10, and a slight increase in eIF2AK3 (107.9%) after treatment with DCE-10 (Fig. 4B1, B2). These results suggested DCE slightly inactivated protein translation by downregulating DOHH and upregulating eIF2AK3.
Effects of DCE on levels of growth factor proteins in mouse livers
Mouse livers treated with DCE-5 showed higher expressions of GHRH (107.2%), HGF-1 (105.3%), and insulin (106.4%) than non-treated controls, and those treated with DCE-10 showed higher insulin levels (108.8%). The expression levels of other growth factors, TGF-β1, TGF-β2, SMAD4, Met, IGF-1, IGFIIR, FGF-1, FGF-2, HER1, HER2, GH, Erβ, and CTGF changed by < ± 5%, as was observed for control housekeeping proteins (Fig. 4C1). However, protein levels of IGF-1, FGF-2, GH, and CTGF were upregulated and of FGF-1 and Erβ were downregulated by DCE-5 (Fig. 4C2). These results suggest DCE-2.5 and DCE-5 slightly increased growth factor levels in murine hepatocytes.
Effects of DCE on the expressions of RAS signaling proteins in mouse livers
Mouse livers treated with DCE-5 showed higher expressions of JNK-1 (106.3%), and those treated with DCE-10 showed higher expression of pAKT1/2/3 (106.3%) than non-treated controls. The expression levels of other RAS signaling proteins, KRAS, NRAS, STAT3, SOS-1, SOS-2, RAF-B, ERK-1, p-ERK-1, PI3K, and MEKK changed by < ± 5% in response to DCE as was observed for control housekeeping proteins (Fig. 4D1), but DCE-5 increased the levels of RAF-B, ERK-1, PI3K, JNK-1, pAKT1/2/3 (Fig. 4D2). These results suggested that DCE-2.5 and DCE-5 slightly enhanced RAS signaling in murine hepatocytes.
Effects of DCE on levels of NFkB signaling proteins in mouse livers
Mouse livers treated with DCE-2.5 showed higher PGC-1α (105.6%) and lower AMPK (93.8%) than non-treated controls, whereas those treated with DCE-5 and DCE-10 showed higher p-38 (107.6%) levels and lower mTOR (94.7%) levels. The levels of other NFkB signaling proteins, that is, NFkB, IKK, ERK-1, p-ERK, GADD45, GADD153, and MDR changed by < ± 5% in response to DCE as was observed for control housekeeping proteins (Fig. 5A1), but DCE-5 increased the level of IKK, p38, p-p38, ERK-1, and PGC-1α (Fig. 5A2). These results suggested DCE-2.5 and DCE-5 slightly inhibited NFkB signaling in murine hepatocytes.
Effects of DCE on cellular stress and adaptation-related protein levels in mouse livers
Mouse livers treated with DCE-2.5 showed a slight increase in MDM2 (105.5%), whereas those treated with DCE-5 or DCE-10 showed higher PKC (110.8%), leptin (109.4%), p38 (105.3%), and pAKT1/2/3 (106.3%) levels and lower mTOR (94.7%) levels than non-treated controls. The expression levels of other cellular stress and adaptation-related proteins, that is, LC3, GADD45, ERK-1, MDR, PLC-β2, AKAP, NFAT5, and HXK II changed by < ± 5% in response to DCE, as was observed for control housekeeping proteins (Fig. 5B1), but DCE-5 tended to increase p38, ERK-1, PI3K, leptin, PLC-β2, PKC, p-PKC, AKAP, NFAT5, and HXK II levels (Fig. 5B2). These results suggested that DCE-2.5 and DCE-5 slightly enhance cellular stress and adaptation-related protein levels in murine hepatocytes.
Effects of DCE on cellular differentiation-related protein levels in mouse livers
Mouse livers treated with DCE-2.5 and DCE-5 showed higher Jagged 2 (106.3%) and GLI-1 (107.6%) levels, whereas those treated with DCE-10 showed higher caveolin (107.3%) and lower SP-3 (93.5%), Notch 1 (90.8%), and Jagged 2 (91.9%) levels than non-treated controls. The expression levels of other cellular differentiation-related proteins, p63, TGase-2, Muc1, Muc4, AP-1, and SP-1 changed by < ± 5% in response to DCE, as was observed for control housekeeping proteins (β-actin, α-tubulin, and glyceraldehyde-3-phosphate dehydrogenase, GAPDH) (Fig. 5C1), but DCE-5 tended to increase caveolin, Jagged 2, Notch 1, GLI-1, Muc4, and SP-1 levels (Fig. 5C2). These results suggested DCE-2.5 and DCE-5 slightly enhance the expressions of cellular differentiation-related proteins in murine hepatocytes.
Effects of DCE on inflammatory proteins upregulated in mouse livers
Mouse livers treated with DCE-5 and DCE-10 showed higher MMP-9 (109%), COX-1 (105.6%), and IL-12 (105.4%) levels than non-treated controls. The expression levels of other inflammatory proteins, that is, IL-8, hepcidin, α1-AT, LL-37, TGF-β1, TGF-β2, CD31, MMP-1, MMP-3, and MMP-10 were upregulated by < ± 5% in response to DCE, as was observed for control housekeeping proteins (Fig. 6A1). However, LL-37, COX-1, and MMP-10 tended to be upregulated by DCE-5 as compared with housekeeping controls (Fig. 6A2). These results suggested DCE-5 and DCE-10 tended to increase the expressions of inflammatory proteins for cellular regeneration in mouse livers.
Effects of DCE on inflammatory proteins downregulated in mouse livers
TNFα (95.1%), IL-10 (90.2%), COX-2 (92.5%), CD68 (93.1%), M-CSF (92%), and CRP-1 (91.7%) were lower in DCE-treated mouse livers than in non-treated controls. Levels of other inflammatory proteins, that is, lysozyme, CD99, lactoferrin, IL-1, IL-6, IL-28, CD56, MMP-2, MMP-12, cathepsin C, and cathepsin G were changed by < ± 5% in response to DCE, as was observed for control housekeeping proteins (Fig. 6B1), but DCE-5 tended to reduce TNFα, IL-10, M-CSF, COX-2, CD68, CD99, cathepsin G, IL-1, and CD56 levels (Fig. 6B2). These results suggested DCE-2.5 and DCE-5 reduced the expressions of inflammatory proteins eliciting inflammatory reaction in mouse livers.
Effects of DCE on p53-mediated apoptosis-related protein levels in mouse livers
Mouse livers treated with DCE-5 or DCE-10 showed higher expressions of BAX (107.7%), BAD (106.2%), APAF-1 (105%), c-PARP (109.4%), and c-caspase 9 (107.9%) than non-treated controls. The expression levels of other p53-mediated apoptosis-related proteins, p53, MDM2, BAK, BCL2, AIF, and BID changed by < ± 5% in response to DCE, as was observed for control housekeeping proteins (Fig. 6C1), but DCE-5 tended to increase the levels of BAX, BCL2, APAF-1, c-caspase-9, and c-PARP (Fig. 6C2). These results suggested that DCE-5 and DCE-10 slightly enhance the expressions of p53-mediated apoptosis-related proteins in murine hepatocytes.
Effects of DCE on FAS-mediated apoptosis-related protein levels in mouse livers
Mouse livers treated with DCE-10 had higher FASL (106.1%), FAS (105.6%), and FLIP (106%) levels than non-treated controls. The levels of other FAS-mediated apoptosis-related proteins, that is, FADD, caspase 8, caspase 3, c-caspase 3, and BID, changed by < ± 5% in response to DCE, as was observed for control housekeeping proteins (Fig. 6D1, D2). These results suggested that DCE-2.5 and DCE-5 did not induce FAS-mediated apoptosis-related protein expressions in murine hepatocytes.
Effects of DCE on angiogenesis-related protein levels in mouse livers
Mouse livers treated with DCE-5 or DCE-10 had higher FLT-4 (106.7%) and COX-1 (105.6%) levels, and those treated with DCE-10 had higher leptin (109.4%) and PAI-1 (107.4%) levels but lower VCAM (94.8%) levels than non-treated controls. The expression levels of other angiogenesis-related proteins, that is, of HIF, VEGF-A, VEGF-C, CMG2, angiogenin, vWF, LYVE-1, CD31, FGF-2, ET-1, and PDGF-A changed by < ± 5% in response to DCE, as was observed for housekeeping controls (Fig. 7A1), but DCE-5 tended to increase CMG2, LYVE-1, PAI-1, ET-1, COX-1, leptin, and VCAM levels (Fig. 7A2). These results suggested DCE-2.5 and DCE-5 slightly enhanced the expressions of angiogenesis-related proteins in mouse livers.
Effects of DCE on antioxidant and protection-related protein levels in mouse livers
Mouse livers treated with DCE-2.5 or DCE-5 had lower NRF2 (90.5%), NOS-1 (91%), and SOD-1 (88.6%) levels than non-treated controls and those treated with DCE-10 had higher leptin (109.4%) levels but consistently lower NRF2 (95.6%), NOS-1 (93.5%), and SOD-1 (90.2%) levels than non-treated controls. The expression levels of other antioxidant and protection-related proteins, that is, HSP-27, HSP-70, HSP-90, HO-1, GST-1, LC3, and hepcidin, changed by < ± 5% in response to DCE, as was observed for housekeeping controls (Fig. 7B1), but DCE-5 tended to increase HO-1, GST-1, and leptin levels (Fig. 7B2). These results suggested DCE-2.5 and DCE-5 reduced free radical concentrations in mouse livers and subsequently reduced antioxidant-related protein levels, but did not affect the expressions of cellular protection-related proteins in murine hepatocytes.
Effects of DCE on oncogenic protein levels in mouse livers
Mouse livers treated with DCE had lower YAP1 (90.3%), ATM (93.8%), and TERT (94.6%) levels, but slightly higher pAKT1/2/3 (106.3%) levels than non-treated controls. The expression levels of other oncogenic proteins, that is, PTEN, DMBT1, CEA, 14-3-3, survivin, Muc1, Muc4, and MBD4 changed by < ± 5% in response to DCE, as was observed for control housekeeping proteins (Fig. 7C1), but DCE-5 tended to reduce CEA, MBD4, YAP1, and TERT levels (Fig.7C2). These results suggested DCE slightly reduced the expressions of oncogenic proteins in murine hepatocytes.
Global protein expressions in mouse livers treated with DCE-5
Global protein expressions induced by DCE in mouse liver changed by < ± 10%, that is, they probably remained in the physiological homeostatic range. The levels of many proteins essential for molecular signaling were altered by ± 5% as was observed for housekeeping controls (β-actin, α-tubulin, and glyceraldehyde-3-phosphate dehydrogenase, GAPDH). These results indicated DCE treatment caused minimal cellular stress and did not induce inflammatory or chemical stress or oncogenic injury.
Generally, mouse livers treated with DCE-5 showed characteristic changes in functional protein levels (Fig. 8). Increases in the 10% range were observed for proliferation-related proteins (MPM2, CDK4, and Ki-67), growth factors (HGF-1, IGF-1, HER2, GH, GHRH, insulin, KRAS, RAF-B, ERK-1, pAKT1/2/3, and JNK-1), cellular adaptation-related proteins (leptin, PKC, AKAP, and HXK II), cellular differentiation-related proteins (Jagged 2, Notch 1, GLI-1, Muc1, Muc4, and SP-1), inflammatory proteins (IL-6, IL-8, IL-12, TGF-β1, COX-1, and LL-37), p53-mediated apoptosis-related proteins (BAX, APAF-1, c-caspase 9, and c-PARP), angiogenesis-related proteins (VEGF-C, CMG2, LYVE-1, FGF-2, and VCAM), and antioxidant and protection-related proteins (HO-1, HSP-70, GST-1, leptin, and hepcidin). On the other hand, decreases in the 10% range were observed for NFkB signaling proteins (mTOR, LC3, and GADD45), inflammatory proteins (TNFα, COX-2, CD68, and cathepsin G), and oncogenic proteins (TERT, MBD4, and YAP1). cMyc/MAX/MAD network proteins, p53/Rb/E2F signaling proteins, Wnt/β-catenin signaling proteins, epigenetic modification-related proteins, protein translation-related proteins, and FAS-mediated apoptosis-related proteins were rarely affected by DCE treatment (Fig. 8).