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Akt pathway
Biomedicine & Pharmacotherapy 97 (2018) 975–984
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Dihydroartemisinin inhibits ER stress-mediated mitochondrial pathway to attenuate hepatocyte lipoapoptosis via blocking the activation of the PI3K/ Akt pathway
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Xingran Chena, Mianli Biana, Chenxi Zhanga, Jun Kaia, Zhen Yaoa, Huanhuan Jina, Chunfeng Lua, ⁎⁎ ⁎ Jiangjuan Shaob, Anping Chenc, Feng Zhanga, , Shizhong Zhenga,d, a
Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Department of Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Department of Pathology, School of Medicine, Saint Louis University, St Louis MO63104, USA d Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, PR China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Alcoholic liver disease Dihydroartemisinin Hepatic steatosis Lipoapoptosis
Alcoholic liver disease (ALD), characterized by accumulation of fatty acids in liver cells, is usually caused by Chronic alcohol consumption. Our previous study has identified that DA protects against alcoholic liver injury in alcohol-fed rats through alleviating hepatocyte steatosis. It has emerged that saturated fatty acids could provoke endoplasmic reticulum (ER) stress and apoptosis in hepatocytes. This study was aimed to explore the impact of DA on ALD and further elaborate the underlying mechanisms. Results demonstrated that DA attenuates alcoholic liver injury in mice. Our results also indicated that DA attenuated lipid accumulation in hepatocytes exposed to ethanol. DA attenuates ethanol-induced hepatocyte apoptosis. Results demonstrated that DA dose-dependently ameliorated activation of mitochondrial pathway activation, which plays a critical role in apoptosis attributed to lipotoxicity. Further, DA suppressed the activation of JNK and the expression of CHOP, attributed to the inhibition of ER stress. It has emerged that activation of ER stress-JNK/CHOP-mitochondria cascade is considered as the key mechanisms underlying hepatocyte lipoapoptosis. In addition, DA attenuates PI3K/Akt Pathway in hepatocytes, consistent with our previous finding in HSCs. DA effects were reinforced by PI3K specific inhibitor LY294002. In summary, DA significantly protected hepatocytes against lipoapoptosis via a PI3K/Akt Pathway inhibition-dependent mechanism.
1. Introduction Alcoholic liver disease (ALD) is a major type of chronic liver disease caused by chronic ethanol consumption, ranging from simple fatty liver to more severe forms of liver injury, which is characterized by accumulation of large lipid droplets and mitochondrial enlargement in hepatocytes [1,2]. Experimental studies indicate that ethanol induced the increased amount of lipid droplets in hepatocytes, and the number of apoptotic cells were increased too [3,4]. Changes in cellular redox state or abnormal accumulation of toxic lipid species is thought to be one of the leading causes of activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress [5,6]. Moreover, the similar effects of obesity and alcohol abuse involve the ER response to cell
stress, but the underlying mechanism remains to be determined [7,8]. Lipotoxicity is a complicated process which have intersections of cell death pathways [9]. Lipoapoptosis occurs by activation of JNK and ER stress, then cooperatively to induce mitochondrial dysfunction that leads to cell death [10]. As a result, lipoapoptosis may be implicated in ethanol-induced hepatocyte injury [9]. Furthermore, Recent scientific reports indicated that mitochondria-mediated apoptosis is caused by dysregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt [11]. Akt is a central regulator of cell fate and also plays a key role in the regulation of lipid metabolism in a wide range of cell types, and this pathway has also been linked to mitochondrial apoptosis under various pathophysiological conditions [12]. Dihydroartemisinin (DA), derived from natural small-molecular
Abbreviations: CPT1, carnitine palmitoyltransferase 1; DMEM, Dulbecco’s modified eagle medium; DMSO, dimethylsulfoxide; FAS, fatty acid synthase; FBS, fetal bovine serum; SREBP1c, sterol regulatory element-binding protein-1c; Bax, bcl-associated X protein; Bcl-2, b-cell lymphoma-2; PUMA, p53-upregulated modulator of apoptosis; Bim, bcl-2-like protein 11; TC, total cholesterol; TG, triglyceride ⁎ Corresponding author at: Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, PR China. ⁎⁎ Corresponding author at: Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, PR China. E-mail addresses: [email protected] (F. Zhang), [email protected] (S. Zheng). http://dx.doi.org/10.1016/j.biopha.2017.11.010 Received 21 August 2017; Received in revised form 3 November 2017; Accepted 3 November 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.
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2.4. Oil red O staining
compound artemisinin, is the most active metabolite of all artemisinin derivatives [13,14]. DA was universally recognized as an anti-malarial drug recommended by World Health Organization (WHO) [15]. DA, which has favorable therapeutic effects on malaria, also exhibits potential pharmacological activities such as anti-tumor, anti-bacterial, and anti-fibrosis properties [16]. Our previous study for the first time confirmed that DA protects against alcoholic liver injury in alcohol-fed rats through alleviating hepatocyte steatosis, highlighting its potential influence on ALD [17]. In this study, we aimed to explore the underlying mechanisms of DA suppression on ethanol-induced hepatocyte apoptosis. We herein hypothesized and verified that DA attenuate Ethanol-induced hepatocyte lipoapoptosis through Modulating the PI3K/Akt Pathway.
Analysis of intracellular lipid droplets was determined using Oil Red O staining kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the culture medium was discarded and the cells were washed three times in phosphate buffer solution, as well as 10-mmthick frozen sections, then fixed with 4% paraformalde-hyde for 30 min and stained with 5% Oil Red O solution for 30 min. They were then washed with 60% isopropanol for 30 s and then rinsed with ddH2O for 30 s, counterstained with hematoxylin for 3 min, rinsed with ddH2O for 5 min, fixed with neutral balata, and then observed under an upright microscope (Leica, Germany). 2.5. Nile red staining
2. Materials and methods
LO2 cells were seeded on aseptic coverslips and incubated in 24well culture plates. After treatment with indicated agents, cells were washed three times with ice-cold phosphate buffer saline and then fixed with 4% paraformaldehyde for 20 min at room temperature. After immobilization, intracellular lipid droplets were stained with Nile Red (3.3 lg/mL) for 15 min at room temperature. Cellular nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI; KeyGEN BioTECH, Nanjing, Jiangsu, China) for 5 min at room temperature. Nile red fluorescent images were collected using a fluorescence microscope (Nikon, Tokyo, Japan).
2.1. Reagents and antibodies DA was purchased from Sigma-Aldrich (St Louis, MO, USA). The primary antibodies against phosphop53, p53, p53-upregulated modulator of apoptosis (PUMA), b-cell lymphoma-2 (Bcl-2), bcl-associated X protein (Bax), cleaved caspase-3, cleaved caspase-9, b-actin, and were purchased from Cell Signaling Technology (Danvers, MA). The primary antibodies against, PPARα, SREBP-1c were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibodies against phosphoPERK, phosphoJNK1 + JNK2 + JNK3, phosphoIRE1, FAS, SCD1, and bcl-2-like protein 11 (Bim) were purchased from Abcam (Cambridge, MA). The primary antibodies against phosphoIRE1, phosphoEIFα and CHOP were purchased from Proteintech Group, Inc. (Rosemont, IL). The primary antibodies against FGF21, FSP27 and VNN1 were purchased from Proteintech Group, Inc. (Rosemont, IL). The horseradish peroxidaseconjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology. The horseradish peroxidase-conjugated anti-goat IgG antibody (SA00001-4) was purchased from Proteintech Group, Inc. (Rosemont, IL).
2.6. Cell proliferation assay Cell viability was determined using a CCK-8 kit (Dojindo, Japan). Cells were seeded into 96-well Plates 200 μL to each well of the plate at 7 × 103 cells/well and cultured for 24 h at 37 °C with 5% CO2. After treatment with 0.1% DMSO as a control or OA at varying concentrations for 24 h, then, following the protocol to add the CCK-8 solutions 20 μL to each well of the plate. The cells were incubated for 2 h, and the absorbance of the samples (450 nm) was determined by using a scanning multiwell spectrophotometer.
2.2. Animals
2.7. Histopathology and immunohistochemistry
twenty-four male ICR mice (ages 6–8 weeks) were purchased from Comparative medicine center of Yangzhou University (Yangzhou, China). and randomly divided into three groups (eight mice/group). All animals received human care according to the National Institutes of Health guidelines. In detail, Red Star wine (56% v/v, 10 mL/kg) was introduced to generate alcoholic fatty liver in mice as we previously described (Lu et al., 2015b). DA was dissolved in olive oil (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China). Group 1 was the vehicle control in which mice were not given ethanol or DA but intraperitoneally (i.p.) injected with equal olive oil. Group 2 was ALD model group in which mice were gavaged with ethanol (56%, v/v, 1 mL/100 g) without DA. Group 3 was the DA-treated group in which mice were intraperitoneally (i.p.) injected with DA at 20 mg/kg once daily for five consecutive days per week.
Liver histology was examined using haematoxylin-eosin (H & E) staining as we previously described [24]. Immunohistochemistry of liver section was performed by using antibody against CD45 and F4/80 according to the previous study [25]. Frozen liver sections were stained with Oil Red O by using standard methods. Typical pictures of liver sections were shown. Image J software was used for quantification. 2.8. Biochemical analysis LO2 cells were incubated in six-well plates for 12 h and treated with corresponding reagents for 24 h. The levels of triglyceride (TG), total cholesterol (TC) were measured according to the protocols from the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The absorbance values were detected using a SPECTRAmaxTM microplate spectrophotometer (Molecular Devices).
2.3. Cell culture 2.9. Assessment of mitochondrial transmembrane potential Human hepatocyte LO2 cells were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). LO2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, Zhejiang, China), 100 U/mL penicillin, and 100 lg/mL streptomycin. Cells were grown in an incubator under a controlled condition of 95% air and 5% CO2 humidified atmosphere at 37 °C.
LO2 cells were incubated on coverslips in 24-well plates and cultured for 12 h, and then treated with correspondingreagents for 24 h. The changes in mitochondrial transmembrane potential (MTP) were evaluated using a JC-1 MTP assay kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) in strict accordance to the protocol. LO2 cells were observed under a fluorescence microscope (Nikon). 976
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2.10. Terminal deoxynucleotidyl transferase-mediated 2.10.1. Deoxyuridine triphosphate nick-end labeling assay LO2 cells were incubated on aseptic coverslips in 24-well culture plates for 12 h. Then cells were treated with indicated agents for 24 h. A terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining kit (Nanjing KeyGEN Biotech Co., Ltd.) was used to evaluate apoptotic hepatocyte according to the protocol. Cellular nuclei were stained with DAPI. Fluorescent photographs were taken under a fluorescence microscope (Nikon). 2.11. Flow cytometry analyses of apoptosis Apoptosis was detected by fluorescein isothiocyanate (FITC)-labeled Annexin-V/propidium iodide (PI) double staining and flow cytometry analysis. LO2 cells were incubated in six-well plates, cultured for 12 h, and then treated with corresponding reagents for 24 h. An AnnexinFITC apoptosis assay kit was used in strict accordance to the protocol from the manufacturer (Nanjing KeyGen Biotech Co., Ltd.). Apoptotic cells were defined as the cells situated in the right two quadrants of each plot and the percentages were determined by flow cytometry (FACS-Cabibur; Becton, Dickinson and Company, Franklin Lakes, NJ). Data were analyzed using CELLQuest software 2.12. Western blot analysis Fig. 1. DA attenuates improves alcoholic liver injury in mouse livers. Mice were grouped into: group 1, control; group 2, alcohol; group 3, alcohol + DA. (A) Liver sections were stained with H & E (original magnification, 20×). (B) Immunohistochemistry for CD45 and F4/80 in liver tissues (original magnification, 20×). (C) Oil Red O staining of liver sections (original magnification, 20×).
Protein extraction and western blot analysis were performed as we previously described [18]. The abundance of target protein bands was densitometrically determined using Quantity Ones 4.4.1 (Bio-Rad Laboratories, Berkeley, CA) and expressed as fold changes after normalization to the invariant control bactin or lamin B or total proteins.
3.2. DA attenuates ethanol-induced lipid accumulation in hepatocytes 2.13. Immunofluorescence staining Nile Red and Oil Red O staining revealed that the increased amount of lipid droplets in hepatocytes exposed to ethanol was almost abolished by DA (Fig. 2A–B). DA compared to the control group, levels of intracellular TG and TC were remarkably increased in model group. However, DA dose-dependently alleviated hepatic lipid accumulation, further confirming the results of Nile Red and Oil Red O staining (Fig. 2C). To study the influence of DA on de novo fatty acid synthesis, we analyzed the expressions of key lipogenic enzymes including FAS and SCD1 in hepatocytes treated with DA at concentrations of 5–20 μM. Western blot analyses indicated that ethanol significantly increased the expression of ethanol induced a significant increased protein expression of both FAS and SCD1, which was significantly reduced in hepatocytes with DA administration (Fig. 2D). It has been well known that chREBP and SREBP-1c are two important enzymes correlated with the regulation of fatty acid synthesis, which lipogenic enzyme genes are commonly trans-activated by. Western blot analyses and Immunofluorescence staining for SREBP-1c showed that SREBP-1c was also significantly decreased in hepatocytes treated with DA (Fig. 2D and F). We noticed that DA had certain influence on the expression of chREBP, but no significant difference, compared with ethanol group (Fig. S2A and C). Next, wondering if DA attenuates ethanol-induced lipid accumulation related regulation of fatty acid oxidation (FAO), we analyzed the expression of PPARα, a nuclear hormone receptor, modulating transport and oxidation of fatty acids. The results showed that protein expression of PPARα had an obvious decrease after ethanol exposure for 24 h, which could be reversed by DA treatment. DA administration also leads to increased expressions of FGF21, FSP27, VNN1, which were strictly PPARα dependent and could be measures of PPARα activity (Fig. 2E). We also investigated protein expressions of PGC-1α, a pivotal regulator of lipid metabolism, and CPT1α, one of canonical target genes (Figs. S2B). Furthermore, a consistent result was obtained from the immunofluorescence staining assay of PPARα (Fig. 2G).
LO2 cells were seeded on coverslips in 24-well plates and treated with corresponding reagents. Immunofluorescence staining was performed as we previously described [20]. Cellular nuclei were stained with DAPI. Immunofluorescence images were taken using a fluorescence microscope (Nikon). 2.14. Statistical analysis All experimental data were presented as mean ± SD, and results were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). The significance of difference was determined by one-way analysis of variance with the post hoc Dunnett’s test. Values of P < 0.05 were considered statistically significant. 3. Result 3.1. DA attenuates alcoholic liver injury H & E staining visually showed the alcohol caused seriously liver injury, characterized by swollen and disorganized hepatocytes, damage of hepatic lobule structure, and lytic necrosis and hepatic steatosis. A sequence of alcohol-induced pathological changes was improved in mice with DA administration (Fig. 1A). Ethanol-induced ROS production can result in lipid peroxidation and depletion of antioxidant defenses. The effects of DA supplementation on ethanol-induced oxidative stress in hepatocytes were shown in Fig. S1. Immunochemical analyses indicated that DA suppressed aggravated inflammatory cell infiltration in liver induced by alcohol, characterized by reduced intrahepatic CD45- and F4/80-positive cells (Fig. 1B). Oil Red O staining showed that DA dose-dependent attenuated the number and size of lipid droplets which is significantly elevated by ethanol (Fig. 1C). 977
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Fig. 2. DA reduces intracellular lipid accumulation in hepatocytes under ethanol stimulation. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A) LO2 cells were performed with were stained with Oil Red O. Lipid droplets were stained red. Representative photographs are shown (original magnification, 20×). (B) LO2 cells were performed with were stained with Nile Red (original magnification, 40×). (C) Determination of TC and TG levels. (D and E) protein expression of lipid metabolism-related genes in hepatocytes. (F and G) Immunofluorescence staining for SREBP-1c and PPAR-α in hepatocytes (original magnification, 40×). Data are expressed as mean ± SD, # P < 0.05 versus DMSO, # # P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P < 0.05, versus DMSO + ethanol, **P < 0.01 versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
lipoapoptosis. Thus we next investigated whether DA had effects on expressions of Bcl-2 family proteins (Fig. 4A–B). Results suggested that expression of pro-apoptotic proteins Bax was enhanced by ethanol, whereas expression of anti-apoptotic protein Bcl-2 was decreased, the Bax/Bcl-2 ratio increasing, leading to mitochondrial apoptosis pathway activation(Fig. 3A). Further, results also found that DA administration could supress the activation of p53 and downregulate pro-apoptotic proteins PUMA and Bim (Fig. 4B). Immunofluorescence staining for pp53 in hepatocytes visually confirmed the results of western blot analyses (Fig. 4D).
3.3. DA attenuates ethanol-induced hepatocyte lipoapoptosis To investigate that whether DA has protective effects on ethanolinduced lipotoxicity, we first employed Annexin v staining to examine the influence of apoptosis in hepatocytes treated with DA at the selected doses. Our results showed that ethanol exposure significantly increased apoptosis in hepatocytes when compared to those in control cells, which could be significantly reduced by DA (Fig. 3A). Consistent with the results above, the number of apoptotic cells stained by TUNEL in hepatocytes treated with ethanol was elevated markedly compared with control, while DA apparently reversed the events (Fig. 3B). Furthermore, ethanol-mediated activation of caspase-9/-3 was significantly reduced by DA (Fig. 3C).
3.5. Suppression of JNK and CHOP by DA is attributed to the inhibition of ER stress It is well established that mitochondrial-dependent apoptosis could be triggered by JNK and CHOP can through regulation of Bcl-2 family proteins. Both JNK and CHOP have a necessary role in ethanol-induced mitochondria-mediated lipoapoptosis. We then wondered if DA inactivated JNK and CHOP pathway to suppress mitochondrial activation. In the presence of DA, We confirmed that ethanol-mediated activations of JNK and CHOP were dramatically decreased (Fig. 5A). Immunofluorescence staining for CHOP verified the results of western blot (Fig. 5C). It has been well documented that downstream events of endoplasmic reticulum (ER) stress includes both JNK activation and CHOP up-regulation, which can be induced by ethanol. We therefore explored effect of DA on ethanol-activated ER stress. compared with
3.4. DA inhibits activation of mitochondrial pathway Mitochondrial pathway activation plays a critical role in apoptosis attributed to lipotoxicity of ethanol. Given that activation of mitochondrial pathway is characterized by disruption of mitochondrial transmembrane potential (MTP), we observed an obvious decline in MTP in ethanol-treated cells. With DA administration, these changes were significantly attenuated, suggesting that ethanol-induced activation of mitochondrial were significantly reduced by DA (Fig. 4C). In addition that Bcl-2 family proteins have been tightly implicated in regulating mitochondrial membrane potential, It also have been found that they might be involved in ethanol-induced hepatocyte 978
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Fig. 3. DA inhibits ethanol-induced apoptosis in hepatocytes. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A) Hepatocyte lipoapoptosis assessed by TUNEL. (B) Flow cytometric analyses of apoptosis. LO2 cells were double stained with FITC-labeled annexin-V/PI. The apoptotic rates were determined by flow cytometry. (C) Suppression of ethanol-activated caspases by DA in hepatocytes analyzed by western blotting. Data are expressed as mean ± SD, # P < 0.05 versus DMSO, # # #P < 0.001 versus DMSO, * P < 0.05, versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
control group, ethanol exposure remarkably enhanced the phosphorylation levels of PERK, EIF2α and IRE1α, which could be significantly reduced by DA (Fig. 5B). Collectively, our data consistently suggested that DA mitigates activation of ER stress, which might be associated with the inhibition of DA on activation of mitochondrial pathway.
hepatocytes, consistent with the effect of DA.Results indicated that apoptosis was significantly enhanced by co-treated LY294002 with DA (Fig. 6C).
3.6. Disruption of PI3K/Akt signaling was associated with the inhibition of lipoapoptosis by DA in hepatocytes
Alcohol consumption are widely prevalent around the world and can cause many health problems [1]. Cell death in the liver occurs mainly by apoptosis or necrosis. Thus, preventing further deterioration of ALD is urgent to develop effective drugs treating hepatocyte apoptosis. Many studies and ours have highlighted that DA had therapeutic effects on ethanol-induced hepatocyte injury [17]. However, nearly all of these studies focused merely on the limited observation of pharmacodynamic action on liver injury or on hepatic steatosis [18,19]. In this study, we hypothesized that DA could suppress hepatocyte lipoapoptosis to protect against alcoholic fatty liver disease through. Moreover, we creatively found that PI3 K/Akt pathway activation was extremely crucial for DA to improve lipid accumulation and hepatocyte lipoapoptosis in hepatocytes. This work confirmed that DA had the desirable therapeutic action
4. Discussion
Our previous work indicated that DA could dose-dependent reduce phosphorylation of PI3 K and Akt in different cell lines. In this study, We observed that ethanol dramatically decreased the activation of PI3 K and Akt in hepatocytes, and this event was significantly attenuated by DA (Fig. 6A). Inhibition of PI3 K/Akt signaling, an intracellular signaling pathway with central roles in the regulation lipid metabolism, reduced the intracellular lipid accumulation. We further used PI3 K inhibitor LY294002 to investigate the connection between disruption of the PI3 K/Akt pathway and DA. LY294002 inhibited the intracellular lipid accumulation, which was significantly enhanced by the administration of DA (Fig. 6B). In addition, LY294002 reduced the number of ethanol-induced apoptotic cells stained by TUNEL in 979
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Fig. 4. DA inhibits activation of mitochondrial pathway. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A and B) Changes of Bcl-2 family proteins by ethanol in the presence or absence of DA analyzed by western blotting in LO2 cells. (C) Protective effect of DA on ethanol −disrupted mitochondrial membrane potential in LO2 cells determined by JC-1 staining. (D) Immunofluorescence staining for p-p53 in hepatocytes (original magnification, 40×). Data are expressed as mean ± SD, # P < 0.05 versus DMSO, ##P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, * P < 0.05, versus DMSO + ethanol, **P < 0.01 versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
hepatocytes. For years, LO2 hepatocyte is especially useful for studying the mechanisms associated with the pathophysiology of hepatocyte such as hepatotoxicity. In this model, the major cause of alcohol hepatotoxicity includes Induction of ER stress and activation of mitochondrial apoptotic pathway. Chronic ethanol consumption can promote the formation of hepatic steatosis, the most remarkable pathological feature of ALD [17].
on alcohol-induced liver injury, inflammation, and steatosis in mice in accordance with previous study. Herein, to determine the effect of DA on ethanol-induced Hepatocyte lipoapoptosis, in our previous studies, LO2 hepatocytes were exposed to ethanol at the concentration of 100 mM for 24 h, recognized as an in vitro model of human ALD [17,20,21]. LO2 cells were initially derived from healthy human and maintained the biological features and ultrastructures of normal adult
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Fig. 5. DA mitigates activation of ER stress. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A) Inhibitory effect of DA on ethanol-mediated JNK and CHOP activation in vitro. The cells were exposed to ethanol in the presence or absence of DA for 24 h and then the changes of JNK phosphorylation and CHOP expression were assessed by western blotting. (B) Inhibitory effect of DA on ethanol mediated ER stress in cell culture. The cells were exposed to ethanol in the presence or absence of DA for 24 h and then the changes of key ER stress markers were assessed by western blotting. (C) Immunofluorescence staining for CHOP in hepatocytes (original magnification, 40×). Data are expressed as mean ± SD, # # P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, * P < 0.05, versus DMSO + ethanol, **P < 0.01 versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
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Fig. 6. DA inhibits hepatocyte lipoapoptosis by disrupting the PI3 K/Akt pathway. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A)Inhibitory effect of DA on ethanol-mediated PI3 K/Akt pathway activation in vitro. The cells were exposed to ethanol in the presence or absence of DA for 24 h and then the activation of PI3 K/Akt pathway were assessed by western blotting. (B) LO2 cells were performed with were stained with Oil Red O. Lipid droplets were stained red. Representative photographs are shown (original magnification, 2×). (C) Hepatocytes lipoapoptosis assessed by TUNEL. Data are expressed as mean ± SD, # P < 0.05 versus DMSO,* P < 0.05, versus DMSO + ethanol.
steatosis in ethanol-induced hepatocytes. Oil Red O staining is universally acknowledged as a vivid and direct assay for representing cell lipid dynamic [23]. Nile red is a lipophilic stain used to localize and quantitate lipids, exhibiting intense red fluorescence under the ultraviolet ligh [24]. Accordant to our previous conclusion, results showed
Previous study exhibited that chronic ethanol exposure may robustly elevate cellular TG accumulation and cause steatosis, a decrease in fatty acid oxidation and an increase in lipogenesis in hepatocytes [22]. To verify our hypothesis that DA can regulate lipid metabolism to alleviate ethanol-induced hepatotoxicity, We initially tested whether DA affected 982
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effect of DA against lipoapoptosis, which might be connected with PI3K/Akt signaling. Our present findings not only revealed that DA was a potential agent for ALD treatment through suppressing hepatocyte lipoapoptosis but also provided novel insights that lipoapoptosis could was involved in the pathological mechanism of ALD, which might be utilized to screen active pharmaceutical ingredients for the treatment of ALD.
that ethanol stimulation obviously increased the number of lipid droplets in hepatocytes. DA diminished lipid deposition in hepatocytes with administration of ethanol. considering that TG and TC are major components of lipid droplets in hepatocytes, we further investigated their contents in hepatocytes. Several studies have indicated that alcohol intake may directly or indirectly regulate lipid metabolism athwart the upregulation of SREBP-1c and downregulation of PPARα [24,25]. PPARα is emerging as a pivotal regulator of fatty acid oxidation through interacting with various transcriptional factors. PGC1α also plays a vital role in β-oxidation [21]. CPT1α, known as a target downstream of PPARα, is a key rate-limiting enzyme of β-oxidation [26]. SREBP-1 regulates gene-encoding proteins that are attached to both cholesterol genesis and lipogenesis, such as FAS and SCD1 [27]. After treatment with DA,the expressions of chREBP and L-PK, its canonical target gene, also had tendency to reduce. There might be some mechanism we had not figured out. In this study, we observed an increase in protein expression of PPARα but a decrease in protein expression of SREBP-1c caused by DA, which supported our hypothesis that DA could inhibit lipogenesis and induce lipolysis. Since excessive lipid accumulation triggers apoptosis in hepatocytes, we next analyzed the apoptosis of steatotic hepatocytes and the effects of DA. The results of Annexin v staining and TUNEL staining visually showed that DA significantly inhibited ethanol-induced hepatocyte apoptosis apparently. Activation of both Caspase-9 and −3 in hepatocytes exposed to ethanol could be reversed by DA treatment, indicating that DA inhibited the stimulative effects of ethanol on caspase family. Our study found that DA could attenuated ethanol-induced hepatocyte staetosis and apoptosis, but the molecular mechanism remains to be explored. Induction of ER stress and activation of mitochondrial apoptotic pathway are considered to be the key mechanisms underlying hepatocyte lipoapoptosis, which is the result of FA overaccumulation (steatosis) [9,28]. Early reports have proved that compared to PA, ethanol could also induce liver cell apoptosis through the intrinsic pathway and promote hepatic lipid accumulation and inflammation by activating ER stress response [4,29]. Therefore, we hypothesized that ethanol could induce hepatocyte lipoapoptosis and DA significantly attenuated the deleterious effects of ethanol by inhibiting ER stressmediated mitochondrial pathway. In this study, we demonstrated that levels of proapoptotic members of Bcl family increased [30], such as Bim, Bax, and PUMA, and levels of antiapoptotic member Bcl-2 decreased in ethanol-treated hepatocytes. All of these genes are components of mitochondrial “intrinsic” machinery that precede the activation of caspase family [31]. Consistent with previous finding, We found an obvious decline in MTP. Activation of JNK and CHOP functions as mediator to convey ER stress signals to mitochondria triggering mitochondrial-dependent apoptosis. Therefore, we comfirmed that JNK activation and CHOP up-regulation were reduced by DA. Moreover, our research also revealed that DA regulated ER stress to restrict activation of mitochondria pathway. All these results validated our previous hypothesis. A variety of signal transduction pathways are also involved in the hepatocytes staetosis and apoptosis. Activation of PI3K, which is important for regulating lipid metabolic homeostasis in vitro and for hepatocyte apoptosis in different cell types, leads to the activation of its key downstream kinase, Akt [32]. In our previous study, we found that DA inhibited PI3K/Akt signaling in HSCs [33]. therefore we confirmed that DA have the same effect on PI3K/Akt signaling in LO2 hepatocyte as in HSCs. Our results further proved that DA obviously inhibited hepatocyte staetosis and apoptosis induced by ethanol and had a synergistic effect with LY294002, a specific inhibitor of PI3K.But It remains elusive whether the current results are applicable to other hepatocyte lines or can be demonstrated by in vivo evidence. In conclusion, our work demonstrated that activation of ER stress −JNK/CHOP-mitochondria cascade was inhibited by DA in ethanoltreated hepatocytes, providing mechanistic support for the protective
Conflict of interest statement We declare that there are no conflicts of interest. Acknowledgments This study was supported by 2015 Program for Graduate Scientific Innovation of Jiangsu Higher Education Institutions (KYLX15_0999), the National Natural Science Foundation of China (81270514, 31401210, 31571455, 31600653, and 81600483), the Youth Natural Science Foundation of Jiangsu Province (BK20140955), the Open Project Program of Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica (JKLPSE 201502), and the Project of the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.biopha.2017.11.010. References [1] F.A. Livero, A. Acco, Molecular basis of alcoholic fatty liver disease: from incidence to treatment, Hepatol. Res.: Off. J. Jpn. Soc. Hepatol. 46 (1) (2016) 111–123. [2] Q.M. Anstee, D. Seth, C.P. Day, Genetic factors that affect risk of alcoholic and nonalcoholic fatty liver disease, Gastroenterology 150 (8) (2016) 1728–1744 (e7). [3] H. Ishii, M. Adachi, J.C. Fernandez-Checa, A.I. Cederbaum, I.V. Deaciuc, A.A. Nanji, Role of apoptosis in alcoholic liver injury, Alcohol. Clin. Exp. Res. 27 (7) (2003) 1207–1212. [4] H.W. Yi, Y.X. Ma, X.N. Wang, C.F. Wang, J. Lu, W. Cao, et al., Ethanol promotes saturated fatty acid-induced hepatoxicity through endoplasmic reticulum (ER) stress response, Chin. J. Nat. Med. 13 (4) (2015) 250–256. [5] L. Dara, C. Ji, N. Kaplowitz, The contribution of endoplasmic reticulum stress to liver diseases, Hepatology 53 (5) (2011) 1752–1763. [6] R. Sano, J.C. Reed, ER stress-induced cell death mechanisms, Biochim. Biophys. Acta 1833 (12) (2013) 3460–3470. [7] J. Xu, K.K. Lai, A. Verlinsky, A. Lugea, S.W. French, M.P. Cooper, et al., Synergistic steatohepatitis by moderate obesity and alcohol in mice despite increased adiponectin and p-AMPK, J. Hepatol. 55 (3) (2011) 673–682. [8] C. Ji, New insights into the pathogenesis of alcohol-Induced ER stress and liver diseases, Int. J. Hepatol. 2014 (2014) 513787. [9] Y. Akazawa, K. Nakao, Lipotoxicity pathways intersect in hepatocytes: endoplasmic reticulum stress, c-Jun N-terminal kinase-1, and death receptors, Hepatol. Res.: Off. J. Jpn. Soc. Hepatol. 46 (10) (2016) 977–984. [10] A.K. Hauck, D.A. Bernlohr, Oxidative stress and lipotoxicity, J. Lipid Res. 57 (11) (2016) 1976–1986. [11] M. Ye, J. Li, J. Gong, PCDH10 gene inhibits cell proliferation and induces cell apoptosis by inhibiting the PI3K/Akt signaling pathway in hepatocellular carcinoma cells, Oncol. Rep. 6 (2017) 3167–3174. [12] J. Li, Q. Huang, X. Long, J. Zhang, X. Huang, J. Aa, et al., CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARalpha pathways, J. Hepatol. 6 (2015) 1378–1389. [13] Z. Zhang, Z. Yao, S. Zhao, J. Shao, A. Chen, F. Zhang, et al., Interaction between autophagy and senescence is required for dihydroartemisinin to alleviate liver fibrosis, Cell Death. Dis. 8 (6) (2017) e2886. [14] X. Shi, L. Wang, X. Li, J. Bai, J. Li, S. Li, et al., Dihydroartemisinin induces autophagy-dependent death in human tongue squamous cell carcinoma cells through DNA double-strand break-mediated oxidative stress, Oncotarget 8 (28) (2017) 45981–45993. [15] X.G. Zhang, G.X. Li, S.S. Zhao, F.L. Xu, Y.H. Wang, W. Wang, A review of dihydroartemisinin as another gift from traditional Chinese medicine not only for malaria control but also for schistosomiasis control, Parasitol. Res. 113 (5) (2014) 1769–1773. [16] L.B. Jiang, D.H. Meng, S.M. Lee, S.H. Liu, Q.T. Xu, Y. Wang, et al., Dihydroartemisinin inhibits catabolism in rat chondrocytes by activating autophagy via inhibition of the NF-kappaB pathway, Sci. Rep. 6 (2016) 38979. [17] W. Xu, C. Lu, L. Yao, F. Zhang, J. Shao, S. Zheng, Dihydroartemisinin protects
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Dihydroartemisinin inhibits ER stress-mediated mitochondrial pathway to attenuate hepatocyte lipoapoptosis via blocking the activation of the PI3K/ Akt pathway
T
Xingran Chena, Mianli Biana, Chenxi Zhanga, Jun Kaia, Zhen Yaoa, Huanhuan Jina, Chunfeng Lua, ⁎⁎ ⁎ Jiangjuan Shaob, Anping Chenc, Feng Zhanga, , Shizhong Zhenga,d, a
Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Department of Pharmacy, College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210023, PR China Department of Pathology, School of Medicine, Saint Louis University, St Louis MO63104, USA d Jiangsu Key Laboratory for Pharmacology and Safety Evaluation of Chinese Materia Medica, Nanjing University of Chinese Medicine, Nanjing, PR China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Alcoholic liver disease Dihydroartemisinin Hepatic steatosis Lipoapoptosis
Alcoholic liver disease (ALD), characterized by accumulation of fatty acids in liver cells, is usually caused by Chronic alcohol consumption. Our previous study has identified that DA protects against alcoholic liver injury in alcohol-fed rats through alleviating hepatocyte steatosis. It has emerged that saturated fatty acids could provoke endoplasmic reticulum (ER) stress and apoptosis in hepatocytes. This study was aimed to explore the impact of DA on ALD and further elaborate the underlying mechanisms. Results demonstrated that DA attenuates alcoholic liver injury in mice. Our results also indicated that DA attenuated lipid accumulation in hepatocytes exposed to ethanol. DA attenuates ethanol-induced hepatocyte apoptosis. Results demonstrated that DA dose-dependently ameliorated activation of mitochondrial pathway activation, which plays a critical role in apoptosis attributed to lipotoxicity. Further, DA suppressed the activation of JNK and the expression of CHOP, attributed to the inhibition of ER stress. It has emerged that activation of ER stress-JNK/CHOP-mitochondria cascade is considered as the key mechanisms underlying hepatocyte lipoapoptosis. In addition, DA attenuates PI3K/Akt Pathway in hepatocytes, consistent with our previous finding in HSCs. DA effects were reinforced by PI3K specific inhibitor LY294002. In summary, DA significantly protected hepatocytes against lipoapoptosis via a PI3K/Akt Pathway inhibition-dependent mechanism.
1. Introduction Alcoholic liver disease (ALD) is a major type of chronic liver disease caused by chronic ethanol consumption, ranging from simple fatty liver to more severe forms of liver injury, which is characterized by accumulation of large lipid droplets and mitochondrial enlargement in hepatocytes [1,2]. Experimental studies indicate that ethanol induced the increased amount of lipid droplets in hepatocytes, and the number of apoptotic cells were increased too [3,4]. Changes in cellular redox state or abnormal accumulation of toxic lipid species is thought to be one of the leading causes of activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress [5,6]. Moreover, the similar effects of obesity and alcohol abuse involve the ER response to cell
stress, but the underlying mechanism remains to be determined [7,8]. Lipotoxicity is a complicated process which have intersections of cell death pathways [9]. Lipoapoptosis occurs by activation of JNK and ER stress, then cooperatively to induce mitochondrial dysfunction that leads to cell death [10]. As a result, lipoapoptosis may be implicated in ethanol-induced hepatocyte injury [9]. Furthermore, Recent scientific reports indicated that mitochondria-mediated apoptosis is caused by dysregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt [11]. Akt is a central regulator of cell fate and also plays a key role in the regulation of lipid metabolism in a wide range of cell types, and this pathway has also been linked to mitochondrial apoptosis under various pathophysiological conditions [12]. Dihydroartemisinin (DA), derived from natural small-molecular
Abbreviations: CPT1, carnitine palmitoyltransferase 1; DMEM, Dulbecco’s modified eagle medium; DMSO, dimethylsulfoxide; FAS, fatty acid synthase; FBS, fetal bovine serum; SREBP1c, sterol regulatory element-binding protein-1c; Bax, bcl-associated X protein; Bcl-2, b-cell lymphoma-2; PUMA, p53-upregulated modulator of apoptosis; Bim, bcl-2-like protein 11; TC, total cholesterol; TG, triglyceride ⁎ Corresponding author at: Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, PR China. ⁎⁎ Corresponding author at: Department of Pharmacology, College of Pharmacy, Nanjing University of Chinese Medicine, 138 Xianlin Avenue, Nanjing, Jiangsu 210023, PR China. E-mail addresses: [email protected] (F. Zhang), [email protected] (S. Zheng). http://dx.doi.org/10.1016/j.biopha.2017.11.010 Received 21 August 2017; Received in revised form 3 November 2017; Accepted 3 November 2017 0753-3322/ © 2017 Published by Elsevier Masson SAS.
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2.4. Oil red O staining
compound artemisinin, is the most active metabolite of all artemisinin derivatives [13,14]. DA was universally recognized as an anti-malarial drug recommended by World Health Organization (WHO) [15]. DA, which has favorable therapeutic effects on malaria, also exhibits potential pharmacological activities such as anti-tumor, anti-bacterial, and anti-fibrosis properties [16]. Our previous study for the first time confirmed that DA protects against alcoholic liver injury in alcohol-fed rats through alleviating hepatocyte steatosis, highlighting its potential influence on ALD [17]. In this study, we aimed to explore the underlying mechanisms of DA suppression on ethanol-induced hepatocyte apoptosis. We herein hypothesized and verified that DA attenuate Ethanol-induced hepatocyte lipoapoptosis through Modulating the PI3K/Akt Pathway.
Analysis of intracellular lipid droplets was determined using Oil Red O staining kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, the culture medium was discarded and the cells were washed three times in phosphate buffer solution, as well as 10-mmthick frozen sections, then fixed with 4% paraformalde-hyde for 30 min and stained with 5% Oil Red O solution for 30 min. They were then washed with 60% isopropanol for 30 s and then rinsed with ddH2O for 30 s, counterstained with hematoxylin for 3 min, rinsed with ddH2O for 5 min, fixed with neutral balata, and then observed under an upright microscope (Leica, Germany). 2.5. Nile red staining
2. Materials and methods
LO2 cells were seeded on aseptic coverslips and incubated in 24well culture plates. After treatment with indicated agents, cells were washed three times with ice-cold phosphate buffer saline and then fixed with 4% paraformaldehyde for 20 min at room temperature. After immobilization, intracellular lipid droplets were stained with Nile Red (3.3 lg/mL) for 15 min at room temperature. Cellular nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI; KeyGEN BioTECH, Nanjing, Jiangsu, China) for 5 min at room temperature. Nile red fluorescent images were collected using a fluorescence microscope (Nikon, Tokyo, Japan).
2.1. Reagents and antibodies DA was purchased from Sigma-Aldrich (St Louis, MO, USA). The primary antibodies against phosphop53, p53, p53-upregulated modulator of apoptosis (PUMA), b-cell lymphoma-2 (Bcl-2), bcl-associated X protein (Bax), cleaved caspase-3, cleaved caspase-9, b-actin, and were purchased from Cell Signaling Technology (Danvers, MA). The primary antibodies against, PPARα, SREBP-1c were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA). The primary antibodies against phosphoPERK, phosphoJNK1 + JNK2 + JNK3, phosphoIRE1, FAS, SCD1, and bcl-2-like protein 11 (Bim) were purchased from Abcam (Cambridge, MA). The primary antibodies against phosphoIRE1, phosphoEIFα and CHOP were purchased from Proteintech Group, Inc. (Rosemont, IL). The primary antibodies against FGF21, FSP27 and VNN1 were purchased from Proteintech Group, Inc. (Rosemont, IL). The horseradish peroxidaseconjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology. The horseradish peroxidase-conjugated anti-goat IgG antibody (SA00001-4) was purchased from Proteintech Group, Inc. (Rosemont, IL).
2.6. Cell proliferation assay Cell viability was determined using a CCK-8 kit (Dojindo, Japan). Cells were seeded into 96-well Plates 200 μL to each well of the plate at 7 × 103 cells/well and cultured for 24 h at 37 °C with 5% CO2. After treatment with 0.1% DMSO as a control or OA at varying concentrations for 24 h, then, following the protocol to add the CCK-8 solutions 20 μL to each well of the plate. The cells were incubated for 2 h, and the absorbance of the samples (450 nm) was determined by using a scanning multiwell spectrophotometer.
2.2. Animals
2.7. Histopathology and immunohistochemistry
twenty-four male ICR mice (ages 6–8 weeks) were purchased from Comparative medicine center of Yangzhou University (Yangzhou, China). and randomly divided into three groups (eight mice/group). All animals received human care according to the National Institutes of Health guidelines. In detail, Red Star wine (56% v/v, 10 mL/kg) was introduced to generate alcoholic fatty liver in mice as we previously described (Lu et al., 2015b). DA was dissolved in olive oil (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China). Group 1 was the vehicle control in which mice were not given ethanol or DA but intraperitoneally (i.p.) injected with equal olive oil. Group 2 was ALD model group in which mice were gavaged with ethanol (56%, v/v, 1 mL/100 g) without DA. Group 3 was the DA-treated group in which mice were intraperitoneally (i.p.) injected with DA at 20 mg/kg once daily for five consecutive days per week.
Liver histology was examined using haematoxylin-eosin (H & E) staining as we previously described [24]. Immunohistochemistry of liver section was performed by using antibody against CD45 and F4/80 according to the previous study [25]. Frozen liver sections were stained with Oil Red O by using standard methods. Typical pictures of liver sections were shown. Image J software was used for quantification. 2.8. Biochemical analysis LO2 cells were incubated in six-well plates for 12 h and treated with corresponding reagents for 24 h. The levels of triglyceride (TG), total cholesterol (TC) were measured according to the protocols from the manufacturer (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu, China). The absorbance values were detected using a SPECTRAmaxTM microplate spectrophotometer (Molecular Devices).
2.3. Cell culture 2.9. Assessment of mitochondrial transmembrane potential Human hepatocyte LO2 cells were purchased from Cell Bank of Chinese Academy of Sciences (Shanghai, China). LO2 cells were cultured in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Sijiqing Biological Engineering Materials Co., Ltd., Hangzhou, Zhejiang, China), 100 U/mL penicillin, and 100 lg/mL streptomycin. Cells were grown in an incubator under a controlled condition of 95% air and 5% CO2 humidified atmosphere at 37 °C.
LO2 cells were incubated on coverslips in 24-well plates and cultured for 12 h, and then treated with correspondingreagents for 24 h. The changes in mitochondrial transmembrane potential (MTP) were evaluated using a JC-1 MTP assay kit (Beyotime Institute of Biotechnology, Haimen, Jiangsu, China) in strict accordance to the protocol. LO2 cells were observed under a fluorescence microscope (Nikon). 976
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2.10. Terminal deoxynucleotidyl transferase-mediated 2.10.1. Deoxyuridine triphosphate nick-end labeling assay LO2 cells were incubated on aseptic coverslips in 24-well culture plates for 12 h. Then cells were treated with indicated agents for 24 h. A terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining kit (Nanjing KeyGEN Biotech Co., Ltd.) was used to evaluate apoptotic hepatocyte according to the protocol. Cellular nuclei were stained with DAPI. Fluorescent photographs were taken under a fluorescence microscope (Nikon). 2.11. Flow cytometry analyses of apoptosis Apoptosis was detected by fluorescein isothiocyanate (FITC)-labeled Annexin-V/propidium iodide (PI) double staining and flow cytometry analysis. LO2 cells were incubated in six-well plates, cultured for 12 h, and then treated with corresponding reagents for 24 h. An AnnexinFITC apoptosis assay kit was used in strict accordance to the protocol from the manufacturer (Nanjing KeyGen Biotech Co., Ltd.). Apoptotic cells were defined as the cells situated in the right two quadrants of each plot and the percentages were determined by flow cytometry (FACS-Cabibur; Becton, Dickinson and Company, Franklin Lakes, NJ). Data were analyzed using CELLQuest software 2.12. Western blot analysis Fig. 1. DA attenuates improves alcoholic liver injury in mouse livers. Mice were grouped into: group 1, control; group 2, alcohol; group 3, alcohol + DA. (A) Liver sections were stained with H & E (original magnification, 20×). (B) Immunohistochemistry for CD45 and F4/80 in liver tissues (original magnification, 20×). (C) Oil Red O staining of liver sections (original magnification, 20×).
Protein extraction and western blot analysis were performed as we previously described [18]. The abundance of target protein bands was densitometrically determined using Quantity Ones 4.4.1 (Bio-Rad Laboratories, Berkeley, CA) and expressed as fold changes after normalization to the invariant control bactin or lamin B or total proteins.
3.2. DA attenuates ethanol-induced lipid accumulation in hepatocytes 2.13. Immunofluorescence staining Nile Red and Oil Red O staining revealed that the increased amount of lipid droplets in hepatocytes exposed to ethanol was almost abolished by DA (Fig. 2A–B). DA compared to the control group, levels of intracellular TG and TC were remarkably increased in model group. However, DA dose-dependently alleviated hepatic lipid accumulation, further confirming the results of Nile Red and Oil Red O staining (Fig. 2C). To study the influence of DA on de novo fatty acid synthesis, we analyzed the expressions of key lipogenic enzymes including FAS and SCD1 in hepatocytes treated with DA at concentrations of 5–20 μM. Western blot analyses indicated that ethanol significantly increased the expression of ethanol induced a significant increased protein expression of both FAS and SCD1, which was significantly reduced in hepatocytes with DA administration (Fig. 2D). It has been well known that chREBP and SREBP-1c are two important enzymes correlated with the regulation of fatty acid synthesis, which lipogenic enzyme genes are commonly trans-activated by. Western blot analyses and Immunofluorescence staining for SREBP-1c showed that SREBP-1c was also significantly decreased in hepatocytes treated with DA (Fig. 2D and F). We noticed that DA had certain influence on the expression of chREBP, but no significant difference, compared with ethanol group (Fig. S2A and C). Next, wondering if DA attenuates ethanol-induced lipid accumulation related regulation of fatty acid oxidation (FAO), we analyzed the expression of PPARα, a nuclear hormone receptor, modulating transport and oxidation of fatty acids. The results showed that protein expression of PPARα had an obvious decrease after ethanol exposure for 24 h, which could be reversed by DA treatment. DA administration also leads to increased expressions of FGF21, FSP27, VNN1, which were strictly PPARα dependent and could be measures of PPARα activity (Fig. 2E). We also investigated protein expressions of PGC-1α, a pivotal regulator of lipid metabolism, and CPT1α, one of canonical target genes (Figs. S2B). Furthermore, a consistent result was obtained from the immunofluorescence staining assay of PPARα (Fig. 2G).
LO2 cells were seeded on coverslips in 24-well plates and treated with corresponding reagents. Immunofluorescence staining was performed as we previously described [20]. Cellular nuclei were stained with DAPI. Immunofluorescence images were taken using a fluorescence microscope (Nikon). 2.14. Statistical analysis All experimental data were presented as mean ± SD, and results were analyzed using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). The significance of difference was determined by one-way analysis of variance with the post hoc Dunnett’s test. Values of P < 0.05 were considered statistically significant. 3. Result 3.1. DA attenuates alcoholic liver injury H & E staining visually showed the alcohol caused seriously liver injury, characterized by swollen and disorganized hepatocytes, damage of hepatic lobule structure, and lytic necrosis and hepatic steatosis. A sequence of alcohol-induced pathological changes was improved in mice with DA administration (Fig. 1A). Ethanol-induced ROS production can result in lipid peroxidation and depletion of antioxidant defenses. The effects of DA supplementation on ethanol-induced oxidative stress in hepatocytes were shown in Fig. S1. Immunochemical analyses indicated that DA suppressed aggravated inflammatory cell infiltration in liver induced by alcohol, characterized by reduced intrahepatic CD45- and F4/80-positive cells (Fig. 1B). Oil Red O staining showed that DA dose-dependent attenuated the number and size of lipid droplets which is significantly elevated by ethanol (Fig. 1C). 977
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Fig. 2. DA reduces intracellular lipid accumulation in hepatocytes under ethanol stimulation. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A) LO2 cells were performed with were stained with Oil Red O. Lipid droplets were stained red. Representative photographs are shown (original magnification, 20×). (B) LO2 cells were performed with were stained with Nile Red (original magnification, 40×). (C) Determination of TC and TG levels. (D and E) protein expression of lipid metabolism-related genes in hepatocytes. (F and G) Immunofluorescence staining for SREBP-1c and PPAR-α in hepatocytes (original magnification, 40×). Data are expressed as mean ± SD, # P < 0.05 versus DMSO, # # P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, *P < 0.05, versus DMSO + ethanol, **P < 0.01 versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
lipoapoptosis. Thus we next investigated whether DA had effects on expressions of Bcl-2 family proteins (Fig. 4A–B). Results suggested that expression of pro-apoptotic proteins Bax was enhanced by ethanol, whereas expression of anti-apoptotic protein Bcl-2 was decreased, the Bax/Bcl-2 ratio increasing, leading to mitochondrial apoptosis pathway activation(Fig. 3A). Further, results also found that DA administration could supress the activation of p53 and downregulate pro-apoptotic proteins PUMA and Bim (Fig. 4B). Immunofluorescence staining for pp53 in hepatocytes visually confirmed the results of western blot analyses (Fig. 4D).
3.3. DA attenuates ethanol-induced hepatocyte lipoapoptosis To investigate that whether DA has protective effects on ethanolinduced lipotoxicity, we first employed Annexin v staining to examine the influence of apoptosis in hepatocytes treated with DA at the selected doses. Our results showed that ethanol exposure significantly increased apoptosis in hepatocytes when compared to those in control cells, which could be significantly reduced by DA (Fig. 3A). Consistent with the results above, the number of apoptotic cells stained by TUNEL in hepatocytes treated with ethanol was elevated markedly compared with control, while DA apparently reversed the events (Fig. 3B). Furthermore, ethanol-mediated activation of caspase-9/-3 was significantly reduced by DA (Fig. 3C).
3.5. Suppression of JNK and CHOP by DA is attributed to the inhibition of ER stress It is well established that mitochondrial-dependent apoptosis could be triggered by JNK and CHOP can through regulation of Bcl-2 family proteins. Both JNK and CHOP have a necessary role in ethanol-induced mitochondria-mediated lipoapoptosis. We then wondered if DA inactivated JNK and CHOP pathway to suppress mitochondrial activation. In the presence of DA, We confirmed that ethanol-mediated activations of JNK and CHOP were dramatically decreased (Fig. 5A). Immunofluorescence staining for CHOP verified the results of western blot (Fig. 5C). It has been well documented that downstream events of endoplasmic reticulum (ER) stress includes both JNK activation and CHOP up-regulation, which can be induced by ethanol. We therefore explored effect of DA on ethanol-activated ER stress. compared with
3.4. DA inhibits activation of mitochondrial pathway Mitochondrial pathway activation plays a critical role in apoptosis attributed to lipotoxicity of ethanol. Given that activation of mitochondrial pathway is characterized by disruption of mitochondrial transmembrane potential (MTP), we observed an obvious decline in MTP in ethanol-treated cells. With DA administration, these changes were significantly attenuated, suggesting that ethanol-induced activation of mitochondrial were significantly reduced by DA (Fig. 4C). In addition that Bcl-2 family proteins have been tightly implicated in regulating mitochondrial membrane potential, It also have been found that they might be involved in ethanol-induced hepatocyte 978
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Fig. 3. DA inhibits ethanol-induced apoptosis in hepatocytes. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A) Hepatocyte lipoapoptosis assessed by TUNEL. (B) Flow cytometric analyses of apoptosis. LO2 cells were double stained with FITC-labeled annexin-V/PI. The apoptotic rates were determined by flow cytometry. (C) Suppression of ethanol-activated caspases by DA in hepatocytes analyzed by western blotting. Data are expressed as mean ± SD, # P < 0.05 versus DMSO, # # #P < 0.001 versus DMSO, * P < 0.05, versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
control group, ethanol exposure remarkably enhanced the phosphorylation levels of PERK, EIF2α and IRE1α, which could be significantly reduced by DA (Fig. 5B). Collectively, our data consistently suggested that DA mitigates activation of ER stress, which might be associated with the inhibition of DA on activation of mitochondrial pathway.
hepatocytes, consistent with the effect of DA.Results indicated that apoptosis was significantly enhanced by co-treated LY294002 with DA (Fig. 6C).
3.6. Disruption of PI3K/Akt signaling was associated with the inhibition of lipoapoptosis by DA in hepatocytes
Alcohol consumption are widely prevalent around the world and can cause many health problems [1]. Cell death in the liver occurs mainly by apoptosis or necrosis. Thus, preventing further deterioration of ALD is urgent to develop effective drugs treating hepatocyte apoptosis. Many studies and ours have highlighted that DA had therapeutic effects on ethanol-induced hepatocyte injury [17]. However, nearly all of these studies focused merely on the limited observation of pharmacodynamic action on liver injury or on hepatic steatosis [18,19]. In this study, we hypothesized that DA could suppress hepatocyte lipoapoptosis to protect against alcoholic fatty liver disease through. Moreover, we creatively found that PI3 K/Akt pathway activation was extremely crucial for DA to improve lipid accumulation and hepatocyte lipoapoptosis in hepatocytes. This work confirmed that DA had the desirable therapeutic action
4. Discussion
Our previous work indicated that DA could dose-dependent reduce phosphorylation of PI3 K and Akt in different cell lines. In this study, We observed that ethanol dramatically decreased the activation of PI3 K and Akt in hepatocytes, and this event was significantly attenuated by DA (Fig. 6A). Inhibition of PI3 K/Akt signaling, an intracellular signaling pathway with central roles in the regulation lipid metabolism, reduced the intracellular lipid accumulation. We further used PI3 K inhibitor LY294002 to investigate the connection between disruption of the PI3 K/Akt pathway and DA. LY294002 inhibited the intracellular lipid accumulation, which was significantly enhanced by the administration of DA (Fig. 6B). In addition, LY294002 reduced the number of ethanol-induced apoptotic cells stained by TUNEL in 979
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Fig. 4. DA inhibits activation of mitochondrial pathway. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A and B) Changes of Bcl-2 family proteins by ethanol in the presence or absence of DA analyzed by western blotting in LO2 cells. (C) Protective effect of DA on ethanol −disrupted mitochondrial membrane potential in LO2 cells determined by JC-1 staining. (D) Immunofluorescence staining for p-p53 in hepatocytes (original magnification, 40×). Data are expressed as mean ± SD, # P < 0.05 versus DMSO, ##P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, * P < 0.05, versus DMSO + ethanol, **P < 0.01 versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
hepatocytes. For years, LO2 hepatocyte is especially useful for studying the mechanisms associated with the pathophysiology of hepatocyte such as hepatotoxicity. In this model, the major cause of alcohol hepatotoxicity includes Induction of ER stress and activation of mitochondrial apoptotic pathway. Chronic ethanol consumption can promote the formation of hepatic steatosis, the most remarkable pathological feature of ALD [17].
on alcohol-induced liver injury, inflammation, and steatosis in mice in accordance with previous study. Herein, to determine the effect of DA on ethanol-induced Hepatocyte lipoapoptosis, in our previous studies, LO2 hepatocytes were exposed to ethanol at the concentration of 100 mM for 24 h, recognized as an in vitro model of human ALD [17,20,21]. LO2 cells were initially derived from healthy human and maintained the biological features and ultrastructures of normal adult
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Fig. 5. DA mitigates activation of ER stress. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A) Inhibitory effect of DA on ethanol-mediated JNK and CHOP activation in vitro. The cells were exposed to ethanol in the presence or absence of DA for 24 h and then the changes of JNK phosphorylation and CHOP expression were assessed by western blotting. (B) Inhibitory effect of DA on ethanol mediated ER stress in cell culture. The cells were exposed to ethanol in the presence or absence of DA for 24 h and then the changes of key ER stress markers were assessed by western blotting. (C) Immunofluorescence staining for CHOP in hepatocytes (original magnification, 40×). Data are expressed as mean ± SD, # # P < 0.01 versus DMSO, ###P < 0.001 versus DMSO, * P < 0.05, versus DMSO + ethanol, **P < 0.01 versus DMSO + ethanol, ***P < 0.001 versus DMSO + ethanol.
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Fig. 6. DA inhibits hepatocyte lipoapoptosis by disrupting the PI3 K/Akt pathway. LO2 cells were exposed to ethanol (100 mM) in the absence or presence of DA for 24 h. DA (97% purity) was dissolved in DMSO. (A)Inhibitory effect of DA on ethanol-mediated PI3 K/Akt pathway activation in vitro. The cells were exposed to ethanol in the presence or absence of DA for 24 h and then the activation of PI3 K/Akt pathway were assessed by western blotting. (B) LO2 cells were performed with were stained with Oil Red O. Lipid droplets were stained red. Representative photographs are shown (original magnification, 2×). (C) Hepatocytes lipoapoptosis assessed by TUNEL. Data are expressed as mean ± SD, # P < 0.05 versus DMSO,* P < 0.05, versus DMSO + ethanol.
steatosis in ethanol-induced hepatocytes. Oil Red O staining is universally acknowledged as a vivid and direct assay for representing cell lipid dynamic [23]. Nile red is a lipophilic stain used to localize and quantitate lipids, exhibiting intense red fluorescence under the ultraviolet ligh [24]. Accordant to our previous conclusion, results showed
Previous study exhibited that chronic ethanol exposure may robustly elevate cellular TG accumulation and cause steatosis, a decrease in fatty acid oxidation and an increase in lipogenesis in hepatocytes [22]. To verify our hypothesis that DA can regulate lipid metabolism to alleviate ethanol-induced hepatotoxicity, We initially tested whether DA affected 982
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effect of DA against lipoapoptosis, which might be connected with PI3K/Akt signaling. Our present findings not only revealed that DA was a potential agent for ALD treatment through suppressing hepatocyte lipoapoptosis but also provided novel insights that lipoapoptosis could was involved in the pathological mechanism of ALD, which might be utilized to screen active pharmaceutical ingredients for the treatment of ALD.
that ethanol stimulation obviously increased the number of lipid droplets in hepatocytes. DA diminished lipid deposition in hepatocytes with administration of ethanol. considering that TG and TC are major components of lipid droplets in hepatocytes, we further investigated their contents in hepatocytes. Several studies have indicated that alcohol intake may directly or indirectly regulate lipid metabolism athwart the upregulation of SREBP-1c and downregulation of PPARα [24,25]. PPARα is emerging as a pivotal regulator of fatty acid oxidation through interacting with various transcriptional factors. PGC1α also plays a vital role in β-oxidation [21]. CPT1α, known as a target downstream of PPARα, is a key rate-limiting enzyme of β-oxidation [26]. SREBP-1 regulates gene-encoding proteins that are attached to both cholesterol genesis and lipogenesis, such as FAS and SCD1 [27]. After treatment with DA,the expressions of chREBP and L-PK, its canonical target gene, also had tendency to reduce. There might be some mechanism we had not figured out. In this study, we observed an increase in protein expression of PPARα but a decrease in protein expression of SREBP-1c caused by DA, which supported our hypothesis that DA could inhibit lipogenesis and induce lipolysis. Since excessive lipid accumulation triggers apoptosis in hepatocytes, we next analyzed the apoptosis of steatotic hepatocytes and the effects of DA. The results of Annexin v staining and TUNEL staining visually showed that DA significantly inhibited ethanol-induced hepatocyte apoptosis apparently. Activation of both Caspase-9 and −3 in hepatocytes exposed to ethanol could be reversed by DA treatment, indicating that DA inhibited the stimulative effects of ethanol on caspase family. Our study found that DA could attenuated ethanol-induced hepatocyte staetosis and apoptosis, but the molecular mechanism remains to be explored. Induction of ER stress and activation of mitochondrial apoptotic pathway are considered to be the key mechanisms underlying hepatocyte lipoapoptosis, which is the result of FA overaccumulation (steatosis) [9,28]. Early reports have proved that compared to PA, ethanol could also induce liver cell apoptosis through the intrinsic pathway and promote hepatic lipid accumulation and inflammation by activating ER stress response [4,29]. Therefore, we hypothesized that ethanol could induce hepatocyte lipoapoptosis and DA significantly attenuated the deleterious effects of ethanol by inhibiting ER stressmediated mitochondrial pathway. In this study, we demonstrated that levels of proapoptotic members of Bcl family increased [30], such as Bim, Bax, and PUMA, and levels of antiapoptotic member Bcl-2 decreased in ethanol-treated hepatocytes. All of these genes are components of mitochondrial “intrinsic” machinery that precede the activation of caspase family [31]. Consistent with previous finding, We found an obvious decline in MTP. Activation of JNK and CHOP functions as mediator to convey ER stress signals to mitochondria triggering mitochondrial-dependent apoptosis. Therefore, we comfirmed that JNK activation and CHOP up-regulation were reduced by DA. Moreover, our research also revealed that DA regulated ER stress to restrict activation of mitochondria pathway. All these results validated our previous hypothesis. A variety of signal transduction pathways are also involved in the hepatocytes staetosis and apoptosis. Activation of PI3K, which is important for regulating lipid metabolic homeostasis in vitro and for hepatocyte apoptosis in different cell types, leads to the activation of its key downstream kinase, Akt [32]. In our previous study, we found that DA inhibited PI3K/Akt signaling in HSCs [33]. therefore we confirmed that DA have the same effect on PI3K/Akt signaling in LO2 hepatocyte as in HSCs. Our results further proved that DA obviously inhibited hepatocyte staetosis and apoptosis induced by ethanol and had a synergistic effect with LY294002, a specific inhibitor of PI3K.But It remains elusive whether the current results are applicable to other hepatocyte lines or can be demonstrated by in vivo evidence. In conclusion, our work demonstrated that activation of ER stress −JNK/CHOP-mitochondria cascade was inhibited by DA in ethanoltreated hepatocytes, providing mechanistic support for the protective
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