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Ligustilide inhibits tumour necrosis factor-alpha-induced autophagy during C2C12 cells differentiation
Biomedicine & Pharmacotherapy 69 (2015) 42–46
Available online at
ScienceDirect www.sciencedirect.com
Short communication
Ligustilide inhibits tumour necrosis factor-alpha-induced autophagy during C2C12 cells differentiation Ying Shi a, Liming Xiao b, Yi Yin a, Lianbo Wei a,* a b
Department of Traditional Chinese Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China The First Affiliated Hospital, Nanchang University, Nanchang 330006, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2014 Accepted 5 November 2014
Ligustilide is widely thought to be the most potent bioactive constituent of Angelica sinensis. We have previously reported the role of ligustilide in preventing TNF-a-induced apoptosis and identified the presence of autophagosome clusters. Then, we hypothesised that autophagy may contribute to muscle loss and that ligustilide could protect cell fibres by regulating the autophagic process. The aim of this study was to identify the effects of ligustilide on autophagy regulation during cell differentiation in the presence of TNF-a. We then observed intracellular morphologic changes and autophagosome formation using transmission electron microscopy. LC3B expression was assessed by immunofluorescence and Atg-7, Atg-5, Atg-12 and LC3B expression levels were detected by western blot. The results revealed a reduction in the number of TNF-a-induced autophagosomes after ligustilide treatment accompanied by a decrease in Atg-7, Atg-5, Atg-12 and LC3B expression, as well as a reduction of the LC3BII/I ratio in a concentration-dependent manner. Our findings provide evidence supporting a protective effect of ligustilide against TNF-a-induced autophagy during myotubes formation. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Ligustilide Myoblast Myotube Autophagy
1. Introduction Autophagy is a process of cellular self-digestion essential in many aspects. An increasing number of studies show the complex roles of autophagy in human health and diseases. Indeed, a basal level of autophagy is required for cellular survival and homeostasis and too little or too much autophagy may result in diseases. Skeletal muscle, which represents 40–50% of the human body mass, constitutes one of the most important organs involved in metabolic regulation. Excessive protein degradation within the skeletal muscle tissue is detrimental for the whole body energy balance and can lead to death. The autophagic and ubiquitinproteasome apoptotic systems are closely interrelated and their coordinated activation contributes to muscle loss [1–3]. Therefore, studying the autophagic and ubiquitin-proteasome systems is of significant importance for the prevention of muscle atrophy. Abbreviations: TNF-a, Tumour necrosis factor-alpha; FBS, Foetal bovine serum; DMEM/F-12, Dulbecco’s modified eagle’s medium F-12 Liquid; DMSO, Dimethyl sulfoxide; GM, Growth medium; DM, Differentiation medium; PBS, Phosphate buffered saline; DAPI, 40 ,6-Diamidino-2-phenylindole; Atg, Autophagy associated gene. ? Corresponding author. Tel./fax: +86 20 6164 3456. E-mail addresses: [email protected] (Y. Shi), [email protected] (L. Xiao), [email protected] (Y. Yin), [email protected] (L. Wei). http://dx.doi.org/10.1016/j.biopha.2014.11.002 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Our group has previously reported that ligustilide could prevent TNF-a-induced apoptosis during C2C12 cell differentiation [4]. Ligustilide exerted a protective effect against the effects of TNF-a by promoting cell proliferation and reducing apoptosis in C2C12 cells. To demonstrate the existence of TNF-a-induced apoptosis, cells were observed under transmission electron microscopy, which revealed the presence of autophagosomes in both mock- and TNF-a-treated groups. TNF-a-mediated upregulation of macroautophagy had been demonstrated previously [5,6]. We then hypothesised that autophagy may contribute to muscle loss and that ligustilide could protect the cells by regulating the autophagic process via a mechanism still not fully understood. The aims of this study were to investigate whether ligustilide could regulate TNF-a-induced cellular autophagy during C2C12 differentiation and to explore the possible mechanisms regulating autophagy-related pathways. 2. Materials and methods 2.1. Reagents FBS, horse serum and DMEM/F-12 were purchased from HyClone Laboratories (Logan, UT, USA). DMSO was purchased
Y. Shi et al. / Biomedicine & Pharmacotherapy 69 (2015) 42–46
from Sigma (Saint-Louis, MO, USA). Ligustilide was purchased from the National Institute for Food and Drug Control (HPLC 98%). RapidStepTM ECL reagent used for western blotting was purchased from Millipore (Bedford, USA). RIPA lysis buffer and BCA protein assay kit were purchased from Shanghai Beyotime Biological Corporation. Atg-7, Atg-5, Atg-12 and LC3B antibodies were purchased from Cell Signaling Technologies (Danvers, USA). Mouse anti-GAPDH, anti-rabbit and anti-mouse antibodies were purchased from EarthOx Biotechnology (EarthOx, CA, USA). The other chemicals and reagents used were of analytical grade. 2.2. Cell culture C2C12 cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and were maintained in growth medium (GM), composed of DMEM/F-12 supplemented with 10% FBS and incubated at 37 8C in a water-saturated atmosphere of 5% CO2. 2.3. Experimental model C2C12 cells, which are derived from mouse skeletal muscle and can be differentiated into myotubes in culture, were maintained in GM at 37 8C under a humid 5% CO2/95% O2 atmosphere. At approximately 60–70% confluency, myoblasts differentiation was initiated by replacing the GM with differentiation medium (DM) composed of DMEM/F-12 supplemented with 2% horse serum. The differentiation medium was changed every 48 h before experimentation. 2.4. Experimental groups and treatments For the mock group, cells were incubated with DM only. For the TNF-a-control group, cells were incubated with 20 ng/mL recombinant murine TNF-a dissolved in DM. Cells in the ligustilide-treated groups were incubated in DM containing 20 ng/mL TNF-a and ligustilide at diverse concentrations. Ligustilide and TNF-a dissolution procedures were previously described [4].
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2.5. Transmission electron microscopy for autophagosome detection To study the effects of the autophagosomes and morphologic changes in the cells, the cells were treated as described above. Treated cells were fixed in PBS with 2.5% cold glutaraldehyde. After two washes in PBS, the cells were post-fixed in 1% osmium tetroxide in PBS, then dehydrated with a graded series of ethanol and propylene oxide. Then, the samples were infiltrated overnight with a mixture of EPON resin/acetone (1:1) and embedded in EPON resin. The blocks were cut into 0.5 mM sections and stained with uranyl acetate and lead citrate [7]. Finally, cells were observed under a transmission electron microscope. 2.6. Immunofluorescence assay Treated cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Then, fixed cells were blocked and permeabilised with 1% FBS containing 0.25% Tween-20 for 30 min at 4 8C and incubated with anti-LC3B (1:200 dilution) overnight at 4 8C. After washing with PBS, the cells were incubated with appropriate secondary antibodies for 1 h at room temperature, then washed with PBS and incubated with DAPI for 15 min. Stained cells were examined by fluorescence microscopy as previously described [8], and images were captured from 6 randomly selected microscopic fields for each treatment (magnification of 400 ). 2.7. Western blotting For immunoblotting, 20 mg aliquots of protein lysate were electrophoretically separated using a 12% SDS-polyacrylamide gel and transferred (Bio-Rad, Hercules, California, USA) to PVDF membranes (Pall, USA). After being placed in blocking buffer, the membranes were incubated with the following primary antibodies: anti-Atg-7 (1:1000 dilution), anti-Atg-5 (1:1000 dilution), anti-Atg-12 (1:1000 dilution), anti-LC3B (1:1000 dilution) or anti-GAPDH (1:5000 dilution). GAPDH protein was used as the internal control. After the membranes were washed with TBST, the appropriate HRP-conjugated secondary antibody (1:5000) was added to the preparation, which was incubated at room
Fig. 1. Ligustilide reduces the number of TNF-a-induced autophagosomes. Ligustilide-mediated effect on autophagy, observed by transmission electron microscopy. The results show cytoplasmic morphology, cellular organelles, and nuclei in C2C12 cells from the mock (a), TNF-a-control (b), 1 mM ligustilide-treated (c), 5 mM ligustilidetreated (d) and 10 mM ligustilide-treated (e) groups. Arrowheads represent autophagic vacuoles.
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temperature for 1 h. Protein immunoblot images were captured and documented using a CCD camera and imaging system (Image Station 2000 MM, Kodak, Rochester, NY, USA). The intensities of the protein bands were analysed using the Molecular Imaging Software Version 4.0 provided with the Kodak 2000 MM System. 2.8. Statistical analyses Statistical significance was calculated using one-way analysis of variance (ANOVA), followed by either LSD’s test or Dunnett’s T3 for multiple comparisons, based on the homogeneity of variances. Values with P < 0.05 were considered significant. The results are expressed as the means SEM and represent assays from at least three independent experiments.
3. Results 3.1. Ligustilide reduces the number of autophagosomes induced by TNF-a A number of free membrane structures and typical doublemembrane vacuoles were found in the cytoplasm in all five groups, as indicated by the black arrowheads in Fig. 1. By definition, autophagosomes are double-membrane limited vacuoles that contain undegraded cytoplasm and no lysosomal proteins [9]. The results from Fig. 1 indicate a tendency towards an elevated number of autophagosomes in the TNF-a-control group, with a dose-dependent reduction in the number of autophagosomes in ligustilide-treated groups.
Fig. 2. Ligustilide inhibits LC3B expression. Immunofluorescence analysis of the autophagosome marker LC3B in C2C12 cells. The cells were stained with an antibody against LC3B (green) and the nuclear stain DAPI (blue) ( 400 magnification).
Y. Shi et al. / Biomedicine & Pharmacotherapy 69 (2015) 42–46
3.2. Ligustilide inhibits LC3B expression To assess the cellular expression levels of the autophagosome marker LC3B, cells were analysed by immunofluorescence. The results revealed that LC3B expression was abundant in the TNF-a-control group, especially in myotubes. As shown in Fig. 2, LC3B expression in the mock- and ligustilide-treated groups was relatively reduced, displaying a dose-dependent relationship with LC3B expression in the ligustilide-treated groups. 3.3. Ligustilide regulates the expression level of autophagy-related proteins To further characterise the effect of ligustilide on TNF-ainduced autophagy, the expression levels of autophagy-related proteins were assessed. Specifically, Atg-7, Atg-5, Atg-12 and LC3B expression levels were affected by TNF-a and ligustilide (as shown in Fig. 3A). Our findings shed light on the anti-autophagy mechanisms of ligustilide, showing that the drug worked via the downregulation of Atg-7, Atg-5 and Atg-12 expression and the reduction of the LC3BII/I ratio in a concentration-dependent manner. Meanwhile, Atg-7, Atg-5 and Atg-12 expression levels as well as the LC3BII/I ratio were increased in the TNF-a-control group compared with the mock group. As shown in Fig. 3A and B, ligustilide treatment led to a decrease in the LC3BII/I ratio and 10 mM ligustilide-treated group reduced the total level of LC3B expression. C2C12 cells incubated in ligustilide at the 5 mM and 10 mM concentrations presented a significantly decreased level of Atg-7 expression compared with TNF-a-treated control cells. In addition, the 10 mM ligustilide-treated group displayed a significantly decreased Atg-12 expression level. However, two ligustilide-treated groups and TNF-a-control group had a significantly increased LC3BII/I ratio compared with the mock group (see Fig. 3B). 4. Discussion The aim of this study was to identify the effect of ligustilide on autophagy during cell differentiation in the presence of TNF-a. The results revealed that ligustilide reduced the number of autophagosomes induced by TNF-a and decreased the expression levels of Atg-7, Atg-5, Atg-12, and LC3B, as well as the LC3BII/I ratio in a concentration-dependent manner.
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Autophagy has homeostatic and biosynthetic functions and serves as a cellular response to stressors, such as nutrient limitation. Autophagy is the major regulated cellular mechanism for degrading long-lived proteins and is the only known pathway of organelle degradation [10–12]. During autophagy, an isolation membrane forms, presumably arising from a vesicular compartment known as the pre-autophagosomal structure. The membrane invaginates and sequesters cytoplasmic constituents, including mitochondria, endoplasmic reticulum, and ribosomes. The edges of the membrane fuse to form a double or multi-membranous structure, known as the autophagosome, autophagic lysosome or vacuole, to deliver the inner membranous vesicle to the lumen of the degradative compartment [13]. As in our study, the presence of autophagosome, detected by transmission electron microscopy, is usually used to identify the autophagic process. The initial step of the autophagic process is the formation of omegasomes and initiation of isolation membranes. During this step, an isolation membrane forms inside the omegasome ring, and the Atg-12–Atg-5/Atg16 complex localises at the isolation membrane. The isolation membrane formed then elongates to engulf cytoplasmic components. In the later stages of isolation membrane elongation, the Atg-12–Atg-5/Atg16 complex progressively dissociates from the isolation membrane, whereas LC3-II gradually localises to both sides of the isolation membrane. During this process, LC3-I, a cytosolic form of LC3, is progressively lipidated by Atg-7, Atg3 and the Atg-12–Atg-5/Atg16 complex to form LC3-II. Finally, the isolation membrane closes to form the autophagosome. Following autolysosome formation, the lysosomal hydrolases degrade the intra-autophagosomal contents [14]. Therefore, during the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, namely Atg-12 conjugation and LC3-modification [15]. Then, we investigated Atg-7, Atg-5, Atg-12 and LC3B expression levels by western blot and detected LC3B expression by immunofluorescence. We demonstrated that ligustilide inhibits TNF-a-induced autophagy during C2C12 differentiation by decreasing Atg-7, Atg-5, Atg-12 and LC3B expression levels, as well as the LC3BII/I ratio in a concentration-dependent manner. During fasting, acute activation of Akt inhibits autophagic process in cell culture and in vivo as Akt identified as the most potent autophagy inhibitor in skeletal muscle [16–19]. MTOR, a nutrient-sensitive kinase downstream of Akt, is important for cell growth. During fasting, IGF1-Akt pathway inhibition stimulates
Fig. 3. Ligustilide regulates the expression levels of autophagy-related proteins. C2C12 cells were incubated for 96 h. A. After treatment, total protein lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against Atg-7, Atg-5, Atg-12, LC3B and GAPDH. B. Data were analysed by densitometric quantification. Data are expressed as the means SEM and are issued from at least three independent experiments (#P < 0.05 compared with the corresponding mock groups; *P < 0.05 compared with the corresponding TNF-a-control groups).
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autophagy mainly via mTOR-independent mechanisms [2]. In addition, expression of FOXO3 is sufficient and is required to activate lysosomal-dependent protein breakdown in cell culture and in vivo. Moreover, several autophagy genes, including LC3, Gabarap, Bnip3, VPS34 and Atg-12, are regulated by FOXO3 [16]. The p38 MAPK pathway was also identified as having a role in regulating the expression of autophagy-related genes independently of FOXO3 during oxidative stress [20]. Therefore, in future work, we will focus on studying autophagy-related signalling pathways and genes to further confirm the function of ligustilide in the autophagic process. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement We acknowledge the financial support from the National Science Foundation of China (No. 81173457). We would like to thank Teacher Cai for his selfless help. References [1] Bao XH, Naomoto Y, Hao HF, Watanabe N, Sakurama K, Noma K, et al. Autophagy: can it become a potential therapeutic target? Int J Mol Med 2010;25:493–503. [2] Sandri M. Autophagy in skeletal muscle. FEBS Lett 2010;584:1411–6. [3] Sandri M. Autophagy in health and disease 3. Involvement of autophagy in muscle atrophy. Am J Physiol Cell Physiol 2010;298:C1291–7. [4] Shi Y, Wang D, Lu L, Yin Y, Wang M, Li C, et al. Ligustilide prevents the apoptosis effects of tumour necrosis factor-alpha during C2C12 cell differentiation. Int Immunopharmacol 2014;19:358–64.
[5] Keller CW, Schmitz M, Munz C, Lunemann JD, Schmidt J. TNF-alpha upregulates macroautophagic processing of APP/beta-amyloid in a human rhabdomyosarcoma cell line. J Neurol Sci 2013;325:103–7. [6] Keller CW, Fokken C, Turville SG, Lunemann A, Schmidt J, Munz C, et al. TNFalpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells. J Biol Chem 2011;286:3970–80. [7] Du B, Zhang Z, Li N. Madecassoside prevents Abeta(25-35)-induced inflammatory responses and autophagy in neuronal cells through the class III PI3K/Beclin-1/Bcl-2 pathway. Int Immunopharmacol 2014;20:221–8. [8] Soares AS, Costa VM, Diniz C, Fresco P. Combination of Cl-IB-MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR-dependent autophagy and mitotic catastrophe on human melanoma cells. J Cancer Res Clin Oncol 2014. [9] Deretic V. Autophagosome and phagosome. Humana Press; 2008. [10] Klionsky DJ. The molecular machinery of autophagy: unanswered questions. J Cell Sci 2005;118:7–18. [11] Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717–21. [12] Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 2004;6:463–77. [13] Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest 2005;115:2679–88. [14] Tanida I. Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal 2011;14:2201–14. [15] Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 2004;36:2503–18. [16] Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del PP, et al. FOXO3 controls autophagy in skeletal muscle in vivo. Cell Metab 2007;6: 458–71. [17] Mammucari C, Schiaffino S, Sandri M. Downstream of Akt: FOXO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 2008; 4:524–6. [18] Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FOXO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 2007;6:472–83. [19] Zhao J, Brault JJ, Schild A, Goldberg AL. Coordinate activation of autophagy and the proteasome pathway by FOXO transcription factor. Autophagy 2008;4: 378–80. [20] McClung JM, Judge AR, Powers SK, Yan Z. p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting. Am J Physiol Cell Physiol 2010;298:C542–9.
Available online at
ScienceDirect www.sciencedirect.com
Short communication
Ligustilide inhibits tumour necrosis factor-alpha-induced autophagy during C2C12 cells differentiation Ying Shi a, Liming Xiao b, Yi Yin a, Lianbo Wei a,* a b
Department of Traditional Chinese Medicine, Zhujiang Hospital of Southern Medical University, Guangzhou 510282, China The First Affiliated Hospital, Nanchang University, Nanchang 330006, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 8 October 2014 Accepted 5 November 2014
Ligustilide is widely thought to be the most potent bioactive constituent of Angelica sinensis. We have previously reported the role of ligustilide in preventing TNF-a-induced apoptosis and identified the presence of autophagosome clusters. Then, we hypothesised that autophagy may contribute to muscle loss and that ligustilide could protect cell fibres by regulating the autophagic process. The aim of this study was to identify the effects of ligustilide on autophagy regulation during cell differentiation in the presence of TNF-a. We then observed intracellular morphologic changes and autophagosome formation using transmission electron microscopy. LC3B expression was assessed by immunofluorescence and Atg-7, Atg-5, Atg-12 and LC3B expression levels were detected by western blot. The results revealed a reduction in the number of TNF-a-induced autophagosomes after ligustilide treatment accompanied by a decrease in Atg-7, Atg-5, Atg-12 and LC3B expression, as well as a reduction of the LC3BII/I ratio in a concentration-dependent manner. Our findings provide evidence supporting a protective effect of ligustilide against TNF-a-induced autophagy during myotubes formation. ß 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Ligustilide Myoblast Myotube Autophagy
1. Introduction Autophagy is a process of cellular self-digestion essential in many aspects. An increasing number of studies show the complex roles of autophagy in human health and diseases. Indeed, a basal level of autophagy is required for cellular survival and homeostasis and too little or too much autophagy may result in diseases. Skeletal muscle, which represents 40–50% of the human body mass, constitutes one of the most important organs involved in metabolic regulation. Excessive protein degradation within the skeletal muscle tissue is detrimental for the whole body energy balance and can lead to death. The autophagic and ubiquitinproteasome apoptotic systems are closely interrelated and their coordinated activation contributes to muscle loss [1–3]. Therefore, studying the autophagic and ubiquitin-proteasome systems is of significant importance for the prevention of muscle atrophy. Abbreviations: TNF-a, Tumour necrosis factor-alpha; FBS, Foetal bovine serum; DMEM/F-12, Dulbecco’s modified eagle’s medium F-12 Liquid; DMSO, Dimethyl sulfoxide; GM, Growth medium; DM, Differentiation medium; PBS, Phosphate buffered saline; DAPI, 40 ,6-Diamidino-2-phenylindole; Atg, Autophagy associated gene. ? Corresponding author. Tel./fax: +86 20 6164 3456. E-mail addresses: [email protected] (Y. Shi), [email protected] (L. Xiao), [email protected] (Y. Yin), [email protected] (L. Wei). http://dx.doi.org/10.1016/j.biopha.2014.11.002 0753-3322/ß 2014 Elsevier Masson SAS. All rights reserved.
Our group has previously reported that ligustilide could prevent TNF-a-induced apoptosis during C2C12 cell differentiation [4]. Ligustilide exerted a protective effect against the effects of TNF-a by promoting cell proliferation and reducing apoptosis in C2C12 cells. To demonstrate the existence of TNF-a-induced apoptosis, cells were observed under transmission electron microscopy, which revealed the presence of autophagosomes in both mock- and TNF-a-treated groups. TNF-a-mediated upregulation of macroautophagy had been demonstrated previously [5,6]. We then hypothesised that autophagy may contribute to muscle loss and that ligustilide could protect the cells by regulating the autophagic process via a mechanism still not fully understood. The aims of this study were to investigate whether ligustilide could regulate TNF-a-induced cellular autophagy during C2C12 differentiation and to explore the possible mechanisms regulating autophagy-related pathways. 2. Materials and methods 2.1. Reagents FBS, horse serum and DMEM/F-12 were purchased from HyClone Laboratories (Logan, UT, USA). DMSO was purchased
Y. Shi et al. / Biomedicine & Pharmacotherapy 69 (2015) 42–46
from Sigma (Saint-Louis, MO, USA). Ligustilide was purchased from the National Institute for Food and Drug Control (HPLC 98%). RapidStepTM ECL reagent used for western blotting was purchased from Millipore (Bedford, USA). RIPA lysis buffer and BCA protein assay kit were purchased from Shanghai Beyotime Biological Corporation. Atg-7, Atg-5, Atg-12 and LC3B antibodies were purchased from Cell Signaling Technologies (Danvers, USA). Mouse anti-GAPDH, anti-rabbit and anti-mouse antibodies were purchased from EarthOx Biotechnology (EarthOx, CA, USA). The other chemicals and reagents used were of analytical grade. 2.2. Cell culture C2C12 cells were purchased from the Chinese Academy of Sciences (Shanghai, China) and were maintained in growth medium (GM), composed of DMEM/F-12 supplemented with 10% FBS and incubated at 37 8C in a water-saturated atmosphere of 5% CO2. 2.3. Experimental model C2C12 cells, which are derived from mouse skeletal muscle and can be differentiated into myotubes in culture, were maintained in GM at 37 8C under a humid 5% CO2/95% O2 atmosphere. At approximately 60–70% confluency, myoblasts differentiation was initiated by replacing the GM with differentiation medium (DM) composed of DMEM/F-12 supplemented with 2% horse serum. The differentiation medium was changed every 48 h before experimentation. 2.4. Experimental groups and treatments For the mock group, cells were incubated with DM only. For the TNF-a-control group, cells were incubated with 20 ng/mL recombinant murine TNF-a dissolved in DM. Cells in the ligustilide-treated groups were incubated in DM containing 20 ng/mL TNF-a and ligustilide at diverse concentrations. Ligustilide and TNF-a dissolution procedures were previously described [4].
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2.5. Transmission electron microscopy for autophagosome detection To study the effects of the autophagosomes and morphologic changes in the cells, the cells were treated as described above. Treated cells were fixed in PBS with 2.5% cold glutaraldehyde. After two washes in PBS, the cells were post-fixed in 1% osmium tetroxide in PBS, then dehydrated with a graded series of ethanol and propylene oxide. Then, the samples were infiltrated overnight with a mixture of EPON resin/acetone (1:1) and embedded in EPON resin. The blocks were cut into 0.5 mM sections and stained with uranyl acetate and lead citrate [7]. Finally, cells were observed under a transmission electron microscope. 2.6. Immunofluorescence assay Treated cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min at room temperature. Then, fixed cells were blocked and permeabilised with 1% FBS containing 0.25% Tween-20 for 30 min at 4 8C and incubated with anti-LC3B (1:200 dilution) overnight at 4 8C. After washing with PBS, the cells were incubated with appropriate secondary antibodies for 1 h at room temperature, then washed with PBS and incubated with DAPI for 15 min. Stained cells were examined by fluorescence microscopy as previously described [8], and images were captured from 6 randomly selected microscopic fields for each treatment (magnification of 400 ). 2.7. Western blotting For immunoblotting, 20 mg aliquots of protein lysate were electrophoretically separated using a 12% SDS-polyacrylamide gel and transferred (Bio-Rad, Hercules, California, USA) to PVDF membranes (Pall, USA). After being placed in blocking buffer, the membranes were incubated with the following primary antibodies: anti-Atg-7 (1:1000 dilution), anti-Atg-5 (1:1000 dilution), anti-Atg-12 (1:1000 dilution), anti-LC3B (1:1000 dilution) or anti-GAPDH (1:5000 dilution). GAPDH protein was used as the internal control. After the membranes were washed with TBST, the appropriate HRP-conjugated secondary antibody (1:5000) was added to the preparation, which was incubated at room
Fig. 1. Ligustilide reduces the number of TNF-a-induced autophagosomes. Ligustilide-mediated effect on autophagy, observed by transmission electron microscopy. The results show cytoplasmic morphology, cellular organelles, and nuclei in C2C12 cells from the mock (a), TNF-a-control (b), 1 mM ligustilide-treated (c), 5 mM ligustilidetreated (d) and 10 mM ligustilide-treated (e) groups. Arrowheads represent autophagic vacuoles.
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temperature for 1 h. Protein immunoblot images were captured and documented using a CCD camera and imaging system (Image Station 2000 MM, Kodak, Rochester, NY, USA). The intensities of the protein bands were analysed using the Molecular Imaging Software Version 4.0 provided with the Kodak 2000 MM System. 2.8. Statistical analyses Statistical significance was calculated using one-way analysis of variance (ANOVA), followed by either LSD’s test or Dunnett’s T3 for multiple comparisons, based on the homogeneity of variances. Values with P < 0.05 were considered significant. The results are expressed as the means SEM and represent assays from at least three independent experiments.
3. Results 3.1. Ligustilide reduces the number of autophagosomes induced by TNF-a A number of free membrane structures and typical doublemembrane vacuoles were found in the cytoplasm in all five groups, as indicated by the black arrowheads in Fig. 1. By definition, autophagosomes are double-membrane limited vacuoles that contain undegraded cytoplasm and no lysosomal proteins [9]. The results from Fig. 1 indicate a tendency towards an elevated number of autophagosomes in the TNF-a-control group, with a dose-dependent reduction in the number of autophagosomes in ligustilide-treated groups.
Fig. 2. Ligustilide inhibits LC3B expression. Immunofluorescence analysis of the autophagosome marker LC3B in C2C12 cells. The cells were stained with an antibody against LC3B (green) and the nuclear stain DAPI (blue) ( 400 magnification).
Y. Shi et al. / Biomedicine & Pharmacotherapy 69 (2015) 42–46
3.2. Ligustilide inhibits LC3B expression To assess the cellular expression levels of the autophagosome marker LC3B, cells were analysed by immunofluorescence. The results revealed that LC3B expression was abundant in the TNF-a-control group, especially in myotubes. As shown in Fig. 2, LC3B expression in the mock- and ligustilide-treated groups was relatively reduced, displaying a dose-dependent relationship with LC3B expression in the ligustilide-treated groups. 3.3. Ligustilide regulates the expression level of autophagy-related proteins To further characterise the effect of ligustilide on TNF-ainduced autophagy, the expression levels of autophagy-related proteins were assessed. Specifically, Atg-7, Atg-5, Atg-12 and LC3B expression levels were affected by TNF-a and ligustilide (as shown in Fig. 3A). Our findings shed light on the anti-autophagy mechanisms of ligustilide, showing that the drug worked via the downregulation of Atg-7, Atg-5 and Atg-12 expression and the reduction of the LC3BII/I ratio in a concentration-dependent manner. Meanwhile, Atg-7, Atg-5 and Atg-12 expression levels as well as the LC3BII/I ratio were increased in the TNF-a-control group compared with the mock group. As shown in Fig. 3A and B, ligustilide treatment led to a decrease in the LC3BII/I ratio and 10 mM ligustilide-treated group reduced the total level of LC3B expression. C2C12 cells incubated in ligustilide at the 5 mM and 10 mM concentrations presented a significantly decreased level of Atg-7 expression compared with TNF-a-treated control cells. In addition, the 10 mM ligustilide-treated group displayed a significantly decreased Atg-12 expression level. However, two ligustilide-treated groups and TNF-a-control group had a significantly increased LC3BII/I ratio compared with the mock group (see Fig. 3B). 4. Discussion The aim of this study was to identify the effect of ligustilide on autophagy during cell differentiation in the presence of TNF-a. The results revealed that ligustilide reduced the number of autophagosomes induced by TNF-a and decreased the expression levels of Atg-7, Atg-5, Atg-12, and LC3B, as well as the LC3BII/I ratio in a concentration-dependent manner.
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Autophagy has homeostatic and biosynthetic functions and serves as a cellular response to stressors, such as nutrient limitation. Autophagy is the major regulated cellular mechanism for degrading long-lived proteins and is the only known pathway of organelle degradation [10–12]. During autophagy, an isolation membrane forms, presumably arising from a vesicular compartment known as the pre-autophagosomal structure. The membrane invaginates and sequesters cytoplasmic constituents, including mitochondria, endoplasmic reticulum, and ribosomes. The edges of the membrane fuse to form a double or multi-membranous structure, known as the autophagosome, autophagic lysosome or vacuole, to deliver the inner membranous vesicle to the lumen of the degradative compartment [13]. As in our study, the presence of autophagosome, detected by transmission electron microscopy, is usually used to identify the autophagic process. The initial step of the autophagic process is the formation of omegasomes and initiation of isolation membranes. During this step, an isolation membrane forms inside the omegasome ring, and the Atg-12–Atg-5/Atg16 complex localises at the isolation membrane. The isolation membrane formed then elongates to engulf cytoplasmic components. In the later stages of isolation membrane elongation, the Atg-12–Atg-5/Atg16 complex progressively dissociates from the isolation membrane, whereas LC3-II gradually localises to both sides of the isolation membrane. During this process, LC3-I, a cytosolic form of LC3, is progressively lipidated by Atg-7, Atg3 and the Atg-12–Atg-5/Atg16 complex to form LC3-II. Finally, the isolation membrane closes to form the autophagosome. Following autolysosome formation, the lysosomal hydrolases degrade the intra-autophagosomal contents [14]. Therefore, during the formation of mammalian autophagosomes, two ubiquitylation-like modifications are required, namely Atg-12 conjugation and LC3-modification [15]. Then, we investigated Atg-7, Atg-5, Atg-12 and LC3B expression levels by western blot and detected LC3B expression by immunofluorescence. We demonstrated that ligustilide inhibits TNF-a-induced autophagy during C2C12 differentiation by decreasing Atg-7, Atg-5, Atg-12 and LC3B expression levels, as well as the LC3BII/I ratio in a concentration-dependent manner. During fasting, acute activation of Akt inhibits autophagic process in cell culture and in vivo as Akt identified as the most potent autophagy inhibitor in skeletal muscle [16–19]. MTOR, a nutrient-sensitive kinase downstream of Akt, is important for cell growth. During fasting, IGF1-Akt pathway inhibition stimulates
Fig. 3. Ligustilide regulates the expression levels of autophagy-related proteins. C2C12 cells were incubated for 96 h. A. After treatment, total protein lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against Atg-7, Atg-5, Atg-12, LC3B and GAPDH. B. Data were analysed by densitometric quantification. Data are expressed as the means SEM and are issued from at least three independent experiments (#P < 0.05 compared with the corresponding mock groups; *P < 0.05 compared with the corresponding TNF-a-control groups).
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autophagy mainly via mTOR-independent mechanisms [2]. In addition, expression of FOXO3 is sufficient and is required to activate lysosomal-dependent protein breakdown in cell culture and in vivo. Moreover, several autophagy genes, including LC3, Gabarap, Bnip3, VPS34 and Atg-12, are regulated by FOXO3 [16]. The p38 MAPK pathway was also identified as having a role in regulating the expression of autophagy-related genes independently of FOXO3 during oxidative stress [20]. Therefore, in future work, we will focus on studying autophagy-related signalling pathways and genes to further confirm the function of ligustilide in the autophagic process. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgement We acknowledge the financial support from the National Science Foundation of China (No. 81173457). We would like to thank Teacher Cai for his selfless help. References [1] Bao XH, Naomoto Y, Hao HF, Watanabe N, Sakurama K, Noma K, et al. Autophagy: can it become a potential therapeutic target? Int J Mol Med 2010;25:493–503. [2] Sandri M. Autophagy in skeletal muscle. FEBS Lett 2010;584:1411–6. [3] Sandri M. Autophagy in health and disease 3. Involvement of autophagy in muscle atrophy. Am J Physiol Cell Physiol 2010;298:C1291–7. [4] Shi Y, Wang D, Lu L, Yin Y, Wang M, Li C, et al. Ligustilide prevents the apoptosis effects of tumour necrosis factor-alpha during C2C12 cell differentiation. Int Immunopharmacol 2014;19:358–64.
[5] Keller CW, Schmitz M, Munz C, Lunemann JD, Schmidt J. TNF-alpha upregulates macroautophagic processing of APP/beta-amyloid in a human rhabdomyosarcoma cell line. J Neurol Sci 2013;325:103–7. [6] Keller CW, Fokken C, Turville SG, Lunemann A, Schmidt J, Munz C, et al. TNFalpha induces macroautophagy and regulates MHC class II expression in human skeletal muscle cells. J Biol Chem 2011;286:3970–80. [7] Du B, Zhang Z, Li N. Madecassoside prevents Abeta(25-35)-induced inflammatory responses and autophagy in neuronal cells through the class III PI3K/Beclin-1/Bcl-2 pathway. Int Immunopharmacol 2014;20:221–8. [8] Soares AS, Costa VM, Diniz C, Fresco P. Combination of Cl-IB-MECA with paclitaxel is a highly effective cytotoxic therapy causing mTOR-dependent autophagy and mitotic catastrophe on human melanoma cells. J Cancer Res Clin Oncol 2014. [9] Deretic V. Autophagosome and phagosome. Humana Press; 2008. [10] Klionsky DJ. The molecular machinery of autophagy: unanswered questions. J Cell Sci 2005;118:7–18. [11] Klionsky DJ, Emr SD. Autophagy as a regulated pathway of cellular degradation. Science 2000;290:1717–21. [12] Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 2004;6:463–77. [13] Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest 2005;115:2679–88. [14] Tanida I. Autophagosome formation and molecular mechanism of autophagy. Antioxid Redox Signal 2011;14:2201–14. [15] Tanida I, Ueno T, Kominami E. LC3 conjugation system in mammalian autophagy. Int J Biochem Cell Biol 2004;36:2503–18. [16] Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del PP, et al. FOXO3 controls autophagy in skeletal muscle in vivo. Cell Metab 2007;6: 458–71. [17] Mammucari C, Schiaffino S, Sandri M. Downstream of Akt: FOXO3 and mTOR in the regulation of autophagy in skeletal muscle. Autophagy 2008; 4:524–6. [18] Zhao J, Brault JJ, Schild A, Cao P, Sandri M, Schiaffino S, et al. FOXO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells. Cell Metab 2007;6:472–83. [19] Zhao J, Brault JJ, Schild A, Goldberg AL. Coordinate activation of autophagy and the proteasome pathway by FOXO transcription factor. Autophagy 2008;4: 378–80. [20] McClung JM, Judge AR, Powers SK, Yan Z. p38 MAPK links oxidative stress to autophagy-related gene expression in cachectic muscle wasting. Am J Physiol Cell Physiol 2010;298:C542–9.