- Email: [email protected]
mTOR pathway
European Journal of Pharmacology 850 (2019) 15–22
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Acetaminophen aggravates fat accumulation in NAFLD by inhibiting autophagy via the AMPK/mTOR pathway Congjian Shia,b, Weiju Xuea,b, Bowen Hana,b, Fengli Yanga,b, Yaping Yina,b, Chengmu Hua,b, a b
T ⁎
Institute for Liver Diseases of Anhui Medical University, School of Pharmacy, Anhui Medical University, Hefei 230032, China Key Laboratory of anti-inflammatory and Immune Medicine, Ministry of Education, Hefei 230032, China
A R T I C LE I N FO
A B S T R A C T
Keywords: NAFLD APAP Autophagy AMPK mTOR Mice
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease which affects millions of people worldwide. Acetaminophen (APAP) overdose is the leading cause of acute liver failure. In this study, APAP (50, 100, 200 mg/kg) were employed on mice fed with a high-fat diet, and APAP (2, 4, 8 mM) were cultured with L02 cells in the presence of alcohol and oleic acid. APAP treatment significantly aggravated hepatic lipid accumulation, increased the serum levels of triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and increased hepatic lipid accumulation in H&E and Oil red O staining results. Transmission electron microscopy (TEM) found fewer number of autophagosomes in APAP (100 mg/kg) treated group. Immunohistochemistry analysis showed the intensity of hepatic mTOR was increased and AMPK was decreased in 200 mg/kg APAP treated group. Western blot analysis showed APAP treatment decreased the levels of LC3-Ⅱ, Beclin1 and AMPK, while increased the levels of mTOR and SREBP-1c, respectively. In vitro study showed APAP treatment obviously increased TG activities in cell supernatant, and Oil red O staining had the same results. Western blot analysis demonstrated APAP treatment decreased the levels of LC3-Ⅱ, Beclin1 and AMPK, increased the levels of mTOR and SREBP-1c, but rapamycin treatment significantly reversed these effects of APAP. In conclusion, therapeutic dosages of APAP aggravates fat accumulation in NAFLD, the potential mechanism might be involved in inhibiting autophagy associated with the AMPK/mTOR pathway, and patients with NAFLD should use a lower dose of APAP.
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is a clinical disease characterized by liver fat exceed 5% of the total liver weight, it can cause severe liver damage and metabolic comorbidities if not controlled (Peng et al., 2018). NAFLD, which encompasses a spectrum from simple hepatic steatosis to steatosis combined with varying degrees of inflammation and fibrosis, is a serious threat to the health of people the world over, with the morbidity rising every year and the age of sufferers getting younger (Zhang et al., 2018; X.Y. Zhao et al., 2018). Patients with steatosis often do not feel that they have the disease for their mild symptoms, when they really realize they are sick, the condition is already very heavy. Acetaminophen (APAP), commonly known as paracetamol, is one of the most frequently used analgesic and antipyretic drugs, because of its effectiveness on reducing fever and pain, the minor irritation to the gastrointestinal tract and the complete oral absorption (Nikravesh et al., 2018). It is safe and effective at therapeutic doses, but an
⁎
overdose can cause hepatotoxicity, acute liver failure (ALF) and even death (Yan et al., 2018). APAP overdose is the leading cause of druginduced acute liver injury. Liver is the main organ that metabolizes APAP in therapeutic doses, approximately 90% is metabolized through hepatic sulfation and glucuronidation pathways (Z. Zhao et al., 2018). Approximately 5% through cytochrome P450 translates into the Nacetyl-p-benzoquinoneimine (NAPQI), which is detoxified by combining with glutathione (GSH) (Shan et al., 2018). However, overdose of APAP can produce excessive NAPQI that could deplete GSH and bind to cellular proteins (APAP protein adducts), result in mitochondrial oxidative stress, liver injury, ROS production and liver failure (Mazraati and Minaiyan, 2018; Wang et al., 2018). APAP-induced hepatotoxicity has drawn lots of attention and solutions, N-acetylcysteine (NAC) is a highly successful approach for treating APAP overdose; however, this protective effect is only observed in the early stage of APAP hepatotoxicity (Shan et al., 2018), and it also has some disadvantages. Autophagy plays an important role in coping with multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia,
Corresponding author at: School of Pharmacy, Anhui Medical University, Hefei 230032, Anhui Province, China. E-mail address: [email protected] (C. Hu).
https://doi.org/10.1016/j.ejphar.2019.02.005 Received 14 November 2018; Received in revised form 2 February 2019; Accepted 8 February 2019 Available online 10 February 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
2.4. Transmission electron microscopy detection
or damaged organelles (Yu et al., 2018). Previous studies showed resveratrol ameliorates alcoholic fatty liver by inducing autophagy (Tang et al., 2016), whether autophagy plays any role during the process of APAP treated NAFLD? Acetaminophen is a commonly used drug that induces serious hepatotoxicity when overdosed, some study found liver affected by NAFLD is more sensitive to acute toxic injury caused by APAP in comparison with non-steatotic liver (Kucera et al., 2012). Other study showed fast food diet-induced non-alcoholic fatty liver disease exerts early protective effect against acetaminophen intoxication in mice (Kim et al., 2017). We are interested in this discrepancy and want to explore the underlying mechanisms. The present study was designed to evaluate whether APAP could affect the level of autophagy, and how can autophagy influence the effects of APAP under NAFLD condition? Therefore, the present study may demonstrate new insights into the rational application of APAP and better avoid its hepatotoxicity.
The liver fragments in the same part of each mouse were fixed in 2.5% glutaraldehyde, then fixed with 1% osmium acid, added gradient alcohol to dehydrate, epoxy resin embedding, and sliced by an ultrathin slicing machine, the sections were stained (Cho et al., 2017) and the images were taken under transmission electron microscopy (HITACHI, Japan). 2.5. Immunohistochemical analysis
2. Materials and methods
The expression of AMPK and mTOR in liver were detected by immunohistochemistry method. The samples from paraffin-embedding tissue were dewaxed, rehydrated, and incubated with the primary antibody (p-AMPK, p-mTOR, 1:200) for overnight at 4 °C. The slides were incubated with secondary antibody (Bioss, Beijing, China) according to the manufacturer's instructions. The brown staining in the cell membrane was considered to be positive.
2.1. Animals and treatments
2.6. Cell culture and treatments
Male C57BL/6 J mice (20–22 g, 6–8 weeks) were purchased from the Animal Center of Anhui Medical University and the experiment was approved by the ethics committee. Mice were housed in plastic cages with food and water ad libitum and maintained at room temperature of (23 ± 2 °C) on a 12 h light/dark cycle. After one week acclimation, the animals were randomly divided into five groups (n = 8 per group): (1) normal control group (NC), (2) high-fat diet control group (MC), (3) MC with 50 mg/kg APAP (Solarbio, Beijing, China) (APAP50), (4) MC with 100 mg/kg APAP (APAP100), and (5) MC with 200 mg/kg APAP (APAP200). Control mice were fed a normal diet (10% calories from fat, 20% calories from protein, 70% calories from carbohydrate; 3.5 Kcal/g diet; TP26312; TROPHIC, Nantong, China), and mice in the other four groups were fed a high-fat diet (42% calories from fat, 15% calories from protein, 43% calories from carbohydrate; 4.5 Kcal/g diet; TP26300; TROPHIC, Nantong, China). The total feeding duration was 56 days. On the last day of study, mice were given different APAP doses (50, 100, 200 mg/kg) by gavage (only once). Mice body weight was recorded every week, and the final liver weight was also recorded. The APAP solution was freshly prepared by dissolving acetaminophen in phosphate-buffered saline (PBS) and warmed to 40 °C. Mice were killed at 24 h after APAP administration and were anesthetized with 10% chloralhydrate solution (3 ml/kg), after blood samples were collected, parts of liver samples were frozen for oil red O staining, fixed in 2.5% glutaraldehyde, or fixed in formalin, and the rest was stored at −80 °C. All procedures were approved by the institutional ethical guidelines for laboratory animal care and use of Anhui Medical University, in accordance with NIH guidelines and were approved by the Animal Care and Use Committee (NO: LLSC20180429).
Human normal liver cell line (L02) cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin solution, cultures were maintained at 37 °C in a humidified 5% CO2 incubator. To induce excessive lipid accumulation in the model of hepatocyte steatosis in vitro, L02 cells were exposed to media supplemented with 100 μM oleic acid and 87 mM alcohol for 48 h-treatment. For the APAP experiments, cells were incubated with different concentrations of APAP during the last 24 h of the 48 htreatment, and were incubated with rapamycin (SIGMA-ALDRICH, USA) during the last 28 h of the 48 h-treatment (Li et al., 2017). Both APAP and rapamycin were dissolved in Dimethyl sulfoxide (DMSO). The cells were divided into six groups: (1) control group (NC), (2) model control group (MC), (3) MC with 2 mM APAP (APAP2), (4) MC with 4 mM APAP (APAP4), (5) MC with 8 mM APAP (APAP8), and (6) MC with 4 mM APAP and 1 μM rapamycin (AR). 2.7. Measurement of intracellular TG and Oil Red O staining Cells were seeded into 6-well plates and treated with designated reagent, then fixed with 4% paraformaldehyde and stained with a freshly prepared working solution of Oil Red O at room temperature for 30 min. To quantify the Oil Red O content, the cell samples were extracted by isopropanol for 5 min, and the optical density of each cell sample was read at 510 nm by a microplate reader (Biotek, USA). The activities of TG in cell supernatant were also measured according to commercial analysis kits (Jiancheng, Nanjing, China) by a microplate reader. 2.8. Western blot analysis Liver specimens and cells were mixed with moderately RIPA buffer to obtain lysates, total protein in the supernatant was measured by using bicinchoninic acid (BCA) Protein Assay Kit (Biyuntian, China). Lysates taken from each sample were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA), blocked with 5% non-fat milk for 2 h at room temperature, and placed overnight at 4 °C with specific antibodies: AMPK, p-AMPK, p-mTOR, Beclin1 (Bioss, Beijing, China) and SREBP-1c (Santa Cruz Bio Technology, Inc) and mTOR, LC3-Ⅱ (Cell Signaling Technology) and β-actin (Zhongshan Jinqiao, Beijing, China). After washing with TBST, the membranes were incubated with a peroxidaselabeled secondary antibody (Zhongshan Jinqiao, Beijing, China) for 1 h. The membranes were detected by an enhanced chemiluminescence (ECL) reaction and exposed to Image Quant LAS 4000 mini (GE Healthcare Bio-Sciences, AB, Uppsala, Sweden). The levels of protein
2.2. Morphological evaluations Formalin-fixed liver samples were paraffin embedded and put on glass slides for hematoxylin and eosin (H&E) staining (Goncalves et al., 2017), 5-µm frozen liver sections were stained with Oil red O with a standard procedure (Lee et al., 2018). Lipid droplets deposition was observed under an optical microscope (Olympus, TH4–200, Japan). 2.3. Serum markers analysis The activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglyceride (TG) were measured according to those commercial assay kits (Jiancheng, Nanjing, China) by a microplate reader (Biotek, USA). 16
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 1. (A) Effects of APAP on mice body weight; (B) Liver index. During 8 weeks of high-fat diet fed, body weights were recorded once a week. APAP were administered after 8 weeks high-fat diet fed, and liver index was measured. Data were mean ± S.D. (n = 8). ##P < 0.01 versus NC; **P < 0.01 versus MC.
expression were quantitatively analyzed with Image J Software. 2.9. Statistical analysis The data were expressed as the (mean ± S.D.). Comparisons between multiple groups were made by one-way analysis of variance (ANOVA) followed by Duncan's test. Differences were considered significant if the P value was less than 0.05. 3. Results 3.1. Body weight and liver index The average body weight did not have significantly difference between the normal group and the other four groups at first week, but from second week, there had significantly increasing body weight in MC and APAP treatment groups compared with that of NC (Fig. 1A). The final liver index of MC and APAP treatment groups were significantly higher than that of NC, meanwhile, the liver index of APAP200 were significantly higher than that of MC (Fig. 1B). 3.2. Serum activities of ALT, AST and TG
Fig. 2. Effects of APAP on the serum levels of ALT, AST and TG in mice. Data were mean ± S.D. (n = 6–8). ##P < 0.01 versus NC; *P < 0.05, **P < 0.01 versus MC.
The serum activities of ALT, AST and TG were markedly increased in MC group, compared to NC. Meanwhile, the activities of ALT, AST and TG were also increased with the dosage of APAP increasing, compared to MC group (Fig. 2).
regulation to autophagy. Current results showed the phosphorylation levels of the mTORC1 were obviously increased in the MC group and APAP groups, compared to the NC and MC group, respectively. As the dosage of APAP increased, the phosphorylation degree of mTORC1 had a further increase (Fig. 4B; Fig. 4D).
3.3. H&E staining and Oil Red O staining pictures Histopathological analysis showed, compare with normal mice, mice in model group developed a higher degree of hepatic steatosis (Fig. 3A) and lipid accumulation (Fig. 3B) in the liver, what's more, with the dosage of APAP increasing, mice treated with APAP developed even more severe hepatic steatosis and more lipid accumulation in the liver.
3.5. APAP decreased autophagosomes As shown in Fig. 5, transmission electron microscopy images reflected that the number of autophagosomes decreased in MC, compared to NC, APAP treatment resulted in further decreasing number of autophagosomes.
3.4. Immunohistochemistry changes of livers 3.6. The effects of APAP on relative protein expression in liver tissues AMPK consumes excess energy by stimulating oxidation of fatty acids, AMPK activation was determined by evaluating the phosphorylation level of the AMPKα subunit at Thr172, which can reflects the activation of AMPK (Yuan et al., 2018). In our study, mice in the MC had a radical decrease in the phosphorylation degree of AMPKα, compared to that in the NC. As the dosage of APAP increased, the phosphorylation degree of AMPKα had a further decrease (Fig. 4A; Fig. 4C). One of the hinge segments in the mTOR mediated lipid accumulation is the activation of mTORC1, and mTORC1 had a negative
As mentioned before, we measured the level of the phosphorylation AMPKα subunit at Thr172 and the level of the phosphorylation mTORC1 to evaluate the activation of AMPK and mTOR, respectively. In liver tissues, as shown in Fig. 6, the protein expressions of mTOR and SREBP-1c were significantly increased in mice treated with APAP, compared with MC group; while the expression of LC3-Ⅱ, Beclin1and AMPK were significantly decreased in mice treated with APAP, compared with MC. 17
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 3. Effects of APAP on accumulation of lipid droplets in liver. Representative (A) H＆E and (B) Oil Red O staining images.
3.7. APAP treatment aggravated lipid accumulation in L02 cells
3.8. The effects of APAP on relative protein expression in L02 cells
Oil Red O staining showed that oleic acid plus alcohol treated obviously increased lipid accumulation in L02 cells, and when treated with APAP, the circumstance became exacerbated. However, rapamycin administration suppressed this effect of APAP (Fig. 7A; Fig. 7B). Intracellular TG result was also in accord with the result of Oil Red O staining (Fig. 7C).
The results observed in vitro were the same as those in liver tissue, as shown in Fig. 8, the expression of LC3-Ⅱ, Beclin1 and AMPK were significantly decreased, while the expressions of mTOR and SREBP-1c were significantly increased in mice treated with APAP, compared with MC. However, rapamycin administrated significantly suppressed the effects of APAP, this evidence implied that autophagy was impaired after administrated APAP and liver lipid deposition became even
Fig. 4. Effects of APAP on Immunohistochemistry changes of livers in mice. (A) Immunohistochemical staining of liver for p-AMPK; (B) Immunohistochemical staining of liver for p-mTOR; (C) densitometric quantification of p-AMPK (n = 3 independent experiments); (D) densitometric quantification of p-mTOR (n = 3 independent experiments). Data were mean ± S.D. (n = 3). #P < 0.05, ##P < 0.01 versus NC; *P < 0.05 versus MC. 18
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 5. Transmission electron microscopy examination of autophagosomes in hepatic tissues. Black arrows denote autophagosomes.
drug-induced acute liver failure. In the UK about 82,000–90,000 people a year went to hospital because of misuse or abuse of APAP (Bateman et al., 2014; Caparrotta et al., 2018), and liver injury caused by APAP has been seriously concerned all over the world. Our studies showed that high-fat diet feeding caused hepatic steatosis and hepatic injuries, evidence as increased serum levels of ALT, AST and TG in mice, and the histopathological changes in liver. ALT and AST are often tested together to illustrate the liver tissue damage (Fu et al., 2018). APAP could aggravate liver damage in mice with fatty liver by increasing serum levels of ALT and AST, what's more, APAP could also increase serum levels of TG, promote the formation of lipid droplets in the liver. To explore the lipid change in each group, we also detected the expression of SREBP-1c, which is the main transcription factor that regulates the genes related to fatty acid and triglyceride synthesis in liver, and mainly expressed in liver and adipocyte (Aragno
heavier. 4. Discussion NAFLD is the main causes of chronic liver disease, and also associated with hepatic and extrahepatic morbidity and mortality, however, the number of effective therapeutic options for curing NAFLD is limited (Kim et al., 2018). People who already have fatty liver disease often don't realize they have it for their mild symptoms, what's worse, some studies found that steatotic liver may be more susceptible to APAP toxicity than non-steatotic liver (Toyoda et al., 2018). APAP served as one of the most frequently used analgesic and antipyretic drugs and a component of many cold medicines. In many countries, APAP is also an over-the counter drug, people can buy APAP without a doctor's prescription. Meanwhile, APAP overdose is the most frequent cause of
Fig. 6. (A) Western blotting analysis of AMPK and mTOR protein levels in mice liver and densitometric quantification of them; (B) Western blotting analysis of LC3-Ⅱ and Beclin1 protein levels in mice liver and densitometric quantification of them; (C) Western blotting analysis of SREBP-1c protein levels in mice liver and densitometric quantification of them. Data were mean ± S.D. (n = 3). ##P < 0.01 versus NC; *P < 0.05, **P < 0.01 versus MC. 19
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 7. (A) Lipid droplets in L02 cells were stained with Oil Red O. (B) The quantification of Oil Red O content. (C) Effects of APAP on TG content in L02 cells induced by oleic acid and alcohol. Data were mean ± S.D. (n = 3). ##P < 0.01 versus NC; *P < 0.05 versus MC; &&P < 0.01 versus APAP.
condition remain to be explored. AMPK acts as a major regulator of metabolism, its activation not only increases the oxidation, but also inhibits the synthesis of fats. Some studies showed the activity of AMPK in liver of rats fed with high-fat diet significantly decreased (Smith et al., 2016). mTOR is an important signal molecule downstream of AMPK, the conserved mTOR protein complex includes mTORC1and mTORC2. mTORC1 acts as a nutrient sensor and thus plays an important role in the regulation of autophagy (Yu et al., 2018). In our studies, western blot and immunohistochemistry were used to detect the expression of AMPK and mTOR, the results showed that high-fat diet feeding inhibited the expression of AMPK, furthermore, APAP treatment significantly decreased the levels of AMPK; but the opposite changes took place in the expression of mTOR, which significantly increased in MC and APAP treated groups. Taken together, we infer that AMPK/mTOR signaling pathway was involved in the effects of APAP on NAFLD. To analyze the role of autophagy in current studies, we used western blot to detect the expression levels of autophagy-related proteins LC3-Ⅱ and Beclin1. LC3-Ⅱ levels are closely connection with the number of autophagosomes and act as a good symbol of autophagosomes formation (Tanida et al., 2004), and Beclin-1 is also one of the marker proteins of autophagy. The results showed that both expression levels of LC3-Ⅱ and Beclin1 were decreased in high-fat diet fed and APAP treated groups, especially in APAP groups. For further verification, we used transmission electron microscopy to detect autophagosomes, because it can directly reflect the activity of autophagy. We got the same results as western blot had shown. mTOR exerts an inhibitory effect on autophagy. The elevated mTOR induced by APAP ultimately led to a reduction in the autophagy-related proteins including Beclin-1 and LC3B II/I (Ueno and Komatsu, 2017), which in turn causes lipid deposition and liver injury further aggravated. The above results indicated that highfat diet fed increased lipid deposition and decreased the activity of autophagy, even the therapeutic dose of APAP aggravates the situation. The metabolites produced by excessive APAP in mice can not be removed effectively through autophagy, other study also reported inhibition of autophagy markedly exacerbated APAP-induced liver injury (Mo et al., 2018). Our previous studies have successfully established a model in which HepG2 cells exposed to oleic and alcohol mixed media to induce excessive lipid accumulation. In current study, L02 cells was selected,
et al., 2009). The results showed expression of SREBP-1c were significantly increased in MC and APAP treated groups. The present results suggested APAP administration further aggravated liver injury and hepatic lipid accumulation in mice with fatty liver. AMPK was an important regulator of cellular energy and metabolism homeostasis, which played an important role in the occurrence and development of NAFLD. Activated AMPK was able to stimulate catabolism and inhibit anabolism, leading to lower fat deposition (Novikova et al., 2015). mTOR was one of the main inhibition of autophagy, which played an important role in the occurrence and development of autophagy (Dai et al., 2018). In addition, AMPK, as upstream protein, when phosphorylation increased, can inhibit the levels of mTOR (Gwinn et al., 2008), thus enhanced autophagy. Autophagy had an intimate connection with fatty liver: the lipid droplets in cells are surrounded by the autophagosome of the double-membrane structure and transferred to the lysosome to combine into the autophagic lysosome, and then degrade into free fatty acids (Wang et al., 2015). Therefore, activation of autophagy could degrade the lipid droplets (LDs) and improve fatty liver. But evidence suggests when in a status of NAFLD, the levels of autophagy decreased significantly (Wu et al., 2018), LDs have been identified as a substrate for autophagy, impaired liver autophagy caused by lipid toxicity is closely related to the pathogenesis of NAFLD (Ueno and Komatsu, 2017). Defects in autophagy homeostasis are implicated in metabolic disorders, including obesity, insulin resistance, diabetes mellitus and atherosclerosis (Madrigal-Matute and Cuervo, 2016). Low level of autophagy can alter cellular lipid metabolism and aggravate fat deposition in the liver. Autophagy, serving as a protective mechanism to maintain cellular homeostasis, is activated in response to severe cellular stress caused by APAP. This is a normal reaction of the body during process of APAPinduced liver injury, the protective function of autophagy in APAP-induced liver injury is mediated through removal of the APAP adducts (Ni et al., 2016). The complexity of liver autophagy not only originates from its different roles in the physiological and pathological processes, but also it is affected by the various mechanisms involved in its regulation. However, the activities of autophagy which changes due to drug targets or exposure to toxic materials cannot be completely predictable (Zeinvand-Lorestani et al., 2018). Under NAFLD status with low levels of autophagy, whether APAP could affect the activities of autophagy? And how autophagy influences the effects of APAP in this 20
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 8. (A) Western blotting analysis of AMPK and mTOR protein levels in L02 cells and densitometric quantification of them; (B) Western blotting analysis of LC3-Ⅱ and Beclin1 protein levels in L02 cells and densitometric quantification of them; (C) Western blotting analysis of SREBP-1c protein levels L02 cells and densitometric quantification of them. Data were mean ± S.D. (n = 3). #P < 0.05, ##P < 0.01 versus NC; *P < 0.05, **P < 0.01 versus MC; &P < 0.05, &&P < 0.01 versus APAP4.
Anhui [grant numbers 1608085MH197], and partially by Scientific Research fund of Anhui Medical University [grant numbers 2012xkj092].
through optimization, this method was also valid. Oil Red O staining results showed that lipid droplets were significantly increased in L02 cells cultured by O + A media, and after APAP treated, lipid droplets accumulation in L02 cells were increased remarkably. However, when treated with rapamycin, an autophagy inducer (Guo et al., 2013), the effect of APAP was significantly reversed. This result was further supported by the quantification of intracellular TG contents. The results above suggested that APAP treatment could aggravated lipid accumulation in L02 cells, and the results of protein expression further verified that APAP treatment aggravated lipid accumulation in L02 cells and inhibited autophagy activity. In conclusion, both in vivo and in vitro studies suggest that APAP treatment aggravates hepatic fat deposition in NAFLD, which may be related to its inhibition of autophagy associated with the AMPK/mTOR pathway. NAFLD is the most common liver disease in the world, and APAP is one of the most frequently drugs that induced liver injuries clinically, current study provides a potentially approach for clinical rational use of APAP under NAFLD status, and patients with NAFLD should use a lower dose APAP.
Conflict of interest The authors declare no conflict of interest with the contents of this article. References Aragno, M., Tomasinelli, C.E., Vercellinatto, I., Catalano, M.G., Collino, M., Fantozzi, R., Danni, O., Boccuzzi, G., 2009. SREBP-1c in nonalcoholic fatty liver disease induced by Western-type high-fat diet plus fructose in rats. Free Radic. Biol. Med. 47, 1067–1074. Bateman, D.N., Carroll, R., Pettie, J., Yamamoto, T., Elamin, M.E., Peart, L., Dow, M., Coyle, J., Cranfield, K.R., Hook, C., Sandilands, E.A., Veiraiah, A., Webb, D., Gray, A., Dargan, P.I., Wood, D.M., Thomas, S.H., Dear, J.W., Eddleston, M., 2014. Effect of the UK's revised paracetamol poisoning management guidelines on admissions, adverse reactions and costs of treatment. Br. J. Clin. Pharmacol. 78, 610–618. Caparrotta, T.M., Antoine, D.J., Dear, J.W., 2018. Are some people at increased risk of paracetamol-induced liver injury? A critical review of the literature. Eur. J. Clin. Pharmacol. 74, 147–160. Cho, H.I., Seo, M.J., Lee, S.M., 2017. 2-Methoxyestradiol protects against ischemia/reperfusion injury in alcoholic fatty liver by enhancing sirtuin 1-mediated autophagy. Biochem. Pharmacol. 131, 40–51.
Acknowledgements This study was supported by Provincial Natural Science Study in 21
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Novikova, D.S., Garabadzhiu, A.V., Melino, G., Barlev, N.A., Tribulovich, V.G., 2015. AMP-activated protein kinase: structure, function, and role in pathological processes. Biochem. Biokhimiia 80, 127–144. Peng, K.Y., Watt, M.J., Rensen, S., Greve, J.W., Huynh, K., Jayawardana, K.S., Meikle, P.J., Meex, R.C.R., 2018. Mitochondrial dysfunction-related lipid changes occur in nonalcoholic fatty liver disease progression. J. Lipid Res. 59, 1977–1986. Shan, S., Shen, Z., Song, F., 2018. Autophagy and acetaminophen-induced hepatotoxicity. Arch. Toxicol. Smith, B.K., Marcinko, K., Desjardins, E.M., Lally, J.S., Ford, R.J., Steinberg, G.R., 2016. Treatment of nonalcoholic fatty liver disease: role of AMPK. Am. J. Physiol. Endocrinol. Metab. 311, E730–E740. Tang, L., Yang, F., Fang, Z., Hu, C., 2016. Resveratrol ameliorates alcoholic fatty liver by inducing autophagy. Am. J. Chin. Med. 44, 1207–1220. Tanida, I., Ueno, T., Kominami, E., 2004. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36, 2503–2518. Toyoda, T., Cho, Y.M., Akagi, J.I., Mizuta, Y., Matsushita, K., Nishikawa, A., Imaida, K., Ogawa, K., 2018. A 13-week subchronic toxicity study of acetaminophen using an obese rat model. J. Toxicol. Sci. 43, 423–433. Ueno, T., Komatsu, M., 2017. Autophagy in the liver: functions in health and disease. Nat. Rev. Gastroenterol. Hepatol. 14, 170–184. Wang, L., Khambu, B., Zhang, H., Yin, X.M., 2015. Autophagy in alcoholic liver disease, self-eating triggered by drinking. Clin. Res. Hepatol. Gastroenterol. 39 (Suppl 1), S2–S6. Wang, L., Li, X., Chen, C., 2018. Inhibition of acetaminophen-induced hepatotoxicity in mice by exogenous thymosinbeta4 treatment. Int. Immunopharmacol. 61, 20–28. Wu, C., Jing, M., Yang, L., Jin, L., Ding, Y., Lu, J., Cao, Q., Jiang, Y., 2018. Alisol A 24acetate ameliorates nonalcoholic steatohepatitis by inhibiting oxidative stress and stimulating autophagy through the AMPK/mTOR pathway. Chem.-Biol. Interact. 291, 111–119. Yan, M., Ye, L., Yin, S., Lu, X., Liu, X., Lu, S., Cui, J., Fan, L., Kaplowitz, N., Hu, H., 2018. Glycycoumarin protects mice against acetaminophen-induced liver injury predominantly via activating sustained autophagy. Br. J. Pharmacol. 175, 3747–3757. Yu, C., Li, W.B., Liu, J.B., Lu, J.W., Feng, J.F., 2018. Autophagy: novel applications of nonsteroidal anti-inflammatory drugs for primary cancer. Cancer Med. 7, 471–484. Yuan, E., Duan, X., Xiang, L., Ren, J., Lai, X., Li, Q., Sun, L., Sun, S., 2018. Aged oolong tea reduces high-fat diet-induced fat accumulation and dyslipidemia by regulating the AMPK/ACC signaling pathway. Nutrients 10. Zeinvand-Lorestani, M., Kalantari, H., Khodayar, M.J., Teimoori, A., Saki, N., Ahangarpour, A., Rahim, F., Alboghobeish, S., 2018. Autophagy upregulation as a possible mechanism of arsenic induced diabetes. Sci. Rep. 8, 11960. Zhang, X., Song, Y., Ding, Y., Wang, W., Liao, L., Zhong, J., Sun, P., Lei, F., Zhang, Y., Xie, W., 2018. Effects of mogrosides on high-fat-diet-induced obesity and nonalcoholic fatty liver disease in mice. Molecules 23. Zhao, X.Y., Xiong, X., Liu, T., Mi, L., Peng, X., Rui, C., Guo, L., Li, S., Li, X., Lin, J.D., 2018a. Long noncoding RNA licensing of obesity-linked hepatic lipogenesis and NAFLD pathogenesis. Nat. Commun. 9, 2986. Zhao, Z., Wei, Q., Hua, W., Liu, Y., Liu, X., Zhu, Y., 2018b. Hepatoprotective effects of berberine on acetaminophen-induced hepatotoxicity in mice. Biomed. Pharmacother. = Biomed. Pharmacother. 103, 1319–1326.
Dai, C., Ciccotosto, G.D., Cappai, R., Wang, Y., Tang, S., Hoyer, D., Schneider, E.K., Velkov, T., Xiao, X., 2018. Rapamycin confers neuroprotection against colistin-induced oxidative stress, mitochondria dysfunction, and apoptosis through the activation of autophagy and mTOR/Akt/CREB signaling pathways. ACS Chem. Neurosci. 9, 824–837. Fu, T., Wang, S., Liu, J., Cai, E., Li, H., Li, P., Zhao, Y., 2018. Protective effects of alphamangostin against acetaminophen-induced acute liver injury in mice. Eur. J. Pharmacol. 827, 173–180. Goncalves, J.L., Lacerda-Queiroz, N., Sabino, J.F.L., Marques, P.E., Galvao, I., Gamba, C.O., Cassali, G.D., de Carvalho, L.M., da Silva, E.S.D.A., Versiani, A., Teixeira, M.M., de Faria, A.M.C., Vieira, A.T., Brunialti-Godard, A.L., 2017. Evaluating the effects of refined carbohydrate and fat diets with acute ethanol consumption using a mouse model of alcoholic liver injury. J. Nutr. Biochem. 39, 93–100. Guo, R., Zhang, Y., Turdi, S., Ren, J., 2013. Adiponectin knockout accentuates high fat diet-induced obesity and cardiac dysfunction: role of autophagy. Biochim. Biophys. Acta 1832, 1136–1148. Gwinn, D.M., Shackelford, D.B., Egan, D.F., Mihaylova, M.M., Mery, A., Vasquez, D.S., Turk, B.E., Shaw, R.J., 2008. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226. Kim, H.M., Kim, Y., Lee, E.S., Huh, J.H., Chung, C.H., 2018. Caffeic acid ameliorates hepatic steatosis and reduces ER stress in high fat diet-induced obese mice by regulating autophagy. Nutrition 55–56, 63–70. Kim, T.H., Choi, D., Kim, J.Y., Lee, J.H., Koo, S.H., 2017. Fast food diet-induced nonalcoholic fatty liver disease exerts early protective effect against acetaminophen intoxication in mice. BMC Gastroenterol. 17, 124. Kucera, O., Rousar, T., Stankova, P., Hanackova, L., Lotkova, H., Podhola, M., Cervinkova, Z., 2012. Susceptibility of rat non-alcoholic fatty liver to the acute toxic effect of acetaminophen. J. Gastroenterol. Hepatol. 27, 323–330. Lee, J.H., Baek, S.Y., Jang, E.J., Ku, S.K., Kim, K.M., Ki, S.H., Kim, C.E., Park, K.I., Kim, S.C., Kim, Y.W., 2018. Oxyresveratrol ameliorates nonalcoholic fatty liver disease by regulating hepatic lipogenesis and fatty acid oxidation through liver kinase B1 and AMP-activated protein kinase. Chem.-Biol. Interact. 289, 68–74. Li, C., Ye, L., Yang, L., Yu, X., He, Y., Chen, Z., Li, L., Zhang, D., 2017. Rapamycin promotes the survival and adipogenesis of ischemia-challenged adipose derived stem cells by improving autophagy. Cell. Physiol. Biochem.: Int. J. Exp. Cell. Physiol., Biochem. Pharmacol. 44, 1762–1774. Madrigal-Matute, J., Cuervo, A.M., 2016. Regulation of liver metabolism by autophagy. Gastroenterology 150, 328–339. Mazraati, P., Minaiyan, M., 2018. Hepatoprotective effect of metadoxine on acetaminophen-induced liver toxicity in mice. Adv. Biomed. Res. 7, 67. Mo, R., Lai, R., Lu, J., Zhuang, Y., Zhou, T., Jiang, S., Ren, P., Li, Z., Cao, Z., Liu, Y., Chen, L., Xiong, L., Wang, P., Wang, H., Cai, W., Xiang, X., Bao, S., Xie, Q., 2018. Enhanced autophagy contributes to protective effects of IL-22 against acetaminophen-induced liver injury. Theranostics 8, 4170–4180. Ni, H.M., McGill, M.R., Chao, X., Du, K., Williams, J.A., Xie, Y., Jaeschke, H., Ding, W.X., 2016. Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice. J. Hepatol. 65, 354–362. Nikravesh, H., Khodayar, M.J., Mahdavinia, M., Mansouri, E., Zeidooni, L., Dehbashi, F., 2018. Protective effect of gemfibrozil on hepatotoxicity induced by acetaminophen in mice: the importance of oxidative stress suppression. Adv. Pharm. Bull. 8, 331–339.
22
Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Acetaminophen aggravates fat accumulation in NAFLD by inhibiting autophagy via the AMPK/mTOR pathway Congjian Shia,b, Weiju Xuea,b, Bowen Hana,b, Fengli Yanga,b, Yaping Yina,b, Chengmu Hua,b, a b
T ⁎
Institute for Liver Diseases of Anhui Medical University, School of Pharmacy, Anhui Medical University, Hefei 230032, China Key Laboratory of anti-inflammatory and Immune Medicine, Ministry of Education, Hefei 230032, China
A R T I C LE I N FO
A B S T R A C T
Keywords: NAFLD APAP Autophagy AMPK mTOR Mice
Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease which affects millions of people worldwide. Acetaminophen (APAP) overdose is the leading cause of acute liver failure. In this study, APAP (50, 100, 200 mg/kg) were employed on mice fed with a high-fat diet, and APAP (2, 4, 8 mM) were cultured with L02 cells in the presence of alcohol and oleic acid. APAP treatment significantly aggravated hepatic lipid accumulation, increased the serum levels of triglyceride (TG), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and increased hepatic lipid accumulation in H&E and Oil red O staining results. Transmission electron microscopy (TEM) found fewer number of autophagosomes in APAP (100 mg/kg) treated group. Immunohistochemistry analysis showed the intensity of hepatic mTOR was increased and AMPK was decreased in 200 mg/kg APAP treated group. Western blot analysis showed APAP treatment decreased the levels of LC3-Ⅱ, Beclin1 and AMPK, while increased the levels of mTOR and SREBP-1c, respectively. In vitro study showed APAP treatment obviously increased TG activities in cell supernatant, and Oil red O staining had the same results. Western blot analysis demonstrated APAP treatment decreased the levels of LC3-Ⅱ, Beclin1 and AMPK, increased the levels of mTOR and SREBP-1c, but rapamycin treatment significantly reversed these effects of APAP. In conclusion, therapeutic dosages of APAP aggravates fat accumulation in NAFLD, the potential mechanism might be involved in inhibiting autophagy associated with the AMPK/mTOR pathway, and patients with NAFLD should use a lower dose of APAP.
1. Introduction Non-alcoholic fatty liver disease (NAFLD) is a clinical disease characterized by liver fat exceed 5% of the total liver weight, it can cause severe liver damage and metabolic comorbidities if not controlled (Peng et al., 2018). NAFLD, which encompasses a spectrum from simple hepatic steatosis to steatosis combined with varying degrees of inflammation and fibrosis, is a serious threat to the health of people the world over, with the morbidity rising every year and the age of sufferers getting younger (Zhang et al., 2018; X.Y. Zhao et al., 2018). Patients with steatosis often do not feel that they have the disease for their mild symptoms, when they really realize they are sick, the condition is already very heavy. Acetaminophen (APAP), commonly known as paracetamol, is one of the most frequently used analgesic and antipyretic drugs, because of its effectiveness on reducing fever and pain, the minor irritation to the gastrointestinal tract and the complete oral absorption (Nikravesh et al., 2018). It is safe and effective at therapeutic doses, but an
⁎
overdose can cause hepatotoxicity, acute liver failure (ALF) and even death (Yan et al., 2018). APAP overdose is the leading cause of druginduced acute liver injury. Liver is the main organ that metabolizes APAP in therapeutic doses, approximately 90% is metabolized through hepatic sulfation and glucuronidation pathways (Z. Zhao et al., 2018). Approximately 5% through cytochrome P450 translates into the Nacetyl-p-benzoquinoneimine (NAPQI), which is detoxified by combining with glutathione (GSH) (Shan et al., 2018). However, overdose of APAP can produce excessive NAPQI that could deplete GSH and bind to cellular proteins (APAP protein adducts), result in mitochondrial oxidative stress, liver injury, ROS production and liver failure (Mazraati and Minaiyan, 2018; Wang et al., 2018). APAP-induced hepatotoxicity has drawn lots of attention and solutions, N-acetylcysteine (NAC) is a highly successful approach for treating APAP overdose; however, this protective effect is only observed in the early stage of APAP hepatotoxicity (Shan et al., 2018), and it also has some disadvantages. Autophagy plays an important role in coping with multiple forms of cellular stress, including nutrient or growth factor deprivation, hypoxia,
Corresponding author at: School of Pharmacy, Anhui Medical University, Hefei 230032, Anhui Province, China. E-mail address: [email protected] (C. Hu).
https://doi.org/10.1016/j.ejphar.2019.02.005 Received 14 November 2018; Received in revised form 2 February 2019; Accepted 8 February 2019 Available online 10 February 2019 0014-2999/ © 2019 Elsevier B.V. All rights reserved.
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
2.4. Transmission electron microscopy detection
or damaged organelles (Yu et al., 2018). Previous studies showed resveratrol ameliorates alcoholic fatty liver by inducing autophagy (Tang et al., 2016), whether autophagy plays any role during the process of APAP treated NAFLD? Acetaminophen is a commonly used drug that induces serious hepatotoxicity when overdosed, some study found liver affected by NAFLD is more sensitive to acute toxic injury caused by APAP in comparison with non-steatotic liver (Kucera et al., 2012). Other study showed fast food diet-induced non-alcoholic fatty liver disease exerts early protective effect against acetaminophen intoxication in mice (Kim et al., 2017). We are interested in this discrepancy and want to explore the underlying mechanisms. The present study was designed to evaluate whether APAP could affect the level of autophagy, and how can autophagy influence the effects of APAP under NAFLD condition? Therefore, the present study may demonstrate new insights into the rational application of APAP and better avoid its hepatotoxicity.
The liver fragments in the same part of each mouse were fixed in 2.5% glutaraldehyde, then fixed with 1% osmium acid, added gradient alcohol to dehydrate, epoxy resin embedding, and sliced by an ultrathin slicing machine, the sections were stained (Cho et al., 2017) and the images were taken under transmission electron microscopy (HITACHI, Japan). 2.5. Immunohistochemical analysis
2. Materials and methods
The expression of AMPK and mTOR in liver were detected by immunohistochemistry method. The samples from paraffin-embedding tissue were dewaxed, rehydrated, and incubated with the primary antibody (p-AMPK, p-mTOR, 1:200) for overnight at 4 °C. The slides were incubated with secondary antibody (Bioss, Beijing, China) according to the manufacturer's instructions. The brown staining in the cell membrane was considered to be positive.
2.1. Animals and treatments
2.6. Cell culture and treatments
Male C57BL/6 J mice (20–22 g, 6–8 weeks) were purchased from the Animal Center of Anhui Medical University and the experiment was approved by the ethics committee. Mice were housed in plastic cages with food and water ad libitum and maintained at room temperature of (23 ± 2 °C) on a 12 h light/dark cycle. After one week acclimation, the animals were randomly divided into five groups (n = 8 per group): (1) normal control group (NC), (2) high-fat diet control group (MC), (3) MC with 50 mg/kg APAP (Solarbio, Beijing, China) (APAP50), (4) MC with 100 mg/kg APAP (APAP100), and (5) MC with 200 mg/kg APAP (APAP200). Control mice were fed a normal diet (10% calories from fat, 20% calories from protein, 70% calories from carbohydrate; 3.5 Kcal/g diet; TP26312; TROPHIC, Nantong, China), and mice in the other four groups were fed a high-fat diet (42% calories from fat, 15% calories from protein, 43% calories from carbohydrate; 4.5 Kcal/g diet; TP26300; TROPHIC, Nantong, China). The total feeding duration was 56 days. On the last day of study, mice were given different APAP doses (50, 100, 200 mg/kg) by gavage (only once). Mice body weight was recorded every week, and the final liver weight was also recorded. The APAP solution was freshly prepared by dissolving acetaminophen in phosphate-buffered saline (PBS) and warmed to 40 °C. Mice were killed at 24 h after APAP administration and were anesthetized with 10% chloralhydrate solution (3 ml/kg), after blood samples were collected, parts of liver samples were frozen for oil red O staining, fixed in 2.5% glutaraldehyde, or fixed in formalin, and the rest was stored at −80 °C. All procedures were approved by the institutional ethical guidelines for laboratory animal care and use of Anhui Medical University, in accordance with NIH guidelines and were approved by the Animal Care and Use Committee (NO: LLSC20180429).
Human normal liver cell line (L02) cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% Penicillin-Streptomycin solution, cultures were maintained at 37 °C in a humidified 5% CO2 incubator. To induce excessive lipid accumulation in the model of hepatocyte steatosis in vitro, L02 cells were exposed to media supplemented with 100 μM oleic acid and 87 mM alcohol for 48 h-treatment. For the APAP experiments, cells were incubated with different concentrations of APAP during the last 24 h of the 48 htreatment, and were incubated with rapamycin (SIGMA-ALDRICH, USA) during the last 28 h of the 48 h-treatment (Li et al., 2017). Both APAP and rapamycin were dissolved in Dimethyl sulfoxide (DMSO). The cells were divided into six groups: (1) control group (NC), (2) model control group (MC), (3) MC with 2 mM APAP (APAP2), (4) MC with 4 mM APAP (APAP4), (5) MC with 8 mM APAP (APAP8), and (6) MC with 4 mM APAP and 1 μM rapamycin (AR). 2.7. Measurement of intracellular TG and Oil Red O staining Cells were seeded into 6-well plates and treated with designated reagent, then fixed with 4% paraformaldehyde and stained with a freshly prepared working solution of Oil Red O at room temperature for 30 min. To quantify the Oil Red O content, the cell samples were extracted by isopropanol for 5 min, and the optical density of each cell sample was read at 510 nm by a microplate reader (Biotek, USA). The activities of TG in cell supernatant were also measured according to commercial analysis kits (Jiancheng, Nanjing, China) by a microplate reader. 2.8. Western blot analysis Liver specimens and cells were mixed with moderately RIPA buffer to obtain lysates, total protein in the supernatant was measured by using bicinchoninic acid (BCA) Protein Assay Kit (Biyuntian, China). Lysates taken from each sample were separated by 10% SDS-PAGE, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, Massachusetts, USA), blocked with 5% non-fat milk for 2 h at room temperature, and placed overnight at 4 °C with specific antibodies: AMPK, p-AMPK, p-mTOR, Beclin1 (Bioss, Beijing, China) and SREBP-1c (Santa Cruz Bio Technology, Inc) and mTOR, LC3-Ⅱ (Cell Signaling Technology) and β-actin (Zhongshan Jinqiao, Beijing, China). After washing with TBST, the membranes were incubated with a peroxidaselabeled secondary antibody (Zhongshan Jinqiao, Beijing, China) for 1 h. The membranes were detected by an enhanced chemiluminescence (ECL) reaction and exposed to Image Quant LAS 4000 mini (GE Healthcare Bio-Sciences, AB, Uppsala, Sweden). The levels of protein
2.2. Morphological evaluations Formalin-fixed liver samples were paraffin embedded and put on glass slides for hematoxylin and eosin (H&E) staining (Goncalves et al., 2017), 5-µm frozen liver sections were stained with Oil red O with a standard procedure (Lee et al., 2018). Lipid droplets deposition was observed under an optical microscope (Olympus, TH4–200, Japan). 2.3. Serum markers analysis The activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and triglyceride (TG) were measured according to those commercial assay kits (Jiancheng, Nanjing, China) by a microplate reader (Biotek, USA). 16
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 1. (A) Effects of APAP on mice body weight; (B) Liver index. During 8 weeks of high-fat diet fed, body weights were recorded once a week. APAP were administered after 8 weeks high-fat diet fed, and liver index was measured. Data were mean ± S.D. (n = 8). ##P < 0.01 versus NC; **P < 0.01 versus MC.
expression were quantitatively analyzed with Image J Software. 2.9. Statistical analysis The data were expressed as the (mean ± S.D.). Comparisons between multiple groups were made by one-way analysis of variance (ANOVA) followed by Duncan's test. Differences were considered significant if the P value was less than 0.05. 3. Results 3.1. Body weight and liver index The average body weight did not have significantly difference between the normal group and the other four groups at first week, but from second week, there had significantly increasing body weight in MC and APAP treatment groups compared with that of NC (Fig. 1A). The final liver index of MC and APAP treatment groups were significantly higher than that of NC, meanwhile, the liver index of APAP200 were significantly higher than that of MC (Fig. 1B). 3.2. Serum activities of ALT, AST and TG
Fig. 2. Effects of APAP on the serum levels of ALT, AST and TG in mice. Data were mean ± S.D. (n = 6–8). ##P < 0.01 versus NC; *P < 0.05, **P < 0.01 versus MC.
The serum activities of ALT, AST and TG were markedly increased in MC group, compared to NC. Meanwhile, the activities of ALT, AST and TG were also increased with the dosage of APAP increasing, compared to MC group (Fig. 2).
regulation to autophagy. Current results showed the phosphorylation levels of the mTORC1 were obviously increased in the MC group and APAP groups, compared to the NC and MC group, respectively. As the dosage of APAP increased, the phosphorylation degree of mTORC1 had a further increase (Fig. 4B; Fig. 4D).
3.3. H&E staining and Oil Red O staining pictures Histopathological analysis showed, compare with normal mice, mice in model group developed a higher degree of hepatic steatosis (Fig. 3A) and lipid accumulation (Fig. 3B) in the liver, what's more, with the dosage of APAP increasing, mice treated with APAP developed even more severe hepatic steatosis and more lipid accumulation in the liver.
3.5. APAP decreased autophagosomes As shown in Fig. 5, transmission electron microscopy images reflected that the number of autophagosomes decreased in MC, compared to NC, APAP treatment resulted in further decreasing number of autophagosomes.
3.4. Immunohistochemistry changes of livers 3.6. The effects of APAP on relative protein expression in liver tissues AMPK consumes excess energy by stimulating oxidation of fatty acids, AMPK activation was determined by evaluating the phosphorylation level of the AMPKα subunit at Thr172, which can reflects the activation of AMPK (Yuan et al., 2018). In our study, mice in the MC had a radical decrease in the phosphorylation degree of AMPKα, compared to that in the NC. As the dosage of APAP increased, the phosphorylation degree of AMPKα had a further decrease (Fig. 4A; Fig. 4C). One of the hinge segments in the mTOR mediated lipid accumulation is the activation of mTORC1, and mTORC1 had a negative
As mentioned before, we measured the level of the phosphorylation AMPKα subunit at Thr172 and the level of the phosphorylation mTORC1 to evaluate the activation of AMPK and mTOR, respectively. In liver tissues, as shown in Fig. 6, the protein expressions of mTOR and SREBP-1c were significantly increased in mice treated with APAP, compared with MC group; while the expression of LC3-Ⅱ, Beclin1and AMPK were significantly decreased in mice treated with APAP, compared with MC. 17
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 3. Effects of APAP on accumulation of lipid droplets in liver. Representative (A) H＆E and (B) Oil Red O staining images.
3.7. APAP treatment aggravated lipid accumulation in L02 cells
3.8. The effects of APAP on relative protein expression in L02 cells
Oil Red O staining showed that oleic acid plus alcohol treated obviously increased lipid accumulation in L02 cells, and when treated with APAP, the circumstance became exacerbated. However, rapamycin administration suppressed this effect of APAP (Fig. 7A; Fig. 7B). Intracellular TG result was also in accord with the result of Oil Red O staining (Fig. 7C).
The results observed in vitro were the same as those in liver tissue, as shown in Fig. 8, the expression of LC3-Ⅱ, Beclin1 and AMPK were significantly decreased, while the expressions of mTOR and SREBP-1c were significantly increased in mice treated with APAP, compared with MC. However, rapamycin administrated significantly suppressed the effects of APAP, this evidence implied that autophagy was impaired after administrated APAP and liver lipid deposition became even
Fig. 4. Effects of APAP on Immunohistochemistry changes of livers in mice. (A) Immunohistochemical staining of liver for p-AMPK; (B) Immunohistochemical staining of liver for p-mTOR; (C) densitometric quantification of p-AMPK (n = 3 independent experiments); (D) densitometric quantification of p-mTOR (n = 3 independent experiments). Data were mean ± S.D. (n = 3). #P < 0.05, ##P < 0.01 versus NC; *P < 0.05 versus MC. 18
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 5. Transmission electron microscopy examination of autophagosomes in hepatic tissues. Black arrows denote autophagosomes.
drug-induced acute liver failure. In the UK about 82,000–90,000 people a year went to hospital because of misuse or abuse of APAP (Bateman et al., 2014; Caparrotta et al., 2018), and liver injury caused by APAP has been seriously concerned all over the world. Our studies showed that high-fat diet feeding caused hepatic steatosis and hepatic injuries, evidence as increased serum levels of ALT, AST and TG in mice, and the histopathological changes in liver. ALT and AST are often tested together to illustrate the liver tissue damage (Fu et al., 2018). APAP could aggravate liver damage in mice with fatty liver by increasing serum levels of ALT and AST, what's more, APAP could also increase serum levels of TG, promote the formation of lipid droplets in the liver. To explore the lipid change in each group, we also detected the expression of SREBP-1c, which is the main transcription factor that regulates the genes related to fatty acid and triglyceride synthesis in liver, and mainly expressed in liver and adipocyte (Aragno
heavier. 4. Discussion NAFLD is the main causes of chronic liver disease, and also associated with hepatic and extrahepatic morbidity and mortality, however, the number of effective therapeutic options for curing NAFLD is limited (Kim et al., 2018). People who already have fatty liver disease often don't realize they have it for their mild symptoms, what's worse, some studies found that steatotic liver may be more susceptible to APAP toxicity than non-steatotic liver (Toyoda et al., 2018). APAP served as one of the most frequently used analgesic and antipyretic drugs and a component of many cold medicines. In many countries, APAP is also an over-the counter drug, people can buy APAP without a doctor's prescription. Meanwhile, APAP overdose is the most frequent cause of
Fig. 6. (A) Western blotting analysis of AMPK and mTOR protein levels in mice liver and densitometric quantification of them; (B) Western blotting analysis of LC3-Ⅱ and Beclin1 protein levels in mice liver and densitometric quantification of them; (C) Western blotting analysis of SREBP-1c protein levels in mice liver and densitometric quantification of them. Data were mean ± S.D. (n = 3). ##P < 0.01 versus NC; *P < 0.05, **P < 0.01 versus MC. 19
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 7. (A) Lipid droplets in L02 cells were stained with Oil Red O. (B) The quantification of Oil Red O content. (C) Effects of APAP on TG content in L02 cells induced by oleic acid and alcohol. Data were mean ± S.D. (n = 3). ##P < 0.01 versus NC; *P < 0.05 versus MC; &&P < 0.01 versus APAP.
condition remain to be explored. AMPK acts as a major regulator of metabolism, its activation not only increases the oxidation, but also inhibits the synthesis of fats. Some studies showed the activity of AMPK in liver of rats fed with high-fat diet significantly decreased (Smith et al., 2016). mTOR is an important signal molecule downstream of AMPK, the conserved mTOR protein complex includes mTORC1and mTORC2. mTORC1 acts as a nutrient sensor and thus plays an important role in the regulation of autophagy (Yu et al., 2018). In our studies, western blot and immunohistochemistry were used to detect the expression of AMPK and mTOR, the results showed that high-fat diet feeding inhibited the expression of AMPK, furthermore, APAP treatment significantly decreased the levels of AMPK; but the opposite changes took place in the expression of mTOR, which significantly increased in MC and APAP treated groups. Taken together, we infer that AMPK/mTOR signaling pathway was involved in the effects of APAP on NAFLD. To analyze the role of autophagy in current studies, we used western blot to detect the expression levels of autophagy-related proteins LC3-Ⅱ and Beclin1. LC3-Ⅱ levels are closely connection with the number of autophagosomes and act as a good symbol of autophagosomes formation (Tanida et al., 2004), and Beclin-1 is also one of the marker proteins of autophagy. The results showed that both expression levels of LC3-Ⅱ and Beclin1 were decreased in high-fat diet fed and APAP treated groups, especially in APAP groups. For further verification, we used transmission electron microscopy to detect autophagosomes, because it can directly reflect the activity of autophagy. We got the same results as western blot had shown. mTOR exerts an inhibitory effect on autophagy. The elevated mTOR induced by APAP ultimately led to a reduction in the autophagy-related proteins including Beclin-1 and LC3B II/I (Ueno and Komatsu, 2017), which in turn causes lipid deposition and liver injury further aggravated. The above results indicated that highfat diet fed increased lipid deposition and decreased the activity of autophagy, even the therapeutic dose of APAP aggravates the situation. The metabolites produced by excessive APAP in mice can not be removed effectively through autophagy, other study also reported inhibition of autophagy markedly exacerbated APAP-induced liver injury (Mo et al., 2018). Our previous studies have successfully established a model in which HepG2 cells exposed to oleic and alcohol mixed media to induce excessive lipid accumulation. In current study, L02 cells was selected,
et al., 2009). The results showed expression of SREBP-1c were significantly increased in MC and APAP treated groups. The present results suggested APAP administration further aggravated liver injury and hepatic lipid accumulation in mice with fatty liver. AMPK was an important regulator of cellular energy and metabolism homeostasis, which played an important role in the occurrence and development of NAFLD. Activated AMPK was able to stimulate catabolism and inhibit anabolism, leading to lower fat deposition (Novikova et al., 2015). mTOR was one of the main inhibition of autophagy, which played an important role in the occurrence and development of autophagy (Dai et al., 2018). In addition, AMPK, as upstream protein, when phosphorylation increased, can inhibit the levels of mTOR (Gwinn et al., 2008), thus enhanced autophagy. Autophagy had an intimate connection with fatty liver: the lipid droplets in cells are surrounded by the autophagosome of the double-membrane structure and transferred to the lysosome to combine into the autophagic lysosome, and then degrade into free fatty acids (Wang et al., 2015). Therefore, activation of autophagy could degrade the lipid droplets (LDs) and improve fatty liver. But evidence suggests when in a status of NAFLD, the levels of autophagy decreased significantly (Wu et al., 2018), LDs have been identified as a substrate for autophagy, impaired liver autophagy caused by lipid toxicity is closely related to the pathogenesis of NAFLD (Ueno and Komatsu, 2017). Defects in autophagy homeostasis are implicated in metabolic disorders, including obesity, insulin resistance, diabetes mellitus and atherosclerosis (Madrigal-Matute and Cuervo, 2016). Low level of autophagy can alter cellular lipid metabolism and aggravate fat deposition in the liver. Autophagy, serving as a protective mechanism to maintain cellular homeostasis, is activated in response to severe cellular stress caused by APAP. This is a normal reaction of the body during process of APAPinduced liver injury, the protective function of autophagy in APAP-induced liver injury is mediated through removal of the APAP adducts (Ni et al., 2016). The complexity of liver autophagy not only originates from its different roles in the physiological and pathological processes, but also it is affected by the various mechanisms involved in its regulation. However, the activities of autophagy which changes due to drug targets or exposure to toxic materials cannot be completely predictable (Zeinvand-Lorestani et al., 2018). Under NAFLD status with low levels of autophagy, whether APAP could affect the activities of autophagy? And how autophagy influences the effects of APAP in this 20
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Fig. 8. (A) Western blotting analysis of AMPK and mTOR protein levels in L02 cells and densitometric quantification of them; (B) Western blotting analysis of LC3-Ⅱ and Beclin1 protein levels in L02 cells and densitometric quantification of them; (C) Western blotting analysis of SREBP-1c protein levels L02 cells and densitometric quantification of them. Data were mean ± S.D. (n = 3). #P < 0.05, ##P < 0.01 versus NC; *P < 0.05, **P < 0.01 versus MC; &P < 0.05, &&P < 0.01 versus APAP4.
Anhui [grant numbers 1608085MH197], and partially by Scientific Research fund of Anhui Medical University [grant numbers 2012xkj092].
through optimization, this method was also valid. Oil Red O staining results showed that lipid droplets were significantly increased in L02 cells cultured by O + A media, and after APAP treated, lipid droplets accumulation in L02 cells were increased remarkably. However, when treated with rapamycin, an autophagy inducer (Guo et al., 2013), the effect of APAP was significantly reversed. This result was further supported by the quantification of intracellular TG contents. The results above suggested that APAP treatment could aggravated lipid accumulation in L02 cells, and the results of protein expression further verified that APAP treatment aggravated lipid accumulation in L02 cells and inhibited autophagy activity. In conclusion, both in vivo and in vitro studies suggest that APAP treatment aggravates hepatic fat deposition in NAFLD, which may be related to its inhibition of autophagy associated with the AMPK/mTOR pathway. NAFLD is the most common liver disease in the world, and APAP is one of the most frequently drugs that induced liver injuries clinically, current study provides a potentially approach for clinical rational use of APAP under NAFLD status, and patients with NAFLD should use a lower dose APAP.
Conflict of interest The authors declare no conflict of interest with the contents of this article. References Aragno, M., Tomasinelli, C.E., Vercellinatto, I., Catalano, M.G., Collino, M., Fantozzi, R., Danni, O., Boccuzzi, G., 2009. SREBP-1c in nonalcoholic fatty liver disease induced by Western-type high-fat diet plus fructose in rats. Free Radic. Biol. Med. 47, 1067–1074. Bateman, D.N., Carroll, R., Pettie, J., Yamamoto, T., Elamin, M.E., Peart, L., Dow, M., Coyle, J., Cranfield, K.R., Hook, C., Sandilands, E.A., Veiraiah, A., Webb, D., Gray, A., Dargan, P.I., Wood, D.M., Thomas, S.H., Dear, J.W., Eddleston, M., 2014. Effect of the UK's revised paracetamol poisoning management guidelines on admissions, adverse reactions and costs of treatment. Br. J. Clin. Pharmacol. 78, 610–618. Caparrotta, T.M., Antoine, D.J., Dear, J.W., 2018. Are some people at increased risk of paracetamol-induced liver injury? A critical review of the literature. Eur. J. Clin. Pharmacol. 74, 147–160. Cho, H.I., Seo, M.J., Lee, S.M., 2017. 2-Methoxyestradiol protects against ischemia/reperfusion injury in alcoholic fatty liver by enhancing sirtuin 1-mediated autophagy. Biochem. Pharmacol. 131, 40–51.
Acknowledgements This study was supported by Provincial Natural Science Study in 21
European Journal of Pharmacology 850 (2019) 15–22
C. Shi, et al.
Novikova, D.S., Garabadzhiu, A.V., Melino, G., Barlev, N.A., Tribulovich, V.G., 2015. AMP-activated protein kinase: structure, function, and role in pathological processes. Biochem. Biokhimiia 80, 127–144. Peng, K.Y., Watt, M.J., Rensen, S., Greve, J.W., Huynh, K., Jayawardana, K.S., Meikle, P.J., Meex, R.C.R., 2018. Mitochondrial dysfunction-related lipid changes occur in nonalcoholic fatty liver disease progression. J. Lipid Res. 59, 1977–1986. Shan, S., Shen, Z., Song, F., 2018. Autophagy and acetaminophen-induced hepatotoxicity. Arch. Toxicol. Smith, B.K., Marcinko, K., Desjardins, E.M., Lally, J.S., Ford, R.J., Steinberg, G.R., 2016. Treatment of nonalcoholic fatty liver disease: role of AMPK. Am. J. Physiol. Endocrinol. Metab. 311, E730–E740. Tang, L., Yang, F., Fang, Z., Hu, C., 2016. Resveratrol ameliorates alcoholic fatty liver by inducing autophagy. Am. J. Chin. Med. 44, 1207–1220. Tanida, I., Ueno, T., Kominami, E., 2004. LC3 conjugation system in mammalian autophagy. Int. J. Biochem. Cell Biol. 36, 2503–2518. Toyoda, T., Cho, Y.M., Akagi, J.I., Mizuta, Y., Matsushita, K., Nishikawa, A., Imaida, K., Ogawa, K., 2018. A 13-week subchronic toxicity study of acetaminophen using an obese rat model. J. Toxicol. Sci. 43, 423–433. Ueno, T., Komatsu, M., 2017. Autophagy in the liver: functions in health and disease. Nat. Rev. Gastroenterol. Hepatol. 14, 170–184. Wang, L., Khambu, B., Zhang, H., Yin, X.M., 2015. Autophagy in alcoholic liver disease, self-eating triggered by drinking. Clin. Res. Hepatol. Gastroenterol. 39 (Suppl 1), S2–S6. Wang, L., Li, X., Chen, C., 2018. Inhibition of acetaminophen-induced hepatotoxicity in mice by exogenous thymosinbeta4 treatment. Int. Immunopharmacol. 61, 20–28. Wu, C., Jing, M., Yang, L., Jin, L., Ding, Y., Lu, J., Cao, Q., Jiang, Y., 2018. Alisol A 24acetate ameliorates nonalcoholic steatohepatitis by inhibiting oxidative stress and stimulating autophagy through the AMPK/mTOR pathway. Chem.-Biol. Interact. 291, 111–119. Yan, M., Ye, L., Yin, S., Lu, X., Liu, X., Lu, S., Cui, J., Fan, L., Kaplowitz, N., Hu, H., 2018. Glycycoumarin protects mice against acetaminophen-induced liver injury predominantly via activating sustained autophagy. Br. J. Pharmacol. 175, 3747–3757. Yu, C., Li, W.B., Liu, J.B., Lu, J.W., Feng, J.F., 2018. Autophagy: novel applications of nonsteroidal anti-inflammatory drugs for primary cancer. Cancer Med. 7, 471–484. Yuan, E., Duan, X., Xiang, L., Ren, J., Lai, X., Li, Q., Sun, L., Sun, S., 2018. Aged oolong tea reduces high-fat diet-induced fat accumulation and dyslipidemia by regulating the AMPK/ACC signaling pathway. Nutrients 10. Zeinvand-Lorestani, M., Kalantari, H., Khodayar, M.J., Teimoori, A., Saki, N., Ahangarpour, A., Rahim, F., Alboghobeish, S., 2018. Autophagy upregulation as a possible mechanism of arsenic induced diabetes. Sci. Rep. 8, 11960. Zhang, X., Song, Y., Ding, Y., Wang, W., Liao, L., Zhong, J., Sun, P., Lei, F., Zhang, Y., Xie, W., 2018. Effects of mogrosides on high-fat-diet-induced obesity and nonalcoholic fatty liver disease in mice. Molecules 23. Zhao, X.Y., Xiong, X., Liu, T., Mi, L., Peng, X., Rui, C., Guo, L., Li, S., Li, X., Lin, J.D., 2018a. Long noncoding RNA licensing of obesity-linked hepatic lipogenesis and NAFLD pathogenesis. Nat. Commun. 9, 2986. Zhao, Z., Wei, Q., Hua, W., Liu, Y., Liu, X., Zhu, Y., 2018b. Hepatoprotective effects of berberine on acetaminophen-induced hepatotoxicity in mice. Biomed. Pharmacother. = Biomed. Pharmacother. 103, 1319–1326.
Dai, C., Ciccotosto, G.D., Cappai, R., Wang, Y., Tang, S., Hoyer, D., Schneider, E.K., Velkov, T., Xiao, X., 2018. Rapamycin confers neuroprotection against colistin-induced oxidative stress, mitochondria dysfunction, and apoptosis through the activation of autophagy and mTOR/Akt/CREB signaling pathways. ACS Chem. Neurosci. 9, 824–837. Fu, T., Wang, S., Liu, J., Cai, E., Li, H., Li, P., Zhao, Y., 2018. Protective effects of alphamangostin against acetaminophen-induced acute liver injury in mice. Eur. J. Pharmacol. 827, 173–180. Goncalves, J.L., Lacerda-Queiroz, N., Sabino, J.F.L., Marques, P.E., Galvao, I., Gamba, C.O., Cassali, G.D., de Carvalho, L.M., da Silva, E.S.D.A., Versiani, A., Teixeira, M.M., de Faria, A.M.C., Vieira, A.T., Brunialti-Godard, A.L., 2017. Evaluating the effects of refined carbohydrate and fat diets with acute ethanol consumption using a mouse model of alcoholic liver injury. J. Nutr. Biochem. 39, 93–100. Guo, R., Zhang, Y., Turdi, S., Ren, J., 2013. Adiponectin knockout accentuates high fat diet-induced obesity and cardiac dysfunction: role of autophagy. Biochim. Biophys. Acta 1832, 1136–1148. Gwinn, D.M., Shackelford, D.B., Egan, D.F., Mihaylova, M.M., Mery, A., Vasquez, D.S., Turk, B.E., Shaw, R.J., 2008. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226. Kim, H.M., Kim, Y., Lee, E.S., Huh, J.H., Chung, C.H., 2018. Caffeic acid ameliorates hepatic steatosis and reduces ER stress in high fat diet-induced obese mice by regulating autophagy. Nutrition 55–56, 63–70. Kim, T.H., Choi, D., Kim, J.Y., Lee, J.H., Koo, S.H., 2017. Fast food diet-induced nonalcoholic fatty liver disease exerts early protective effect against acetaminophen intoxication in mice. BMC Gastroenterol. 17, 124. Kucera, O., Rousar, T., Stankova, P., Hanackova, L., Lotkova, H., Podhola, M., Cervinkova, Z., 2012. Susceptibility of rat non-alcoholic fatty liver to the acute toxic effect of acetaminophen. J. Gastroenterol. Hepatol. 27, 323–330. Lee, J.H., Baek, S.Y., Jang, E.J., Ku, S.K., Kim, K.M., Ki, S.H., Kim, C.E., Park, K.I., Kim, S.C., Kim, Y.W., 2018. Oxyresveratrol ameliorates nonalcoholic fatty liver disease by regulating hepatic lipogenesis and fatty acid oxidation through liver kinase B1 and AMP-activated protein kinase. Chem.-Biol. Interact. 289, 68–74. Li, C., Ye, L., Yang, L., Yu, X., He, Y., Chen, Z., Li, L., Zhang, D., 2017. Rapamycin promotes the survival and adipogenesis of ischemia-challenged adipose derived stem cells by improving autophagy. Cell. Physiol. Biochem.: Int. J. Exp. Cell. Physiol., Biochem. Pharmacol. 44, 1762–1774. Madrigal-Matute, J., Cuervo, A.M., 2016. Regulation of liver metabolism by autophagy. Gastroenterology 150, 328–339. Mazraati, P., Minaiyan, M., 2018. Hepatoprotective effect of metadoxine on acetaminophen-induced liver toxicity in mice. Adv. Biomed. Res. 7, 67. Mo, R., Lai, R., Lu, J., Zhuang, Y., Zhou, T., Jiang, S., Ren, P., Li, Z., Cao, Z., Liu, Y., Chen, L., Xiong, L., Wang, P., Wang, H., Cai, W., Xiang, X., Bao, S., Xie, Q., 2018. Enhanced autophagy contributes to protective effects of IL-22 against acetaminophen-induced liver injury. Theranostics 8, 4170–4180. Ni, H.M., McGill, M.R., Chao, X., Du, K., Williams, J.A., Xie, Y., Jaeschke, H., Ding, W.X., 2016. Removal of acetaminophen protein adducts by autophagy protects against acetaminophen-induced liver injury in mice. J. Hepatol. 65, 354–362. Nikravesh, H., Khodayar, M.J., Mahdavinia, M., Mansouri, E., Zeidooni, L., Dehbashi, F., 2018. Protective effect of gemfibrozil on hepatotoxicity induced by acetaminophen in mice: the importance of oxidative stress suppression. Adv. Pharm. Bull. 8, 331–339.
22