Abstract

   The senescence of mesenchymal stem cells (MSCs) impairs their
   regenerative capacity to maintain tissue homeostasis. Numerous studies
   are focusing on the interventions and mechanisms to attenuate the
   senescence of MSCs. C-phycocyanin (C-PC) is reported to have multiple
   functions such as antitumor, antioxidation, anti-inflammation and
   anti-aging roles, but there is little research about the effects of
   C-PC on the senescence of MSCs. Here we investigated the roles and
   mechanism of C-PC on MSCs senescence. In vitro results showed that C-PC
   could reduce senescence, enhance proliferation, promote the adipogenic
   and osteogenic differentiation in senescent MSCs induced by oxidative
   stress. In vivo D-Galactose (D-Gal) induced rats aging models showed
   C-PC also increased the viability and differentiation of intrinsic
   senescent bone marrow derived MSCs (BMSCs). Furthermore, C-PC also
   decreased the levels of oxidative stress markers ROS or MDA, elevated
   the SOD activity, and increased the anti-inflammatory factors.
   Proteomic chip analysis showed that C-PC interacted with ZDHHC5, and
   their interaction was verified by pull down assay. Overexpression of
   ZDHHC5 aggravated the senescence of MSCs and greatly lessened the
   beneficial effects of C-PC on senescence. In addition, we found ZDHHC5
   regulated autophagy by altering LC3, Beclin1 and PI3K/AKT/mTOR pathway.
   In summary, our data indicated that C-PC ameliorates the senescence of
   MSCs through zinc finger Asp-His-His-Cys (DHHC) domain-containing
   protein 5 (ZDHHC5) mediated autophagy via PI3K/AKT/mTOR pathway. The
   present study uncovered the key role of autophagy in MSCs senescence
   and PI3K/AKT/mTOR pathway may be a potential target for anti-senescence
   studies of MSCs.

   Keywords: MSCs, Senescence, C-PC, ZDHHC5, Autophagy

INTRODUCTION

   Aging is the biological process of gradual decline in physiological
   functions over time. The cellular and molecular hallmarks of aging
   process include genomic instability, telomere attrition, epigenetic
   alterations, loss of proteostasis, deregulated nutrient-sensing,
   mitochondrial dysfunction, cellular senescence, stem cell exhaustion,
   and altered intercellular communication [[67]1]. Cell senescence is the
   cellular basis of biological aging, which leads to the development and
   progression of aging-related diseases such as cancer, diabetes,
   cardiovascular disorders and neurodegenerative diseases [[68]2-[69]5].
   Nowadays the investigation and intervention of cell senescence have
   attracted more and more attention.

   The senescence and exhaustion of stem cells are considered important
   drivers of organism aging [[70]6]. Stem cell senescence is a cellular
   response to endogenous or exogenous stresses [[71]7]. Adult stem and
   progenitor cells are responsible for tissue maintenance, repair, and
   regeneration. During the process of aging, this population of cells is
   decreased, and their activities are reduced which compromise the tissue
   integrity and cause pathologies [[72]8]. Age-associated stem cell
   senescence has been confirmed as an important factor for the
   pathogenesis of multiple disorders. For example, hematopoietic stem
   cells (HSCs) gradually become senescent with aging, lose their
   self-renewal and regenerative potential, and weaken the adaptive immune
   system [[73]9]. BMSCs aging may contribute to aging-related
   osteoporosis [[74]10]. BMSCs extracted from osteoporosis patients
   acquired several abnormalities including reduced proliferation and
   osteogenic differentiation potential. Therefore BMSCs senescence is
   regarded as a major driver in the pathogenesis of age-related bone loss
   [[75]11].

   MSCs are tissue-specific progenitor cells with self-renewal and
   multi-potent differentiation potentials. They were found not only in
   bone marrows, but also in various tissues like adipose tissue, skeletal
   muscle, umbilical cord blood, placenta, and dental pulp. MSCs can
   differentiate into several different cell types including osteocytes,
   chondrocytes, adipocytes and so on [[76]7, [77]12]. MSCs can
   automatically migrate toward injury areas and spontaneously
   differentiate into desired tissues to perform regenerative functions
   [[78]12]. However, the regenerative capacity of MSCs to maintain tissue
   homeostasis declines progressively and the senescence of MSCs increases
   with aging [[79]13-[80]15]. Senescent MSCs not only hamper tissue
   repair but also mediate tissue degeneration by initiating and spreading
   senescence-associated inflammation [[81]7].

   In order to maintain MSCs from being senescent, pharmacological
   interventions are being explored [[82]1]. For example, ascorbic acid
   could inhibit the senescence of MSCs through ROS and Akt/mTOR signaling
   [[83]16]. Trans-cinnamaldehyde (TC) treatment reduced the senescence of
   the adipose derived MSCs (AMSCs) induced by H[2]O[2] and restored their
   functionality in vivo [[84]17]. Likewise, many other small molecular
   compounds such as quercetin, rapamycin, resveratrol and melatonin are
   reported to inhibit stem cell senescence [[85]18].

   C-PC is a natural active protein extracted from Spirulina platensis
   which is composed of α and β subunits. Recent studies showed that C-PC
   has pharmaceutical potentials in anticancer [[86]19], anti-inflammation
   [[87]20] and antioxidation [[88]21]. For example, Zhang et al. found
   that C-PC could elicit antitumor efficacy via causing cell-cycle arrest
   and apoptosis of esophageal squamous cell carcinoma [[89]22]. Ji et al.
   reported C-PC inhibited epithelial-to-mesenchymal transition (EMT) in
   caski cervical cancer cells [[90]23]. Besides, C-PC has been shown to
   be effective in improving tissue and organ inflammation including
   osteoarthritis, colitis etc. [[91]24, [92]25]. In particular, C-PC
   could serve as a promising reproductive system protective agent against
   low fertility by inhibiting ROS in D-Gal-induced aging mice [[93]26,
   [94]27] . Similarly, C-PC also prevents acute myocardial
   infarction-induced oxidative stress, inflammation, and cardiac damage
   [[95]28]. Whether C-PC could be applied against MSCs senescence is not
   known. Herein, we evaluated the effects of C-PC on oxidative
   stress-induced MSCs senescence in vitro and D-Gal-induced aging rat
   model in vivo. Moreover, we investigated the underlying molecular
   mechanism by using proteome chip and transcriptome sequencing. Our
   results suggested that C-PC could act as an anti-senescence agent via
   ZDHHC5 mediated PI3K/AKT/mTOR pathway to induce autophagy and finally
   prevent MSCs senescence induced by oxidative stress.

MATERIALS AND METHODS

Extraction, cultivation, and identification of primary MSCs

   Primary MSCs were obtained by separating adipose tissue discarded from
   normal people after liposuction as reported previously[[96]29]. All
   experiments were approved by the Qingdao University Ethics Committee.
   After collagenase (Sigma Aldrich, USA) digestion, purified MSCs were
   cultured in DME/F-12 complete medium (HyClone, USA) supplemented with
   fetal bovine serum (ExCell Bio, Shanghai, China),
   penicillin-streptomycin (New cell& Molecular Biotech, Suzhou, China)and
   incubated in a humid incubator at 37°C and 5% CO[2]. At the fourth
   passage, MSCs were collected and incubated with different phenotypic
   antibodies at 4°C for 30min (For intracellular molecules, MSCs were
   fixed with 4% paraformaldehyde at room temperature, and then ruptured
   with 0.1% Triton X-100 solution). The manufacturer and catalog of
   antibodies we used were listed in [97]Supplement Table. After
   incubation, the cells were washed and re-suspended. Analysis was
   performed on Accuri C6 Flow Cytometers with CFlow software (Accuri
   Cytometers, Inc.).

Cell Counting Kit-8 Assay

   Cells were seeded in 96-well plates (5000/well) and cultured with
   different concentrations of hydrogen peroxide solution reagent (3%)
   (Sigma Aldrich, USA) and C-PC (Binmei Biotechnology, Taizhou, China)
   for appropriate time. After incubation, the drugs were removed and then
   100 μl of CCK-8 mixture (1:10) (Biosharp, Beijing, China) was added.
   The cells were incubated in a cell incubator for an additional 2 h and
   the absorbance at 450 nm was measured.

Senescence β-Galactosidase Stain

   Cells were cultured in 6-well plates and washed with PBS after drug
   treatment. Then 1 ml of β-galactosidase staining fixative (Solarbio,
   Beijing, China) was added for 15 min at room temperature. After removal
   of the fixative, the cells were washed 3 times with Phosphate Buffered
   Saline (PBS) (Biosharp, Beijing, China) for 3 min each. PBS was
   aspirated and 1 ml of staining working solution was added to each well.
   Preparation method of staining solution: 10 μl of β-galactosidase
   staining solution A, 10 μl of β-galactosidase staining solution B, 930
   μl of β-galactosidase staining solution C and 50 μl of X-Gal solution.
   Cells are incubated overnight at 37°C and 6-well plates were covered
   with plastic wrap to prevent evaporation. Cells were observed and
   counted under a common optical microscope. Green indicates senescent
   cells. The percentage of senescence (%) was calculated by counting the
   number of stained cells and dividing them into the total number of
   cells.

Reactive Oxygen Species Assay

   The fluorescent probe DCFH-DA (1:1000) (Beyotime Biotechnology,
   Beijing, China) was diluted with serum-free medium to a final
   concentration of 10 μM. The original liquid in each well of the
   six-well plate was removed and 1ml of DCFH-DA diluent was added. The
   incubation lasted for 20min in a 37ºC cell incubator. Then the cells
   were washed three times with serum-free cell culture medium to
   sufficiently remove DCFH-DA that did not enter the cells. Finally, the
   cells were observed under a fluorescence microscope.

SOD activity assay

   SOD activity was determined by xanthine oxidase method (hydroxylamine).
   MSCs were lysed by RIPA lysis buffer and the supernatant was collected
   by centrifugation. The samples were prepared according to the
   manufacturer’s instructions of SOD Test Kit (Nanjing Jiancheng,
   Bioengineering Institute, Nanjing, China). The maximum absorbance at
   550 nm was measured to calculate the SOD activity [[98]30].

MDA assay

   The concentration of MDA was used to indicate lipid peroxidation. BMSCs
   were lysed by RIPA lysis buffer and the supernatant was collected by
   centrifugation. The samples were prepared according to the
   manufacturer’s instructions of MDA Test Kit (Nanjing Jiancheng,
   Bioengineering Institute, Nanjing, China). The concentrations of MDA
   were calculated by measuring the maximal absorbance at 532 nm [[99]30].

Adipogenic differentiation

   The 4^th passage of AMSCs or BMSCs were seeded into 6-well plates and
   cultured to 80% confluence at 37°C, 5% CO[2] cell incubator. The
   adipogenic induction medium was composed of 100 μg/ml isobutyl
   methylxanthine, 1 μM dexamethasone, 50 μg/ml ascorbate acid, 100 IU
   penicillin, 100 μg/ml streptomycin (Sigma Aldrich, USA) and DMEM high
   glucose medium (HyClone, USA) containing 10% FBS. During the induction,
   the adipogenic induction medium was changed every 3 days. On the 12th
   day, total protein was extracted to detect the expression of PPAR-γ. In
   addition, lipid droplets were detected by Oil Red O staining.

Oil red O staining

   Cells were washed twice with PBS and fixed with 4% paraformaldehyde for
   10 min at room temperature. Then, the cells were washed three times
   with PBS and stained with filtered oil red O solution (Sigma-Aldrich,
   USA) (stock solution: 3 mg/ml in isopropanol; working solution: 60% oil
   red O stock solution and 40% distilled water) for 30 min at 37 °C.
   After staining, the cells were washed under flowing water for 1 min and
   then visualized and imaged by light microscopy.

Osteogenic differentiation

   The 4th passage of AMSCs or BMSCs were seeded into 6-well plates and
   cultured to 80% confluence at 37°C, 5% CO[2] cell incubator. The
   components of osteogenic differentiation induction medium included 10mM
   β-glycerophosphate sodium, 100nM dexamethasone, 50 µM ascorbate acid,
   100 IU penicillin, 100 μg/ml streptomycin (Sigma Aldrich, USA) and DMEM
   high glucose medium (HyClone, USA) containing 10% FBS. The osteogenic
   induction medium was changed every 3 days. On the 12th day, proteins
   were extracted to detect the expression of RUNX-2. The calcium
   mineralization deposition was detected by Alizarin Red S staining.

Alizarin Red S staining

   Cells in culture were washed twice with PBS, fixed in 4%
   paraformaldehyde for 10 min, and then washed with distilled water and
   stained with 1% Alizarin red (Leagene, Beijing, China) for 30 min at
   room temperature. Then, cells were washed with distilled water to
   remove the unbound dye, and calcification was visualized as red
   deposits under light microscopy.

Proteome chip

   HuProt™ arrays covering approximately 20,000 full-length human proteins
   were purchased from CDI Laboratories Inc. (CDI, USA). Each protein was
   printed in duplicate onto Hydrogel Shot slides which was immobilized
   with GST tag antibody. To identify potential interacting proteins,
   purified C-PC proteins were probed to the HuProt arrays. Briefly, the
   chip was stored at -80 °C and transferred to -20 °C. Then it was placed
   at 4 °C for 30 min. Next it was immersed in a precooled blocking
   solution (5% BSA/1×PBS-T) in a dish on a shaker at 50 rpm and blocked
   for 5 min at room temperature. The blocking solution was replaced, and
   the blocking continued for 1.5 h. After a brief wash with 1×PBS-T for 5
   min, the chip was placed in a hybridization chamber containing C-PC
   proteins diluted in blocking buffer and the incubation lasted for 1 h
   at room temperature. After washing with 1×PBS-T solution and deionized
   water, the chip was dried by centrifugation at 1000 rpm for 2 min.
   Finally, the chip was scanned with a microarray scanner (GenePix
   4000B). All procedures were in dark and each experiment was performed
   in triplicates [[100]31].

GST pull down assay

   GST-V, GST-C-PC α, GST-C-PC β proteins were expressed and purified as
   reported previously and adjusted to 5 μg/μl as the final concentration
   [[101]32, [102]33]. AMSCs (3×10^7) cells were lysed with 2ml RIPA lysis
   buffer, and 200 μl was used as input. The remaining lysates were
   incubated with 30 μg of GST-V beads at 4°C for 1 h. After
   co-incubating, the samples were divided into 3 equal volumes and
   incubated overnight with 30 μg of GST-V, GST-α, GST-β beads (Sangon
   Biotech,China) on a shaker at 4°C. Then, the samples were centrifuged
   at 2000 rpm, 4 °C for 2 min. The supernatant was discarded, and the
   pre-prepared magnetic bead wash was used for gradient washing. Finally,
   the proteins were further detected by western blot.

ZDHHC5 overexpression

   AMSCs were transfected with ZDHHC5 expressing lentivirus using HiTransG
   A (Shanghai Genechem, Shanghai, China) for 72 h. Following infection,
   the cells were selected with puromycin (10 μg/ml) (Sigma-Aldrich, USA)
   for 24 h. The overexpression efficiency was detected under fluorescence
   microscope or by western blot. The cells were further treated with C-PC
   for 24 h and were divided into the following four groups: Vector group,
   Vector+C-PC group, ZDHHC5 group and ZDHHC5+C-PC group for functional
   experiments.

Western blot analysis

   After lysis with RIPA (Epizyme, Shanghai, China), the proteins were
   extracted and centrifuged. The supernatants were collected, and the
   total protein concentrations were measured using BCA (Epizyme,
   Shanghai, China) assay. Proteins were separated using SDS-PAGE
   (Epizyme, Shanghai, China) and transferred to PVDF membranes
   (Millipore, Germany) and blocked with protein free rapid blocking
   buffer for 1 h. The membranes were immunoblotted with the target
   primary antibodies at 4°C overnight. After washing with TBST, the
   membranes were treated with HRP-conjugated secondary antibody at room
   temperature for 1 h. Bands were visualized using chemiluminescence, and
   gray value analysis of the bands was performed using Image J software.
   The manufacturer and catalog of antibodies we used were listed in the
   [103]Supplementary Table 1.

Transcriptome sequencing

   Total RNA was extracted using TRIzol reagent (New cell& Molecular
   Biotech, Suzhou, China)according to the manufacturer’s protocol. RNA
   purity and quantification were evaluated using NanoDrop 2000
   spectrophotometer (Thermo Scientific, USA). RNA integrity was assessed
   using the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara,
   CA, USA). Then the libraries were constructed using TruSeq Stranded
   mRNA LT Sample Prep Kit (Illumina, San Diego, CA, USA) according to the
   manufacturer’s instructions. The transcriptome sequencing and analysis
   were conducted by OE Biotech Co. Ltd. in Shanghai, China. The libraries
   were sequenced on an Illumina HiSeq X Ten platform and 150 bp
   paired-end reads were generated for subsequent differentially expressed
   genes analysis.

D-Gal-induced accelerated aging rat model

   30 SD male rats (8 weeks old) (Charles River, Jinan, China) were used
   for animal experiments. 10 of them were injected with saline as the
   control group and 20 were subcutaneously injected with D-Gal (250
   mg/kg) (Aladdin, Shanghai, China) for 40 days. After the establishment
   of the animal aging model, 10 mice were given saline by gavage as the
   D-Gal group. The remaining 10 were given C-PC (100 mg/kg) (Binmei
   Biotechnology, Taizhou, China) for 30 days as the D-Gal +C-PC group.
   Finally, the rats were sacrificed. BMSCs were extracted from bone
   marrows as reported previously [[104]34]. The animal study was reviewed
   and approved by the Ethics Committee of Qingdao University.

Enzyme-linked immunosorbent assay

   Enzyme-linked immunosorbent assay (ELISA) kits (FANKEWEI, Shanghai,
   China) were used to detect the levels of interleukin-10 (IL-6) and
   transforming growth factor β (TGF-β) in MSCs. Samples were collected
   and tested according to the manufacturer's instructions. Analysis was
   performed within 15 min after adding the stop solution.

Statistical analysis

   Statistical analyses were performed using SPSS Statistics (Version:
   25). All variables were described using mean ± standard deviation.
   Kruskal-Wallis test, a non-parametric alternative was used to calculate
   the comparisons between multiple groups. Further multiple comparisons
   were conducted by Bonferroni correction. The number of replicates and
   other statistical details were reported in the figure legends. P values
   less than 0.05 were considered statistically significant.

RESULTS

Isolation, characterization, and senescence model induction of MSCs

   MSCs were isolated from human adipose tissue as reported previously
   [[105]29]. Under microscope, MSCs show a spindle-shape, plastic
   adherent growth, and a vortex-like distribution ([106]Fig. 1A). The
   phenotypic molecular analysis by flow cytometry showed that MSCs were
   positive for CD73^+ and CD90^+ expression with a positive rate above
   95%. The expression of CD14^– and CD34^– in MSCs was negative with a
   positive rate below 3% ([107]Fig. 1B). To investigate the roles of C-PC
   on MSCs senescence, we established a H[2]O[2]-induced MSCs senescence
   model. First, MSCs were exposed to different concentrations of H[2]O[2]
   (0, 50, 100, 150 and 200 μM) for 2 h. The cell activity of MSCs
   decreased gradually with the increase of H[2]O[2] concentration.
   Statistical results showed that 150 μM was the half-maximal inhibitory
   concentration (IC50) of H[2]O[2] on MSCs ([108]Fig. 1C). Next, several
   well-established senescence biomarkers were analyzed in these cells.
   Compared with the control group, the percentage of
   senescence-associated β-galactosidase (SA-β-gal) positive cells were
   significantly increased after exposure to oxidative stress. And the
   senescence rate of MSCs showed positive correlation to the
   concentration of H[2]O[2] ([109]Fig. 1D). The protein level of
   senescence marker, SIRT1 was decreased while the protein levels of P53,
   P21 and P16^INK4a were increased in a H[2]O[2]-concentration dependent
   manner ([110]Fig. 1E). In conclusion, H[2]O[2] can induce the
   senescence of MSCs and 150 μM was used as the optimal concentration for
   subsequent experiments.

Figure 1.

   [111]Figure 1.
   [112]Open in a new tab

   The effects of H[2]O[2] on Morphology, phenotype and senescence
   analysis of MSCs. (A) The morphology of the 4th passage of MSCs. Scale
   bars, 40 μm. (B) Flow cytometry analysis of MSCs phenotype including
   CD73, CD90, CD14 and CD34. (C) Cell Counting Kit-8 (CCK-8) assay to
   detect the cell viability of MSCs in different groups. (D)
   Representative images of senescence-associated β-Galactosidase Staining
   and the quantification analysis of the percentage of senescence MSCs.
   Scale bars, 30 μm. (E) Western blot analysis of senescence related P53,
   P21, P16^INK4a and SIRT1 in MSCs and the quantification of western
   blot. Protein levels were normalized to β-actin. All results were
   presented as the mean ± SEM (n = 3). The P values were determined by
   Kruskal-Wallis test.

C-PC inhibits senescence of MSCs in vitro

   To detect the effect of C-PC on the senescence of MSCs, 250 μg/ml C-PC
   was added to the culture medium of senescent MSCs for 24h. Afterwards,
   the proliferation of MSCs was examined. The results showed that the
   proliferative activity of MSCs was increased after C-PC treatment. The
   statistical results showed that C-PC could restore the proliferation
   activity of senescent MSCs to a certain extent ([113]Fig. 2A). The
   senescence rate of MSCs was markedly reduced as shown by SA-β-gal
   staining indicating that C-PC could improve the senescence of MSCs
   ([114]Fig. 2B). Compared with the senescence group, C-PC group also
   increased the expression of SIRT1 and decreased the expression of P53,
   P21 and P16^INK4a ([115]Fig. 2C). Since C-PC was reported to have
   anti-oxidation roles, the levels of ROS and SOD activities were
   detected. As expected, C-PC reduced the ROS levels ([116]Fig. 2D) and
   increased SOD activities in senescent MSCs ([117]Fig. 2E). These
   results indicate that C-PC has antioxidant capacity to repair
   H[2]O[2]-induced oxidative stress damage. Next, we examined the effects
   of C-PC on MSCs functions. MSCs were differentiated with adipogenic or
   osteogenic induction media, respectively. As shown by Oil Red O
   staining, more lipid droplets were present in C-PC-treated MSCs than
   senescent MSCs ([118]Fig. 2F). Besides, the protein level of peroxisome
   proliferator-activated receptor γ (PPAR-γ), a specific transcription
   factor for adipocyte differentiation was also increased ([119]Fig. 2G).
   Similarly, more calcium deposits were detected in C-PC-treated MSCs
   than senescent MSCs by Alizarin Red S staining ([120]Fig. 2H). C-PC
   treatment also elevated the level of osteogenic transcription factor,
   RUNX-2 ([121]Fig. 2I). These results showed that C-PC could enhance the
   adipogenic and osteogenic differentiation potential of MSCs. In
   summary, C-PC inhibited the senescence of MSCs in vitro.

Figure 2.

   [122]Figure 2.
   [123]Open in a new tab

   C-PC inhibits senescence of MSCs in vitro. (A) CCK-8 assay to show the
   proliferation ability of senescent MSCs after C-PC treatment. (B)
   Representative images of β-Galactosidase staining and the
   quantification analysis of the percentage of senescence MSCs. Scale
   bars, 30 μm. (C) Western blot analysis of senescence related P53, P21,
   P16^INK4a and SIRT1 in MSCs and the quantification analysis of western
   blot. Protein levels were normalized to β-actin. (D) ROS staining of
   control, senescent and C-PC treated MSCs. Scale bars, 40 μm. (E) SOD
   activity assay of control, senescent and C-PC treated MSCs. (F)
   Representative images of Oil Red O staining and the quantification
   analysis of Oil Red O staining by Image-J software. Scale bars, 20 μm.
   (G) Western blot was performed to analyze the protein levels of
   adipogenic differentiation marker after treatment with C-PC. The
   quantification analysis of western blot was carried out to show the
   changes. Protein levels were normalized to β-actin. (H) Representative
   images of Alizarin Red S staining and the quantification analysis of
   Alizarin Red S staining by Image-J software. The red dots indicate the
   calcium deposits. Osteogenic differentiation potential was evaluated
   using calcium nodules. Scale bars, 20 μm. (I) The protein level of
   RUNX2, an osteogenic differentiation marker was analysed by Western
   blot. The quantification analysis of western blot was also performed to
   show the changes. Protein levels were normalized to β-actin. All
   results were expressed as the mean ± SEM (n = 3). The P values were
   determined by Kruskal-Wallis test.

C-PC attenuates MSC senescence by interfering with ZDHHC5-mediated autophagy

   To further illustrate the molecular mechanisms of C-PC in senescent
   MSCs, we performed a human proteome chip assay to screen the candidate
   proteins interacting with C-PC. Gene Ontology (GO) functional
   enrichment analysis showed the interacting candidate proteins were
   especially enriched in regulation of mRNA metabolic process and the
   cellular component such as microtube ([124]Fig. 3A). Among all these
   candidates, ZDHHC5, a member of palmitoyltransferase family, was among
   the top hits with strong specificity ([125]Fig. 3B). To verify the
   interaction between C-PC and ZDHHC5 and find the specific binding
   sites, the α and β subunit of C-PC were expressed and purified as
   described previously [[126]19]. According to the results of pull-down
   assay, ZDHHC5 could bind to the α subunit of C-PC ([127]Fig. 3C).
   Interestingly, the protein level of ZDHHC5 in senescent MSCs was
   increased ([128]Fig. 3D). To assess the potential effect of ZDHHC5,
   MSCs were transfected with ZDHHC5 expressing-lentivirus. The senescence
   was aggravated in MSCs overexpressing ZDHHC5, which was shown by
   stronger SA-β-gal staining ([129]Fig. 3E). A decrease in the activities
   of SOD was also detected ([130]Fig. 3F). C-PC administration reversed
   all these results caused by ZDHHC5 overexpression. Since autophagy is
   involved in cell senescence, we wondered whether ZDHHC5 affected
   autophagy in MSCs. As expected, ZDHHC5 overexpression decreased the
   protein level of autophagy related Beclin1 and LC3. Autophagy related
   PI3K/AKT/mTOR signaling activation was also observed. What’s more, C-PC
   treatment reversed all the roles of ZDHHC5 in MSCs ([131]Fig. 3G).
   Besides, the autophagosomes were observed in C-PC treated MSCs
   ([132]Fig. 3H). The blockade of autophagy flux by bafilomycin A1
   (Baf-A1) (200 nM, 6 h) was shown by accumulated LC3-II. C-PC
   administration increased the autophagy flux in ZDHHC5-overexpressed
   MSCs ([133]Fig. 3I). These results showed that ZDHHC5 inhibited
   autophagy and caused senescence of MSCs. C-PC inhibited senescence of
   MSCs by reversing the roles of ZDHHC5.

Figure 3.

   [134]Figure 3.
   [135]Open in a new tab

   C-PC attenuates MSC senescence by interfering with ZDHHC5-mediated
   autophagy. (A) GO enrichment analysis showed that the differentially
   expressed genes were enriched in several biological processes. (B)
   Representative proteome chip results. Protein-protein interaction was
   detected (blue boxes) between C-PC and ZDHHC5 by red fluorescence.
   Representative images of protein chip showed positive control (red
   arrow), negative control (blue arrow) and ZDHHC5 spot (yellow arrow) on
   the enlarged images. (C) Pull down assay and the quantification
   analysis to show the interactions of α and β subunits of C-PC with
   ZDHHC5. (D) Western blot was performed to detect the protein levels of
   ZDHHC5 in different groups and the corresponding quantification
   analysis. β-actin was used as the internal control. (E) Representative
   images of β-Galactosidase staining and the quantification analysis of
   the percentage of senescence MSCs. Scale bars, 50 μm. (F) SOD activity
   assay of MSCs in different groups. (G)The protein levels of senescence,
   autophagy and PI3K/AKT/mTOR signaling pathway related proteins detected
   by Western blot and the corresponding quantification analysis. Protein
   levels were normalized to GAPDH. (H) Representative images of the
   observed autophagosomes by transmission electron microscope. One red
   arrow pointed to autophagosomes while two arrows pointed to
   autolysosome. Scale bars were shown in Figure. (I) Western blot
   analysis of the protein levels involved in autophagy and mTOR signaling
   after autophagy block by Baf-A1 (200 nM, 6 h) in MSCs. The
   quantification analysis of western blot was calculated by Image-J.
   Protein levels were normalized to GAPDH. Results were expressed as the
   mean ± SEM (n = 3). The P values were determined by Kruskal-Wallis
   test.

C-PC inhibited MSCs senescence by downregulating PI3K/AKT/mTOR pathway

   To further explore whether C-PC inhibits senescence by regulating
   autophagy and the underling signaling pathway, we systematically
   assessed the differentially expressed genes by transcriptome
   sequencing. The heat map analysis showed that there were significant
   gene expression differences between senescent MSCs group and the C-PC
   treated MSCs group ([136]Fig. 4A). A total of 399 differentially
   expressed mRNA (absolute fold-change≥1.5; P-value≤0.05) was detected.
   Among them, 46 mRNAs were upregulated in MSCs treated with C-PC
   compared to the senescent group, while 353 mRNAs were downregulated.
   Next GO functional enrichment analysis was performed on these
   differentially expressed genes. A total of 49 terms were enriched
   including 22 biological processes, 16 cellular components and 11
   molecular functions. Biological adhesion, biological regulation and
   cellular component organization were the top 3 enriched biological
   processes. Cell, cell junction and cell part were listed at the front
   of cellular components. Antioxidant activity, binding and catalytic
   activity were mostly enriched in molecular functions ([137]Fig. 4B).
   Meanwhile, KEGG pathway enrichment analysis disclosed 20 signaling
   pathways such as mTOR signaling pathway, longevity regulating pathway,
   autophagy-animal, regulating pluripotency of stem cells and so on
   ([138]Fig. 4C). PI3K is involved in multiple signaling pathways such as
   mTOR signaling pathway and autophagy-animal cascade, it also plays a
   role in regulating stem cell pluripotency and longevity. Therefore, we
   speculated that C-PC might inhibit MSCs senescence by PI3K/AKT/mTOR
   signaling pathway. Indeed, the level of p-PI3K was increased in
   senescent MSCs which were inhibited by C-PC ([139]Fig. 4D). To further
   validate the regulatory mechanism of PI3K, normal MSCs were treated
   with PI3K activator, 740Y-P (20 μM) for 24 h. The senescence of MSCs
   was observed and C-PC attenuated the senescence caused by PI3K
   activation in MSCs ([140]Fig. 4E). The activity of SOD from MSCs was
   also improved by C-PC in 740Y-P activated MSCs ([141]Fig. 4F). Further
   research showed that C-PC reduced the levels of p-PI3K, p-AKT and
   p-mTOR, enhanced the autophagy level and therefore attenuated
   senescence of 740Y-P activated MSCs ([142]Fig. 4G). In addition, the
   number of autophagosomes was increased in C-PC treated groups
   ([143]Fig. 4H). Baf-A1 was used to block the autophagy flux. C-PC
   administration increased the autophagy flux in 740Y-P activated MSCs
   ([144]Fig. 4I). These results showed that activation of PI3K promoted
   senescence of MSCs and C-PC inhibited senescence by inhibiting the
   autophagy related PI3K/AKT/mTOR pathway.

Figure 4.

   [145]Figure 4.
   [146]Open in a new tab

   C-PC inhibited MSCs senescence by downregulating PI3K/AKT/mTOR pathway.
   (A) Transcriptome sequencing analysis. A heat map of differentially
   expressed genes in senescent and C-PC treated MSCs. (B) GO enrichment
   analysis to show the enriched biological processes of differentially
   expressed genes. (C) KEGG pathway analysis to show the signaling
   pathway which differentially expressed genes were enriched in. (D) The
   protein levels of p-PI3K by Western blot and the quantification
   analysis. Protein levels were normalized to β-actin. (E) Representative
   images of β-Galactosidase Staining and the quantification analysis by
   Image J software. Scale bars, 50μm. (F) SOD activity assay of MSCs in
   different groups. (G) Western blot and the quantification analysis of
   senescence, autophagy and PI3K/AKT/mTOR pathway related proteins.
   β-actin was used as the internal control. (H) Representative images of
   the observed autophagosomes by transmission electron microscope. One
   red arrow pointed to autophagosomes while two arrows pointed to
   autolysosome. Scale bars were shown in figure. (I) Western blot
   analysis of the protein levels involved in autophagy and mTOR signaling
   after autophagy block by Baf-A1 (200 nM, 6 h)in MSCs of different
   groups. β-actin was used as the loading control. Results were expressed
   as the mean ± SEM (n = 3). The P values were determined by
   Kruskal-Wallis test.

C-PC attenuated MSCs senescence in vivo

   The therapeutic effects of C-PC on oxidative stress-induced MSCs
   senescence in vitro prompt us to investigate their role in D-Galactose
   (D-Gal) -induced accelerated aging rat model in vivo. Briefly, rats
   were induced to aging with subcutaneous injection with D-Gal (250
   mg/kg) for 40 days, and then treated with C-PC (100 mg/kg) for 30 days
   by gavage [[147]35]. Since bone marrow is one important source of MSCs
   and aging impairs proliferation, induces senescence and chondrogenic
   response of BMSCs while it has no negative effects on AMSCs [[148]36] ,
   we chose BMSCs to validate the therapeutic effects of C-PC in vivo. The
   isolation and cultivation of BMSCs were carried out as reported
   previously [[149]37]. The morphology ([150]Fig. 5A) and immunological
   phenotypes ([151]Fig. 5B) validated its identity as BMSCs. We found
   that C-PC improved the viability ([152]Fig. 5C) and hindered the
   senescence rate of BMSCs in aging rats ([153]Fig. 5D). Compared with
   the aging group, C-PC administration increased the level of
   senescence-related protein SIRT1, while decreased the expression of
   P53, P21 and P16^INK4a in BMSCs ([154]Fig.5E). These results indicated
   that C-PC could delay the senescence of BMSCs in vivo. C-PC are
   reported to exert anti-oxidation and anti-inflammation roles in aging
   models. In order to better study the therapeutic effect of C-PC on
   BMSCs in aging rats, we also detected the level of oxidative stress
   marker MDA and SOD activity in BMSCs. C-PC decreased the level of MDA
   and enhanced SOD activity from BMSCs ([155]Fig. 5F). Besides, the
   levels of anti-inflammatory cytokines IL-10 and TGF-β were also
   elevated in BMSCs ([156]Fig. 5G). BMSCs were differentiated with
   adipogenic or osteogenic induction media, respectively. As shown by Oil
   Red O staining, more lipid droplets were present in BMSCs of
   C-PC-treated aging rats ([157]Fig. 5H). Besides, the protein level of
   PPAR-γ was also increased ([158]Fig. 5I). Similarly, more calcium
   deposits were detected in BMSCs of C-PC-treated aging rats by Alizarin
   Red S staining ([159]Fig. 5J). C-PC treatment also elevated the level
   of RUNX-2 ([160]Fig. 5K). Finally, the autophagy and PI3K/AKT/mTOR
   signaling pathway related proteins were measured in BMSCs from
   different groups. The results showed that C-PC treatment also promoted
   autophagy by inhibiting the autophagy related PI3K/AKT/mTOR signaling
   pathway ([161]Fig. 5L). The above data suggest C-PC could attenuate
   MSCs senescence in vivo.

Figure 5.

   [162]Figure 5.
   [163]Open in a new tab

   C-PC attenuated MSCs senescence in vivo. (A) The morphology of the 4th
   passage of rat BMSCs. Scale bars, 40 μm. (B) Flow cytometry analysis of
   rat BMSCs immuno-phenotype including CD44, CD90, CD14 and CD34. (C)
   CCK-8 assay was performed to detect the viability of MSCs in different
   groups. (D) Representative images of β-Galactosidase Staining and the
   quantification analysis of the percentage of senescent BMSCs. Scale
   bars, 20 μm. (E) Western blot analysis of senescence related P53, P21,
   P16^INK4a and SIRT1 in different groups. Protein levels were normalized
   to β-actin. (F) The content detection of MDA in BMSCs and SOD activity
   detection of BMSCs. (G) Enzyme-linked immunosorbent assay (ELISA) was
   carried out to detect the levels of interleukin-10 (IL-10) and
   transforming growth factor β (TGF-β) in BMSC. (H) Representative images
   of Oil Red O staining and the quantification analysis of Oil Red O
   staining by Image-J software. Scale bars, 40 μm. (I) The protein level
   of PPAR-γ,an adipogenic differentiation marker was detected by Western
   blot. The quantification analysis of western blot was carried out to
   show the changes. Protein levels were normalized to β-actin. (J)
   Representative images of Alizarin Red S staining and the quantification
   analysis of Alizarin Red S staining by Image-J software. The red dots
   indicate the calcium deposits. Osteogenic differentiation potential was
   evaluated using calcium nodules. Scale bars, 40 μm. (K) The protein
   level of RUNX2, an osteogenic differentiation marker was analysed by
   Western blot. The quantification analysis of western blot was also
   performed to show the changes. Protein levels were normalized to
   β-actin. (L) Western blot and quantification analysis of the protein
   levels involved in autophagy, PI3K/AKT/mTOR signaling. β-actin was used
   as a loading control. Results were expressed as the mean ± SEM (n = 3).
   The P values were determined by Kruskal-Wallis test.

DISCUSSION

   In this study, we aim to find a rational drug intervention to delay
   MSCs senescence. C-PC is a compound isolated from a variety of seaweeds
   such as Spirulina which is a well-known for its anti-aging effect
   [[164]38]. C-PC has been reported to have multiple functions such as
   antitumor, antioxidation, anti-inflammatory and anti-aging roles
   [[165]39]. So far, no research about the roles of C-PC on stem cell
   senescence has been conducted. Here, we systematically study the roles
   and mechanisms of C-PC on senescent MSCs. We demonstrated that C-PC
   could protect against H[2]O[2]-induced senescence of MSCs and improve
   their proliferation and differentiation. The therapeutic role of C-PC
   on preventing MSCs senescence was also observed in D-Gal-induced
   accelerated aging rat model in vivo. Our research is the first study,
   to our knowledge, to show that C-PC could inhibit MSCs senescence in
   vitro and in vivo.

   Oxidative stress model constructed by H[2]O[2] is often used in cell
   senescence research [[166]40] and the current best practice to identify
   senescent cells are used in our study including the altered expression
   of senescence markers, positive staining for SA-β-gal and cell function
   abnormities. The altered levels of several proteins such as SIRT1, P53,
   P21 and P16^INK4a are typical characteristics of senescent cells
   [[167]17, [168]41]. We also observed the increased expression of P53,
   P21 and P16^INK4a in our senescent MSCs model.

   Many researchers use D-galactose-induced aging animals as an
   experimental model for aging research [[169]16]. In this model, D-Gal
   is long-term supplied excessively which causes changes similar to
   natural aging [[170]5]. Naturally aging model is a time-consuming
   process while D-Gal-induced aging is more preferred due to its easy
   application, shorter duration of study and higher survival rate of
   animals throughout the experimental period. We also chose D-Gal-induced
   aging to study the potential therapeutic roles of C-PC on senescent
   MSCs in vivo. Since oral administration of health care products is
   prevalent nowadays and therefore, we tested the roles of C-PC in aging
   rats by gavage.

   MSCs are differentially influenced by aging. Some researchers found
   that BMSCs from aging animals had impaired proliferation, induced
   senescence and chondrogenic response, whereas muscle-derived stem cells
   and AMSCs exhibited no negative effects [[171]36]. Thus, we chose MSCs
   from bone marrow to check the roles of C-PC in vivo. Someone may wonder
   how C-PC attenuates MSCs senescence through gavage. C-PC can be
   digested into peptides and absorbed into blood by intestine. They may
   carry out their potential roles as biological active peptides in vivo.
   Besides, many studies found C-PC decreased the inflammatory factors in
   serum of aging animals. The level of MDA and the activity of SOD in
   serum were also improved by C-PC [[172]27]. The gut microbiota may also
   be involved in these inflammation diseases [[173]42]. Altogether, it’s
   a complex process to delay senility in vivo for C-PC which needs
   further research in future.

   Next, we focused on the mechanism of C-PC on MSCs senescence. We
   performed a human proteome chip assay to screen out the interacting
   proteins. Currently, the HuProt™ Human Proteome Chip is the highest
   throughput human protein chip, covering approximately 20,000
   full-length human proteins. ZDHHC5, a member of palmitoyltransferases
   family, was screened out by GO functional enrichment analysis and chip
   scanning data analysis. Molecular docking and other experiments are
   needed in future to find the specific interacting sites between C-PC
   and ZDHHC5. Besides, we found that C-PC could also down-regulate the
   expression of ZDHHC5 which also needs further work.

   ZDHHC5, one of 23 ZDHHC protein acyltransferase family members, was
   found to localize on the plasma membrane, endoplasmic reticulum, and
   the Golgi apparatus. ZDHHC5 could catalyze protein palmitoylation as a
   dynamic and reversible post-translational modification [[174]43,
   [175]44]. ZDHHC5 is highly expressed in a variety of embryonic cells
   including neural progenitor cells with an undifferentiated state, but
   is rapidly degraded during differentiation [[176]45]. It is also
   upregulated in glioma stem cells (GSC) and contributes to the tumor
   development and progression [[177]44, [178]45]. These results revealed
   that ZDHHC5 played important roles in stem cells. Therefor we chose
   ZDHHC5 for further study.

   Back to MSCs senescence, what’s the relationship between ZDHHC5 and
   MSCs senescence?How did ZDHHC5 affect MSCs senescence? To answer these
   questions, we overexpressed ZDHHC5 in MSCs. We noticed that ZDHHC5 was
   closely related to MSCs senescence and autophagy which might be
   involved in this process. Autophagy is a degradation mechanism that
   attenuates senescence of cells through degrading long-lived proteins,
   pathogens, damaged DNA elements, and damaged organelles. It is
   activated especially during metabolic stress conditions [[179]6]. Young
   quiescent MSCs have a constitutive autophagy activity that diminishes
   with aging. The impairment occurs in old cells which is related to the
   lack of autophagosome formation capacity [[180]46]. The
   microtubule-associated protein 1 light chain 3 (LC3) is a key regulator
   of autophagy. LC3 controls the growth of autophagic membranes, the
   recognition of autophagic cargoes and the fusion of autophagosomes with
   lysosomes in the autophagic pathway [[181]47].

   In order to demonstrate the molecular mechanisms, we performed
   transcriptome sequencing of C-PC treated MSCs and senescent MSCs
   meantime. Based on GO and KEGG pathway enrichment analysis, we selected
   the differentially expressed key protein PI3K and its mediated
   PI3K/AKT/mTOR autophagy pathway. Activation of PI3K/AKT/mTOR pathway
   not only contributed to the regulation of apoptosis, oxidative stress
   and autophagy but is also considered central to aging. mTOR activation
   in various tissues during aging is considered a major driving force
   [[182]48, [183]49]. Additionally, studies have also reported the
   regulatory role of ZDHHC5 in controlling target protein stability
   through the crosstalk with autophagy, and further revealed the
   association between S-palmitoylation-based autophagy [[184]50]. All
   these results and references proved that autophagy-related