Ligustilide Prevents The Apoptosis Effects Of Tumour Necrosis Factor-alpha During C2C12Cell Differentiation

BackgroundSkeletal muscle atrophy is a condition which occurs as the result of disuse, starvation, aging and a multitude of disease states, such as diabetes, cancer, and AIDS[1]. The pro-inflammatory cytokine TNF-a 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-a 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-a have been described:inhibition of myogenin in myoblasts and apoptosis of myoblasts and myotubes[3]. Thus, delivery of TNF-a to primary human myoblasts or murine C2C12myoblasts can inhibit myogenic differentiation and result in cell apoptosis.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-2family 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-2family 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 capable of repairing tissue damage in Chinese traditional medicine. The chemical constituents of Angelica 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 hemorrhage by reducing apoptotic damage through decreased expression of p53and cleavage of caspase-3. Although no change in Bax expression was detected, ligustilide treatment after injury resulted in a dose-dependent increase in Bcl-2levels, 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 anti-apoptotic properties of ligustilide, specifically through its contribution to the up-regulation of Bcl-2and the down-regulation of Bax and caspase-3. Thus, the regulation of Bcl-2family proteins and caspases is usually considered to be the main mechanism of action of ligustilide against apoptosis. Because the effectiveness of ligustilide against apoptosis of muscles cells remains unclear, this study was conducted to examine the anti-apoptosis effects of ligustilide on TNF-a-induced C2C12cells during differentiation.ObjectiveThe aim of the study was to investigate the anti-apoptosis effects of ligustilide on TNF-a-induced C2C12cells during differentiation.Method1Cell CultureC2C12cells were purchased from the Chinese Academy of Sciences (Shanghai, China). C2C12cells were maintained in growth medium (GM, DMEM/F-12with10%FBS) and incubated at37℃in a water-saturated atmosphere of5%CO2.2Cytotoxicity of ligustilide assayC2C12myoblasts were seeded in GM in96-well plates for24h, and then exposed to GM with1,5,10,20,30,40, or50μM of ligustilide, which was dissolved in a vehicle solution of0.1%DMSO. The control group cells were cultured in GM only. Cell viability was analysed at24hours and48hours post-treatment via MTT assay.3Experimental modelC2C12cells derived from mouse skeletal muscle were maintained in GM at37℃under a humid5%CO2/95%O2atmosphere. When the myoblasts were approximately60-70%confluent, myotube differentiation was initiated by replacing the GM with differentiation medium (DM, DMEM/F-12with2%horse serum). Medium was changed every48hours before experimentation.4Treatment of cells in groupsStock solutions of TNF-α were diluted with sterile DM to a concentration of500ng/mL and stored at-20℃until required for use. Cells of the TNF-a-control group and of the ligustilide-treated experimental groups were incubated with20ng/mL recombinant murine TNF-a dissolved in DM. The concentration of TNF-a 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 with1%DMSO. Cells in ligustilide-treated groups were incubated in DM with TNF-a and ligustilide at diverse concentrations.5Apoptosis assayFor the Hoechst33342staining, the cells were grown in12-well plates and treated as described above. Nuclear DNA was visualised in cells by staining with the DNA-specific fluorescent dye, Hoechst33342, at a final concentration of6μg/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. 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 a2%agarose gel, stained with ethidium bromide (EB), and visualised under UV light.6Western blottingFor immunoblotting,20mg aliquots of the lysates were separated on a12%SDS-polyacrylamide gel and transferred electrophoretically (Bio-Rad, Hercules, california, USA) to a PVDF membrane (Pall, USA). After being placed in blocking buffer, the membranes were incubated with the primary antibodies. After the membranes were washed with TBST, the appropriate HRP-conjugated secondary antibody was added to the preparation. The blot was incubated at37℃for1h. The protein bands were captured and documented using a CCD camera and imaging system (Image Station2000MM, Kodak, Rochester, NY, USA). The intensities of the protein bands were analysed using Molecular Imaging Software Version4.0, which was provided with the Kodak2000MM System.7Morphology and diameter of myotubesMyotube cultures were photographed under a phase contrast microscope at200x magnification and stained with hematoxylin-eosin staining (H&E). Cells in different experimental groups were treated as described above. The medium was changed every48hours before experimentation.8Statistical analysesStatistical analyses were performed using SPSS13.0(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 T3for 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 by either Bonferroni’s test or Dunnett’s T3for multiple comparisons, based on the homogeneity of variances. Values with p<0.05were considered significant. The results are expressed as means±S.E.M. and represent assays from at least three independent experiments.Results1Ligustilide showed cytotoxic effects on C2C12cellsTo examine the cellular tolerance to the cytotoxicity of ligustilide, C2C12cells were treated with the drug in increasing concentrations ranging from1to50μM for respective24hours and48hours. 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 were10μM or above, in both the24hours and the48hours groups. No significant differences were identified between the two time points.2Ligustilide reduced apoptosis induced by TNF-a during C2C12cell differentiation2.1Hoechst33342assayBased on the results above, which confirmed that ligustilide induced significant cell death at a concentration of20μM because of its cytotoxicity, as in preliminary experiments, there were6experimental groups:the mock group, the DMSO-control group, the TNF-a-control group, and the ligustilide-treated groups (1μM,5μM,10μM). To investigate ligustilide’s anti-apoptotic effects on TNF-a during C2C12-cell differentiation, C2C12cells incubated for96hours, then stained with Hoechst33342. The number of cells exhibiting typical apoptotic morphology in the TNF-a-control 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. Based on the calculated AI, the DMSO-control groups showed no significant differences from the mock groups, while the TNF-a-control groups displayed a significantly 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-a-control groups, and the groups treated with ligustilide at the10μM concentration exhibited the lowest AI scores. Moreover, incubation time also impacted the AI, as results at24hours and48hours were significantly different from those at72hours and96hours. In contrast, there were no significant differences between the incubation times of24hours and48hours.2.2DNA ladders assayBased on these results, there were5groups tested in the subsequent experiments, as the DMSO-control group had no significant differences from the mock group and we chose the48hour-and96hour-time points for further investigating. 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. Featuring the same tendency as observed in the AI, the TNF-a-control group showed significantly increased apoptotic DNA fragmentation in the extracted DNA compared with the mock group. All ligustilide-treated groups (1,5, and10μM) showed decreased apoptotic DNA fragmentation compared to the TNF-a-control groups, and the group treated with10μM ligustilide showed the least fragmentation.3Ligustilide regulated the expression of apoptosis-related proteinsTo further characterise ligustilide’s inhibition of TNF-a-mediated apoptosis, the expression levels of apoptosis-related proteins were assessed. Specifically, the expression levels of Bcl-2, Bax, pro-caspase-8, pro-caspase-3were affected by TNF-a and ligustilide. 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-3expression and pro-caspase-8expression in a concentration-dependent manner. Furthermore, the Bcl-2/Bax ratio, pro-caspase-3expression and pro-caspase-8expression were reduced in the TNF-a-control group compared to the mock group. Ligustilide treatment led to a shift of Bcl-2/Bax ratio in favour of Bcl-2. In addition, ligustilide-treated group manifested increased pro-caspase-3expression and pro-caspase-8.4Ligustilide increased the level of phosphorylated AktLigustilide also up-regulated the levels of phosphorylated Akt, while the TNF-a-control groups had lower phosphorylated Akt levels, compared to the mock groups. The level of phosphorylated Akt in the TNF-a-control group and that of the ligustilide-treated groups both decreased significantly compared to that of the mock group, in both the48hours and the96hours groups. In48hours, significant differences were also detected between the TNF-a-control group and the5μM and10μM ligustilide-treated groups. In96hours, ligustilide significantly increased the level of phosphorylated Akt in the1μM and10μM ligustilide-treated groups compared to TNF-α-control group.5Ligustilide inhibited myogenic differentiationC2C12cells cultured for96hours 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 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. Cells treated with ligustilide at all 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.ConclusionsIn the present study, we investigated the anti-apoptotic effects of ligustilide on C2C12cells induced by TNF-α during differentiation. Ligustilide reduced apoptosis by shifting the Bcl-2/Bax ratio, increasing anti-apoptotic Bcl-2levels, upregulating pro-caspases3and8, and by inhibiting the activation of caspase-3and8. Meanwhile, ligustilide activated Akt-mediated cell survival pathways, which resulted in cell proliferation rather than apoptosis.