Abstract
Background/Aim: Particular matter 2.5 (PM2.5) pollution is associated with senescence induction. Since the impact of PM2.5 on stem cell senescence and potential compounds capable of reversing this process are largely unknown, this study aimed to examine the senescence effects of PM2.5 on dermal papilla (DP) stem cells. Additionally, we explored the reversal of these effects using natural product-derived substances, such as resveratrol (Res) or Emblica fruits, soybean, and Thunbergia Laurifolia (EST) extract. Materials and Methods: Cell senescence was determined using the β-Galactosidase (SA-β-gal) assay. The senescence-associated secretory phenotype (SASP) was detected using real-time RT-PCR. For senescence markers, the mRNA and protein levels of p21 and p16 were measured using real-time RT-PCR and immunofluorescence analysis. Results: Subtoxic concentration of PM2.5 (50 μg/ml) induced senescence in DP cells. Resveratrol (50, 100 μM) and plant extracts (400, 800 μg/ml) reversed PM2.5-induced cell senescence. Treatment with Res or EST significantly decreased SA-β-gal staining in PM2.5-treated cells. Furthermore, Res and EST decreased the mRNA levels of SASP, including IL1α, IL7, IL8, and CXCL1. DP cells exposed to PM2.5 exhibited an increase in p21 and p16 mRNA and protein levels, which could be reversed by the addition of Res or EST. Res and EST could reduce p21 and p16 in senescent cells approximately 3- and 2-fold, respectively, compared to untreated senescent cells. Conclusion: PM2.5 induced senescence in human DP stem cells. Res and EST extract potentially reverse the senescence phenotypes of such cells.
Particulate matter 2.5 (PM2.5) poses health risks to the human body. Such tiny particles, measuring 2.5 micrometers or smaller, have the potential to be inhaled into the lungs and enter the bloodstream (1, 2). When PM2.5 particles translocate into the blood, they potentially affect the cardiovascular system by causing cellular injury and the production of pro-inflammatory cytokines both in vascular and heart tissues. They have been associated with an increased risk of heart attacks, strokes, and other cardiovascular-related complications (3, 4). Studies have shown that exposure to PM2.5 can induce cell senescence through a mechanism dependent on reactive oxygen species (ROS) generation (5, 6). However, while research has extensively examined the impact of PM2.5 on differentiated cells (7), there’s less information available regarding its effects on stem cells.
Senescence refers to a state of cell cycle arrest, where cells lose their ability to divide and function optimally (8, 9). This can contribute to aging and various age-related diseases (10). Cellular senescence can be monitored by several means including p21 and p16, indicators for cell cycle arrest (11). Besides, the cellular production of Senescence-Associated Secretory Phenotype (SASP) has been widely used for determining the phenotype associated with senescent cells (12).
Resveratrol (Res) (3,5,4′-trihydroxystilbene), is a polyphenolic bioactive compound with health-promoting attributes. Res effectively reduces the expression levels of IL-1α/β, IL-6, IL-8, GROα, and VEGF in senescent human lung fibroblasts (13). Furthermore, a prior study demonstrated that Res effectively suppresses SASP factors like IL1β, IL-8, TNFα, MVP-1, and VEGFR by blocking NF-
B activation in arterial vascular smooth muscle cells from aged rhesus monkeys (14). Likewise, Emblica fruits (Phyllanthus emblica L) extract is rich in flavonoids, glycosides, terpenoids, and phenolic compounds, which exhibit antioxidant properties (15). Soybeans (Glycine max L.) are rich in isoflavones, saponins, anthocyanins, and phytophenols. Soybeans are recognized for their antioxidant properties, primarily attributed to the presence of key constituents, such as isoflavones (16). Thunbergia Laurifolia, a member of the Acanthaceae family, is known to contain apigenin, caffeic acid, catechin, quercetin, and isoquercetin (17).
Given the information on the adverse impact of PM2.5-induced senescence, researchers are investigating bioactive compounds with the ability to counteract or even reverse this process. Consequently, we established experimental cellular models to induce cellular senescence in hair dermal papilla stem cells using PM2.5 or ROS exposure. The cell senescence was monitored by several senescence markers. This was done to explore the potential of plant-derived compounds and extracts in protecting and reversing senescence in these cell types.
Materials and Methods
PM2.5 sampling. In this study, PM2.5 levels were measured at the west side of the city center of Phitsanulok, Thailand, at 16° 49′ N 100° 12′ E. The sampling of PM2.5 was conducted using a low-volume air sampler (Low Volume Sampler, LVS) and portable sampler (MiniVol TAS 5.0, Airmetrics, Springfield, OR, USA). The sampler was equipped with the PTEF microfiber filter paper (47-mm, Whatman™, Kent, UK). Sampling was carried out during a period of 24 h every day with a flow rate of 5 l/min. The filter paper was weighed before and after sampling using a micro analytical balance (MC5, Sartorius, Johnson Avenue Bohemia, NY, USA). The filters were stored in a refrigerator with a temperature of 4°C. The PTFE filters were extracted according to the method of Aileen Yang et al. (18). The filters were immersed in ethanol, and extracted in an ultrasonic bath for 30 min. The extracts were evaporated and the remaining solution was placed in an Eppendorf tube and dried under nitrogen.
Cells and reagents. Human dermal papilla cells (DP) were obtained from Applied Biologicl Materials Inc. (Richmond, BC, Canada). DP cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Gibco, Grand Island, NY, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was acquired from Sigma-Aldrich, Co. (St. Louis, MO, USA). All primers were obtained from Marcogen (Gangnam-gu, Seoul, Republic of Korea). The rabbit mono-clonal antibodies for p21 Waf1/Cip1 (cat no: 2947) and p16 INK4A (cat no: 92803) were sourced from Cell Signaling (Beverly, MA, USA). The rabbit secondary antibody anti-rabbit (cat no: 7074) was provided by Cell Signaling.
Preparation of plant extract. The EST mixture comprises a blend of extracts from Emblica fruits, Soybean, and Thunbergia Laurifolia in equal proportions (1:1:1). The plant materials from agricultural sources in Nan province, Thailand are being utilized as part of the Kasikornthai Foundation’s project to enhance the value of Thai medicinal plants for sustainable development.
Cell viability assay. Cells were exposed to PM2.5 (0-200 μg/ml), Res (0-800 μM), and EST (0-1,200 μg/ml) for 24 h. Then, MTT reagent (4 mg/ml in PBS) was added and incubated for 3 h at 37°C in the dark. The MTT reagent was replaced with 100 μl of DMSO to dissolve the formazan crystals. The measurement of the formazan product was conducted at 570 nm utilizing a microplate reader.
Senescence-associated β-galactosidase (SA-β-gal) activity. The β-Galactosidase staining kit (cat no: #9860) was from Cell Signaling. The DP cells were treated with PM2.5 for 4 days to induce senescence. After that the senescent DP cells population was treated with Res or EST. The following day, the fixative solution (1×) was placed on each well and incubated for 15 min at room temperature. The β-Galactosidase staining solution was added to the cells for overnight incubation at 37°C. The senescent cells developed blue color and photographed at 20×magnification.
Real-time polymerase chain reaction (RT-PCR). Cells were treated with 50 μg/ml PM2.5 for 4 days. After that, Res (50, 100 μM) or EST (400, 800 μg/ml) was added to PM2.5 treated cells for 24 h. The RNA was extracted using GENEzol reagent (Geneaid, Shijr District, New Taipei, Taiwan, ROC). SuperScript® III First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, Waltham, MA USA) was utilized to synthesize cDNA from total RNA. After the cDNA synthesis, 100 ng of the cDNA was employed in RT-PCR using Luna Universal qPCR Master Mix (New England Biolabs NEB, Ipswich, MA, USA), with a total volume of 20 μl. The reaction was performed in a CFX 96 Real-time PCR system (Bio-Rad, Hercules, CA, USA). For the RT-PCR, the protocol included an initial denaturation step at 95°C for 1 min, followed by 45 cycles involving denaturation at 95°C for 15 s and primer annealing at 60°C for 30 s. The gene expression levels were normalized using the GAPDH gene as an internal control. The relative mRNA gene expression level for each gene was determined based on the comparative Cq values.
Immunofluorescence. The cells were fixed with 4% paraformaldehyde for 15 min and permeabilization with 0.5% Triton-X for 5 min, followed by blocking non-specific binding with 10% FBS in 0.1% Triton-X PBS for 1 h. The cells were incubated with primary antibodies at 4°C overnight and then, were incubated with secondary antibodies. Nuclei were stained with Hoechst 33342. The images were captured using fluorescence microscope (Olympus IX 51 with DP70, Olympus America Inc., Center valley, PA, USA). The fluorescence intensity was measured using Image J software (Image J 1.52a, Rasband, W., National Institutes of Health, Bethesda, MD, USA).
Statistical analysis. The results are reported as the mean±standard deviation (SD) based on a minimum of three independent biological experiments. Multiple comparisons were conducted using one-way ANOVA analysis with a post hoc test in GraphPad Prism software version 9.0 (GraphPad Software, La Jolla, CA, USA). Differences between groups were considered significant at a p-value <0.05.
Results
PM2.5 exposure induces cellular senescence and the effect of resveratrol on senescent cells. The appropriate concentrations of PM2.5 and plant substances were first characterized in human DP cells. Cells were exposed to different concentrations of PM2.5 (0-200 μg/ml) for 24 h, and their viability was assessed using the MTT assay. The findings indicated that the IC50 value for PM2.5 was 1,208 μg/ml (Figure 1A). DP cells were exposed to a range of Res concentrations (0-800 μM) for 24 h. The findings indicated that Res had minimal effects on cell survival at concentrations up to 100 μM (Figure 1B).
Cytotoxicity of PM2.5 and Res on human DP cells. (A, B) The cells were treated with PM2.5 (0-200 μg/ml) or Res (0-800 μM) for 24 h. The MTT assay was performed. Data are presented as mean±SD (n=3). Significant compared to the control group, ***p<0.001 versus untreated control cells.
SA-β-gal is an important indicator that has been widely used for identifying cellular senescence (12). DP cells were subjected to a 4-day treatment with 50 μg/ml of PM2.5, followed by staining with SA-β-gal. As shown in Figure 2, the proportion of SA-β-gal positive cells increased at 4 days of treatment. To investigate whether Res can reverse senescence in PM2.5-treated cells, the cells were first treated with PM2.5 for 4 days. Then, the senescent cells were treated with 50 μM or 100 μM Res for a further 24 h. The data presented in Figure 2 indicate that treatment with Res dramatically reduced SA-β-gal-positive cells compared to control PM2.5-treated senescent cells.
Res reverses senescence in PM2.5-treated cells as assayed using staining for senescence-associated-β-galactosidase (SA-β-gal). The blue color indicates senescent cells. DP cells were subjected to PM2.5 treatment at a concentration of 50 μg/ml for 4 days. Then, PM2.5-induced senescent cells were treated with Res (50, and 100 μM) for 24 h.
Effect of PM2.5 on senescence-associated secretory phenotype (SASP) production of DP cells. The secretion of various cytokines, chemokines, and proteinases by senescent cells is primarily associated with the development of diseases (14). PM2.5-treated senescent DP cells exhibited SASP as assessed by examining the mRNA levels of SASP-related genes. DP cells induced by PM2.5 showed a significant increase in the expression of SASP-related genes, with IL1α, IL7, IL8, and CXCL1 displaying a 3-, 4-, 2.5-, and 4-fold increase in mRNA levels, respectively (Figure 3A-D).
PM2.5 induces senescence-associated secretory phenotype (SASP), which was reduced following treatment with Res. DP cells were subjected to PM2.5 treatment at a concentration of 50 μg/ml for four days. Then, PM2.5-treated senescent cells were treated with Res (50, and 100 μM) for 24 h. The mRNA expression level of SASP cytokines was assessed using RT-PCR; (A) IL1α, (B) IL7, (C) IL8, (D) CXCL1. The mRNA level was normalized by the mRNA of the housekeeping GAPDH gene. The relative mRNA expression was calculated using comparative CT cycles. Data are presented as mean±SD (n=3). Significant compared to the control group, *p<0.05, **p<0.01, ***p<0.001 versus untreated control cells #p<0.05, ##p<0.01, ###p<0.001 versus PM2.5-induced senescence in DP cells.
Resveratrol reduces the level of SASP in PM2.5-induced cellular senescence. The senescent cells were generated as described previously, and were similarly treated with Res. The results of the SASP investigation showed that Res can cause a 50% reduction in the mRNA expression level of IL1α (Figure 3A). Treatment with Res (100 μM) led to a 3-fold reduction in the mRNA expression level of IL7 and a 1.5-fold reduction in the mRNA expression level of IL8 in PM2.5-induced senescent DP cells (Figure 3B and C). Another marker that indicates SASP phenotypes is CXCL1. The findings indicated that Res treatment significantly reduced the mRNA expression level of CXCL1 in PM2.5-induced senescent cell populations (Figure 3D).
Effect of resveratrol on PM2.5-induced cell cycle arrest. Cellular senescence was linked to the arrest of the cell cycle (12). The senescence-related markers p21, p16, and p57 mRNA levels were measured using RT-PCR. The results showed that PM2.5 exposure of DP cells increased the p21, p16, and p57 mRNA levels (Figure 4A-C). Immunofluorescence analysis was used to assess the protein expression levels of p21 and p16 in PM2.5-treated senescent cells. According to the results, the population of senescent cells induced by PM2.5 showed a 3-fold increase in the protein expression level of p21 (Figure 4D) and a 2.5-fold increase in the protein expression level of p16 (Figure 4E). To examine the ability of Res to reverse senescence in PM2.5-induced DP senescent cells, we used RT-PCR to measure the mRNA levels of p21, p16, and p57. PM2.5-treated senescent DP cells were exposed to Res (50, 100 μM) for 24 h, and their mRNA levels were subsequently measured. The findings indicated that treatment with Res resulted in a 4-fold reduction of the p21 mRNA level, compared to the non-treated senescent DP cell population (Figure 4A). Res was able to reverse the PM2.5-induced senescence in DP cells, leading to a decrease in the p16 and p57 mRNA levels (Figure 4B and C).
Effect of PM2.5 on cellular senescence through DNA damage response via p21/p16 level and reversal of senescence by Res. DP cells were subjected to PM2.5 treatment at a concentration of 50 μg/ml for 4 days. Then, PM2.5-treated senescent cells were treated with Res (50, and 100 μM) for 24 h. The mRNA expression levels of p21, p16, and p57 was assayed using RT-PCR;(A) p21 (B) p16 (C) p57. The mRNA level was normalized by the mRNA of the housekeeping GAPDH gene. The relative mRNA expression was calculated by using comparative CT cycles. The fluorescence of (D) p21 (E) p16 was captured by fluorescence microscope and the fluorescence intensity was determined using Image J software. Data are presented as mean±SD (n=3). Significant compared to the control group, *p<0.05, **p<0.01, ***p<0.001 versus untreated control cells and ###p<0.001 versus PM2.5-induced senescence in DP cells.
Res treatment of PM2.5-treated senescent DP cells for 24 h was followed by an assessment of p21 and p16 protein expression levels using immunofluorescence analysis. The findings indicated that Res treatment of PM2.5-treated senescent cells led to a significant 3-fold reduction in p21 protein expression level and a 2-fold reduction in p16 protein expression (Figure 4D and E).
Plant extracts reverse PM2.5-induced DP senescent cells. The cytotoxicity of the plant extract was first investigated. Cells were treated with different concentrations of EST extracts (0-1,200 μg/ml) for 24 h. The results showed that these concentrations of EST extracts did not influence the cell viability of DP cells (Figure 5A).
EST reverses senescence in PM2.5-treated DP cells. DP cells were subjected to PM2.5 treatment at a concentration of 50 μg/ml for four days. Then, PM2.5-treated senescent cells were treated with EST (400, and 800 μg/ml) for 24 h. (A) Viability of DP cells treated with EST (0-1,200 μg/ml). (B) images of SA-β-gal staining revealing reversal of senescence of PM2.5-induced senescent DP cells by EST. (C, D) The mRNA expression levels of p21 and p16 in EST-treated PM2.5-induced senescent DP cells assayed using RT-PCR. (E, F) The protein expression levels of p21 and p16 in EST-treated PM2.5-induced senescent DP cells assayed using immunofluorescence analysis. Data are presented as mean±SD (n=3). Significant compared to the control group, ***p<0.001 versus untreated control cells and ###p<0.001 versus PM2.5-induced senescence in DP cells.
To investigate whether EST reverses senescence in PM2.5-treated cells, PM2.5-treated cells were treated with 400 μg/ml and 800 μg/ml of EST for 24 h. Figure 5B indicates that treatment with EST significantly decreased SA-β-gal-positive cells. In addition, we measured the p21CIP/WAF1 and p16INK4a mRNA and protein levels using RT-PCR and immunofluorescence assays, respectively. EST was able to cause a substantial decrease in the p21 and p16 mRNA levels compared to the control PM2.5-treated cells (Figure 5C and D).
Furthermore, when the PM2.5-treated senescent cell population was treated with EST, it displayed a significant 3.5-fold reduction in p21 protein level (Figure 5E) and a 50% reduction in p16 protein expression levels (Figure 5F).
Discussion
PM2.5 poses significant health risks to the human body. Senolytics, drugs designed to remove senescent cells from the body, are the focus of investigation in both preclinical and clinical studies (19). The potential of these drugs to reverse or eliminate senescent cells holds significant promise in combating certain aging effects and potentially extending individuals’ health spans (20). Here we demonstrated the potential use of a well-known antioxidant compound named resveratrol in the reversal of senescence in human stem cells. It was shown that the senescence of stem cells like mesenchymal cells not only hampers tissue regeneration and repair via cell cycle arrest and senescence-associated stem cell exhaustion but also contributes to tissue collapse. The senescent stem cells in the tissue can spread senescence-associated inflammation and induce senescence of stem cells via the production of SASP (21).
In the present study, PM2.5 increased the proportion of SA-β-gal blue stained cells and the expression of p21 and p16 in DP cells, accompanied by increased secretion of SASP cytokines, including IL1α, IL7, IL8, and CXCL1. Overall, these results indicate that PM2.5 promotes senescence in DP stem cells (Figure 2). Res, a polyphenol present in different plants, possesses anti-aging attributes, and extends the lifespan of various species by influencing numerous cellular processes (22). Res was shown to decrease SASP cytokine levels in rats exposed to cigarette smoke and hinder lung aging in mouse models that experience accelerated aging (23-25). Furthermore, Res hinders the secretion of SASP cytokines and the induction of senescence markers like p53, p21, and p16 in bone marrow stromal stem cells when induced in vivo by tert-butyl hydroperoxide (26).
Studying PM2.5 pollution and exploring ways to enhance health and well-being aligns with Sustainable Development Goals (SDGs) being a global priority (27). PM2.5 pollution is significantly associated with respiratory illnesses and health complications (1). As part of this research, there is a suggestion about the potential use of plant-derived substances for prevention or therapy. This approach directly addresses Sustainable Development Goals 3 (SDG 3), which aims to ensure healthy lives and promote well-being for all (27). Research into natural remedies derived from plants to mitigate the health impacts of PM2.5 pollution exemplifies an innovative strategy in alignment with this goal.
Focusing on mesenchymal stem cells, we found that PM2.5 can induce cellular senescence as indicated by the increase in β-galactosidase enzyme level, p21 and p16 proteins. Moreover, we found that PM2.5 could increase the cellular production of IL1α, IL7, IL8, and CXCL1, which may in turn induce inflammation and lead to senescence of nearby stem cells. Interestingly, our results revealed that Res can reverse senescence in DP cells. One noteworthy outcome indicated that treatment with Res reduced the expression of β-galactosidase enzyme in PM2.5-induced senescent DP cells compared to those not treated with Res (Figure 2). In an addition, treatment of PM2.5-induced senescent DP cells with Res decreased secretion of SASP, such as IL1α, IL7, IL8, and CXCL1 (Figure 3). In addition, we found that Res treatment of PM2.5-induced senescent DP cells reduced the factors involved in regulating senescence markers, including p21Cip1/WAF1 and p16INK4a, compared to untreated senescent cells with Res (Figure 4).
Previous literature revealed that the Thunbergia Laurifolia extract suppresses oxidative stress triggered by PM2.5 through the modulation of the p62–KEAP1–NRF2 signaling pathway (28). In our findings, the treatment of PM2.5-induced senescent DP cells with EST reversed senescence in these cells. The results revealed that EST-treated PM2.5-induced senescent DP cells showed a significant reduction in the expression levels of β-galactosidase enzyme and senescence markers p21 and p16 compared to senescent cells not treated with EST (Figure 5).
These results provided direct evidence that Res or EST extract reverses senescence in PM2.5-treated DP cells by modulating the senescence markers. Therefore, natural products represent significant therapeutic approaches for reversing senescence in the aging process.
Conclusion
In summary, PM2.5 induces senescence in mesenchymal DP cells leading to their dysfunction. Additionally, Res and EST could reverse the senescence in PM2.5-treated senescent DP cells. This study represents novel research on the effect of PM2.5 on mesenchymal stem cells as well as the potential use of plant-derived compounds and extracts in treating senescent stem cells.
Acknowledgements
The Author Z.Z.E is grateful to the Second Century Fund (C2F) for postdoctoral fellowship, Chulalongkorn University. The Authors thank Pharma-Agroforestry District and Kasikornthai foundation for their support.
Footnotes
Authors’ Contributions
Conceptualization and validation, P.Chan.; methodology, Z.Z.E., T.S., P. Chun., C.C., W.A., P.P. and P. Chan., formal analysis, Z.Z.E. and P. Chan.; investigation, Z.Z.E. and P. Chan.; resources, T.S., P. Chun. and P. Chan.; writing-original draft preparation, Z.Z.E., T.S. and P. Chan.; writing-review and editing, P. Chan.; Funding, T.S., P. Chan.; supervision, P. Chan. All Authors have read and agreed to the published version of the manuscript.
Funding
This research was supported by the Fundamental Fund and Ratchadaphisek Somphot Fund. This work was also partly funded by Pibulsongkram Rajabhat University for Fundamental Fund RDI-1-64-1.
Conflicts of Interest
The Authors declare that there are no conflicts of interest in relation to this study.
- Received November 10, 2023.
- Revision received December 13, 2023.
- Accepted December 14, 2023.
- Copyright © 2024 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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