Abstract
Coenzyme Q10 (CoQ10) which acts as an electron transporter in the mitochondrial respiratory chain has many beneficial effects on liver diseases. In our previous research, CoQ10 has been found to attenuate acetaminophen (APAP)-induced acute liver injury (ALI). However, whether CoQ10 administration is still effective at the late stage of APAP overdose is still unknown. In this study, we aimed to test CoQ10 efficacy at the late stage of APAP overdose. C57BL/6J mice were intraperitoneally treated with APAP to induce liver injury. CoQ10 (5 mg/kg) was given to mice at 16 h after APAP treatment. The results showed that while CoQ10 treatment at 16 h post-APAP overdose had no effects on the expression of ROS generated genes or scavenged genes, it still significantly decreased necrosis of hepatocytes following APAP-induced ALI. Moreover, CoQ10 increased MerTK+ macrophages accumulation in the APAP-overdose liver and inhibition of MerTK signaling partly abrogated the protective role of CoQ10 treatment on the hepatic necrosis. CoQ10 treatment also significantly enhanced hepatocytes proliferation as shown in the increased 5-bromodeoxyuridine incorporation in the APAP-intoxicated mice liver section. In addition, CoQ10 treatment increased hepatic Proliferating Cell Nuclear Antigen (PCNA) and Cyclin D1 expression and promoted activation of the β-catenin signaling in APAP-overdose mice. To conclude, these data provide evidence that CoQ10 treatment is still effective at the late stage of APAP-induced ALI and promotes resolution of necrosis and liver regeneration following ALI.
Acetaminophen (APAP) overdose is the leading cause of drug-induced acute liver injury (ALI; Larson et al., 2005). APAP overdose can further induce fulminant liver failure (FLF) which is a major health problem worldwide. Most of previous research have focused on the mechanism and treatment of the APAP hepatoxicity. And the most successful therapeutic approach of APAP-induced ALI is the administration of N-acetylcysteine (NAC) as antidote. However, the NAC treatment is effective only at early stage (Athuraliya and Jones, 2009; Bernal et al., 2015). APAP overdose patients who fail to treat with NAC timely may further develop FLF and need suitable liver for transplantation. The shortage of suitable livers for transplantation, the large cost resulting from transplantation and associated lifelong immunosuppression drive the search to novel therapies to treat development of APAP-induced FLF. Compensatory liver regeneration occurs after liver injury in APAP overdose, which is a critical factor to influence the survival of APAP overdose (Apte et al., 2009; Horn et al., 1999). Liver regeneration capacity can be regulated even at the late stage of APAP overdose (Bhushan et al., 2017). Therefore, stimulating liver regeneration may have a great therapeutic potential to treat APAP-induced FLF.
Coenzyme Q10 (CoQ10) which acts as an electron transporter in the mitochondrial respiratory chain has many beneficial effects on liver diseases. CoQ10 supplementation prevents inflammation, hepatic fibrosis, and hepatocarcinogenesis in rat model (Abdel-Latif et al., 2020; Mohamed et al., 2019). Moreover, studies also demonstrate that CoQ10 has therapeutic potential in metabolic stress-induced liver injury as well as APAP caused hepatoxicity (Chen et al., 2019; Elshazly et al., 2020; Fouad and Jresat, 2012). Our previous research finds that hepatic CoQ10 levels decreased with the progression of APAP-induced liver injury (Zhang et al., 2021). And CoQ10 treatment at early stage of APAP overdose attenuates liver injury via scavenging the reactive oxygen species (ROS) and enhancing mitophagy (Zhang et al., 2021). However, whether CoQ10 administration is still effective at the late stage of APAP overdose is still unknown. In this study, we further test CoQ10 efficacy in the late stage of APAP overdose and find CoQ10 treatment promotes resolution of necrosis and increase liver regeneration capacity which suggests CoQ10 administration may have therapeutic potential to improve APAP-induced FLF.
MATERIALS AND METHODS
Reagents and antibodies
APAP (A105808), CoQ10 (C111044), and soybean lecithin (L105732) were obtained from Aladdin. UNC2250 (S7342) and Polyethylene glycol (PEG300; S6704) were purchased from Selleck. Antibodies against MerTK (ab184086), CD163 (ab182422), and Ly6G (ab238132) were purchased from Abcam. Antibody against Proliferating Cell Nuclear Antigen (PCNA; sc-56) was obtained from Santa Cruz. Antibodies against phosphor-GSK-3β (9322), cyclin D1 (55506), β-catenin (8480), and phosphor-β-catenin (9561) were purchased from Cell Signaling Technology.
Animal models
Mice were maintained in the specific pathogen-free facility at the School of Public Health, Sun Yat-sen university. This study was approved by the Animal Care and Protection Committee of Sun Yat-sen University. All animal procedures were conducted in accordance with the National Institutes of Health guide for the care and use of Laboratory animals (NIH Publications No. 8023, revised 1978). Eight-week-old male C57BL/6J mice were purchased from the Experimental Animal Center of Guangdong Province. For examining the changes of CoQ10 levels at the late stage of APAP overdose, mice were injected with 400 mg/kg APAP and sacrificed at 0, 16, 32, 48, and 72 h after APAP overdose (n = 5/group). In order to investigate CoQ10 role on the late stage of APAP overdose, mice were divided into 4 groups: (1) normal saline + lecithin group, (2) normal saline + CoQ10 group, (3) APAP + lecithin group, and (4) APAP + CoQ10 group. APAP and CoQ10 were dissolved in normal saline or soybean lecithin, respectively. Mice were received intraperitoneal injections of APAP (400 mg/kg) after being fasted overnight. CoQ10 administered to the mice intraperitoneally at 16 h after APAP overdose at the dose of 5 mg/kg. After euthanasia, mice were sacrificed at 32, 48, or 72 h after APAP overdose. To test the NAC efficacy at the late stage of APAP overdose, mice were received NAC (100 mg/kg) at 16 h after APAP overdose and sacrificed at 32 h after APAP overdose (n = 5/group). For analyzing MerTK signal on the protective role of CoQ10, UNC2250, dissolved in a solvent containing 5% dimethyl sulfoxide (DMSO), 30% PEG300 and ddH2O, was used to inhibit MerTK signaling. Mice were divided into 4 groups: (1) APAP group, (2) APAP + UNC2250 group, (3) APAP + CoQ10 group, and (4) APAP + CoQ10 + UNC2250 group (n = 8/group). C57BL/6 mice were pretreated with UNC2250 (5 mg/kg) or vehicle control, administrated CoQ10 or lecithin at 16 h after APAP overdose and sacrificed at 32 h after APAP overdose. Serum and liver samples were collected for further analysis.
Histological examination
Liver samples were fixed in 4% formalin, embedded in paraffin, sectioned at 4-μm thickness. The sections were further stained with hematoxylin and eosin (H&E), and then observed the histopathological changes by light microscopy.
CoQ10 measurement
CoQ10 levels in serum or liver were measured by high-performance liquid chromatography, as previous described (Zhang et al., 2021). C18 column (3.5 μm, 2.1 × 100 mm, WAT058965, Waters) was used to separate CoQ10. The mobile phase consisted of 40% methanol and 60% 2-propanol. The flow rate was 1 ml/min. CoQ10 levels were determined and calculated by using peak areas.
Immunofluorescence staining
Liver tissues embedded in Tissue-tek embedding medium (Sakura Finetek) were cut into 10-μm sections. After blocked with 2% bovine serum albumin (BSA) for 30 min at room temperature, the slides were incubated with primary antibodies including CD163, Ly6G, and MerTK (Abcam) overnight at 4°C and then with the corresponding secondary antibody for an additional 1 h at room temperature in dark. Slides were mounted using an antiquenching agent (Beyotime) prior to observation. The fluorescent intensity was analyzed using ImageJ software.
Immunohistochemistry
Liver tissues embedded in paraffin were cut into 4-μm sections. After deparaffinized, rehydrated and received antigen retrieval, sections were blocked with 2% BSA for 30 min at room temperature. The slides were then incubated with primary antibodies including PCNA (Abcam) overnight at 4°C and then with the biotinylated immunoglobulin G (IgG) secondary antibody for an additional 1 h at room temperature. After washed by PBS buffer, the slides were then incubated with Vectastain ABC reagent for 1 h and diaminobenzidine (DAB) substrate solution for 3 min. Finally, the slides were counterstained with hematoxylin and observed by light microscopy.
5-Bromodeoxyuridine cell proliferation assay
5-Bromodeoxyuridine (BrdU; Sigma-Aldrich, St Louis, Missouri) was injected intraperitoneally with 100 mg/kg (body weight) in PBS at 1 h before sacrifice. The liver in Tissue-Tek was cut to 10-μm sections to detect the cell proliferation.
ALT/AST detection
Commercial kits (C009-1 and C010-1, Jiancheng Bioengineering) were used to detect the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST).
Real-time RT-PCR
Trizol reagent (Invitrogen) was used to extract total RNA from liver tissues, and reverse transcription was performed with a PrimeScript RT Reagent Kit (TaKaRa, Tokyo, Japan) according to the manufacturer’s instructions. The mRNA expression was quantified in a 96-well plate using a 7500 Real-Time PCR System (Applied Biosystems, Inc., Foster City, California) with SYBR Green Master Mix (TaKaRa, Tokyo, Japan). The relative mRNA expression levels were determined using the method with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control for normalization.
Western blotting
Nuclear extracts were obtained using a commercially available nuclear extraction buffer (Beyotime, China). Following cytoplasmic and nuclear extraction, protein concentrations were determined. Total cellular, mitochondrial or nuclear lysates (30 μg) were separated by 8–12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto an Immobilon-P membrane (Millipore, Bedford, Massachusetts). After blocking with 5% nonfat milk, blots were incubated with various primary antibodies, followed by horseradish peroxidase (HRP)-labeled secondary antibodies (Santa Cruz). The protein bands were detected with an enhanced chemiluminescence kit (Roche). GAPDH was used for normalization.
Statistical analysis
The data are presented as the means ± SD. One-way analysis of variance with the Student-Newman-Keuls test was applied for analyzing data. Prism software (Prism 7.0, GraphPad Software) was used for all analyses. The difference was considered significant for p values < .05.
Results
Hepatic CoQ10 Levels Were Replenished at the Late Stage of APAP Overdose
First, we measured the temporal changes of hepatic and serum CoQ10 levels at the late stage of APAP overdose. Hepatic CoQ10 levels were decreased significantly at 16 h after APAP overdose and gradually replenished at 32 h after APAP overdose (Figure 1A). At 72 h, the content of CoQ10 in liver returned to the level before APAP treatment (Figure 1A). And there were no changes in the serum CoQ10 levels (Figure 1B).
Changes in hepatic Coenzyme Q10 (CoQ10) levels and serum CoQ10 levels at the late stage of APAP overdose. Eight-week-old male C57BL/6J mice were intraperitoneally injected with acetaminophen (APAP; 400 mg/kg). Hepatic CoQ10 levels (A) and serum CoQ10 concentrations (B) were measured at 0, 16, 32, 48, and 72 h after APAP overdose. The data are expressed as the mean ± SD, n = 5, *p < .05 versus 0-h level; #p < .05 versus 16-h level.
CoQ10 Decreased Necrosis of Hepatocytes Following APAP-Induced ALI
In order to investigate CoQ10 role on the late stage of APAP overdose, C57BL/6 mice were administrated CoQ10 or NAC at 16 h after APAP overdose when ALI is established (Starkey Lewis et al., 2020). At 32 h after APAP overdose, mice were sacrificed and we found that NAC or CoQ10 treatment had no effects on the serum ALT or AST activity (Figs. 2A and 2B and Supplementary Figs. 1A and 1B). However, while NAC treatment did not improve the necrosis, H&E staining results showed that CoQ10 treatment significantly reduced necrotic area compared with lecithin controls (Figure 2C and Supplementary Figure 1C).
Coenzyme Q10 (CoQ10) improved necrosis of hepatocytes in acetaminophen (APAP)-treated mice. Eight-week-old male C57BL/6J mice were divided into 4 groups: (1) normal saline + lecithin control group, (2) normal saline + CoQ10 group, (3) APAP + lecithin control group, and (4) APAP + CoQ10 group. CoQ10 was administered to the mice intraperitoneally 16 h after APAP (400 mg/kg) treatment at the dose of 5 mg/kg. Mice were sacrificed at 32 h after the APAP injection. Serum ALT (A) and AST (B) levels were measured. Representative images of hematoxylin and eosin staining (C) were shown (magnification, ×200). The data are expressed as the mean ± SD, n =8. Labeled means without a common letter differ, p < .05.
To determine whether the administration of CoQ10 at 16 h provides sustained protection at later time points, C57BL/6 mice administrated with CoQ10 at 16 h were sacrificed at 48 or 72 h after APAP overdose. H&E staining showed that with the passage of time, the necrotic area in the liver of APAP-overdose mice decreased (Supplementary Figure 2). And compared with controls, the administration of CoQ10 further reduced the area of the necrosis (Supplementary Figure 2).
CoQ10 Increased MerTK+ Macrophages Accumulation in Liver Following APAP-Induced ALI
Macrophages with the expression of clearance receptors such as MerTK have the ability of clearance of necrotic cells which is important to resolve the inflammation and injury effectively (Lemke, 2019). Immunofluorescence results showed that CoQ10 treatment compared with lecithin control markedly increased the hepatic MerTK+CD163+ macrophages accumulation in the APAP-overdose mice (Figure 3A). Furthermore, CoQ10 treatment also attenuated Ly6G+ hepatic neutrophil infiltration after APAP-induced ALI (Figure 3B). To next, interrogate the causal effects of MerTK signal on the protective role of CoQ10, UNC2250 was used to inhibit MerTK signaling. C57BL/6 mice were pretreated with UNC2250 (5 mg/kg) and administrated CoQ10 at 16 h after APAP overdose. UNC2250 treatment significantly decreased the hepatic MerTK+CD163+ macrophages accumulation in the APAP-overdose mice (Figure 3C). Moreover, UNC2250 treatment remarkedly increased hepatic necrotic area (Figure 3D) and the levels of serum ALT and AST (Supplementary Figure 3) in the APAP-overdose mice and partly abrogated the protective role of CoQ10 treatment on the hepatic necrosis.
Coenzyme Q10 (CoQ10) increased MerTK+ macrophages accumulation in liver following acetaminophen (APAP)-induced acute liver injury. C57BL/6J mice were divided into 4 groups: (1) normal saline + lecithin control group, (2) normal saline + CoQ10 group, (3) APAP + lecithin control group, and (4) APAP + CoQ10 group. CoQ10 was administered to the mice intraperitoneally 16 h after APAP (400 mg/kg) treatment at the dose of 5 mg/kg. Mice were sacrificed at 32 h after the APAP injection. A, Representative images of MerTK (red) and CD163 (green) immunofluorescence were shown (magnification, ×200). B, Representative images of Ly6G (red) were shown (magnification, ×200). C57BL/6J mice were divided into 4 groups: (1) APAP group, (2) APAP + UNC2250 group, (3) APAP + CoQ10 group, and (4) APAP + CoQ10 + UNC2250 group (n = 5/group). C57BL/6 mice were pretreated with UNC2250 (5 mg/kg), administrated CoQ10 at 16 h after APAP overdose and sacrificed at 32 h after APAP overdose. C, Representative images of MerTK (red) and CD163 (green) immunofluorescence were shown (magnification, ×200). D, Representative images of hematoxylin and eosin staining were shown (magnification, ×200). The data are expressed as the mean ± SD, n =8. Labeled means without a common letter differ, p < .05.
CoQ10 Treatment at 16 h post-APAP Overdose Had No Effects on the ROS Levels
The APAP-induced imbalance of ROS production and scavenging leads to the accumulation of ROS in the hepatocytes which plays a central role in the APAP hepatoxicity. Previous studies have shown that CoQ10 pretreatment or treatment at the beginning stage of APAP hepatoxicity can reduce the ROS levels and protect against liver injury (Amimoto et al., 1995; Zhang et al., 2021). Next, we further examined whether CoQ10 treatment at the late stage of APAP overdose still affected ROS levels in hepatocytes. And CoQ10 treatment at 16 h post-APAP overdose did not change the total glutathione (GSH) or oxidized glutathione (GSSG) levels (Figs. 4A and 4B). Furthermore, qPCR results showed that CoQ10 had no significant effects on the expression of ROS generated genes or scavenged genes (Figs. 4C and 4D).
Coenzyme (CoQ10) treatment at 16 h post- acetaminophen (APAP) overdose had no effects on the ROS levels. C57BL/6J mice were divided into 4 groups: (1) normal saline + lecithin control group, (2) normal saline + CoQ10 group, (3) APAP + lecithin control group, and (4) APAP + CoQ10 group. CoQ10 was administered to the mice intraperitoneally 16 h after APAP (400 mg/kg) treatment at the dose of 5 mg/kg. Mice were sacrificed at 32 h after the APAP injection. The hepatic GSH levels (A) and GSSG levels (B) were measured. C, The mRNA expression of ROS generation-related genes was determined by q-PCR. D, The mRNA expression of ROS scavenging-related genes was determined by q-PCR. The data are expressed as the mean ± SD, n = 8. Labeled means without a common letter differ, p < .05.
CoQ10 Treatment Stimulated Hepatocellular Proliferation After APAP-Induced ALI
Compensatory liver regeneration which is occurred after liver injury has a critical role in the survival after APAP overdose. So next, we examined whether CoQ10 affected the hepatocellular proliferation process after APAP-induced ALI. BrdU was given to mice 1 h before sacrifice to track the proliferation cells. As Figure 5A shown, CoQ10 treatment significantly increased the BrdU incorporation in liver tissue compared with lecithin control. PCNA plays an important role in the cell cycle regulation and DNA replication (Bhushan et al., 2014). Immunohistochemical analysis of PCNA showed that CoQ10 treatment significantly increased the PCNA-positive cells surrounding the necrotic zones (Figure 5B). Furthermore, Western blot analysis was consistent with that of immunohistochemistry. CoQ10 elevated the PCNA expression compared with lecithin control in the APAP-overdose mice (Figure 5C). Cyclin D1 induction is a central event that controls cell cycle progression in hepatocytes (Wu et al., 2020). And we also found that cyclin D1 protein expression was significantly higher in the CoQ10-treated group compared with the lecithin control group in the APAP-overdose mice (Figure 5C). Overall, these data indicated that CoQ10 treatment had potential to stimulate hepatocellular proliferation after APAP overdose.
Coenzyme (CoQ10) treatment stimulated hepatocellular proliferation after acetaminophen (APAP)-induced acute liver injury. CoQ10 was administered to the mice intraperitoneally 16 h after APAP (400 mg/kg) treatment at the dose of 5 mg/kg. Mice were sacrificed at 32, 48, or 72 h after the APAP injection. BrdU was given to mice 1 h before sacrifice to track the proliferation cells. A, The representative images of BrdU incorporation in liver tissue were shown (magnification, ×200). B, The representative images of Proliferating Cell Nuclear Antigen (PCNA) immunohistochemistry were shown (magnification, ×400). C, Hepatic expression levels of PCNA and Cyclin D1 were detected by western blot. The data are expressed as the mean ± SD, n =8. *p < .05 versus control group at the same time point.
CoQ10 Activates the β-Catenin Signaling After APAP-Induced ALI
β-catenin is a multifunctional protein which interacts with lymphoid enhancer binding factor (LEF) or transcription factor (TCF) family of transcription factors and activates expression of target genes such as cyclin D1 to promote cells division (Yang et al., 2017). So next we measured the β-catenin activity by CoQ10 treatment in APAP-overdose mice. Phosphorylation of β-catenin can lead to proteosomal-mediated degradation of itself (Taurin et al., 2006). And Western blot analysis showed that while CoQ10 treatment did not change the total β-catenin levels in the livers of APAP-induced ALI mice, CoQ10 significantly decreased the phosphorylation of β-catenin (Figure 6A). Phosphorylation of glycogen synthase kinase-3β (GSK-3β) at Ser9 activate β-catenin signal and promote its nuclear translocation. CoQ10 treatment also remarkedly increased phosphorylation of GSK-3β at Ser9 (Figure 6A). Furthermore, the nuclear β-catenin levels were also higher in CoQ10 treatment group compared with lecithin control, whereas cytosolic β-catenin levels were significantly lower in CoQ10 treatment group (Figure 6B). These results suggested that CoQ10 may activate β-catenin to enhance hepatocellular proliferation at the late stage of APAP overdose.
Coenzyme (CoQ10) activates the β-catenin signaling after acetaminophen (APAP)-induced acute liver injury. C57BL/6J mice were divided into 4 groups: (1) normal saline + lecithin control group, (2) normal saline + CoQ10 group, (3) APAP + lecithin control group, ad (4) APAP + CoQ10 group. CoQ10 was administered to the mice intraperitoneally 16 h after APAP (400 mg/kg) treatment at the dose of 5 mg/kg. Mice were sacrificed at 32 h after the APAP injection. A, Western blot analysis of β-catenin, p-β-catenin, and p-GSK-3β in mice liver lysates. B, Western blot analysis of β-catenin in the nuclear or cytosolic extract from mice liver. The data are expressed as the mean ± SD, n = 8. Labeled means without a common letter differ, p < .05.
DISCUSSION
In the past, the research about APAP-induced liver pathophysiology has mainly focused on the mechanism of APAP hepatotoxicity and targeting liver injury for therapeutic strategies. However, many patients who have APAP overdose often get medical treatment too late that liver injury is already established. Nowadays, NAC is still only treatment standard for APAP overdose. And NAC fail to protect mice from APAP overdose when administered 4 h after APAP (James et al., 2003). In human, NAC efficacy is also diminished when liver injury has already been established and prolonged treatment with NAC may even have detrimental effects on the prognosis of APAP overdose (Yang et al., 2009). Therefore, new therapeutic approaches are needed to treat APAP-overdose patients with established liver injury. CoQ10 is a nutrient which is found in variety of food and can be synthesized in all tissues. In cells, CoQ10 is coenzyme for mitochondrial enzymes and acts as an electron transporter. Furthermore, CoQ10 is also a powerful lipid-soluble antioxidant which has beneficial role in the liver diseases. Our previous research finds that hepatic CoQ10 levels decreased with the progression of APAP-induced liver injury (Zhang et al., 2021). And we and other research verify that CoQ10 can prevent the initiation of APAP-induced hepatotoxicity and ameliorate the liver injury caused by the APAP overdose (Amimoto et al., 1995; Fouad and Jresat, 2012; Zhang et al., 2021). However, as far as we know, whether CoQ10 treatment is effective at the late stage of APAP overdose is first reported in this study. Here, our results demonstrate that CoQ10 treatment at the late stage of APAP overdose can also decrease the necrosis, promote the regeneration of hepatocytes and accelerate resolution of necrosis.
A large amount of hepatocyte death and severe liver inflammation is the key feature of acute liver failure (Dong et al., 2020). Macrophages with the expression of clearance receptors such as MerTK are critical in liver homeostasis and the response to APAP-induced liver injury (Triantafyllou et al., 2018). MerTK+ macrophages are critical in the phagocytosis of apoptotic cells. APAP-treated Mer−/− mice showed severe liver injury and inflammation (Triantafyllou et al., 2018). Induction of MerTK+ macrophages promote the resolution of necrosis after APAP overdose and decrease number of neutrophils (Triantafyllou et al., 2018). In our study, we also observed that CoQ10 treatment increased MerTK+ macrophages accumulation in the areas of centrilobular necrosis and decreased hepatic neutrophil infiltration at the late stage of APAP overdose. Moreover, MerTK signaling inhibition increased hepatic necrotic area in the APAP-overdose mice and partly abrogated the protective role of CoQ10 treatment on the hepatic necrosis, which suggests CoQ10 may promote resolution of APAP-induced liver necrosis via expanding MerTK+ macrophages. However, further research is needed to verify the CoQ10 role on the MerTK+ macrophages function.
Liver regeneration is an important aspect to influence the outcome of APAP overdose especially at the late stage of APAP overdose when the liver injury has been established (Bhushan and Apte, 2019). In our study, CoQ10 treatment significantly enhanced hepatocytes proliferation as shown in the increased BrdU incorporation in the liver section. PCNA which acts as a processivity factor for DNA polymerase delta and is essential for DNA replication was markedly upregulated by CoQ10 treatment. Furthermore, cyclin D1 is necessary in the cell cycle. In the G1 phase, cyclin D1 expression is induced rapidly and accumulates in the nucleus to regulate G1/S phase transition which is a critical event in the cell proliferation (Fausto, 2000). At the late stage of APAP overdose, cyclin D1 expression is positively correlated with liver regeneration and benign outcome (Bhushan et al., 2014). And in this study, we also found that CoQ10 treatment increased the cyclin D1 expression. Overall, these results demonstrate CoQ10 treatment at the late stage of APAP overdose stimulates the hepatocytes proliferation and promotes the liver regeneration which may have beneficial effects in the outcome of APAP overdose.
The cell proliferation process is modulated by many transcriptional factors. β-catenin is one of the most important transcriptional factors to regulate cell proliferation. β-catenin expression and transcriptional activity is precisely regulated by its phosphorylation. When β-catenin is phosphorylated, it will further be ubiquitinated by β-Trcp and degraded through proteasomal pathway (Stamos and Weis, 2013). Hypophosphorylated β-catenin can translocate to the nucleus, associate with TCF/LEF family of transcriptional factors and displaces the negative regulatory elements from TCF/LEF to activate target genes expression including cyclin D1 (Karim et al., 2004; Novak and Dedhar, 1999). In the partial hepatectomy model, overexpression β-catenin stimulate the liver regeneration at the early time with the increased cyclin D1 expression (Nejak-Bowen et al., 2010). In line with this finding, liver-specific knockout of β-catenin lead to delayed liver regeneration after partial hepatectomy with the lower cyclin D1 expression (Sekine et al., 2007; Tan et al., 2006). Liver regeneration after APAP overdose is also modulated by β-catenin signaling. Activation of β-catenin promotes liver regeneration after APAP-induced liver injury (Apte et al., 2009). And inhibition of GSK-3 also promotes the liver regeneration in APAP-overdose mice by activation of β-catenin (Bhushan et al., 2017). In this study, we found that CoQ10 treatment decreased phosphorylated β-catenin expression and increased the nuclear β-catenin accumulation at the late stage of APAP overdose. These results suggest CoQ10 may stimulate liver regeneration via β-catenin activation.
To conclude, in this study, we test CoQ10 role on the late stage of APAP overdose and find that CoQ10 treatment can still promote the resolution of necrosis and liver regeneration after APAP overdose. It suggests that CoQ10 treatment may be a potential therapeutic choice for APAP-overdose patients when the liver injury has been established.
SUPPLEMENTARY DATA
Supplementary data are available at Toxicological Sciences online.
AUTHOR CONTRIBUTIONS
S.C.: methodology, investigation, writing—original draft; Y.T.: formal analysis, data curation, investigation; W.F.: investigation; T.H.: supervision; X.C.: conceptualization, supervision, funding acquisition, writing—review and editing; P.Z.: conceptualization, supervision, funding acquisition, writing—review and editing.
DECLARATION OF CONFLICTING INTERESTS
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
FUNDING
Guangdong Basic and Applied Basic Research Foundation (2020A1515111148), Discipline construction project of Guangdong Medical University (4SG21016G), and China Postdoctoral Science Foundation (2020M683134).
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