cAMP Agonist Forskolin Disrupts Mitochondrial Metabolism and Induces Senescence in Human Mesenchymal Cells

Backgroud: Adult-derived mesenchymal stem cells (MSCs) can be used in therapies for the treatment of various diseases. However, MSCs derived from aging tissues or long-term MSC cultures could have diminished therapeutic effects compared with MSCs derived from younger tissues, but the underlying mechanism has not been completely established. Dysfunction of energy metabolism is one of the main mechanisms underlying cell senescence. Although cyclic adenosine monophosphate (cAMP) is known to inhibit cell division and proliferation in vitro, its impact on MSC senescence has not been described. Methods: In this study, we used forskolin, an adenylate cyclase agonist and cAMP inducer, to disrupt metabolism in human adipose-derived MSCs and investigate the effects of metabolic dysfunction on MSC senescence. cAMP and ATP levels were identied by its ELISA kits. The identication of MSCs senescence was assessed by MTS assay, cell cycle tests, β-galactosidase staining, and genes evaluation through qRT-PCR and Western blotting. Functions of the forskolin treated-MSCs were evaluated by oil Red O staining and alkaline phosphatase staining, as well as mRNA tests and wound healing assay. Mitochondrial morphology and function tests were performed by transmission electron microscopy and oxygen consumption analysis, mitochondrial superoxide assay, quantication of nitric oxide. Results: Treatment of human MSCs with forskolin resulted in senescence phenotypes, including reduced proliferation, cell-cycle arrest, and enhanced expression of the cell aging markers P16 and P21. Furthermore, the senescent MSCs exhibited increased adipogenesis capacity and decreased osteogenesis capacity as well as a senescence-associated secretory phenotype characterized by increased expression of several inammatory factors. Forskolin-associated MSC senescence was mainly caused by oxidative stress–induced disruption of mitochondrial metabolism, and the senescent MSCs had high levels of reactive oxygen species and reduced sirtuin gene expression. Lastly, we found that cAMP inhibitor SQ22536 protects MSCs from forskolin-induced senescence and senescence-related-inammatory-phenotype (SASP). Conclusions: Our results indicate that forskolin can cause

Furthermore, the senescent MSCs exhibited increased adipogenesis capacity and decreased osteogenesis capacity as well as a senescence-associated secretory phenotype characterized by increased expression of several in ammatory factors. Forskolin-associated MSC senescence was mainly caused by oxidative stress-induced disruption of mitochondrial metabolism, and the senescent MSCs had high levels of reactive oxygen species and reduced sirtuin gene expression. Lastly, we found that cAMP inhibitor SQ22536 protects MSCs from forskolin-induced senescence and senescence-related-in ammatoryphenotype (SASP).
Conclusions: Our results indicate that forskolin can cause senescence of human MSCs through oxidative stress-induced mitochondrial metabolic dysfunction.

Background
Adult mesenchymal stem cells (MSCs) have multiple applications for the treatment of various diseases, mainly owing to their powerful immunomodulatory and regenerative features, paracrine effects, multilineage differentiation potential, and migration ability. Because the human body has relatively few MSCs, MSCs are typically expanded in vitro to obtain a su cient amount of cells for clinical applications. During culture in vitro, MSCs may undergo senescence owing to their limited capacity for continuous cell division [1][2][3][4][5]. Aged MSCs generally have relatively reduced e cacy compared with younger MSCs in various disease models. And the donor's age and/or health status may limit the therapeutic potential of the harvested MSCs [6,7]. This can be partly attributable to the fact that senescent MSCs may produce in ammatory cytokines, chemokines, and/or extracellular matrix-remodeling factors that can disrupt tissue structure and function.
Cyclic adenosine monophosphate (cAMP) is a second-messenger molecule that plays important roles in various cellular physiological and pathological processes including metabolism, cell differentiation, and in ammation [15]. Forskolin, a natural compound produced in the roots of the Indian plant Coleus forskohlii, directly activates adenylate cyclase, which generates cAMP from ATP, thereby increasing the intracellular concentration of cAMP [16]. As an adenylate cyclase agonist, forskolin inhibits cell proliferation, motility, colony formation, and migration and promotes apoptosis and improves the sensitivity of various types of tumor cells to chemotherapeutic drugs; therefore, forskolin has applications for the treatment of several cancers [17][18][19][20][21].
MSCs are present in almost every tissue and are essential for tissue longevity. Because cAMP is generated from ATP, we carried out experiments to determine the effects of the cAMP agonist forskolin on MSC senescence and studied the underlying mechanism.

Preparation and treatment of MSCs
MSCs were isolated from discarded adipose tissue removed from healthy patients undergoing liposuction surgery, as described [22]. Informed consent was obtained from all patients. The procedure was approved by the Ethics Committee of the Chinese Academy of Medical Sciences and Peking Union Medical College. The isolated cells were seeded in T75 asks containing 12
One hour before each time point, 20 µL MTS reagent was added into each well of the 96-well plate containing 100 µL culture medium. After incubation at 37°C for 1 h, absorbance at 490 nm was measured using a 96-well plate reader (Bio-Rad, Hercules, CA, USA).
Assessment of senescence using β-galactosidase staining A senescence β-Galactosidase staining kit (Beyotime) was used for staining senescent cells based on the upregulation of senescence-associated β-galactosidase activity during senescence. Culture medium was aspirated from 6-well plates, and the wells were washed once with phosphate-buffered saline (PBS). The cells were then xed at room temperature for 15 min with 1 mL β-galactosidase staining xative solution (from the kit) per well and then washed three times with PBS. Finally, 1 mL of working solution of the stain was added to each well, and the plates were incubated overnight at 37°C in a CO 2 -free incubator.
The cells were then observed and counted under an optical microscope.

Quanti cation of cAMP
The intracellular concentration of cAMP was determined with a cAMP ELISA kit (Elabscience, Wuhan, China), which uses a competitive ELISA method. Brie y, wells of a microtiter plate were coated with cAMP (the antigen), and the cAMP present in each cell extract or standard solution was allowed to compete with the immobilized cAMP for binding to a biotin-labeled monoclonal antibody against cAMP. Unbound components were then washed away. Horseradish peroxidase (HRP) conjugated avidin was then added, resulting in the formation of an immune complex; the solution in each well was aspirated, and the plates were washed three times with PBS. The chromogenic substrate TMB was then added, which turned the solution blue upon catalysis by HRP and then yellow after the stop solution was added. Finally, absorbance at 450 nm was measured with a microplate reader, and the cAMP concentration in each sample was calculated based on a standard curve.

Cell-cycle analysis
Cell-cycle phase was determined by ow cytometry using propidium iodide staining. MSCs were seeded into 6-well plates at a density of 2 × 10 5 cells per well and then treated with DMSO (vehicle control) or forskolin (60 µM nal concentration) for 24 h. Cells were then harvested and xed in 1 mL of 75% ethanol at 4°C for 24 h. The xed cells were then washed twice and resuspended in 50 µL PBS, followed by addition of 5 µL RNase A (10 mg/mL) and incubation at 37°C for 30 min. Next, 450 µL PBS with 10 µL propidium iodide (1 mg/mL) was added, and the samples were incubated at 4°C in the dark for 30 min.
Cells were detected with a ow cytometer (Accuri™ C6, Becton Dickinson, Franklin Lakes, NJ, USA), and the data were analyzed using CFlow Plus software (Becton Dickinson) and ModFit (Verity Software House, Topsham, ME, USA).

Transmission electron microscopy
MSCs were treated with DMSO (control) or forskolin and then xed in 2% glutaraldehyde solution in 0.1 M cacodylate buffer for 24 h, post-xed in 1% osmium tetroxide (OsO 4 ) for 1 h, and dehydrated in sequential dilutions of acetone (25%, 50%, 75%, and 100% twice) before impregnation in resin of increasing concentration (25%, 50%, 75%, and 100% three times) in acetone over a 24 h period. Each sample was subjected to transmission electron microscopy, and mitochondrial morphology was assessed and imaged at magni cations of 8000× and 10,000×.

Measurement of intracellular ATP content
Intracellular ATP content was measured with an ATP assay kit (Beyotime). Brie y, cells were lysed and then centrifuged at 12,000 ⋅ g for 10 min at 4°C. The supernatant was removed and mixed with dilution buffer from the kit, after which the relative light units were measured using a luminometer. A standard curve was prepared for each round of assays to calculate ATP content.

Measurement of intracellular reactive oxygen species (ROS)
Mitochondrial ROS formation was assayed using 2,7-dichloro uorescein diacetate, a uorescent probe, as part of a ROS assay kit (Beyotime) with a slight procedural modi cation. Brie y, MSCs were incubated with the probe (10 µM) dissolved in serum-free DMEM at 37°C for 20 min.

Oxygen consumption analysis
The Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) was used to measure oxygen consumption in MSCs. Forskolin-treated and control MSCs were plated onto 24 well XF cell culture microplates at a density of 50,000 cells/well in 250 µL culture medium on the day before analysis. XF assay medium was supplemented with 250 mM glucose and 2 mM sodium pyruvate. Hydrate a sensor cartridge in XF Calibrant at 37°C in a non-CO2 incubator overnight. After basal respiration measurements, cells were treated sequentially with oligomycin (1 mM) to measure the nonphosphorylating OCR, carbonyl cyanide-4-(tri uoromethoxy) phenylhydrazone (0.5 mM) to determine maximal OCR, and rotenone and antimycin A mixture (0.5 mM) to measure the residual mitochondrial OCR, and changes in respiration were recorded. Use the default software settings analyzed in Wave or XF software.
Wound healing assay Wound healing assays were performed using Culture-Insert two-well inserts (Ibidi, Munich, Bavaria, Germany). Brie y, the inserts were placed in 6-well plates prior to seeding the cells and were removed after the cells formed a con uent layer. The remaining medium was replaced with fresh serum-free medium, and images were acquired at the indicated time points. After 24 h, the distances traveled by cells were measured in forskolin-treated and non-treated MSCs.
Alkaline phosphatase staining Alkaline phosphatase (ALP) staining was performed using an ALP staining kit (Institute of Hematology RNA isolation and quantitative real-time polymerase chain reaction (qPCR) analysis Total RNA was extracted from cultured cells with TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and the RNA was quanti ed with a Model ND1000 spectrophotometer from NanoDrop Technologies (Wilmington, DE, USA). Reverse transcription was performed using a Reverse Transcription kit (Takara, Tokyo, Japan). qPCR was performed with SYBR premix Ex Taq (Takara) with the Step One Plus Real-Time PCR Detection System (Applied Biosystems, Foster City, CA, USA). The relative expression of mRNA was evaluated by the 2 −ΔΔCt method and normalized to the expression of GAPDH. Supplementary Table 1 lists the primers used in this study.

Western blotting
Cells were harvested in RIPA lysis buffer (Beyotime) supplemented with 1 mM phenylmethanesulfonyl uoride. Total protein was quanti ed using a BCA Protein Assay kit (Beyotime). Cell extracts (20 µg protein) were subjected to SDS-PAGE (10% acrylamide gels) and then transferred to a 0.45-µm polyvinylidene di uoride membrane (Millipore, Boston, MA, USA). After blocking with 5% fat-free milk in Tris-buffered saline Tween-20 (TBST) for 1 h at room temperature, each membrane was incubated with a speci c primary antibody at 4°C overnight. The next day, each membrane was incubated with a HRPconjugated secondary antibody for 1 h at room temperature. Immunopositive bands were visualized with HRP substrate (Proteintech, Rosemont, IL, USA) and quanti ed using an Image Quant LAS 4000 mini imaging system (GE Healthcare, Chicago, IL, USA). GAPDH was used as an internal control. Primary antibodies (rabbit IgG, from Proteintech) were used that targeted the following proteins: P16 (diluted 1:1000), P21 (1:1000), P53 (1:2000), and lamin B1 (1:2000).

Statistical analysis
Data were analyzed using Prism 8 software (GraphPad Software Inc. USA). The Student's t-test was used for comparisons between groups. P-values of < 0.05 were considered to re ect statistically signi cant differences between data. Error bars indicate standard deviation.

Forskolin increases cAMP level and reduced ATP level in MSCs
To study the effects of forskolin on MSCs, we rst treated MSCs with 60 mM forskolin and monitored changes in cAMP and ATP levels. cAMP production in forskolin-treated MSCs (12 or 24 h) was ~ 3-fold greater than that in control MSCs, and this increase subsided after 48 h (Fig. 1a). Forskolin signi cantly reduced intracellular ATP level after 24 h (data not shown) and 48 h (Fig. 1b). Thus, forskolin affected cAMP and ATP levels in MSCs.

Forskolin leads to a senescence phenotype in MSCs
Certain cAMP-elevating agents and cAMP analogs can inhibit cell proliferation [23]. We found that a 24-h incubation with forskolin signi cantly decreased MSC proliferation compared with control MSCs (Fig. 2a). Cell-cycle analysis of forskolin-treated MSCs revealed a signi cant arrest at the S and G2 + M phases (Fig. 2b). With regard to MSC senescence, after one day of treatment with forskolin, βgalactosidase staining was observed in 82.33 ± 4.04% of forskolin-treated MSCs and 7.77 ± 2.08% of control cells, indicating that a forskolin-induced increase in cAMP level could induce senescence (Fig. 2c). This conclusion was supported by the forskolin-induced upregulation of molecular markers associated with cell aging, namely P16 and P21, and downregulation of lamin B1 (Fig. 2d, e).

Forskolin alters the differentiation capacity of MSCs
We next studied the immunophenotype of the MSCs after forskolin treatment. Both the forskolin-treated and control MSCs expressed CD44 and CD29 but not HLA-DR, CD45, and CD31 (Fig. 3a). Oil red O staining is commonly used to detect lipogenesis. And Alkaline phosphatase staining is commonly used to detect osteogenesis. Forskolin-treated MSCs showed strong Oil red O staining yet weak ALP staining (Fig. 3b), and the early adipocyte genes LPL, C/EBPα, and PPARγ were signi cantly upregulated compared with the control groups (Fig. 3c), and the osteogenesis genes ALP, RUNX2, and OPN were downregulated (Fig. 3d). Thus, forskolin-treated MSCs had a normal immunophenotype but greater adipogenic capacity and lesser osteogenic capacity than the control MSCs.

Forskolin induces senescence-related transcriptional reprogramming
To determine the mechanism underlying the effects of forskolin on MSCs, we exposed MSCs to 60 µM forskolin for 24 h and identi ed 3154 upregulated and 2731 downregulated genes, indicating that forskolin triggered global transcriptional reprogramming in MSCs (Fig. 4a). Gene Ontology enrichment of differentially expressed genes revealed cAMP catabolic processes and cAMP-mediated signaling as two of the top four activated processes (Fig. 4b), and thus forskolin indeed targeted the expected pathways in our experiment. Two other activated processes, namely apoptosis and oxidative stress response, also supported our result that forskolin triggered MSC senescence (Fig. 4b). Several senescence-and differentiation-related processes also differed between forskolin-treated and control MSCs, such as osteoblast differentiation, cell proliferation, and cell-cycle processes (Fig. 4b). Moreover, several of the top 15 enriched KEGG pathways were related to senescence (Fig. c). Interestingly, certain in ammationrelated pathways differed signi cantly between the two groups of cells, such as the NF-κB signaling and TNF signaling pathways (Fig. 4c).
Because cell-cell adhesion was the most signi cantly downregulated Gene Ontology process (Fig. 4b), we analyzed the effect of forskolin on MSC migration. In wound healing assays, the average distance traveled by cells in the forskolin group was signi cantly less than that of control MSCs (Fig. 4d). Senescent cells typically exhibit characteristics of producing in ammatory factors, and thus they are often referred to as having a senescence-related secretory phenotype (SASP) [24,25]. qPCR was thus performed to assess whether forskolin treatment resulted in a SASP. MSCs treated with forskolin expressed lower levels of the pluripotent genes encoding Nanog and Sox2 (Fig. 4e) but much higher levels of several proin ammatory factors, including IL-6, IL-8, IL-1β, and TNF-α (Fig. 4g), than did the control MSCs. Further, the longevity genes encoding SIRT1, 2, 3, and 6 were downregulated in forskolintreated cells, con rming cellular aging (Fig. 4f). Together, these results suggested that forskolin caused global transcriptional reprogramming in MSCs that contributed to senescence-related cellular functions.

Forskolin disrupts mitochondrial metabolism in MSCs
We next studied mitochondrial function, which is key for regulating cell viability, in forskolin-treated MSCs. Forskolin-treated MSCs had atypical mitochondria that were untethered, swollen, yet small, compared with mitochondria in control MSCs. (Fig. 5a). Mitochondrial metabolism-associated functions were downregulated in forskolin-treated MSCs, including the processes of ATP production, basal and maximal respiration, and the proton gradient (Fig. 5b-d). However, there was no statistically signi cant difference in coupling e ciency and non-mitochondrial oxygen consumption between the two groups (Fig. 5e). Forskolin did not affect the total number of mitochondria ( Fig. 5f) but increased the production of ROS and SOX (Fig. 5g, h), NO (Fig. 5i), and calcium ux (Fig. 5j). Moreover, forskolin-treated MSCs exhibited upregulated expression of the NO-induced genes HO-1 and FOXO-1 (Fig. 5k), the latter being a ROS-regulated gene expressed at higher levels in older muscle than in younger muscle [26]. The expression of BACH-1, an inhibitor of HO-1, was also activated in forskolin-treated MSCs (Fig. 5k). These results suggested that mitochondria of forskolin-treated senescent MSCs were impaired with respect to both morphology and metabolism." An adenylate cyclase inhibitor blocks forskolin-induced MSC senescence The NF-κB pathway is essential for SASPs [27], and thus we treated MSCs with the NF-κB inhibitor pyrrolidine dithiocarbamic acid (PDTC) 100µM to determine whether NF-κB inhibition could rescue forskolin-induced senescence. To our surprise, the cAMP content increased after 24 h of PDTC treatment (Fig. 6a). Although the levels of IL-6 and IL-8 mRNAs decreased, IL-1β expression was increased (Fig. 6b). Moreover, the senescence-related gene P16 was downregulated whereas P21 was upregulated (Fig. 6c). On the other hand, the adenylate cyclase inhibitor SQ22536 30µM strikingly reversed the senescent state of forskolin-treated MSCs, exempli ed by the decreased expression of each of P16, P21, and the in ammatory factors IL-8, IL-1β, and TNF-α (Fig. 6d). Thus, the adenylate cyclase inhibitor SQ22536, but not PDTC, could effectively reverse forskolin-induced senescence.

Discussion
MSCs have shown potential in clinical trials for the treatment of several human diseases. However, preventing MSC senescence during in vitro culture remains a challenge for clinical applications of MSCs. Thus, deciphering the critical factors that contribute to MSC senescence is of great importance for developing effective strategies to prolong the viability of MSCs destined for clinical use. In this study, we treated MSCs with forskolin in vitro and demonstrated that forskolin signi cantly induces MSC senescence and abnormal differentiation. Forskolin triggered global transcriptional reprogramming of MSCs, thereby reducing their viability, disrupting mitochondrial metabolism, and producing other senescence-related phenotypes.
An increasing body of evidence has shown that metabolic changes within individual cells affect cell-fate decisions including entry into an apoptosis or senescence program [28][29][30][31][32]. cAMP is generated in both the mitochondria and cytoplasm by adenylate cyclase in response to metabolically generated carbon dioxide [33], and cAMP signaling is important for regulating mitochondrial biogenesis. The natural cAMPelevating compound forskolin raises intracellular cAMP levels by activating adenylate cyclase, which generates cAMP from ATP. Because forskolin alters cellular energy metabolism, it has been proposed as an effective anticancer agent for several cancer types [34]. However, the role of forskolin in MSC senescence remains unknown. We found that forskolin-induced senescence of MSCs resulted in abnormal mitochondrial morphology and impaired metabolism-associated functions, particularly increased production of both ROS and NO and increased Ca 2+ ux. Surprisingly, although both RNA-seq and qPCR data revealed that forskolin-treated MSCs had a SASP, the NF-κB inhibitor PDTC did not prevent forskolin-induced MSC senescence (rather, it only partially reduced the expression of genes encoding proin ammatory cytokines). These results indicate that the NF-κB pathway is not the primary mediator of forskolin-induced MSC senescence and that the observed SASP is a consequence of events occurring downstream of senescence. One the other hand, SQ22536, an adenylyl cyclase inhibitor that inhibits forskolin-induced cAMP elevation [35], remarkably reversed forskolin-induced MSC senescence.
Research has shown that forskolin at 4 µM promotes mitochondrial biogenesis and at 4 µM upregulates fatty-acid oxidation [36]. In contrast, use of a higher dose (60 µM) in our experiments revealed that forskolin alters mitochondrial morphology and function in MSCs and triggers their senescence (Fig. 7). This phenomenon suggests that forskolin may have contrasting effects in different contexts or at different doses. For example, eye drops containing forskolin have passed all clinical trial phases, and 10 mg/day of forskolin could prevent asthma in children and adults with mild/moderate persistent asthma via an increase in FEV1 and a decrease frequency in asthmatic attacks [37][38][39]. Moreover, our study may provide probable references of forskolin for clinical use, such as cumulative dose repeatedly and drug resistance mechanism. Additionally, forskolin has also been proposed for anticancer treatments. Thus, our results that forskolin can induce MSC senescence improves our knowledge of the role forskolin plays in cellular energy metabolism yet raises caution regarding its clinical applications.
Preventing senescence has been a long-standing goal of developmental biologists, and MSCs have proved promising for anti-aging strategies countering the aging process. Our study reveals that forskolin can potently induce MSC senescence. Moreover, an adenylyl cyclase inhibitor could potentially be an effective means of countering forskolin-induced MSC senescence, thus providing a basis for further research toward the goal of optimizing cAMP signaling to minimize the impact of senescence on MSCs. However, our study has certain limitations that require further investigation. For example, although our results provide solid evidence that forskolin induces MSC senescence via transcriptional reprogramming and disrupting mitochondrial metabolism, any potential interactions between these two aspects remains unclear. Because mitochondrial metabolism is key for controlling cell fate, further studies to reveal subtle changes in both the nuclear and mitochondrial transcriptomes may suggest practical means for preventing forskolin-induced senescence in vitro. Furthermore, the opposing effects of low-dose and highdose forskolin-as demonstrated in our current study and previous studies-raises the necessity to perform in vivo studies to further investigate the dose-related effects of forskolin on mitochondrial metabolism and senescence.

Conclusions
We found that forskolin can cause senescence of human MSCs with distinctive differentiation abilities and secretory functions through oxidative stress-induced mitochondrial metabolic dysfunction. These results suggest the mechanism of senescence induced by cAMP metabolic disorders in MSC, and thus the results provide a basis for developing strategies for improving the quality and e cacy of cultured MSCs for clinical use.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Competing interests
The authors declare that they have no competing interests.     forskolin-treated cells as assessed with qPCR. f Expression of the longevity genes encoding SIRT1-7 in control and forskolin-treated cells as assessed with qPCR. g Expression of the proin ammatory factors IL-6, IL-8, IL-1β, and TNF-α in control and forskolin-treated cells as assessed with qPCR. Mean ± SD values are presented in the graph; Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 100 μm.

Figure 5
Analysis of mitochondrial metabolism in senescent MSCs. a Representative transmission electron microscopy images of control and forskolin-treated MSCs. b-e Assessment of mitochondrial respiration metabolism with the Seahorse XF cell Mito Stress test. f Total number of mitochondria stained by MitoTracker Red and examined by ow cytometry. g ROS generation in control and forskolin-treated MSCs. h Oxidative respiration in mitochondria as assessed by MitoSOX Red mitochondrial SOX indicator staining. i Changes in intracellular NO generation analyzed by DAF-FM diacetate in the NO indication test. j Ca2+ ux analyzed by Fluo-3 AM staining. k Expression of HO-1, BACH-1, and FOXO-1 as assessed with qPCR. Mean ± SD values are presented in the graph; Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001, ns: not signi cant. Scale bars left = 1 μm, right = 220 nm.

Figure 6
Effects of an NF-κB inhibitor and a cAMP inhibitor on forskolin-induced senescence in MSCs. a cAMP content analysis in the PDTC (P) + forskolin (F) group and the F group. b, c Expression of in ammationrelated genes b and P16 and P21 c in the P+F and F groups as assessed with qPCR. d Expression of in ammation-related genes and senescence-related genes in the F + SQ22536 (S) group and the F group as assessed with qPCR. PDTC, Pyrrolidine dithiocarbamic acid. Mean ± SD values are presented in the graph; Student's t-test, *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7
Schematic diagram of the mechanism by which forskolin induces senescence in MSCs. Forskolin induces an increase in cAMP level and a decrease in ATP level, leading to dysfunction of mitochondrial metabolism in MSCs and an increase in the levels of ROS, NO, FOXO1, and HO-1. Additionally, expression of longevity genes sirtuins is suppressed. These changes lead to a senescence phenotype in MSCs characterized by cell-cycle arrest, a SASP, reduced chemotaxis, and abnormalities in pluripotency and differentiation.

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