A novel cytoprotective peptide protects mesenchymal stem cells against mitochondrial dysfunction and apoptosis induced by starvation via Nrf2/Sirt3/FoxO3a pathway

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.

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.

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.

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.

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.

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.

To examine the possible protection of CHBP, cell viability of nutrient-starved MSCs treated with different concentrations of CHBP (0, 0.1, 1, 10 nmol/L) was analyzed using CCK-8 assay. Figure 1a exhibited the dynamics of cell viability during starvation. Serum deprivation, just as expected, caused significant decrease of cell viability compared with normal control. CHBP were able to partially alleviate the decrease of viability in a dose-dependent manner, and the protective effect was observed for up to 72 h of starvation. We observed that starvation resulted in the detachment of cells, which usually represented cell death. Therefore, we further used Trypan blue staining to determine loss of cell membrane integrity and found that CHBP could obviously inhibit starvation-induced cell death (Fig. 1b). To exclude a possible protective effect of peptide molecule itself, a scrambled peptide was used and identified no detectable protection. To examine whether CHBP itself has an effect on the proliferation of MSCs, we subjected MSCs to CHBP at various concentrations in complete medium. As shown in Fig. 1c, CHBP had no detectable impact on the proliferation of MSCs at the concentration used in the present study. Collectively, our data suggested that CHBP could preserve cell viability against starvation by alleviating cell death.

To further delineate the effect of CHBP in starvation-induced cell death, apoptosis and necrosis was qualitatively analyzed using Annexin V/PI assay by flow cytometry. Annexin V−/PI− represents live cells; Annexin V+/PI− or Annexin V−/PI+ represents early or late apoptosis phase respectively; Annexin V+/PI+ reflects necrosis. As suggested in Figs. 2a, 3a, starvation induced significant apoptosis (Annexin V+) that exacerbated over time and CHBP, in contrast, markedly inhibited apoptosis in a concentration dependent manner. The highest concentration (10 nmol/L) was therefore applied for the following experiments. The protective effect of CHBP was also confirmed using Hoechst staining. Nuclear condensation observed in starved MSCs was obviously prevented by CHBP treatment (Fig. 3b). Taken together, these results indicated that CHBP attenuated starvation-induced apoptosis in MSCs.

Mitochondria are the main energy-producing organelles within cells and also play a key role in regulating cell bioactivity. Previous studies have shown that serum deprivation could result in mitochondrial dysfunction, which acted as an important contributor to apoptosis [28‐30]. Therefore, we explored whether CHBP were able to diminish mitochondrial stress. Mitochondria are the main source of intracellular ROS, which is an inevitable product of the respiratory chain during oxidative phosphorylation. Excessive ROS accumulate during pathological conditions and cause oxidative damage. Several studies indicated that ROS might serve as an initiator of the mitochondrial change [31, 32]. As shown in Fig. 4a, serum deprivation led to an obvious increase of ROS, which was significantly alleviated by CHBP. MMP is recognized as a marker of mitochondrial statement. Physiologically, JC-1 is prone to form aggregates that are fluorescent red depending on the polarization of MMP; in contrast, when MMP collapses, JC-1 disperses into monomers that are fluorescent green. Our data suggested that CHBP could help maintain a normal polarized MMP (Fig. 4b). Mitochondrial injury damages the integrity of mitochondrial membrane and leads to the leakage of cytochrome c, a promoter of inner apoptosis pathway. Normally, as shown in Fig. 4c, cytochrome c labeled with FITC colocalized with MitoRed-stained mitochondria. Under starvation, green fluorescence dramatically increased, indicating a release of cytochrome c from mitochondria. Consistent with the ROS generation and MMP results, CHBP inhibited mitochondrial membrane breakdown. However, CHBP did not prevented the decrease in starvation-associated mitochondrial mass (Fig. 4d).

Previous studies have shown that erythropoietin could upregulate nuclear factor erythroid 2-related factor 2 (Nrf2), which could further induce Sirt3 expression to exert protective effect. Therefore, we presumed that CHBP, as an erythropoietin deviant, might protect MSCs against starvation stress via Nrf2/Sirt3 related pathway. To confirm this hypothesis, we detect the expression of Nrf2, Sirt3, total FoxO3a, and p-FoxO3a by western blot. As shown in Fig. 5a, Nrf2 and Sirt3 were significantly upregulated by CHBP, while p-FoxO3a was suppressed. It is well accepted that FoxO3a plays a crucial role in mitochondrial protection and the phosphorylation of FoxO3a can prevent its activation [33]. To further determine the role of Sirt3/FoxO3a pathway in the effect of CHBP, Sirt3 siRNA was used. Compared with negative control (NC) siRNA-treated group, Sirt3 siRNA increased the level of p-FoxO3a and thereby inhibited the activation of FoxO3a. The NC siRNA were prone to downregulate p-FoxO3a, which, however, did not reach the statistic significance (Fig. 5a). Furthermore, we repeated the apoptosis assay by flow cytometry using Sirt3 siRNA-treated MSCs. As indicated in Fig. 5b, Sirt3 knockdown partially abolished the protection of CHBP, indicating a certain role of sirt3 in the protective effect of CHBP. Taken together, all these results suggested that Nrf2/Sirt3/FoxO3a pathway might participate in the protection of CHBP on MSCs against serum deprivation.

Pharmacological preconditioning is a well-accepted strategy to improve poor survival of transplanted cells. To investigate the potential protection of CHBP in a pretreatment manner, MSCs were pretreated with CHBP for 24 h and then subjected to serum-free medium. The results demonstrated that preconditioning of CHBP could also significantly inhibit apoptotic cell death in MSCs during serum deprivation (Fig. 6a, b). The protective effect was observed for up to 48 h of starvation, although the decrease of apoptosis at 48 h did not reach a statistical significance (P = 0.054). However, preconditioning MSCs with CHBP for 24 h failed to improve the viability at 72 h (data not shown).

Multipotential differentiation and self-renewal capacities are hallmarks to define a stem cell. MSCs, as adult stem cells, are characterized by their differentiations into osteoblasts and adipocytes. To test whether treatment of CHBP could affect these biological activities, differentiation and CFU assay were performed. As shown in Fig. 7a, b, preconditioning MSCs with CHBP at 10 nmol/L in normal medium for 3 days has no significant impact on the differential and self-renewal capability.