Microplastics accelerates the premature aging of blood vessels though ROS-mediated CDK5 signaling pathway

doi:10.21203/rs.3.rs-3222853/v1

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Research Article

Microplastics accelerates the premature aging of blood vessels though ROS-mediated CDK5 signaling pathway

https://doi.org/10.21203/rs.3.rs-3222853/v1

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Background

Microplastic has become a kind of pollutant widely existing in soil, atmosphere, fresh water and marine environment. At present, microplastics have been found in many tissues and organs of organisms. Research shows that as a new environmental pollutant, microplastics has shown a health hazard to human and animal. Aging and aging-related diseases are major social and medical problems facing the world. However, up to now, the effect of microplastic exposure on premature aging of blood vessels has not been evaluated. Therefore, we investigated the health damage of microplastics to blood vessels in vivo and in vitro experiments.

Methods

ELISA, indirect immunofluorescence, SiRNA, laser confocal microscopy, and Flow cytometry were performed to evaluate the effect of microplastics on premature aging of blood vessels.

Results

In vitro experiments, we found that microplastics can internalize into vascular cells, and the internalized microplastics cause damage to organelles. Further biochemical experiments showed that microplastics stimulation caused the premature aging of blood vessels by detecting a series of aging markers. Further mechanism research indicates that microplastics could increase ROS level of mitochondria mediated by calcium overload, and then ROS leads to the LaminA degradation by CDK5 mediation, further resulting in genomic instability, thus finally causing the aging of vascular cells/tissues. In vivo model, we found that microplastics induced aging damage on vascular tissue, the expression of aging maker molecules were significantly increased. Furthermore, the level of inflammation and oxidative stress was also significantly increased.

Conclusion

In summary, in this work, we evaluated the effect of microplastic exposure on premature aging of blood vessels, and we also revealed the molecular mechanism by which microplastics cause premature aging of the cardiovascular system.

microplastics

Vascular tissue

Aging

Oxidative stress

lamina A

Microplastic has become a new type of pollutant widely existing in soil, atmosphere, fresh water and marine environment (1). At present, studies have shown that microplastics widely exist in soil, atmosphere, ocean and freshwater water due to their small volume and small density (2). The microplastics in the environment will be aged and degraded by external factors, resulting in the change of particle characteristics, which will cause more extensive pollution of microplastics. Microplastic can enter animal body through inhalation, ingestion and direct contact. Because of its wide distribution and small size, microplastics can rapidly penetrate the cell membrane and distribute and accumulate in human tissues and organs, thus causing health damage (2). After entering the body, the micro plastic will rapidly interact with the biological molecules in the body fluid, and flow to various tissues and organs with blood, thus causing health risks. Therefore, the research on microplastics and health risks has become the focus of attention and research in the field of environment (3). Microplastic generally refers to plastic particles with particle size less than 5mm. This concept was first proposed by Thompson et al. in 2004 (4). The most commonly detected microplastics in the environment mainly include polypropylene (pp), styrene (ps), polyvinyl chloride (PVC), polyethylene (PE) and polyethylene terephthalate (PET) (5). At present, micro-plastic pollution has become one of the major environmental problems in the world.

In recent years, the existence of microplastics has been found in the ocean, soil and atmospheric environment, which means that human beings are exposed to the omni-directional environmental exposure risk of microplastics pollution (6). Relevant research shows that microplastics enter the human body and accumulate in tissues and organs mainly through diet (food and drinking water contaminated by microplastics), or inhalation and direct contact (7). Scientists have conducted a series of in vitro and in vivo studies to evaluate the potential toxicity of microplastics. Some studies show that microplastics cause damage to intestinal cells and change the internal microenvironment (8–9). Microplastic can also induce endoplasmic reticulum and oxidative stress effects in lung epithelial cells (10). Further more, microplastics can induce cytotoxic and inflammatory effects in lung cells (11). The study also showed that microplastics caused damage to chicken cardiac myocytes (12). In short, microplastics cause a series of health damage in vivo and in vitro models (2).

Once the microplastics pass through the gastrointestinal barrier, they can enter the blood and distribute to various tissues and organs through the human blood system, thus posing a potential threat to the tissues or organs of the whole body. Relevant studies have reported that MPs can interact with red blood cells and be absorbed by cells through hydrogen bonding, van der Waals force and electrostatic interaction (13). Microplastic can also penetrate secondary barriers such as placenta and blood brain through blood transport (14). Yang et al. confirmed that 20 nM microplastics have obvious blood-brain barrier permeability. Therefore, once the microplastics break through the tissue barrier, it is very easy to carry out extensive accumulation and distribution in human tissues and organs, and cause the activation of the immune system to trigger the occurrence of inflammatory reaction (15). However, up to now, the potential biological effects of microplastics on blood vessels have not been revealed.

Aging is an important scientific problem facing the world. As a circulatory organ, blood vessel is responsible for the transport of blood throughout the body, and it is one of the most important circulatory organs. Therefore, the aging-caused damage of blood vessels will induce the corresponding damage of tissues and organs of the whole body. Therefore, in the current study, we evaluated the impact of microplastics on the health damage of blood vessels. In vitro cell model, we found that microplastics led to the aging of blood vessels by detecting a series of aging marker molecules. The mechanism study shows that the micro-plastics may activate ROS through calcium overload, and then ROS activates CDK5. The activated CDK5 regulates the degradation of Lamin A, and finally causes the aging damage of blood vessels. In vivo research, we also found that microplastics induce the aging of blood vessels, and also trigger the inflammation reaction and oxidative stress. This study lays a foundation for further research of health hazards of microplastics on circulatory system.

Microplastic caused aging damage of blood vessels in vivo

We first evaluated the effect of microplastics on vascular histopathological changes. As shown in Fig. 1A, the intima of the aorta in control group was intact and the endothelial cells were orderly arranged. In the microplastic treatment group, aortic endothelial cells proliferated with inflammatory infiltration. The thickness of the arterial vessels was increased in the microplastics treatment group.

In addition, we evaluated the expression of p16 and p21 in blood vessels, and found that their expression was up-regulated in the microplastic treatment group compared with the control group (Fig. 1B). Furthermore, the expression of p65 and α-SMA was significantly up-regulated (Fig. 1B). Additionally, we also evaluated the effect of microplastics on H3K27me3 (heterochromatin maker) and γH2AX (DNA damage marker), Fig. 1C shows that the expression of H3K27me3 decreased, but The expression of γH2AX increased under the treatment of microplastics. These experiment results shows that microplastics can induce the aging of blood vessels in vivo.

Studies have shown that aging is closely related to oxidative stress. Therefore, the effect of microplastics on oxidative stress of blood vessels was evaluated. The results showed that microplastic treatment induced oxidative stress in vascular tissue, and the fluorescence signal of ROS increased significantly compared with the control group (Fig. 2A). Besides, the level of MDA increased, but the expression level of SOD, CAT, GSH and CAT decreased significantly. The expression level of 8-OHdG (a marker of the oxidative stress response) was increased. Additionally, the expression of Nrf2 was significantly reduced (Fig. 2C).

Furthermore, we evaluated the effect of microplastics on vascular inflammation. ELISA results showed that microplastics caused TNFα, IL-1β and IL-6 level significantly increased (Fig. 3A). Moreover, the results of immunohistochemistry also showed that the expression of IL-6, TNFα and IL-1β was significantly increased in the microplastic treatment group (Fig. 3B). Additionally, the expression of IL-8 (Fig. 3C) and MCP-1 (Fig. 3D) was also significantly reduced.

Microplastic internalized into vascular endothelial cells

Here, we first evaluated whether microplastics can internalize into HCAECs and HUVEC. Therefore, cells were treated with different concentrations of microplastics (0, 0.1, 0.3, 0.6, or 0.9 mg/mL) for 90 min, and results exhibited that microplastic can internalize into HCAECs and HUVEC with a dose-dependant manner. Furthermore, the cells were incubated with 0.6 mg/mL microplastics at different time points, we found that the internalization of microplastics into the cells showed a time-dependent manner (Fig. 4B), and the absorption of microplastics by the cells reached the highest point at 40–60 min.

Microplastic caused vascular cell aging in vitro

In vitro experiments, we used two cell models (HCAECs and HUVEC) to evaluate the effect of microplastics on cell senescence. Sa-β-gal staining displays that the proportion Sa-β-gal-positive cell increased significantly (Fig. 5A). Furthermore, Western-blot showed that the expression of p16, p21 and p53 (aging marker) increased significantly in HCAECs and HUVEC cells (Fig. 5B). These results indicates that the vascular cells were obviously senescent. Further experiments showed that the cell cycle was blocked in the microplastic treatment group (Fig. 5C). According to the above research results, in the subsequent experiment, we selected 0.6 mg of microplastics for the following experiment.

Oxidative stress is key factor for microplastic-induced cell senescence

Previous studies have shown that microplastics can cause oxidative stress reaction (16). In the current study, we also found a similar phenomenon. The microplastics treatment led to a significant increase in ROS level (Fig. 6A), indicating that mitochondria were subject to obvious oxidative stress under microplastic treatment. In vivo, we also found that ROS increased in the microplastics treatment group (Fig. 2). To verify whether ROS-mediated oxidative stress is the main factor leading to cell senescence, we used ROS inhibitor (ROS scavenger NAC, 20 µM; mitochondrial-specific ROS scavenger, Mito-TEMPO with 10µM for 1 h). The results showed that ROS significantly decreased through MitoSOX staining (mitochondrial ROS-specific probe) (Fig. 6B, upper panel). We also found that cell senescence was significantly alleviated (Fig. 6B, down-panel). These experimental results showed that cell senescence induced by microplastics was caused by ROS-mediated oxidative stress.

So what mechanism does microplastics cause mitochondrial ROS accumulation? Previous studies have shown that ROS accumulation is related to the calcium overload in mitochondria, which can induce oxidative stress (17). Furthermore, research shows that microplastics can improve Ca²⁺ signaling (18). To sum up, we propose the hypothesis that microplastics cause ROS accumulation (oxidative stress) through calcium overload. For this, we first detected the calcium level using calcium fluorescent probe (Fluo 4AM). The confocal microscope observation results showed that the signal of Ca²⁺ increased significantly under microplastics stimulation (Fig. 6C), which is consistent with previous research report [18]. Further, we studied whether calcium overload caused ROS accumulation in mitochondria. We found that when calcium was inhibited by using calcium chelating agent (BAPTA, 20 µM). It was found that the mitochondria ROS decreased significantly. These research results indicated that mitochondrial calcium overload caused ROS accumulation (Fig. 6C, down panel). Furthermore, we detected the mitochondrial calcium level using the mitochondrial calcium-specific fluorescence probe (Rhod-2 AM), and found that the mitochondrial- specific calcium fluorescence signal also significantly increased under microplastics treatment, indicating that the mitochondrial calcium overload occurred under the treatment of microplastics (Fig. 6D). We further found that the cell senescence was significantly alleviated after using calcium chelating agent (BAPTA), suggesting that calcium overload was the upstream factor of mitochondrial ROS accumulation (Fig. 6E).

The endoplasmic reticulum (ER) is the largest intracellular calcium pool. Whether the entry of calcium from endoplasmic reticulum into mitochondria is the main cause of mitochondrial oxidative stress (ROS accumulation)? Therefore, we further explore whether the calcium is derived from endoplasmic reticulum. Previous studies have shown that the release of endoplasmic reticulum calcium is closely related to IP3R of ER (19). Therefore, after using IP3R-specific inhibitor (Xestospongin C: XSC, 5 µM), we found that the calcium level in mitochondria was significantly reduced (Fig. 6F), indicating that calcium entered the cytoplasm from the endoplasmic reticulum, and then transfers into mitochondria. In order to verify that calcium enters into the mitochondria through the channel on the mitochondria, we have carried out relevant experiments. VDAC has been reported to be able to regulate calcium transport into mitochondria (20). For this, the mitochondrial VDAC was inhibited by using the VDAC-specific inhibitor (the cells were pretreated with VBIT-4 (10 mM) for 12 h). The results showed that the amount of calcium located in mitochondria was significantly reduced (Fig. 6G), indicating that calcium entered into mitochondria through VDAC mediation, which in turn induced oxidative stress.

Microplastic leads to the degradation of nuclear fiber layer mediated by ROS

Cell senescence is closely related to the nuclear lamina. The instability of the nuclear lamina will directly cause the instability of the genome, and then resulting in cell aging (21). In the current study, we found that Lamin A showed obvious downregulation, and the nuclear morphology also changed, and the cell nuclei significantly enlarged in the microplastic treatment group (Fig. 7A-C). In addition, we found that the expression of Lamin A/C (nuclear lamina is closely related to cell senescence) was significantly down-regulated in the microplastic treatment group (Lamin A participated in cell senescence, and it participated in cell senescence process by controlling the stability of genome). In order to study whether oxidative stress (ROS) causes Lamin A to lose its homeostasis. After the cells were treated with ROS inhibitor, the expression of Lamin A was restored (Fig. 7D). Therefore, we assume that ROS induced by microplastics can lead to the loss of stability of Lamin A, resulting in cell senescence. So, the molecular mechanism of ROS inducing Lamin A down-regulation is a scientific question that needs to be answered. For this reason, we first look for the answer at the gene level, and found that the expression of Lamin A gene did not change after microplastic treatment compared with the control group (Fig. 7E). However, Lamin A did decrease significantly at the protein level, suggesting that the decrease of Lamin A expression caused by microplastics may be achieved by affecting protein degradation rather than by regulating gene expression. Previous studies have shown that the dissociation/degradation of Lamin A is induced by Caspae 6 or autophagy (22). Therefore, Caspae 6-specific inhibitor (10 µM Z-VEID-FMK) or the autophagy inhibitor (40 µM 3-methyladenine, 3-MA) were used to treat cells, respectively. The results showed that Lamin A expression was not significantly increased compared with the control group. In contrast, when proteasome inhibitor MG132 (10µM) was used, the Lamin A expression was significantly restored, indicating that the degradation of Lamin A might depend on the ubiquitin-proteasome pathway (Fig. 7F). To further validate this question, the the inhibitor of ubiquitin activating enzyme E1 (1 µM PYR-41) was used, the results showed that they could increase the expression of Lamin A. Taken together, these evidences show that Lamin A is degraded by ubiquitin protease system (Fig. 7G).

Next, we asked the molecular pathway by which ROS induced the degradation of Lamin A. Recent studies demonstrated that lamin A phosphorylation is required for its degradation (23). It has been reported that Cdk1, Cdk5, and Akt may induce Lamin A phosphorylation, and in turn promote Lamin A degradation. For this, Cdk1, Cdk5 or AKT inhibitor was used, respectively. The experimental results showed that inhibition of Cdk1/2/5 by rosivitine completely blocked lamin A degradation, whereas the Cdk1/2 inhibitor (BMS-265246) and the Akt inhibitor (MK-2206) did not inhibit lamin A degradation (Fig. 8A), suggesting that CDK5 plays an important role for lamin A degradation. Recent studies have shown that CDK5 can phosphorylate Lamin A and lead to lamin A degradation (24). In order to further study and prove whether CDK5 is required for lamin A degradation under microplastic treatment, CDK5 was silenced using SiRNA, and results found that the lamin A degradation was significantly relieved (Fig. 8B). In addition, γ-H2AX (DNA damage maker) and H3K27me3 (heterochromatin marker) were also significantly reduced, and cell aging was also significantly reduced (Fig. 8C). These studies showed that the degradation of CDK5-induced Lamin A was involved in the cell senescence induced by microplastics. Moreover, research shows that ROS can induce the activation of CDK5 (25). To this end, ROS was removed using ROS scavenger (NAC, 20 µM), and found that the activity of CDK5 was inhibited (Fig. 9, Histone1 as the substrate of CDK5, which can be used to evaluate the activity of CDK5), and the degradation of Lamin A was significantly reduced (Fig. 9). To sum up, in the current study, we propose the molecular mechanism by which microplastic-induced vascular cell senescence (Fig. 10).

Microplastic is called a new pollutant. A series of studies show that most marine fish contain microplastic (even deep-sea fish) (1). So far, microplastics have been found in many organs of different animal species (2). Research shows that the average human intake of plastic is 5000 mg per week (26). The previous research shows that there may be a risk of long-term accumulation of micro-plastics in human body due to food chain pollution. Human body fluid is a complex physiological environment, including a variety of bioactive molecules, such as proteins. However, microplastics can easily interacted with biological active molecules such as proteins to form "protein crowns" (27). The interaction between microplastics and bioactive molecules is mainly through hydrophobic bonds, hydrogen bonds or electrostatic electricity (28). Although microplastics have been found in the blood circulation system, up to now, the effect of microplastics on vascular aging damage has not been reported. Therefore, in the current study, we carried out experimental research on this problem, and found that microplastics lead to vascular aging, inflammation and oxidative stress in vivo and in vitro models.

First of all, in vivo research, we found that microplastics can cause the aging of vascular cells by evaluating a series of aging-related marker molecules, such as p16 and p21, etc. The dosage of microplastics used in this work is 0.3 mg and 0.6 mg twice per week for 8 weeks. It has been reported that the average human ingests 5,000 mg of plastic per week [26]. This is equivalent to 80.64 mg/kg of micro-plastic per week. In the current study, the dose of microplastics used is about 40 mg/kg. We found that microplastics caused the senescence of vascular cells. In addition, microplastics also cause inflammation of vascular tissue. Besides, previous studies have shown that microplastics also cause damage to other tissues, for example, Karbalaei et al reported that microplastics cause inflammatory damage to kidney cells (3).

Next, in vitro cell model, we found that microplastics can internalize into vascular cells, and found that some microplastics enter the cell nuclei. Previous studies have also found that microplastics can internalize into cells in different cell lines (29). (7) found that the internalization of microplastics entered into human kidney proximal tubular epithelial cells. Additionally, research indicates that microplastics can also be found to enter different tissues and organs in vivo. However, up to now, it is still not clear how microplastics are internalized into cells. Furthermore, it is still not clear which intracellular molecule(s) interact with internalized micro-plastic. These scientific problems need to be solved in future research.

In addition, we found that microplastics can cause oxidative stress of vascular cells. The experimental results showed that the ROS level increased significantly when the cells were stimulated with microplastics, indicating that the cell undergoes oxidative stress reaction. It is well known that ROS is one of the important inducements of cell aging [30]. Moreover, previous studies have shown that the mitochondrial calcium overload can promote ROS production (31). Therefore, we detected the intracellular calcium level. Under the stimulation of microplastics, we found that the calcium was significantly up-regulated. Furthermore, we found that the micro-plastics induced ROS production by inducing mitochondrial Ca²⁺ overload through a series of biochemical experiments.

SASP is one of the markers of senescent cell, secreted by senescent cell. SASP secreted by aging cells has high heterogeneity in different types of cells, but NF- κ B is considered to be one of the core regulators of SASP. In the current work, we found that the expression of inflammatory factors was significantly up-regulated, including TNF α, IL-6 and IL-1 β. Previous studies have also found that microplastics can also cause inflammatory reactions in cells or tissues (32).

It is well known that cellular senescence is closely related to the stability of the lamin A. Premature aging is a typical example [33–34]. Here, we found that microplastics caused a significant decrease in Lamin A, microplastic-induced Lamin degradation may be important factor for vascular cell senescence. We continue to pursue the potential molecular mechanism of the down-regulation of Lamin A caused by microplastics, and found that the degradation of Lamin A is closely related to CDK5. ROS can activate CDK5 (as the upstream of CDK5), which may be the potential molecular mechanisms of Lamin A degradation induced by microplastics. Previous studies have shown that in addition to CDK5, there are many molecules that can activate Lamin A degradation, such as CDK1/2, but these possibilities are excluded from the current study (22). Interestingly, the relationship between Cdk5 and ROS is mutual. CDK5 can activate ROS, and ROS can also activate CDK5, but its molecular mechanism is still unclear. In addition, there are many factors that cause aging, such as telomere shortening and DNA damage. DNA damage is one of the typical mechanisms of inducing cell senescence. DNA double-strand break (DSB) triggers DNA damage response (DDR) (35), thereby promoting cell senescence. Here, we have detected the expression of γH2AX was significantly increased in the micro-plastic treatment group. However, its specific molecular mechanism needs further explanation in future work.

To sum up, in this study, we found that microplastics caused aging damage of blood vessels in vivo and in vitro models. In terms of mechanism, we found that microplastics may play its biological role through the signal axis of Ca²⁺/ROS/CDK5/laminA. This work has laid a research foundation for further exploring the health damage of microplastics to blood vessels.

Antibodies and reagents

The 1- μM PS-MPs were obtained from BaseLine company (Tianjin, China). The microplastics were characterized by laser confocal microscopy and electron microscopy (please see supplementary Figure 1). Superoxide dismutase (SOD) kit, Catalase (CAT) kit, glutathione peroxidase (GSH-Px) kit, and Malondialdehyde (MDA) kit were purchased from Solarbio Biotechnology. Human IL-1β ELISA Kit (PI305), Mouse IL-1β ELISA Kit (PI301), Mouse IL-6 ELISA Kit (PI326), human IL-6 ELISA Kit (PI330), human TNF-α ELISA Kit (PT518), Mouse TNF-α ELISA Kit (PT512) were purchased from Beyontime (Shanghai, China). anti-p16 (Cat no, AF1672, 1: 1500) was purchased form Beyotime company (Shanghai, China). β-actin (Cat no. AC006, 1:1500) and anti-CDK5 antibody (A18080) were purchased from abclonal company (UK). SOD detection kits (No. S0033S) and MDA detection kits (S0131S) were purchased from Beyotime biotechnology Company (China). Anti-Lamin A antibody (ab226198), p16^INK4aand anti-γ-H2A.X antibody (Cat no. ab81299, 1:1000) were purchased from Abcam (UK). Anti-IL-1 beta (Cat no. ab254360), anti-TNF alpha (Cat no. ab307164, 1:1000), and anti-p21 antibody (Cat no. ab109199) were obtained from Abcam company (UK). The galactosidase staining kit (C0602) was purchased from Biyuntian Company.

Cell culture

HCAECs and human umbilical vein endothelial cell line (HUVEC) were used as cell models in vitro in current experiments. The cells were incubated in 37 ℃ constant temperature carbon dioxide (5%) incubator. The cells were cultured in DMEM medium containing 10% serum.

Detection of cellular reactive oxygen species

The cells were digested, centrifuged, resuspended and counted. The number of 1×10⁵ cells per well was inoculated into the 6-well cell culture plate, and the cell culture medium was added (the final volume of each well was 2 mL). After cells were stimulated with micro-plastics. The 6-well plate cells were removed from the incubator and the cell culture solution was discarded. After washing with PBS for one time, the DCFH-DA probe diluted with serum-free culture medium was added to the cell at 37℃ for incubation for 20 minutes. After washing, 1 mL of serum-free cell culture medium was added. Cell samples were analyzed using laser confocal microscopy or Flow cytometry.

Western-blot

Cells were rinsed twice with precooled PBS, 1mL cell lysate (1% SDS; 50mM Tris-Cl PH7.5; 5mM EDTA; 120nM NaCl), was added and reacted on ice for 30 min. The cells were collected and transferred into a 1 mL centrifuge tube. After centrifugation at 15000 rpm for 15 min, the supernatant was collected. The protein was quantified using BCA method. After SDS-PAGE electrophoresis, protein transfer was performed using BIO-RAD mini Trans-Blot Electrophoretic Transfer cell. PVDF membrane was immersed in 5% skimmed milk powder to block non-specific binding sites. The sealed PVDF was immersed in the first antibody and incubated at 4 ℃ overnight. The PVDF membrane was washed three times (5min each time), and the PVDF membrane was immersed in TBST diluted secondary antibody (1:3000) and incubated at room temperature for 60 min. ECL (Merck Millipore) was added to detect immunoprotein band.

Sa-β Gal staining

The Sa-β- gal experiment was carried out according to the instructions of the kit. In short, washing cells using 2mL PBS, 1mL fixed solution was added and fixed for 15 minutes. After the fixed solution was discarded, 2mL PBS was used to wash cells. 1mL of staining solution was added to each well of the cell culture plate. The culture plate was incubated in an incubator with a constant temperature of 37℃ (no CO₂) for 12-16h. The 6-well plate is sealed with a sealing film to prevent the evaporation of the dye solution in the culture plate. Cell samples were observed under microscope.

qRT-PCR analysis

The cells were rinsed twice with PBS at 4℃, and 1mL Trizol was added. The cells were lysed. 200 uL of chloroform was added at 4 ℃ and centrifuged at 15000 rpm for 15 min. The supernatant was sucked out and transferred to a new centrifuge tube. An equal volume of isopropanol is added and mixed at room temperature for 10 min. The cells were centrifuged at 4℃ 15000 rpm for 10 min. The supernatant was discarded, and 75% ethanol was added for 12 h overnight. After centrifugation at 7500g for 5 min at 4℃, 10 ul of DEPC H₂O was added and mixed. RNA concentration was detected by Nano Drop2000. RNA was reverse transcribed into cDNA, RT-PCR was then performed.

SiRNA transfection

2×10⁵ cells were evenly inoculated in a six-well cell culture plate. When the confluence of cells reached 50%~60%, the prepared polyplus and siRNA mixture (buffer 200 μ l+Polyplus 4 μ L+siRNA 50nM) was added into the medium, and the cells were put back into the cell incubator for further culture. The transfection efficiency was observed by fluorescence microscope after the cells were cultured for 48-72 hours.

Indirect immunofluorescence

After the cells were inoculated into the cell culture plate for 24h. After washing, 4% paraformaldehyde was added to fix cell for 15 minutes. After three washes (5 minutes each time), 0.5% Triton X-100 was added to the cells for 30 minutes. After washing, 5% BSA solution was added to the cells for 1 hour. The blocking solution was discarded. After PBS was used to wash the cells three times (5 minutes each time), the first antibody was added, and the samples were placed at 4 ℃ overnight. After the PBS was washed three times (5 min each time), the fluorescent secondary antibody was added (1% BSA diluted fluorescent secondary antibody) and incubated in dark at room temperature for 2 h. DAPI was used to stain the nucleus for 10 min. The sealing solution containing anti-fluorescence quenching agent was dripped, and the sample was observed using a laser confocal microscope.

Extraction of subcellular tissue protein

Nuclear protein and cytoplasmic protein were extracted using commercial kits, and the specific operation process was carried out according to the manufacturer's instructions.

Detection of cell cycle

The cells were inoculated in the 6-well cell culture plate, and wait for the cells to fully adhere to the wall. The cells were washed once with PBS, and the serum-free cell culture medium was added to cells for 12 hours to synchronize the cell cycle. The cells were washed once using PBS. The normal cell culture medium containing microplastics (the final concentration of microplastics was 0.1-0.9 mg) was added. After microplastic treatment, cells were collected and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and 1ml PBS was added to wash cells for 3 times. After the cells were centrifuged again, 1 ml of pre-cooled 70% ethanol was added and mixed at -20 ℃ for 12 hours. Cell samples were centrifuged for 5 min (1000 rpm). After the supernatant was discarded, 1 ml of pre-cooled PBS was added to wash cells. The propidium iodide staining solution containing 10 ul of RNase A was added and resuspended for cell precipitation, and incubated in dark at 37 ℃ for 30 minutes. Flow cytometry was used to analyze and detect cell samples (BD).

Detection of apoptosis

Cells were collected, 1-5×10⁶/mL cells were centrifuged by 1000 r/min (5 min).The culture medium was discarded. The cells were washed with PBS for three times, and the cells were fixed with pre-cooled 70% ethanol at 4 ℃ for 8 hours. After centrifugation, 3mL PBS was added to the resuspended cells. The cells were filtered with 300 nylon mesh and centrifuged by 1000 r/min for 5 min. 1ml of PI dye solution was added and dyed in dark at 4℃ for 30 min. Cell samples were detected by Flow cytometry (BD Accuri ® C6).

Analysis of microplastic internalization

The cells were inoculated on the coverslip, the fluorescent labeled PS-MPs was added (0.1-0.9 mg/mL) and incubated for 90 minutes. After washing (5 minutes each time), 4% paraformaldehyde was added for 15 minutes. After washing with TBST containing 0.05% Tween-20 three times, DAPI was used to stain the nucleus, and the cell samples were then observed by using Leica laser confocal microscope.

Detection of intracellular SOD activity

After the cells were treated with microplastics, the cells were washed with precooled PBS once, 150 ul of precooled PBS solution was added to the resuspended cells. The samples were treated with ultrasound (power 200 W, ultrasound 3 s, interval 10 s, repeating 30 times). Cell samples were centrifuged at 12000 rpm for 15 min at 4℃, and the supernatant was collected. The BCA protein concentration determination kit was used to detect the protein concentration. The protein sample was then placed in a 96-well plate at 37ºC and incubated in dark for 40 minutes. The absorbance of the cell sample was detected using the ELISA reader at 560nm wavelength.

Animal administration

The 6-week-old male C57BL/6 mice were purchased from Huafukang Experiment Animal Company. All procedures were approved by the institutional animal Care and Use Committee of Guangzhou Medical University. The mice were cultured in 12 hours/dark cycle and 55% ±10% relative humidity at 22 ± 2℃for one week. In the current work, the experimental animals were administered with PS-MPs (1 μm) by oral gavage at 0.3 mg and 0.6 mg twice per week for 8 weeks. The mice in control group were administrated with water. The mice were randomly divided into three groups with 8 in each group. After the experiment, cardiovascular tissues were collected, and some samples were fixed with 10% formalin for further histological analysis. At the same time, blood was also collected.

HE staining analysis

The section was subjected to routine dewaxing treatment, and the specific treatment process is as follows: xylene I 15min, xylene II 15min, anhydrous ethanol I 5min, anhydrous ethanol II 5min, 95% ethanol 5min, and 80% ethanol 5min. After the sample was washed with running water, distilled water was used to soak the tissue slices. After the sections were stained with hematoxylin for 5 min, running water was used to wash the sections for 5 min. 0.5% eosin dye solution was added to the section and incubated for 30s. After washing the sample with running water for 15 min, the slice is dehydrated according to the following process (80% ethanol for 5 min, 95% ethanol for 5 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, xylene for 5 min, xylene for 5 min). After neutral gum sealing, HE-stained section tissue was observed under microscope.

Immunohistochemical analysis

The slices were incubated at RT for 0.5 h with 3% H₂O₂ to block endogenous peroxidase. The slices was rinsed with running water for 15 min and 0.1M PBS for 5min (for 3 times). After the antigen was repaired, the sections were washed with for 3 times (5 min each time). After the antibody was evenly dropped and completely covered the tissue, the sections were placed in a 4℃ refrigerator for incubation overnight. In the negative control group, PBS was used instead of the first antibody. After washing, the second antibody was dropped to cover the section sample and incubated in a 37 ℃ incubator for 30 minutes. After washing (5 min each time), DAB color kit was used for color development. The section sample was observed under a microscope.

Detection of pro-inflammatory factors

ELISA kit was used to detect IL-1β, IL-6 and TNF- α Level. The serum samples were added and incubated in the micro-well plate (96 well) for 2 hours at 37 ℃. The specific enzyme-linked immune antibody was added and incubated at 37℃ for 1 h. TMB was added, and the absorbance of OD450 wavelength was detected using a multi-function enzyme marker.

Statistical analysis

SPSS22.0 was used for data analysis. The experimental data was expressed by mean± standard deviation. The comparison between multiple groups was performed by one-way ANOVA. The difference between the two groups was tested by t-test. P<0.05 indicated that the difference was statistically significant.

Acknowledgments

None.

Credit authorship contribution statement

Kaihao Wang: Experiment design and Paper writing.

Zheng Huang：Guidance and supervision.

Yipeng Du, Peixin Li, Chang Guan，Zengfu Liu：cell experiments.

Min Zhou，Lanlan Wu：statistical analysis of data.

All authors discussed theresults and assisted in the preparation of the paper.

Funding

This work is financially supported by the Guangzhou Medical and Health Technology Project (20171A010301)；

Guangzhou Science and Technology Project (202102010177);

Guangzhou Medical and Health Technology Project (20221A011075).

Availability of data and materials

Data analyzed during the current study are available from the corresponding author upon request.

Ethics approval and consent to participate

All procedures were approved by the institutional animal Care and Use Committee of Guangzhou Medical University.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Author details

¹Department of Cardiology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China

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No competing interests reported.

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Microplastics accelerates the premature aging of blood vessels though ROS-mediated CDK5 signaling pathway

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Abstract

Figures

Background

Results

Microplastic caused aging damage of blood vessels in vivo

Microplastic internalized into vascular endothelial cells

Microplastic caused vascular cell aging in vitro

Oxidative stress is key factor for microplastic-induced cell senescence

Microplastic leads to the degradation of nuclear fiber layer mediated by ROS

Discussion

Conclusion

Methods

Declarations

References

Additional Declarations

Supplementary Files

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