Curcumin Alleviates The Senescence Of Canine Bone Marrow Mesenchymal Stem Cells During In Vitro Expansion

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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. cistanche dosage reddit 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 uM) 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 (pl6 and p21)and pro-inflammatory molecules(tumor necrosis factor-α(TNF-a)and interleukin-6(IL-6)). Furthermore, Cur（0.1 uM~10μM） 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 on ameliorating cBMSC senescence and provides new insight for delaying senescence and improving the therapeutic potential of MSCs.

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

1. Introduction

Aging and longevity have always been seductive topics for organisms. As a type of adult stem cell with the capacity for self-renewal, immunosuppression, differentiation, and migration, mesenchymal stem cells (MSCs)extensively reside within various tissues and organs, such as bone marrow, adipose tissue, amniotic fluid, placenta, umbilical cord, and muscle [1,2], and play an essential role in maintaining tissue homeostasis throughout the lifespan of an organism [3,4]. Accumulating evidence has revealed that biological aging and the development of many degenerative diseases can attribute to MSC senescence [5,6]. In addition, MSCs are promising sources of cell-based regenerative therapy, but usually need to be expanded in vitro to achieve the minimum transplantation number of MSCs (20-100 million) for treatments [7]. However, in vitro culture is not able to completely simulate the in vivo microenvironment of MSCs, and inevitably triggers cellular senescence [8,9]Senescence in mitotic cells occurs in response to a variety of stressors, such as telomere dysfunction [10], DNA damage [11], oxidative stress [12], and oncogene activation [13], and ultimately leads to the termination of cell division. Moreover, senescent MSCs exhibit a negative effect on biological functions, including immunomodulation, differentiation, and migration [14]. Much work has attempted to establish effective strategies to retard senescence in MSCs. Recently, several herb-derived products have been suggested to promote organism health and longevity and may be promising tools for delaying MSC senescence [15].

Cistanche can anti-aging

Curcumin(Cur), a hydrophobic polyphenol extracted from traditional Chinese medicine turmeric, has attracted great attention due to its anti-inflammatory, antioxidant, anti-apoptotic, anti-tumor, and anti-aging effects in tissue cells and some disease models [16-19]. Numerous studies have demonstrated that Cur has health-related benefits in age-related diseases, including osteoarticular, neurological, reproductive, and cardiovascular diseases [20-24]. Aging is associated with musculoskeletal changes and the initiation and progression of osteoarticular diseases. Chronic dietary intervention with Cur was found to ameliorate the motor function of the hand and digits in middle-aged monkeys and had beneficial effects on aged skeletal muscle [25,26]. Available data from Buhrmann and colleagues have revealed that Cur can attenuate environment-derived osteoarthritis by balancing chondrocyte survival and inflammatory responses [27,28]. Additionally, Cur exerts neuroprotective effects via mediating autophagy and inflammation and may therefore be an effective therapy for patients with neurodegenerative diseases, such as Alexander disease, Alzheimer's disease, and Parkinson's disease [29,30].

Indeed, the fact that Cur possesses pleiotropic activity could be attributed to their activation and protection effects on MSCs [31,32]. Several studies have confirmed that Cur is involved in regulating the immunomodulatory capabilities; the differential potential of MSCs to different cell lineages (neurocyte, chondrocyte, adipocyte, and osteoblast), and in multiple signaling mechanisms is involved in this process [2831,33-35]. Moreover, Cur is also regarded as a good antioxidant by which to improve the lifespan of rat adipose tissue-derived mesenchymal stem cells(ADSCs) due to the increase in the telomerase reverse transcriptase(TERT)gene expression [36]. A recent study showed that the inhibition of p65 by Cur can prevent cellular senescence and inflammatory activation in human umbilical cord-derived MSCs [37]. cistanche extract benefits Obviously, more attention has been focused on the anti-aging effects of Cur in MSCs due to their enormous therapeutic potential, but the mechanisms underlying this effect are still unclear. Some evidence from in vitro studies has confirmed that the beneficial effects exerted by Cur should be attributed to its modulation of autophagy [29,38-41]. Han and colleagues demonstrated that Cur can protect human umbilical vein endothelial cells (HUVECs) from H2O2-induced oxidative damage and that positive effects occur by the enhancement of autophagy via phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) inhibition [40]. Another study showed that Cur is able to promote cell survival by inhibiting hypoxia/reoxygenation (H/R)-induced apoptosis and excessive autophagy among cardiomyocytes [41]. Therefore, whether Cur performs as a positive or negative regulator of autophagy depends on the stress-producing stimulus and cellular setting, and more details are required to determine whether the modulation of autophagy by Cur is an effective strategy for delaying MSC senescence. As a principle of degradation and recycling pathway for intracellular substances, autophagy plays a crucial role in maintaining cellular homeostasis and withstanding environmental pressure and has exhibited the enormous potential for delaying cellular senescence and treating age-related diseases. Organelle dysfunction and toxic metabolite accumulation are the key characteristics of senescent MSCs [42], thus suggesting a potential link between autophagy and senescence. Ma and colleagues found that the autophagic activity of aged BMSCs was diminished in comparison with young BMSCs and that autophagy inhibitor 3-methyladenine (3-MA) could accelerate senescence in young MSCs; in contrast, they found that autophagy activator rapamycin (RAP) could partially restore the biological properties of aged BMSCs[5]. A recent study examined the role of modulated autophagy through the employment of RAP and 3-MA in MSC senescence induced by D-galactose(D-gal) and showed that RAP remarkably alleviated MSC senescence [43]. Interestingly, several studies have demonstrated that an increase in autophagy is observed in senescent MSCs and that autophagy is indispensable for maintaining the MSC senescence processes [44,45]. Therefore, whether autophagy could be regarded as a positive or negative regulator of senescence in MSCs remains conflicting and ambiguous. Consequently, it would be interesting to investigate the relationship between autophagy, MSC senescence, and Cur, which could provide a wider perspective for delaying senescence and improving the therapeutic potential of MSCs.

In this study, our primary purpose was to elucidate whether Cur treatment could delay canine bone marrow-derived MSC(cBMSC)senescence and whether the underlying mechanism was correlated with the modulation of autophagy. To this end, we evaluated the phenotypic characterization, autophagic activity, and gene expression after exposure to Cur in senescent cBMSCs, as well as whether the relationship between autophagy and its effects on cBMSCs senescence is determined by using 3-MA and RAP.

2. Results

2.1. Characteristics of cBMSCs

cBMSCs were expanded in vitro with standard procedures and were shown to be stably passaged over nine generations (data not shown). The cBMSCs displayed a plastic-adherent and fibroblast-like appearance throughout the culturing process (Figure 1A). The differentiation capacity of cBMSCs was confirmed by adipogenic and osteogenic induction (Figure 1B, C). The immunophenotype of cBMSCs was evaluated using flow cytometry. The results showed that the cell population positively expressed CD90, CD105, and ITGB1, and negatively expressed CD31, CD34, and CD45, suggesting their mesenchymal rather than hematopoietic origin (Figure 1 and Table 1).

2.2.cBMSCs Progressively Display Senescent Features along Expansion In Vitro

Early-passage cBMSCs (P1 and P3) display a long fusiform or triangular shape and are arranged into a whirlpool after growth in vitro (Figure 2A). Subsequently, cBMSCs gradually displayed senescence-associated phenotypes over the course of long-term cultivation in vitro, characterized by an enlarged and flat morphology and a reduction in proliferation (Figure 2A, B). Additionally, the results from a colony-forming unit-fibroblast (CFU-F) assay showed that both the number of CFU-F and the size of colonies were signifi-cantly diminished along culture passages (Figure 2C), consistent with the downregulated mRNA expression of pluripotent transcription factors Nanog and SOX-2 in the 6th and 9th passage (Figure 2G).

Senescence-associated β-galactosidase (SA-β-gal) is a lysosomal enzyme, the activity of which is strongly correlated with cellular senescence [46]. The results of SA-β-gal staining indicated that the number of SA-β-gal positive cells grew from 7.5± 2.4%(P3) to 65.0 ±5.0%(P6) and 87.3± 3.3%(P9), respectively(Figure 2D). cistanche genghis khan Additionally, increases in the expression of cell cycle kinase inhibitors (p21 and p16) and pro-inflammatory molecules (TNF-α and IL-6), which act as components of a senescence-associated secretory phenotype (SASP), were detected in the 6th and 9th passage (Figure 2E, F). Therefore, cBMSCs exhibit a passage-dependent increase in senescent phenotypes and cease proliferation at the 9th passage.

2.3. Cur Alleviates the Senescent State of cBMSCs

The 6th passage cBMSCs were incubated in the absence or presence of Cur at doses of 0.1，0.5，1，5， or 10μmol/mL（μM） for 12h，24 h， 48 h， and 72h， and their cellular viability was assessed using CCK-8. The results showed that Cur (0.1-10 uM) does not induce a toxic effect in cBMSCs and has palpable effects on increasing the viability of cBMSCs after24 h of treatment (Figure 3A). To evaluate the effect of Cur on cellular senescence, cBMSCs were exposed to different concentrations of Cur(0.1,1, and 10 uM) for 24 h. The result showed that the numbers of SA-β-gal-positive cells were significantly decreased in cBMSCs after treatment with Cur（1 μM and 10uM） compared with the control group (Figure 3B). CFU-F assays had been used to evaluate the effects of Cur on the self-renewal efficiency of cBMSCs(P6). The dose of 1 uM of Cur was found to be the most effective concentration for improving the efficiency of the CFU-F of cBMSCs. However， the dose of 10 μM Cur had adverse effects on the self-renewal efficiency of cBMSCs(Figure 3C), In addition, the senescence-alleviating effect of Cur was observed at a dose of 1 μM， manifested by the downregulation of p16, IL-6, and TNF-α and upregulation of the expression of Nanog and SOX-2 (Figure 3D-F). cistanche life extension Taken together, these results indicate that Cur at a dose of 1 uM can effectively exert cytoprotective effects on senescent cBMSCs.

2.4. Cur Treatment Enhanced Autophagic Activity in cBMSCs

Lysosome functional activation plays a critical role in the course of autophagy, and disorders of lysosomal acidification are adverse to the lysosomal degradation function [38]. Therefore, autophagic activity was first detected by LysoTracker staining, which can monitor lysosomal acidification. The result indicated that preconditioning with Cur dose-dependently enhanced the intensity of red fluorescence in cBMSCs, which suggested that lysosome acidification had been increased (Figure 4D).

To further evaluate the effects of Cur on the autophagy of cBMSCs, we made use of the autophagosome markers microtubule-associated protein 1A/1Belight chain 3 (LC3)and p62/SQSTM1, which are pivotal markers in for evaluating autophagic flux[47,48]. The increased autophagic activity was observed in cBMSCs after exposure to Cur, as shown by a dose-dependent increase in the conversion of LC3-I to LC3-II, alongside the accelerating p62 degradation(Figure 4A).In addition, the mRNA expression of ATG7, ATG12.LC3. and ULK1 were upregulated after Cur treatment(Figure 4B). The ultrastructure of cBMSCs was examined using transmission electron microscopy. We then observed an increase in the formation of autophagosomes and autolysosomes in cBMSCs after treatment with Cur at doses of 0.1， 1， and 10 μM for 24 h（Figure 4C）. cistanche nz Immunofluorescence staining also showed that the number of characteristic punctate fluorescent dots of LC3 was significantly increased by Cur treatment (Figure 4E). These results indicated that Cur can promote lysosomal acidification and autophagy activation in a dose-dependent manner.

This article is extracted from Int. J. Mol. Sci. 2021, 22, 11356. https://doi.org/10.3390/ijms222111356 https://www.mdpi.com/journal/ijms