Curcumin Alleviates the Senescence of Canine Bone Marrow Mesenchymal Stem Cells during In Vitro Expansion by Activating the Autophagy Pathway

Abstract

Senescence in mesenchymal stem cells (MSCs) not only hinders the application of MSCs in regenerative medicine but is also closely correlated with biological aging and the development of degenerative diseases. In this study, we investigated the anti-aging effects of curcumin (Cur) on canine bone marrow-derived MSCs (cBMSCs), and further elucidated the potential mechanism of action based on the modulation of autophagy. cBMSCs were expanded in vitro with standard procedures to construct a cell model of premature senescence. Our evidence indicates that compared with the third passage of cBMSCs, many typical senescence-associated phenotypes were observed in the sixth passage of cBMSCs. Cur treatment can improve cBMSC survival and retard cBMSC senescence according to observations that Cur (1 μM) treatment can improve the colony-forming unit-fibroblasts (CFU-Fs) efficiency and upregulated the mRNA expression of pluripotent transcription factors (SOX-2 and Nanog), as well as inhibiting the senescence-associated beta-galactosidase (SA-β-gal) activities and mRNA expression of the senescence-related markers (p16 and p21) and pro-inflammatory molecules (tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6)). Furthermore, Cur (0.1 μM~10 μM) was observed to increase autophagic activity, as identified by upregulation of microtubule-associated protein 1 light chain 3 (LC3), unc51-like autophagy-activating kinase-1 (ULK1), autophagy-related gene (Atg) 7 and Atg12, and the generation of type II of light chain 3 (LC3-II), thereby increasing autophagic vacuoles and acidic vesicular organelles, as well as causing a significant decrease in the p62 protein level. Moreover, the autophagy activator rapamycin (RAP) and Cur were found to partially ameliorate the senescent features of cBMSCs, while the autophagy inhibitor 3-methyladenine (3-MA) was shown to aggravate cBMSCs senescence and Cur treatment was able to restore the suppressed autophagy and counteract 3-MA-induced cBMSC senescence. Hence, our study highlights the important role of Cur-induced autophagy and its effects for ameliorating cBMSC senescence and provides new insight for delaying senescence and improving the therapeutic potential of MSCs.

Keywords: autophagy; canine bone marrow-derived mesenchymal stem cells; curcumin; senescence.

Figure 1

Characteristics of MSCs isolated from canine bone marrow. (A) The morphology of third-generation canine bone marrow-derived mesenchymal stem cells (cBMSCs) cultured at 48 h was observed using a light microscope; scale bars = 100 μm. (B) Alizarin Red S stained calcium deposition; scale bars = 100 μm. (C) Oil Red O staining was used to observe the lipid droplet accumulation; scale bars = 100 μm. (D) The 3rd passage cBMSCs were stained with FITC, PE, or APC fluorescent-labeled antibodies and analyzed using flow cytometry.

Figure 2

Senescence characteristics of cBMSCs after expansion in vitro. (A) Cell morphology of cBMSCs (P1, P3, P6, and P9) cultured in vitro for 24 h, 48 h, and 72 h; scale bars = 100 μm. (B) Growth curve of cBMSCs (P3, P6, and P9) was determined by cell counting. The proliferation capacity of cBMSCs was gradually decreased over the process of long-term cultivation and they underwent growth arrest at P9. (C) CFU-F assays of cBMSCs at P3, P6, and P9. The number of colonies was estimated after culturing in vitro for 14 d. (D) Representative image of SA-β-gal staining and percentage of SA-β-gal positive cells in cBMSCs at P3, P6, and P9. The expression of senescence-related genes of p21 and p16 (E), pro-inflammatory cytokine genes TNF-α and IL-6 (F), and stemness markers Nanog and SOX-2 (G) measured by RT-qPCR. Gene expression was normalized relative to the expression of GAPDH. These values are the mean ± SD of triplicate experiments. ** p < 0.01 compared with P3. ^# p < 0.05, ^## p < 0.01 for intragroup comparisons.

Figure 3

Effects of Cur on senescent cBMSCs (P6). (A) Effect of Cur on the viability of cBMSCs. Effects of different doses (0.1, 0.5, 1, 5, and 10 μmol/L) and time points (12 h, 24 h, 48 h, and 72 h) of the responses of Cur on the viability of P6 cBMSCs were evaluated using a CCK-8 assay. (B) SA-β-gal expression levels in the P6 cBMSCs in the absence (control) or presence of Cur (0.1, 1, and 10 μmol/L) for 24 h; scale bars = 100 μm. (C) The rate of colony formation in CFU-F after treatment with Cur for 24 h. The expression of senescence-related genes of p21 and p16 (D), pro-inflammatory cytokines genes TNF-α and IL-6 (E), and stemness markers Nanog and SOX-2 (F), as measured by RT-qPCR. The values are the means ± SDs of triplicate experiments. Gene expression was normalized relative to the expression of the GAPDH. * p < 0.05, ** p < 0.01 compared with the control group. ^# p < 0.05, ^## p < 0.01 for intragroup comparisons.

Figure 4

Effects of Cur on the autophagy of cBMSCs (P6). (A) Immunoblots showing the protein expression levels of p62, LC3-I, and LC3-II in BMSCs treated or untreated Cur (0.1, 1, and 10 μM), β-actin was used as the loading control. (B) The relative expression of LC3, ATG12, ATG7, and ULK1 in cBMSCs were analyzed using RT-qPCR. (C) The ultrastructure of cBMSCs with or without Cur treatment was examined by transmission electron microscopy. The red arrows show autophagosomes and autolysosomes; scale bars = 2 μm. (D) cBMSCs were treated or untreated with Cur (0.1, 1, and 10 μM) and then stained with LysoTracker Red DND-99 (60 nM). The relative fluorescence intensity at the intracellular level was quantified using the Image Pro Plus software; scale bars = 50 μm. (E) Representative immunofluorescence images show LC3 punctae (red) in BMSCs; scale bars = 20 μm. * p < 0.05, ** p < 0.01 compared with the control group. ^# p < 0.05, ^## p < 0.01 for intragroup comparisons.

Figure 5

The effects of modulated autophagy through the employment of RAP (100 nM) and 3-MA (5 mM) in cBMSCs (P6). (A) Immunoblots showing the protein expression levels of p62, LC3-I, and LC3-II in cBMSCs, β-actin was used as the loading control. (B) The relative expressions of LC3, ATG12, ATG7, and ULK1 in cBMSCs were analyzed using RT-qPCR. (C) cBMSCs were treated or untreated with Cur (0.1, 1, and 10 μM) and then stained with LysoTracker Red DND-99 (60 nM); scale bars = 50 μm. (D) Representative immunofluorescence images showing LC3 punctae (red) in cBMSCs; scale bars = 50 μm. (E) The ultrastructure of cBMSCs was examined with transmission electron microscopy. The red arrows show autophagosomes and autolysosomes; scale bars = 500 nm. * p < 0.05, ** p < 0.01 compared with the control group; ^Δ p < 0.05, ^ΔΔ p < 0.01 compared with the Cur group; ^# p < 0.05, ^## p < 0.01 for intragroup comparisons.

Figure 6

The effects of the modulation of autophagy through the employment of RAP (100 nM) and 3-MA (5 mM) on cBMSC (P6) senescence. (A) SA-β-gal expression levels in cBMSCs; scale bars = 100 μm (B) The rate of colony formation in CFU-F. The expression of senescence-related genes p21 and p16 (C), pro-inflammatory cytokine genes TNF-α and IL-6 (D), and stemness markers Nanog and SOX-2 (E), as measured by RT-qPCR. (F) The potential relationship between Cur-induced autophagy and its effects on cBMSC senescence. The values are the means ± SDs of triplicate experiments; * p < 0.05, ** p < 0.01 compared with the control group; ^Δ p < 0.05, ^ΔΔ p < 0.01 compared with the Cur group; ^# p < 0.05, ^## p < 0.01 for intragroup comparisons.

Figure 7

Schematic diagram of the replicative senescence of cBMSCs and the potential mechanisms by which Cur delays cBMSC senescence. cBMSCs (P6) inevitably acquire a senescent phenotype after in vitro expansion, such as a hypertrophic and flat morphology, the activation of cell cycle kinase inhibitors p16 and p21, a decreased expression of pluripotent transcription factors (SOX-2 and Nanog), an enhanced secretion of pro-inflammatory molecules (IL-6 and TNF-α), and an enhanced activity of SA-β-gal and intraluminal pH in lysosomes. Treatment with curcumin can delay the course of cBMSC senescence, while enhancing autophagic activity and promoting lysosomal acidification. All in all, Cur-induced autophagy is a potential molecular mechanism for ameliorating cBMSC senescence.