International Immunopharmacology 19 (2014) 358–364
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Ligustilide prevents the apoptosis effects of tumour necrosis factor-alpha during C2C12 cell differentiation Ying Shi a,b, Dongtao Wang a,b, Lu Lu a,b, Yi Yin a,b, Ming Wang a,b, Chengjie Li a,b, Jianxin Diao b, Yanjing Wang a, Lianbo Wei a,b,⁎ a b
Department of Traditional Chinese Medicine, ZhuJiang Hospital, Southern Medical University, Guangzhou 510280, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
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Article history: Received 17 September 2013 Received in revised form 26 January 2014 Accepted 6 February 2014 Available online 21 February 2014 Keywords: Ligustilide Apoptosis Myoblast Myotube
a b s t r a c t Ligustilide, the major component of Angelica sinensis, is also thought to be the most potent bioactive constituent of this plant. Ligustilide has been reported to markedly protect neural tissue against apoptosis. However, little is known regarding ligustilide's anti-apoptosis effect in muscle tissue. The aim of the study was to investigate the anti-apoptosis effects of ligustilide on TNF-α-induced C2C12 cells during differentiation. It was revealed that ligustilide at various concentrations significantly prevented the apoptosis of C2C12 cells incubated in TNF-α as assessed by apoptosis index and DNA fragmentation. Moreover, ligustilide-treated groups exhibited a significant increase in the bcl-2/bax ratio, pro-caspase 3 and pro-caspase 8 compared with the TNF-α-control group in a dose-dependent manner. Meanwhile, ligustilide-treated groups presented a significantly increased level of phosphorylated Akt and suppressed expression of the myogenin protein. Therefore, the findings derived suggested that ligustilide protected C2C12 cells from TNF-α-induced apoptosis during differentiation by reducing apoptosis and inducing cell proliferation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Skeletal muscle atrophy is a condition which occurs as the result of disuse, starvation, ageing and a multitude of disease states, such as diabetes, cancer, and AIDS [1]. The pro-inflammatory cytokine TNF-α plays a critical role in muscle atrophy and in a broad range of diverse activities, including cell growth and differentiation, inflammation, apoptosis and necrosis. Moreover, a pathologic level of TNF-α has been identified as a significant mechanism associated with skeletal muscle wasting [2]. At least two pathways for the skeletal muscle-wasting effects of TNF-α have been described: inhibition of myogenin in myoblasts and apoptosis of myoblasts and myotubes [3]. Thus, delivery of TNF-α to primary human myoblasts or murine C2C12 myoblasts can inhibit myogenic differentiation and result in cell apoptosis.
Abbreviations: TNF-α, tumour necrosis factor alpha; FBS, foetal bovine serum; DMEM/ F-12, Dulbecco's Modified Eagle's Medium F12 Liquid; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazo-lium bromide; DMSO, dimethyl sulfoxide; GM, growth medium; DM, differentiation medium; OD, optical density; AI, apoptosis index; EB, ethidium bromide; bHLH, helix–loop–helix; MRFs, myogenic regulatory factors. ⁎ Corresponding author at: Department of Traditional Chinese Medicine, ZhuJiang Hospital, Southern Medical University, Guangzhou 510280, China. Tel./fax: +86 20 6164 3456. E-mail addresses: [email protected] (Y. Shi), [email protected] (D. Wang), [email protected] (L. Lu), [email protected] (Y. Yin), [email protected] (M. Wang), [email protected] (C. Li), [email protected] (J. Diao), [email protected] (Y. Wang), [email protected] (L. Wei).
http://dx.doi.org/10.1016/j.intimp.2014.02.007 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Apoptosis, a physiological form of cell suicide, ensures the elimination of superfluous tissues in development and is critical for the maintenance of tissue homeostasis in adulthood [4]. The initial stage of apoptosis involves death-inducing signals, including the release of reactive oxygen and nitrogen species, the expression of ligands for ‘death receptors’ and altered levels of bcl-2 family protein [5]. Following this early stage, nuclear activators, cell surface receptors, and mitochondrial pathways become activated, cells become committed to cell death, and specific cytoplasmic and nuclear events occur [6]. During this phase, caspases, which are responsible for proteolytic cleavage of a broad spectrum of cellular targets, are activated in the cytosol [5] and orchestrate the changes associated with apoptosis (DNA fragmentation, nuclear condensation, cell shrinkage, membrane blebbing, etc.) [7]. Therefore, treatments aimed at inhibiting the changes in the levels of bcl-2 family proteins, blocking caspase expression and reducing typical apoptotic morphology (e.g., DNA fragmentation, nuclear condensation, and cell shrinkage) may have potential therapeutic advantages with regard to apoptosis. Angelica sinensis, known as Danggui in China, is thought to be capable of repairing tissue damage in Chinese traditional medicine. The chemical constituents of A. sinensis extract are classified into essential oils containing the main pharmacologically active compounds [8] and water soluble components. Ligustilide, the major component, is thought to be the most potent bioactive constituent [9] and has been previously demonstrated to be effective at providing neuroprotection against subarachnoid haemorrhage by reducing apoptotic damage through
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Because the effectiveness of ligustilide against apoptosis of muscles cells remains unclear, this study was conducted to examine the antiapoptosis effects of ligustilide on TNF-α-induced C2C12 cells during differentiation. 2. Materials and methods 2.1. Reagents
Fig. 1. Ligustilide showed cytotoxic effects on C2C12 cells. C2C12 cells were treated with ligustilide (1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, or 50 μM) for 24 h and 48 h. All data are reported as the means ± S.E.M. from more than three independent experiments. *, p b0.05 compared with control cells in 24 h; #, p b 0.05 compared with control cells in 48 h.
decreased expression of p53 and cleavage of caspase-3. Although no change in bax expression was detected, ligustilide treatment after injury resulted in a dose-dependent increase in bcl-2 levels, which led to a large shift of the bcl-2/bax ratio in favour of the anti-apoptotic bcl-2 [10]. Moreover, other published data shed further light on the antiapoptotic properties of ligustilide, specifically through its contribution to the up-regulation of bcl-2 and the down-regulation of bax and caspase3 [11]. Thus, the regulation of bcl-2 family proteins and caspases is usually considered to be the main mechanism of action of ligustilide against apoptosis.
FBS, horse serum and DMEM/F-12 were purchased from HyClone Laboratories (Logan, UT, USA). MTT, Hoechst33342 and DMSO were purchased from Sigma (St. Louis, MO, USA). Ligustilide was purchased from the National Institute for Food and Drug Control (HPLC ≥ 98%). RapidStep™ 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. Bcl2, bax, caspase-3, caspase-8, phosphor-Akt (Ser473), and total Akt antibodies were purchased from Cell Signaling Technologies (Danvers, USA). Myogenin antibodies were purchased from Santa Cruz Biotechnology (Texas, USA). Mouse anti-GAPDH, anti-rabbit and anti-mouse antibodies were purchased from EarthOx Biotechnology (EarthOx, CA, USA). 2.2. Cell culture C2C12 cells were purchased from the Chinese Academy of Sciences (Shanghai, China). C2C12 cells were maintained in growth medium (GM, DMEM/F-12 with 10% FBS) and incubated at 37 °C in a watersaturated atmosphere of 5% CO2.
Fig. 2. Ligustilide reduced apoptosis induced by TNF-α during C2C12 cell differentiation. A. C2C12 cells incubated for 96 h, then stained with Hoechst 33342 and observed under a fluorescence microscope at 400× magnification. B. A semi-quantitative analysis was performed by calculating an AI from observed images. Data are expressed as means ± S.E.M. from more than three independent experiments.
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2.3. Cytotoxicity of ligustilide assay C2C12 myoblasts were seeded in GM in 96-well plates for 24 h, and then exposed to GM with 1, 5, 10, 20, 30, 40, or 50 μM of ligustilide, which was dissolved in a vehicle solution of 0.1% DMSO. The control group cells were cultured in GM only. Cell viability was analysed at 24 h and 48 h post-treatment via MTT assay. 2.4. Experimental model C2C12 cells derived from mouse skeletal muscle were maintained in GM at 37 °C under a humid 5% CO2/95% O2 atmosphere. When the myoblasts were approximately 60–70% confluent, myotube differentiation was initiated by replacing the GM with differentiation medium (DM, DMEM/F-12 with 2% horse serum). Medium was changed every 48 h before experimentation. 2.5. Treatment of cells in groups Stock solutions of TNF-α were diluted with sterile DM to a concentration of 500 ng/mL and stored at −20 °C until required for use. Cells of the TNF-α-control group and of the ligustilide-treated experimental groups were incubated with 20 ng/mL recombinant murine TNF-α dissolved in DM. The concentration of TNF-α was described in [3] and confirmed in preliminary experiments. For the mock group, the cells were incubated with DM only. Cells in the DMSO-control group were incubated in DM with 1% DMSO. Cells in ligustilide-treated groups were incubated in DM with TNF-α and ligustilide at diverse concentrations.
2000 MM, Kodak, Rochester, NY, USA). The intensities of the protein bands were analysed using Molecular Imaging Software Version 4.0, which was provided with the Kodak 2000 MM System. 2.8. Morphology and diameter of myotubes Myotube cultures were photographed under a phase contrast microscope at 200× magnification and stained with hematoxylin–eosin staining (H&E). As myotubes were not formed in a sufficient number in a shorter time, and cells in the TNF-α-control group would be almost dead in 5 days, we chose an incubation time of 96 h to detect myotubes. Cells in different experimental groups were treated as described above. The medium was changed every 48 h before experimentation. 2.9. Statistical analyses Statistical analyses were performed using SPSS 13.0 (SPSS, Chicago, IL, USA). Because the incubation times and different interventions all contributed to the outcomes simultaneously, tests of between-subjects effects were needed to examine group effects and incubation time effects on the variable by the univariate analysis. The data were then further analysed post-hoc using either Bonferroni's test or Dunnett's T3 for multiple comparisons, depending on the homogeneity of variances. For detecting single variable effects, statistical significance was estimated using one-way analysis of variance (ANOVA), followed
2.6. Apoptosis assay For the Hoechst 33342 staining, the cells were grown in 12-well plates and treated as described above. Nuclear DNA was visualised in cells by staining with the DNA-specific fluorescent dye, Hoechst 33342, at a final concentration of 6 μg/mL. The cells were immediately observed with a light microscope, using filters for blue fluorescence. To quantify the extent of apoptosis, an apoptosis index (AI) was calculated, as described in [3], from 6 images of randomly selected microscopic fields for each treatment. The AI was calculated as the percentage of the total number of nuclei that were apoptotic. Apoptotic nuclei were identifiable because they appeared brightly fluorescent and condensed compared to normal nuclei. For the extraction of DNA and the detection of DNA fragments, cells were treated as described above and the total DNA was isolated according to the protocol provided with the Genomic DNA Mini Preparation Kit with Spin Column (Beyotime, Shanghai, China). DNA fragments were separated in a 2% agarose gel, stained with ethidium bromide (EB), and visualised under UV light. 2.7. Western blotting For immunoblotting, 20 mg aliquots of the lysates were separated on a 12% SDS-polyacrylamide gel and transferred electrophoretically (Bio-Rad, Hercules, California, USA) to a PVDF membrane (Pall, USA). As cleavage of actin has been observed under apoptotic conditions in vitro and in cardiac and skeletal muscles [12–14], GAPDH protein was used as the internal control. After being placed in blocking buffer, the membranes were incubated with the following primary antibodies: anti-bcl-2 (1:1000 dilution), anti-bax (1:1000 dilution), anti-caspase-3 (1:1000 dilution), anti-caspase-8 (1:1000 dilution), anti-myogenin (1:200 dilution), anti-Akt (1:1000 dilution), anti-phospho-Akt (1:1000 dilution), and anti-GAPDH (1:5000 dilution). After the membranes were washed with TBST, the appropriate HRP-conjugated secondary antibody (1:5000) was added to the preparation. The blot was incubated at 37 °C for 1 h. The protein bands were captured and documented using a CCD camera and imaging system (Image Station
Fig. 3. Ligustilide decreased apoptotic DNA fragmentation induced by TNF-α. C2C12 cells were incubated for 48 h and 96 h and their total DNA was isolated. DNA fragments were separated in an agarose gel, stained with EB, and visualised under UV light. A. Cells were incubated for 48 h and 96 h. B. Densities of the bands following the DNA fragmentation shown in A. Data are expressed as means ± S.E.M. from more than three independent experiments.
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by either Bonferroni's test or Dunnett's T3 for multiple comparisons, based on the homogeneity of variances. Values with p b 0.05 were considered significant. The results are expressed as means ± S.E.M. and represent assays from at least three independent experiments.
3. Results 3.1. Ligustilide showed cytotoxic effects on C2C12 cells To examine the cellular tolerance to the cytotoxicity of ligustilide, C2C12 cells were treated with the drug in increasing concentrations ranging from 1 to 50 μM for respective 24 h and 48 h. Cytotoxicity of ligustilide was evaluated by an MTT assay. The results showed that ligustilide exerted significant inhibitory effects on the growth of the cells when the drug concentrations were 10 μM or above, in both the 24 hour and the 48 hour groups (see Fig. 1). No significant differences were identified between the two time points.
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3.2. Ligustilide reduced apoptosis induced by TNF-α during C2C12 cell differentiation Based on the results above, which confirmed that ligustilide induced significant cell death at a concentration of 20 μM because of its cytotoxicity, as in preliminary experiments, there were 6 experimental groups: the mock group, the DMSO-control group, the TNF-α-control group, and the ligustilide-treated groups (1 μM, 5 μM, 10 μM). To investigate ligustilide's anti-apoptotic effects on TNF-α during C2C12-cell differentiation, C2C12 cells were incubated for 96 h and then stained with Hoechst 33342 as apoptotic cells showed nuclear condensation and DNA fragmentation. The number of cells exhibiting typical apoptotic morphology in the TNF-αcontrol group was higher than the number of apoptotic cells in the other groups. In contrast, the ligustilide-treated groups presented reduced numbers of apoptotic cells in a dose-dependent manner (as shown in Fig. 2A). Based on the calculated AI (as shown in Fig. 2B), the DMSO-control groups showed no significant differences from the mock groups, while the TNF-α-control groups displayed a significantly
Fig. 4. Ligustilide regulated the apoptosis-related proteins expression. C2C12 cells were incubated for 48 h and 96 h. A. After treatment, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against bcl-2, bax, pro-caspase 3, pro-caspase 8 and GAPDH. B. Data were analysed by densitometric quantification. Data are expressed as means ± S.E.M. from at least three independent experiments (#, p b 0.05 compared with the corresponding mock groups; *, p b 0.05 compared with the corresponding TNF-α-control groups).
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increased AI compared with the mock groups and the DMSO-control groups. The cells treated with ligustilide exhibited a lower AI compared with the TNF-α-control groups, and the groups treated with ligustilide at the 10 μM concentration exhibited the lowest AI scores (p = 0.01664206). Moreover, incubation time also impacted the AI, as results at 24 h and 48 h were significantly different from those at 72 h and 96 h. In contrast, there were no significant differences between the incubation times of 24 h and 48 h. Based on these results, there were 5 groups tested in the subsequent experiments, as the DMSO-control group had no significant differences from the mock group and we chose the 48 hourand 96 hour-time points for further investigation. Considered one of the hallmarks of apoptosis, DNA ladders were chosen to show the anti-apoptotic effects of ligustilide. For DNA extraction and the detection of DNA fragments, DNA fragmentation was measured using DNA electrophoresis and fluorescent staining (as displayed in Fig. 3A and B). The DNA marker used in the experiments ranged from 8 kb to 500 bp, and had an enhanced band at 3 kb. Featuring the same tendency as observed in the AI, the TNF-α-control group showed significantly increased apoptotic DNA fragmentation in the extracted DNA compared with the mock group (shown in Fig. 3B). All ligustilidetreated groups (1, 5, and 10 μM) showed decreased apoptotic DNA fragmentation compared to the TNF-α-control groups, and the group treated with 10 μM ligustilide showed the least fragmentation (p = 0.008).
ratio, pro-caspase 3 expression and pro-caspase 8 expression were reduced in the TNF-α-control group compared to the mock group. As shown in Fig. 4A and B, ligustilide treatment led to a shift of bcl-2/bax ratio in favour of bcl-2. C2C12 cells incubated in ligustilide at the 5 μM and 10 μM concentrations for 96 h presented a significantly increased bcl-2/bax ratio compared with those in the TNF-α-control group. In addition, the 10 μM ligustilide-treated group manifested significantly increased pro-caspase 3 expression at 48 h and the 1 μM ligustilide-treated group had significantly increased pro-caspase 8 at 96 h (see Fig. 4B). 3.4. Ligustilide increased the level of phosphorylated Akt Fig. 5 shows that ligustilide also up-regulated the levels of phosphorylated Akt, while the TNF-α-control groups had lower phosphorylated Akt levels, compared to the mock groups. The level of phosphorylated Akt in the TNF-α-control group and that of the ligustilide-treated groups both decreased significantly compared to that of the mock group, in both the 48 hour and the 96 hour groups. In 48 h, significant differences were also detected between the TNF-α-control group and the 5 μM and 10 μM ligustilide-treated groups. In 96 h, ligustilide significantly increased the level of phosphorylated Akt in the 1 μM and 10 μM ligustilide-treated groups compared to TNF-α-control group. 3.5. Ligustilide inhibited myogenic differentiation
3.3. Ligustilide regulated the expression of apoptosis-related proteins To further characterise ligustilide's inhibition of TNF-α-mediated apoptosis, the expression levels of apoptosis-related proteins were assessed. Specifically, the expression levels of bcl-2, bax, pro-caspase 8 and pro-caspase 3 were affected by TNF-α and ligustilide (as shown in Fig. 4A). Our findings shed light on the anti-apoptotic mechanisms of ligustilide, showing that the drug worked via up-regulation of the bcl-2/bax ratio, pro-caspase 3 expression and pro-caspase 8 expression in a concentration-dependent manner. Furthermore, the bcl-2/bax
As shown in Fig. 6A, C2C12 cells cultured for 96 h were stained with H&E. TNF-α and ligustilide both inhibited myogenic differentiation compared to the mock group. The mock group's cells formed myotubes which remarkably outnumbered those in the TNF-α-control group and the ligustilide-treated groups. However, the ligustilide-treated groups had more cells and most of the cells remained in the myoblast state. To confirm ligustilide's inhibitory effect on myogenic differentiation, the expression of the protein myogenin was measured in various groups. As shown in Fig. 6B, cells treated with ligustilide at all
Fig. 5. Ligustilide increased the level of phosphorylated Akt. C2C12 cells were incubated for 48 h and 96 h. A. After treatment, the proteins from the total cell lysates were separated by SDSPAGE gel electrophoresis and immunoblotted with antibodies against total Akt and phosphorylated Akt. B. Data were analysed by densitometric quantification. Data are expressed as means ± S.E.M. from at least three independent experiments (#, p b 0.05 compared with corresponding the mock groups; *, p b 0.05 compared with the corresponding TNF-α-control groups).
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Fig. 6. Ligustilide inhibited myogenic differentiation. A. C2C12 cells cultured for 96 h were stained with H&E and observed under a fluorescence microscope at 200× magnification. B. C2C12 cells were treated with ligustilide (1 μM, 5 μM, or 10 μM) for 96 h. Cells were lysed, and expression of myogenin was assayed by Western blot analysis. GAPDH was used as a loading control, and the blots were quantified by densitometry. Data are expressed as means ± S.E.M. from at least three independent experiments (#, p b 0.05 compared with the corresponding mock groups; *, p b 0.05 compared with the corresponding TNF-α-control groups).
concentrations had significantly inhibited myogenin expression compared with the TNF-α-control group. Meanwhile, the TNF-α-control group and ligustilide-treated groups manifested a significant suppression of myogenic differentiation compared with the mock group. 4. Discussion In the present study, we investigated the anti-apoptotic effects of ligustilide on C2C12 cells induced by TNF-α during differentiation. Ligustilide reduced apoptosis by shifting the bcl-2/bax ratio, increasing anti-apoptotic bcl-2 levels, upregulating pro-caspases 3 and 8, and inhibiting the activation of caspase-3 and -8. Meanwhile, ligustilide activated Akt-mediated cell survival pathways, which resulted in cell proliferation rather than apoptosis. The early stage of apoptosis involves death-inducing signals [4]. In this stage, the bcl-2 protein forms heterodimers with bax, which is considered to be a promoter of cell apoptosis and plays a central role in controlling cell death. The expression of bax and bcl-2 and the balance of these two proteins might lead to induction of cell apoptosis [15]. After this induction phase, nuclear activators, cell surface receptors, or mitochondrial pathways become activated during the commitment to cellular death, followed by cytoplasmic and nuclear events [4]. In the nucleus, DNA fragmentation is caused by activated endonucleases, the chromatin condenses, and the nuclear envelope breaks down and eventually the cell itself disintegrates into apoptotic bodies
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[6]. The nuclear alterations recognised as a “DNA ladder” by conventional agarose gel electrophoresis have long been considered to be a biochemical hallmark of apoptosis. These DNA ladders are derived from large fragments of DNA of 30 ± 50 and 200 ± 300 kb, which may represent loops and rosettes of DNA, in terms of higher-order chromatin structure [16]. TNF-induced apoptosis has all of the above characteristics of apoptosis. TNF-induced apoptosis is mediated primarily through the activation of type I receptor. TNF is known to activate caspase-8, caspase-9 and caspase-3. Activation of an upstream caspase can lead to activation of a downstream caspase which means that caspase-8 can activate caspase-3. There are some reports that various members of the bcl-2 family, including bcl-2, can block TNF-induced apoptosis [17]. In this study, we chose the proteins bcl-2, bax, caspase-3, and caspase-8 and DNA fragmentation to be the measurable indicators to indicate apoptosis. We demonstrated that ligustilide prevents TNFα-induced apoptosis during C2C12 cell differentiation by shifting the bcl-2/bax ratio and increasing pro-caspases to attenuate the activation of caspases. DNA fragmentation, apparent as DNA laddering, was reduced as the result of ligustilide's effect on apoptosis. Akt is involved in many cell survival pathways. Activated Akt phosphorylates various substrates in the cytosol, and one of its major functions is to act indirectly as an anti-apoptotic protein [18]. The increased expression of phosphorylated Akt and the increased number of myoblasts in the ligustilide-treated group indicate that ligustilide could induce cellular proliferation and exert anti-apoptotic effects by up-regulating phosphorylated Akt. Myoblasts cultured in DM make one of three choices, either fusing to form myotubes, arresting as satellite cells, or undergoing apoptosis. Of these three choices, satellite cells cycle when re-exposed to GM [19]. This means that proliferating cells cannot fuse into myotubes. In skeletal muscle, regeneration involves successive steps of satellite cell activation, proliferation, and differentiation, and finally leads to formation of regenerated myofibres. The process is regulated by basic helix–loop– helix (bHLH) myogenic regulatory factors (MRFs). These factors constitute the so-called MyoD family of proteins, which contain four members: Myf5, MyoD, myogenin and MRF4 [20]. One of the MyoD family of proteins, myogenin, which we detected in experiments, was inhibited by ligustilide. This finding indirectly supports the conclusion that ligustilide induced C2C12 cells to arrest as satellite cells and proliferate instead of undergoing apoptosis induced by TNF-α. As shown in Fig. 3A, apoptosis was not the only response to TNF-α. DNA extracted from TNF-α-control group was degraded into a smear, a characteristic of the random cleavage of DNA that is found in cells undergoing necrotic cell death [21]. Moreover, as there was a small amount of myotubes in both the treated groups and the TNF-αcontrol groups, autophagy may play an important role in maintaining homeostasis. One study reported that the presence of nucleardependent “territorial” death domains in the syncytium could explain a slower death of myotubes compared to mononucleated cells. In addition, autophagy could preserve and protect muscle cell integrity against chemical stimuli, making C2C12 cells, in particular myotubes, more resistant to the induction of apoptosis [22]. In future work, we will focus on the relationship between autophagy, necrosis and ligustilide's effect on C2C12 cells, and further study the role of Akt-related signalling pathways in mediating the effect of ligustilide. Acknowledgement We acknowledge the financial support from the National Science Foundation of China (No. 81173457). We would like to thank Prof. Wang, Teacher Cai and Minghan Li for their suggestions and help. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.02.007.
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International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp
Ligustilide prevents the apoptosis effects of tumour necrosis factor-alpha during C2C12 cell differentiation Ying Shi a,b, Dongtao Wang a,b, Lu Lu a,b, Yi Yin a,b, Ming Wang a,b, Chengjie Li a,b, Jianxin Diao b, Yanjing Wang a, Lianbo Wei a,b,⁎ a b
Department of Traditional Chinese Medicine, ZhuJiang Hospital, Southern Medical University, Guangzhou 510280, China School of Traditional Chinese Medicine, Southern Medical University, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 17 September 2013 Received in revised form 26 January 2014 Accepted 6 February 2014 Available online 21 February 2014 Keywords: Ligustilide Apoptosis Myoblast Myotube
a b s t r a c t Ligustilide, the major component of Angelica sinensis, is also thought to be the most potent bioactive constituent of this plant. Ligustilide has been reported to markedly protect neural tissue against apoptosis. However, little is known regarding ligustilide's anti-apoptosis effect in muscle tissue. The aim of the study was to investigate the anti-apoptosis effects of ligustilide on TNF-α-induced C2C12 cells during differentiation. It was revealed that ligustilide at various concentrations significantly prevented the apoptosis of C2C12 cells incubated in TNF-α as assessed by apoptosis index and DNA fragmentation. Moreover, ligustilide-treated groups exhibited a significant increase in the bcl-2/bax ratio, pro-caspase 3 and pro-caspase 8 compared with the TNF-α-control group in a dose-dependent manner. Meanwhile, ligustilide-treated groups presented a significantly increased level of phosphorylated Akt and suppressed expression of the myogenin protein. Therefore, the findings derived suggested that ligustilide protected C2C12 cells from TNF-α-induced apoptosis during differentiation by reducing apoptosis and inducing cell proliferation. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Skeletal muscle atrophy is a condition which occurs as the result of disuse, starvation, ageing and a multitude of disease states, such as diabetes, cancer, and AIDS [1]. The pro-inflammatory cytokine TNF-α plays a critical role in muscle atrophy and in a broad range of diverse activities, including cell growth and differentiation, inflammation, apoptosis and necrosis. Moreover, a pathologic level of TNF-α has been identified as a significant mechanism associated with skeletal muscle wasting [2]. At least two pathways for the skeletal muscle-wasting effects of TNF-α have been described: inhibition of myogenin in myoblasts and apoptosis of myoblasts and myotubes [3]. Thus, delivery of TNF-α to primary human myoblasts or murine C2C12 myoblasts can inhibit myogenic differentiation and result in cell apoptosis.
Abbreviations: TNF-α, tumour necrosis factor alpha; FBS, foetal bovine serum; DMEM/ F-12, Dulbecco's Modified Eagle's Medium F12 Liquid; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazo-lium bromide; DMSO, dimethyl sulfoxide; GM, growth medium; DM, differentiation medium; OD, optical density; AI, apoptosis index; EB, ethidium bromide; bHLH, helix–loop–helix; MRFs, myogenic regulatory factors. ⁎ Corresponding author at: Department of Traditional Chinese Medicine, ZhuJiang Hospital, Southern Medical University, Guangzhou 510280, China. Tel./fax: +86 20 6164 3456. E-mail addresses: [email protected] (Y. Shi), [email protected] (D. Wang), [email protected] (L. Lu), [email protected] (Y. Yin), [email protected] (M. Wang), [email protected] (C. Li), [email protected] (J. Diao), [email protected] (Y. Wang), [email protected] (L. Wei).
http://dx.doi.org/10.1016/j.intimp.2014.02.007 1567-5769/© 2014 Elsevier B.V. All rights reserved.
Apoptosis, a physiological form of cell suicide, ensures the elimination of superfluous tissues in development and is critical for the maintenance of tissue homeostasis in adulthood [4]. The initial stage of apoptosis involves death-inducing signals, including the release of reactive oxygen and nitrogen species, the expression of ligands for ‘death receptors’ and altered levels of bcl-2 family protein [5]. Following this early stage, nuclear activators, cell surface receptors, and mitochondrial pathways become activated, cells become committed to cell death, and specific cytoplasmic and nuclear events occur [6]. During this phase, caspases, which are responsible for proteolytic cleavage of a broad spectrum of cellular targets, are activated in the cytosol [5] and orchestrate the changes associated with apoptosis (DNA fragmentation, nuclear condensation, cell shrinkage, membrane blebbing, etc.) [7]. Therefore, treatments aimed at inhibiting the changes in the levels of bcl-2 family proteins, blocking caspase expression and reducing typical apoptotic morphology (e.g., DNA fragmentation, nuclear condensation, and cell shrinkage) may have potential therapeutic advantages with regard to apoptosis. Angelica sinensis, known as Danggui in China, is thought to be capable of repairing tissue damage in Chinese traditional medicine. The chemical constituents of A. sinensis extract are classified into essential oils containing the main pharmacologically active compounds [8] and water soluble components. Ligustilide, the major component, is thought to be the most potent bioactive constituent [9] and has been previously demonstrated to be effective at providing neuroprotection against subarachnoid haemorrhage by reducing apoptotic damage through
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Because the effectiveness of ligustilide against apoptosis of muscles cells remains unclear, this study was conducted to examine the antiapoptosis effects of ligustilide on TNF-α-induced C2C12 cells during differentiation. 2. Materials and methods 2.1. Reagents
Fig. 1. Ligustilide showed cytotoxic effects on C2C12 cells. C2C12 cells were treated with ligustilide (1 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, or 50 μM) for 24 h and 48 h. All data are reported as the means ± S.E.M. from more than three independent experiments. *, p b0.05 compared with control cells in 24 h; #, p b 0.05 compared with control cells in 48 h.
decreased expression of p53 and cleavage of caspase-3. Although no change in bax expression was detected, ligustilide treatment after injury resulted in a dose-dependent increase in bcl-2 levels, which led to a large shift of the bcl-2/bax ratio in favour of the anti-apoptotic bcl-2 [10]. Moreover, other published data shed further light on the antiapoptotic properties of ligustilide, specifically through its contribution to the up-regulation of bcl-2 and the down-regulation of bax and caspase3 [11]. Thus, the regulation of bcl-2 family proteins and caspases is usually considered to be the main mechanism of action of ligustilide against apoptosis.
FBS, horse serum and DMEM/F-12 were purchased from HyClone Laboratories (Logan, UT, USA). MTT, Hoechst33342 and DMSO were purchased from Sigma (St. Louis, MO, USA). Ligustilide was purchased from the National Institute for Food and Drug Control (HPLC ≥ 98%). RapidStep™ 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. Bcl2, bax, caspase-3, caspase-8, phosphor-Akt (Ser473), and total Akt antibodies were purchased from Cell Signaling Technologies (Danvers, USA). Myogenin antibodies were purchased from Santa Cruz Biotechnology (Texas, USA). Mouse anti-GAPDH, anti-rabbit and anti-mouse antibodies were purchased from EarthOx Biotechnology (EarthOx, CA, USA). 2.2. Cell culture C2C12 cells were purchased from the Chinese Academy of Sciences (Shanghai, China). C2C12 cells were maintained in growth medium (GM, DMEM/F-12 with 10% FBS) and incubated at 37 °C in a watersaturated atmosphere of 5% CO2.
Fig. 2. Ligustilide reduced apoptosis induced by TNF-α during C2C12 cell differentiation. A. C2C12 cells incubated for 96 h, then stained with Hoechst 33342 and observed under a fluorescence microscope at 400× magnification. B. A semi-quantitative analysis was performed by calculating an AI from observed images. Data are expressed as means ± S.E.M. from more than three independent experiments.
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2.3. Cytotoxicity of ligustilide assay C2C12 myoblasts were seeded in GM in 96-well plates for 24 h, and then exposed to GM with 1, 5, 10, 20, 30, 40, or 50 μM of ligustilide, which was dissolved in a vehicle solution of 0.1% DMSO. The control group cells were cultured in GM only. Cell viability was analysed at 24 h and 48 h post-treatment via MTT assay. 2.4. Experimental model C2C12 cells derived from mouse skeletal muscle were maintained in GM at 37 °C under a humid 5% CO2/95% O2 atmosphere. When the myoblasts were approximately 60–70% confluent, myotube differentiation was initiated by replacing the GM with differentiation medium (DM, DMEM/F-12 with 2% horse serum). Medium was changed every 48 h before experimentation. 2.5. Treatment of cells in groups Stock solutions of TNF-α were diluted with sterile DM to a concentration of 500 ng/mL and stored at −20 °C until required for use. Cells of the TNF-α-control group and of the ligustilide-treated experimental groups were incubated with 20 ng/mL recombinant murine TNF-α dissolved in DM. The concentration of TNF-α was described in [3] and confirmed in preliminary experiments. For the mock group, the cells were incubated with DM only. Cells in the DMSO-control group were incubated in DM with 1% DMSO. Cells in ligustilide-treated groups were incubated in DM with TNF-α and ligustilide at diverse concentrations.
2000 MM, Kodak, Rochester, NY, USA). The intensities of the protein bands were analysed using Molecular Imaging Software Version 4.0, which was provided with the Kodak 2000 MM System. 2.8. Morphology and diameter of myotubes Myotube cultures were photographed under a phase contrast microscope at 200× magnification and stained with hematoxylin–eosin staining (H&E). As myotubes were not formed in a sufficient number in a shorter time, and cells in the TNF-α-control group would be almost dead in 5 days, we chose an incubation time of 96 h to detect myotubes. Cells in different experimental groups were treated as described above. The medium was changed every 48 h before experimentation. 2.9. Statistical analyses Statistical analyses were performed using SPSS 13.0 (SPSS, Chicago, IL, USA). Because the incubation times and different interventions all contributed to the outcomes simultaneously, tests of between-subjects effects were needed to examine group effects and incubation time effects on the variable by the univariate analysis. The data were then further analysed post-hoc using either Bonferroni's test or Dunnett's T3 for multiple comparisons, depending on the homogeneity of variances. For detecting single variable effects, statistical significance was estimated using one-way analysis of variance (ANOVA), followed
2.6. Apoptosis assay For the Hoechst 33342 staining, the cells were grown in 12-well plates and treated as described above. Nuclear DNA was visualised in cells by staining with the DNA-specific fluorescent dye, Hoechst 33342, at a final concentration of 6 μg/mL. The cells were immediately observed with a light microscope, using filters for blue fluorescence. To quantify the extent of apoptosis, an apoptosis index (AI) was calculated, as described in [3], from 6 images of randomly selected microscopic fields for each treatment. The AI was calculated as the percentage of the total number of nuclei that were apoptotic. Apoptotic nuclei were identifiable because they appeared brightly fluorescent and condensed compared to normal nuclei. For the extraction of DNA and the detection of DNA fragments, cells were treated as described above and the total DNA was isolated according to the protocol provided with the Genomic DNA Mini Preparation Kit with Spin Column (Beyotime, Shanghai, China). DNA fragments were separated in a 2% agarose gel, stained with ethidium bromide (EB), and visualised under UV light. 2.7. Western blotting For immunoblotting, 20 mg aliquots of the lysates were separated on a 12% SDS-polyacrylamide gel and transferred electrophoretically (Bio-Rad, Hercules, California, USA) to a PVDF membrane (Pall, USA). As cleavage of actin has been observed under apoptotic conditions in vitro and in cardiac and skeletal muscles [12–14], GAPDH protein was used as the internal control. After being placed in blocking buffer, the membranes were incubated with the following primary antibodies: anti-bcl-2 (1:1000 dilution), anti-bax (1:1000 dilution), anti-caspase-3 (1:1000 dilution), anti-caspase-8 (1:1000 dilution), anti-myogenin (1:200 dilution), anti-Akt (1:1000 dilution), anti-phospho-Akt (1:1000 dilution), and anti-GAPDH (1:5000 dilution). After the membranes were washed with TBST, the appropriate HRP-conjugated secondary antibody (1:5000) was added to the preparation. The blot was incubated at 37 °C for 1 h. The protein bands were captured and documented using a CCD camera and imaging system (Image Station
Fig. 3. Ligustilide decreased apoptotic DNA fragmentation induced by TNF-α. C2C12 cells were incubated for 48 h and 96 h and their total DNA was isolated. DNA fragments were separated in an agarose gel, stained with EB, and visualised under UV light. A. Cells were incubated for 48 h and 96 h. B. Densities of the bands following the DNA fragmentation shown in A. Data are expressed as means ± S.E.M. from more than three independent experiments.
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by either Bonferroni's test or Dunnett's T3 for multiple comparisons, based on the homogeneity of variances. Values with p b 0.05 were considered significant. The results are expressed as means ± S.E.M. and represent assays from at least three independent experiments.
3. Results 3.1. Ligustilide showed cytotoxic effects on C2C12 cells To examine the cellular tolerance to the cytotoxicity of ligustilide, C2C12 cells were treated with the drug in increasing concentrations ranging from 1 to 50 μM for respective 24 h and 48 h. Cytotoxicity of ligustilide was evaluated by an MTT assay. The results showed that ligustilide exerted significant inhibitory effects on the growth of the cells when the drug concentrations were 10 μM or above, in both the 24 hour and the 48 hour groups (see Fig. 1). No significant differences were identified between the two time points.
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3.2. Ligustilide reduced apoptosis induced by TNF-α during C2C12 cell differentiation Based on the results above, which confirmed that ligustilide induced significant cell death at a concentration of 20 μM because of its cytotoxicity, as in preliminary experiments, there were 6 experimental groups: the mock group, the DMSO-control group, the TNF-α-control group, and the ligustilide-treated groups (1 μM, 5 μM, 10 μM). To investigate ligustilide's anti-apoptotic effects on TNF-α during C2C12-cell differentiation, C2C12 cells were incubated for 96 h and then stained with Hoechst 33342 as apoptotic cells showed nuclear condensation and DNA fragmentation. The number of cells exhibiting typical apoptotic morphology in the TNF-αcontrol group was higher than the number of apoptotic cells in the other groups. In contrast, the ligustilide-treated groups presented reduced numbers of apoptotic cells in a dose-dependent manner (as shown in Fig. 2A). Based on the calculated AI (as shown in Fig. 2B), the DMSO-control groups showed no significant differences from the mock groups, while the TNF-α-control groups displayed a significantly
Fig. 4. Ligustilide regulated the apoptosis-related proteins expression. C2C12 cells were incubated for 48 h and 96 h. A. After treatment, proteins from total cell lysates were separated by SDS-PAGE gel electrophoresis and immunoblotted with antibodies against bcl-2, bax, pro-caspase 3, pro-caspase 8 and GAPDH. B. Data were analysed by densitometric quantification. Data are expressed as means ± S.E.M. from at least three independent experiments (#, p b 0.05 compared with the corresponding mock groups; *, p b 0.05 compared with the corresponding TNF-α-control groups).
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increased AI compared with the mock groups and the DMSO-control groups. The cells treated with ligustilide exhibited a lower AI compared with the TNF-α-control groups, and the groups treated with ligustilide at the 10 μM concentration exhibited the lowest AI scores (p = 0.01664206). Moreover, incubation time also impacted the AI, as results at 24 h and 48 h were significantly different from those at 72 h and 96 h. In contrast, there were no significant differences between the incubation times of 24 h and 48 h. Based on these results, there were 5 groups tested in the subsequent experiments, as the DMSO-control group had no significant differences from the mock group and we chose the 48 hourand 96 hour-time points for further investigation. Considered one of the hallmarks of apoptosis, DNA ladders were chosen to show the anti-apoptotic effects of ligustilide. For DNA extraction and the detection of DNA fragments, DNA fragmentation was measured using DNA electrophoresis and fluorescent staining (as displayed in Fig. 3A and B). The DNA marker used in the experiments ranged from 8 kb to 500 bp, and had an enhanced band at 3 kb. Featuring the same tendency as observed in the AI, the TNF-α-control group showed significantly increased apoptotic DNA fragmentation in the extracted DNA compared with the mock group (shown in Fig. 3B). All ligustilidetreated groups (1, 5, and 10 μM) showed decreased apoptotic DNA fragmentation compared to the TNF-α-control groups, and the group treated with 10 μM ligustilide showed the least fragmentation (p = 0.008).
ratio, pro-caspase 3 expression and pro-caspase 8 expression were reduced in the TNF-α-control group compared to the mock group. As shown in Fig. 4A and B, ligustilide treatment led to a shift of bcl-2/bax ratio in favour of bcl-2. C2C12 cells incubated in ligustilide at the 5 μM and 10 μM concentrations for 96 h presented a significantly increased bcl-2/bax ratio compared with those in the TNF-α-control group. In addition, the 10 μM ligustilide-treated group manifested significantly increased pro-caspase 3 expression at 48 h and the 1 μM ligustilide-treated group had significantly increased pro-caspase 8 at 96 h (see Fig. 4B). 3.4. Ligustilide increased the level of phosphorylated Akt Fig. 5 shows that ligustilide also up-regulated the levels of phosphorylated Akt, while the TNF-α-control groups had lower phosphorylated Akt levels, compared to the mock groups. The level of phosphorylated Akt in the TNF-α-control group and that of the ligustilide-treated groups both decreased significantly compared to that of the mock group, in both the 48 hour and the 96 hour groups. In 48 h, significant differences were also detected between the TNF-α-control group and the 5 μM and 10 μM ligustilide-treated groups. In 96 h, ligustilide significantly increased the level of phosphorylated Akt in the 1 μM and 10 μM ligustilide-treated groups compared to TNF-α-control group. 3.5. Ligustilide inhibited myogenic differentiation
3.3. Ligustilide regulated the expression of apoptosis-related proteins To further characterise ligustilide's inhibition of TNF-α-mediated apoptosis, the expression levels of apoptosis-related proteins were assessed. Specifically, the expression levels of bcl-2, bax, pro-caspase 8 and pro-caspase 3 were affected by TNF-α and ligustilide (as shown in Fig. 4A). Our findings shed light on the anti-apoptotic mechanisms of ligustilide, showing that the drug worked via up-regulation of the bcl-2/bax ratio, pro-caspase 3 expression and pro-caspase 8 expression in a concentration-dependent manner. Furthermore, the bcl-2/bax
As shown in Fig. 6A, C2C12 cells cultured for 96 h were stained with H&E. TNF-α and ligustilide both inhibited myogenic differentiation compared to the mock group. The mock group's cells formed myotubes which remarkably outnumbered those in the TNF-α-control group and the ligustilide-treated groups. However, the ligustilide-treated groups had more cells and most of the cells remained in the myoblast state. To confirm ligustilide's inhibitory effect on myogenic differentiation, the expression of the protein myogenin was measured in various groups. As shown in Fig. 6B, cells treated with ligustilide at all
Fig. 5. Ligustilide increased the level of phosphorylated Akt. C2C12 cells were incubated for 48 h and 96 h. A. After treatment, the proteins from the total cell lysates were separated by SDSPAGE gel electrophoresis and immunoblotted with antibodies against total Akt and phosphorylated Akt. B. Data were analysed by densitometric quantification. Data are expressed as means ± S.E.M. from at least three independent experiments (#, p b 0.05 compared with corresponding the mock groups; *, p b 0.05 compared with the corresponding TNF-α-control groups).
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Fig. 6. Ligustilide inhibited myogenic differentiation. A. C2C12 cells cultured for 96 h were stained with H&E and observed under a fluorescence microscope at 200× magnification. B. C2C12 cells were treated with ligustilide (1 μM, 5 μM, or 10 μM) for 96 h. Cells were lysed, and expression of myogenin was assayed by Western blot analysis. GAPDH was used as a loading control, and the blots were quantified by densitometry. Data are expressed as means ± S.E.M. from at least three independent experiments (#, p b 0.05 compared with the corresponding mock groups; *, p b 0.05 compared with the corresponding TNF-α-control groups).
concentrations had significantly inhibited myogenin expression compared with the TNF-α-control group. Meanwhile, the TNF-α-control group and ligustilide-treated groups manifested a significant suppression of myogenic differentiation compared with the mock group. 4. Discussion In the present study, we investigated the anti-apoptotic effects of ligustilide on C2C12 cells induced by TNF-α during differentiation. Ligustilide reduced apoptosis by shifting the bcl-2/bax ratio, increasing anti-apoptotic bcl-2 levels, upregulating pro-caspases 3 and 8, and inhibiting the activation of caspase-3 and -8. Meanwhile, ligustilide activated Akt-mediated cell survival pathways, which resulted in cell proliferation rather than apoptosis. The early stage of apoptosis involves death-inducing signals [4]. In this stage, the bcl-2 protein forms heterodimers with bax, which is considered to be a promoter of cell apoptosis and plays a central role in controlling cell death. The expression of bax and bcl-2 and the balance of these two proteins might lead to induction of cell apoptosis [15]. After this induction phase, nuclear activators, cell surface receptors, or mitochondrial pathways become activated during the commitment to cellular death, followed by cytoplasmic and nuclear events [4]. In the nucleus, DNA fragmentation is caused by activated endonucleases, the chromatin condenses, and the nuclear envelope breaks down and eventually the cell itself disintegrates into apoptotic bodies
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[6]. The nuclear alterations recognised as a “DNA ladder” by conventional agarose gel electrophoresis have long been considered to be a biochemical hallmark of apoptosis. These DNA ladders are derived from large fragments of DNA of 30 ± 50 and 200 ± 300 kb, which may represent loops and rosettes of DNA, in terms of higher-order chromatin structure [16]. TNF-induced apoptosis has all of the above characteristics of apoptosis. TNF-induced apoptosis is mediated primarily through the activation of type I receptor. TNF is known to activate caspase-8, caspase-9 and caspase-3. Activation of an upstream caspase can lead to activation of a downstream caspase which means that caspase-8 can activate caspase-3. There are some reports that various members of the bcl-2 family, including bcl-2, can block TNF-induced apoptosis [17]. In this study, we chose the proteins bcl-2, bax, caspase-3, and caspase-8 and DNA fragmentation to be the measurable indicators to indicate apoptosis. We demonstrated that ligustilide prevents TNFα-induced apoptosis during C2C12 cell differentiation by shifting the bcl-2/bax ratio and increasing pro-caspases to attenuate the activation of caspases. DNA fragmentation, apparent as DNA laddering, was reduced as the result of ligustilide's effect on apoptosis. Akt is involved in many cell survival pathways. Activated Akt phosphorylates various substrates in the cytosol, and one of its major functions is to act indirectly as an anti-apoptotic protein [18]. The increased expression of phosphorylated Akt and the increased number of myoblasts in the ligustilide-treated group indicate that ligustilide could induce cellular proliferation and exert anti-apoptotic effects by up-regulating phosphorylated Akt. Myoblasts cultured in DM make one of three choices, either fusing to form myotubes, arresting as satellite cells, or undergoing apoptosis. Of these three choices, satellite cells cycle when re-exposed to GM [19]. This means that proliferating cells cannot fuse into myotubes. In skeletal muscle, regeneration involves successive steps of satellite cell activation, proliferation, and differentiation, and finally leads to formation of regenerated myofibres. The process is regulated by basic helix–loop– helix (bHLH) myogenic regulatory factors (MRFs). These factors constitute the so-called MyoD family of proteins, which contain four members: Myf5, MyoD, myogenin and MRF4 [20]. One of the MyoD family of proteins, myogenin, which we detected in experiments, was inhibited by ligustilide. This finding indirectly supports the conclusion that ligustilide induced C2C12 cells to arrest as satellite cells and proliferate instead of undergoing apoptosis induced by TNF-α. As shown in Fig. 3A, apoptosis was not the only response to TNF-α. DNA extracted from TNF-α-control group was degraded into a smear, a characteristic of the random cleavage of DNA that is found in cells undergoing necrotic cell death [21]. Moreover, as there was a small amount of myotubes in both the treated groups and the TNF-αcontrol groups, autophagy may play an important role in maintaining homeostasis. One study reported that the presence of nucleardependent “territorial” death domains in the syncytium could explain a slower death of myotubes compared to mononucleated cells. In addition, autophagy could preserve and protect muscle cell integrity against chemical stimuli, making C2C12 cells, in particular myotubes, more resistant to the induction of apoptosis [22]. In future work, we will focus on the relationship between autophagy, necrosis and ligustilide's effect on C2C12 cells, and further study the role of Akt-related signalling pathways in mediating the effect of ligustilide. Acknowledgement We acknowledge the financial support from the National Science Foundation of China (No. 81173457). We would like to thank Prof. Wang, Teacher Cai and Minghan Li for their suggestions and help. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.intimp.2014.02.007.
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