A novel cytoprotective peptide protects mesenchymal stem cells against mitochondrial dysfunction and apoptosis induced by starvation via Nrf2/Sirt3/FoxO3a pathway
- Shuo Wang†1, 2,
- Chao Zhang†1, 2,
- Sidikejiang Niyazi†1, 2,
- Long Zheng1, 2,
- Jiawei Li1, 2,
- Weitao Zhang1, 2,
- Ming Xu1, 2,
- Ruiming Rong1, 2, 4,
- Cheng Yang1, 2Email author and
- Tongyu Zhu1, 2, 3Email author
© The Author(s) 2017
Received: 12 December 2016
Accepted: 8 February 2017
Published: 15 February 2017
Mesenchymal stem cell (MSC) has been widely explored in the past decade as a cell-based treatment for various diseases. However, poor survival of adaptively transferred MSCs limits their clinical therapeutic potentials, which is largely ascribed to the nutrient starvation. In this study, we determined whether a novel kidney protective peptide CHBP could protect MSCs against starvation and invested the underlying mechanisms.
MSCs were subjected to serum deprivation and CHBP of graded concentrations was administered. Cell viability and apoptosis were detected by CCK-8, Annexin V/PI assay and Hoechst staining. ROS generation, mitochondrial membrane potential indicated by JC-1 and mitochondrial mass were measured by flow cytometry. The location of cytochrome c within cells was observed under fluorescence microscopy. Expressions of Nrf2, Sirt3, and FoxO3a were analyzed by western blot. In addition, preconditioning MSCs with CHBP was applied to test the possible protection against starvation. Finally, the effect of CHBP on the differentiation and self-renewal capacity of MSCs was also examined.
CHBP improved cell viability and suppressed apoptosis in a dose dependent manner. Starvation resulted in the mitochondrial dysfunction and treatment of CHBP could alleviate mitochondrial stress by diminishing oxidative injury of ROS, restoring mitochondrial membrane potential and maintaining mitochondrial membrane integrity. Importantly, Nrf2/Sirt3/FoxO3a pathway was activated by CHBP and Sirt3 knockdown partially abolished the protection of CHBP. Moreover, MSCs pretreated with CHBP were more resistant to starvation. Under normal condition, CHBP exerted little effects on the differential and self-renewal capacity of MSCs.
The present study demonstrated the efficient protection of CHBP upon MSCs against starvation-induced mitochondrial dysfunction and apoptosis and indicated possible involvement of Nrf2/Sirt3/FoxO3a pathway in the protective effect.
KeywordsCyclic helix B peptide Mesenchymal stem cell Starvation Nrf2 Sirt3 FoxO3a
Mesenchymal stem cell (MSC) is a common adult stem cell originating from the mesoderm and characterized by its multi-potentiality and unique immune-modulatory property [1, 2]. Besides their classic tri-lineage differentiation into adipocytes, osteoblasts, and chondrocytes, it is reported that MSCs can also differentiate into ectodermal and endodermal cells, such as endothelial cells, smooth muscle cells, hepatocytes, and cardiomyocytes [3, 4]. Importantly, as adult stem cells, MSCs can be easily isolated and feasibly expanded ex vivo . In this respect, MSC is considered as an ideal cell-based therapy.
An increasing body of evidence has demonstrated that MSCs hold great promise for the therapy of tissue injuries, inflammatory diseases, and allograft rejections [6–12]. However, in most cases the survival rate of adoptively transferred MSCs is poor, which significantly hampers the treatment efficiency [13–15]. Although the therapeutic capability of MSCs, in the settings of some diseases, might rely on regulating inflammation and enhancing endogenous tissue repair [16, 17], longer survival and retention time would optimize MSC-based therapy efficacies. The reason that transplanted MSCs are short-lived could be largely ascribed to the complex microenvironments. After adoptive transfer, MSCs will encounter with various undesirable factors including nutrient deprivation, oxidative stress, inflammation reactions and so on, all of which could decrease the cell viability and thereby compromise their therapeutic activities [18–21]. Nutrient starvation is one of the major obstacles confronted by engrafted MSCs, especially within injured tissues. In spite that appropriate preconditioning with hypoxia and serum starvation could augment the cellular viability, prolonged exposure to starvation will be a lethal disaster for MSCs [22, 23]. Therefore, improving survival of MSCs under starvation conditions is of vital importance for the MSC-based cell therapies.
Cyclic helix B peptide (CHBP) is a novel erythropoietin-derived peptide synthesized by our group recently with prominent renoprotective effect [24–27]. Our previous studies have demonstrated that CHBP could alleviate kidney ischemia–reperfusion injury through inhibiting apoptosis as well as inflammatory responses [25, 26]. However, whether CHBP is able to exert cytoprotection in the background of starvation remains elusive. In the present study, we adopted the serum deprivation strategy to mimic the starvation during MSCs’ administration. Treatment of CHBP significantly improved mitochondrial dysfunction, prevented apoptosis and prolonged cell survival. Moreover, we revealed that CHBP exerted its cytoprotection through Nrf2/Sirt3/FoxO3a pathway. This research provided a novel pharmacological recognition of CHBP for the cytoprotective effects and suggested CHBP as a promising adjuvant agent in cell-based treatments.
Cell culture and treatment
Primary bone marrow-derived MSCs of C57/BL6 mice were purchased from Cyagen Biosciences Inc. (Guangzhou, China). MSCs were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12) medium with 10% heat-inactivated fetal bovine serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a humidified incubator containing 5% CO2. All experiments were performed on MSCs from 6th to 10th passages. Apoptosis was induced by serum deprivation. Briefly, after MSCs were seeded into 6-well plates or 96-well plates, the culture medium was replaced with serum-free DMEM low glucose with or without CHBP at various concentrations (0.1, 1, 10 nmol/L). MSCs maintained in complete medium were used as the normal control. To exclude a nonspecific impact of peptide itself, a scrambled control peptide (LSEARNQSEL) was also used. Both CHBP and scrambled peptide were dissolved in PBS.
Cell viability in response to starvation was examined by Cell Counting Kit-8 (CCK-8, Dojindo, Shanghai, China). Cells were seeded in 96-well plates at a density of 1 × 104 cells/well. After different treatment, 10ul of CCK-8 reagent was added to each well of the 96-well plates and incubated at 37 °C for 2 h. The absorbance of each sample at 450 nm was measured by a microplate reader.
Meanwhile, trypan blue staining was used to distinguish cell death. MSCs were seeded in 6-well plates 1 × 106 cells/well cells and treated differently. Then non-adherent and adherent cells were harvested and resuspended in 1 ml complete medium. 5 μl of these cell suspensions were aspirated and mixed with an equal volume of 0.4% trypan blue solution, and finally counted using a hemacytometer under microscope.
Annexin V/PI assay
To further explore the protection of CHBP against starvation, cell death was analyzed using Annexin V–FITC/PI apoptosis detection kit (Vazyme Biotech, Nanjing, China). According to the manufacturer’s protocol, cells were harvested and resuspended in 100 μl binding buffer mixed with 5 μl Annexin V-FITC reagent and 5 μl PI reagent. After incubation for 15 min at room temperature in the dark, another 400 μl binding buffer was added, and cells were measured by flow cytometry (Beckman Coulter). Data was analyzed with FlowJo software.
Hoechst 33258 (Beyotime, Nanjing, China) was used for to examine the morphological changes of apoptosis. MSCs with different treatment were fixed in 4% paraformaldehyde for 2 h and subsequently stained with Hoechst 33258 reagent for 30 min at room temperature. Nuclear alterations were then observed under fluorescence microscopy. Fragmentation and condensation of the nucleus were recognized as the characteristics of apoptotic cells.
Reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and mitochondrial mass detection
Briefly, for quantitative detection of mitochondria-derived ROS, MMP and mitochondrial mass, cells were incubated with MitoSOX reagent (2.5 mmol/L, Invitrogen), JC-1 reagent (5 mg/mL, Beyotime), and MitoRed (50 nmol/L, KeyGEN Biotech) for 30 min at 37 °C. Subsequently, MSCs were collected, washed with PBS and then analyzed by flow cytometry.
Cells were first incubated with 50 nmol/L MitoRed for 30 min at 37 °C. To examine cytochrome c location, cells were fixed in 4% paraformaldehyde for 2 h, permeabilized with 0.2% Triton X-100 for 5 min. After blocked with 10% bovine serum albumin for 1 h at room temperature, cells were incubated with primary antibody against cytochrome c (1:500, Beyotime) overnight at 4 °C, and subsequently incubated with FITC-conjugated secondary antibodies (1:1000, Beyotime). Locations of mitochondria and cytochrome c were visualized under fluorescence microscopy.
Knockdown of Sirt3 by small interfering RNA (siRNA)
For knockdown of Sirt3 in vitro, siRNA targeting Sirt3 (ThermoFisher Scientific, AM16708) was used. The siRNA was transfected into MSCs using Lipofectamine 3000 (Invitrogen) according the manufacturer’s instructions. Briefly, MSCs were seeded in 6-well plates with serum-free DMEM medium, and then subjected to the mixture of siRNA and Lipofectamine 3000 reagent. After incubation for 6 h, medium was changed and the cells were harvested for the further experiments.
Western blotting analysis
Briefly, total proteins from MSCs were separated on SDS–polyacrylamide gels, transferred onto nitrocellulose membranes, blocked and incubated with anti-Nrf2, anti-Sirt3, anti-total FoxO3a, and phosphorylated-FoxO3a (p-FoxO3a, Ser253 and Ser318/321) antibodies (1:1000, Cell Signaling Technology) and anti-tubulin antibodies (1:10000, Abcam) overnight at 4 °C. Membranes were then washed 3 times and incubated with secondary antibodies for 1 h at room temperature. The semi-quantitative analysis (AlphaView Software 3.3, Cell Biosciences, Inc.) results were expressed as the optical volume densities (OD × mm2) normalized to tubulin.
For osteogenic differentiation, MSCs were cultured in osteogenic differentiation medium containing 10% FBS, 0.2 mmol/L ascorbate, 10 mmol/L b-glycerolphosphate, 0.1 mmol/L dexamethasone, 100 U/ml penicillin and 100 mg/ml streptomycin (all from Cyagen, Guangzhou, China) at the confluence of 60–70%. Osteogenic medium was changed every 3 days. After induction for 21 days, cells were fixed by 4% formaldehyde, and then stained with Alizarin Red S to assess calcium deposits.
MSCs at 100% confluence were used for adipogenic differentiation. Cells were first cultured in adipogenic induction medium supplemented with 10% FBS, 1 mmol/L dexamethasone, 100 mmol/L indomethacin, 0.5 mmol/L methyisobutylxanthine, 10 mmol/L insulin, 100 U/ml penicillin and 100 mg/ml streptomycin for 3 days, followed by cultivating in maintenance medium consisting of 10% FBS, 5 mg/ml insulin, 100 U/ml penicillin and 100 mg/ml streptomycin for 24 h. After 4 cycles of induction/maintenance exchange, MSCs were cultured in maintenance medium for 6 days and finally fixed by 4% formaldehyde. Lipid droplets were examined using oil red O solution staining.
Colony forming unit (CFU) assay
To test the self-renewal capacity of CHBP-treated MSCs, CFU assay was conducted. In brief, MSCs were plated in 10 cm dishes at a density of 50 cells/cm2 and cultured in normal complete medium in the presence or absence of 10 nM CHBP for 14 days, with medium exchange every 3 days. At the end of cultivation, cells were washed with PBS, fixed by 4% formaldehyde for 30 min and stained with 2% crystal violet for 10 min. Subsequently, cells were washed with PBS for 3 times and left drying. Colonies consisting of more than 50 cells were defined as CFUs and were counted.
All data are presented as mean ± standard deviation. Statistical analysis was performed using the Student’s t test or one-way ANOVA by SPSS 19.0 software (SPSS Inc., Armonk, NY, USA). P < 0.05 was recognized as statistically significant.
CHBP preserved the viability of MSCs during starvation
CHBP attenuated starvation-induced apoptosis in MSCs
CHBP improved mitochondrial dysfunction in response to serum deprivation
Nrf2/Sirt3/FoxO3a pathway may involve in the protection of CHBP against serum deprivation
MSCs pretreated with CHBP were more resistant to starvation
CHBP exerted little effects on the differential and self-renewal capacity of MSCs
In the past decade, there has been considerable interest in the exploration of MSCs as cell-based treatment for various diseases. However, poor survival of adaptively transferred MSCs limits their clinical therapeutic potential, which to a great extent is due to the nutrient starvation. Therefore, it is of vital importance to discover regimens that improve the viability of starved MSCs. This study aims to determine whether the organ-protective peptide CHBP can rescue MSCs that were subjected to serum deprivation. Serum deprivation is a widely used model of starvation that could initiate the intrinsic apoptotic pathway . In the present study, we confirmed that serum deprivation could result in apoptosis and exacerbate cell death as starvation time increased. It is demonstrated that CHBP could limit apoptosis of MSCs in a concentration-dependent manner. To detect whether this protection is a by-product effect of peptide molecule itself, a scrambled peptide is used as a control. No detectable protection was recognized for the scrambled peptide. Although erythropoietin and its deviants have been shown cytoprotection in the background of starvation, the underlying mechanisms are not fully elucidated [35–38].
Mitochondria, in addition to the well-known energy-producing function, also serve as a crucial mediator of cell injury and death [39, 40]. Physiologically, the homeostasis of mitochondria is finely orchestrated by a complex network consisting of intracellular proteins and microenvironments. Upon severe stresses, such homeostasis can be dramatically damaged, leading to mitochondrial dysfunction and consequent cell death. Consistent with previous studies [28–30, 41], out data showed that serum deprivation induced mitochondrial dysfunction, resulting in increased ROS, impaired MMP and release of cytochrome c. ROS is an inevitable product of the respiratory chain during oxidative phosphorylation. Pathological stimuli such as nutrient starvation cause a functional disruption of mitochondria and consequently result in excessive ROS generation, which induced undesirable oxidative damage to DNA, proteins, and organelles and even lead to cell death [42–44]. Simultaneously, as the main source of ROS, mitochondria itself acts as a preferable target for the deleterious effect of ROS. Oxidative stress in turn deteriorates mitochondrial dysfunction and amplifies mitochondrial collapse in a positive feedback loop. In contrast, CHBP could alleviate mitochondrial stress by diminishing oxidative injury of ROS, restoring MMP and maintaining mitochondrial membrane integrity.
Our results further indicated that antioxidant Nrf2/Sirt3/FoxO3a axis might involve in the protection of CHBP against serum deprivation. Nrf2, also known as GA-binding protein, is a transcription factor belonging to E26 transformation-specific factor family . Nrf2 interacts with PGC-1α to regulate key functional signaling within mitochondria and actives the expression of many cytoprotective proteins including Sirt3 [46–48]. Previous studies have shown that erythropoietin could induce Nrf2 expression [49–51]. Accordingly, we found that CHBP was also able to upregulate Nrf2 as well as Sirt3 in the background of starvation. Very recently, increasing evidence demonstrated that Sirt3/FoxO3a pathway served as an important contributor to the mitochondrial protection in response to cellular stresses [52–54]. Sirt3 is a mitochondrial deacetylase involving in several important metabolic processes [55, 56]. The Sirt3-mediated deacetylation of FoxO3a further reduces FoxO3a phosphorylation . The function of FoxO3a is critically modulated by phosphorylation: phosphorylated FoxO3a retains in the cytoplasm and keeps functionally inactive; in contrast, dephosphorylation leads to its transfer to the nucleus to activate target genes . Our results provided direct evidence for the activation of FoxO3a by Sirt3 and revealed that Sirt3 knockdown partially abolished the protection of CHBP.
Scientific community has paid much attention to improve the viability of MSCs in response to nutrient deprivation. Treatment of various reagents is considered as a feasible procedure to protect MSCs from starvation. Among them, there are hormone, endogenous substance, clinically approved drug, peptide and energy resource ATP [57–62]. Neuropeptide substance P (SP) is a peptide containing 11 amino acids and has been suggested to inhibit starvation-induced apoptosis by enhancing Wnt pathway . Moreover, another study shows that the glucose-dependent insulinotropic peptide (GIP) exerts an anti-apoptotic function by regulating adenylate cyclase . These studies suggested that peptides serve as effective adjuvant agents for improving MSC-based therapies. As a peptide, CHBP is relatively low-cost, feasible for large-scale production and stable under various pathophysiologic conditions. In addition, no obvious effect of CHBP is observed upon the osteogenic and adipogenic differentiation, as well as the self-renewal capacity that are crucial hallmarks of MSCs. In this regard, CHBP shows a promising prospect in the clinical practice.
In conclusion, the present study demonstrates the efficient protection of CHBP upon MSCs against starvation-induced apoptosis and unveils the underlying mechanism of this protective effect. These results provide a better understanding of the multiple pharmacologic functions of CHBP and indicate a possible role of CHBP in regulating nutrient-associated biological processes. Our study suggests that CHBP holds great promise for sustaining cell survival under nutrient-deprived conditions and improving the therapeutic effectiveness of MSC-based treatment. However, the efficacy of CHBP-treated MSCs needs further confirmation in vivo in the future.
cyclic helix B peptide
Dulbecco’s modified Eagle’s medium
mitochondrial membrane potential
mesenchymal stem cell
reactive oxygen species
nuclear factor erythroid 2-related factor 2
small interfering RNA
CY and TZ conceived and designed the study and analyzed the data. SW performed cell proliferation and apoptosis assay, conducted siRNA infection and wrote the draft. CZ performed mitochondria-related experiments and revised the manuscript. SN performed western blotting and revised the manuscript. LZ and JL performed differentiation and CFU assay. MX and RR supported the study. WZ revised the manuscript. All authors read and approved the final manuscript.
Availability of data and materials
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.
The authors declare that they have no competing interests.
This study was supported by National Natural Science Foundation of China (Grants 81400752 to CY, 81270832 to RR, 81370852 to MX, 81270833, 81570674 to TZ).
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