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ROS-AKT-mTOR axis mediates autophagy of human umbilical vein endothelial cells induced by cooking oil fumes-derived fine particulate matters in vitro
Free Radical Biology and Medicine 113 (2017) 452–460
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Original article
ROS-AKT-mTOR axis mediates autophagy of human umbilical vein endothelial cells induced by cooking oil fumes-derived fine particulate matters in vitro
MARK
Rui Dinga,1, Chao Zhanga,1, Xiaoxia Zhub,1, Han Chenga, Furong Zhua, Yachun Xua, Ying Liuc, ⁎ ⁎⁎ Longping Wend, , Jiyu Caoa,e, a
Department of Occupational and Environmental Health, School of Public Health, Anhui Medical University, Hefei, Anhui, China Department of Chronic Disease Control and Prevention, Shanghai Putuo District Center for Disease Control and Prevention, Shanghai, China Department of Maternal, Child and Adolescent Health, School of Public Health, Anhui Medical University, Hefei, Anhui, China d School of Medicine, South China University of Technology of China, Guangzhou, Guangdong, China e Teaching Center for Preventive Medicine, School of Public Health, Anhui Medical University, Hefei, Anhui, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Cooking oil fumes PM2.5 Reactive oxygen species Autophagy Human umbilical vein endothelial cells
Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) exposure can induce oxidative stress and cytotoxic effects. Here we investigated the role of ROS-AKT-mTOR axis in COFs-derived PM2.5-induced autophagy in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with different concentrations of COFs-derived PM2.5, together with or without N-acetyl-L-cysteine (NAC, a radical scavenger) or 3-methyladenine (3-MA, an autophagy inhibitor). Cell viability was assessed with MTT assay, and ROS level was measured with DCFH-DA assay after the treatment. Transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes, while immunofluorescent assay and western blot were used to assess the expression of LC3-I/II and beclin 1. Proteins involved in the PI3K-AKT-mTOR signaling pathway were measured with western blot. The results showed that the treatment of COFs-derived PM2.5 dose-dependently reduced the viability of HUVECs and increased the ROS levels in the cells. Both immunofluorescent assay and western blot showed that treatment with COFs-derived PM2.5 significantly increased LC3-II and beclin 1 levels, as well as the ratio of LC3-II/LC3-I, which could be rescued by the co-incubation with NAC or 3-MA. TEM also confirmed the increased formation of autophagosomes in the cells treated with COFs-derived PM2.5, while co-treatment with NAC evidently decreased autophagosomes formation. In addition, western blot also showed that the phosphorylation of PI3K, AKT, and mTOR all decreased by the treatment of COFs-derived PM2.5, which was effectively rescued by the co-treatment with NAC. These findings demonstrate ROS-AKT-mTOR axis plays a critical role in HUVECs autophagy induced by COFs-derived PM2.5.
1. Introduction Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) is an important source of indoor air pollution, especially in East Asian countries such as China. In contrast to ambient PM2.5, COFs-derived PM2.5 contains more noxious components, such as polycyclic aromatic hydrocarbons, and could persistently exist in the microenvironment at
relatively high concentration [1]. Although the China Food-Based Dietary Guidelines (FBDGs, 2008) issued by the Ministry of Health recommended that the daily intake of fats and oils should be 25–30 g per reference man [2], the actual consumption in China is much higher [3], thus the contribution of COFs-PM2.5 to indoor air pollution in Chinese families is substantial. In a previous survey, we found that the COFsderived PM2.5 level was as high as 1.679–7.023 mg/m3 in domestic
Abbreviations: PM2.5, Fine particulate matter of < 2.5 µm in aerodynamic diameter; COFs, Cooking oil fumes; LBW, Low birth weight; ROS, Reactive oxygen species; PI3K, Phosphatidylinositol 3-kinase; mTOR, Mechanistic target of rapamycin; HUVECs, Human umbilical vein endothelial cells; DMSO, Dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide; DCFH-DA, 2’,7’-dichlorofluorescin diacetate; NAC, N-acetyl-L-cysteine; TEM, Transmission electron microscopy; DAPI, 4',6-diamidino-2-phenylindole; PI, Propidium iodide; 3-MA, 3-methyladenine; TBST, Tris-Buffered-Saline with Tween; PAHs, Polycyclic aromatic hydrocarbons; BaP, Benzo(α)pyrene; ANOVA, Analysis of variances ⁎ Corresponding author. ⁎⁎ Corresponding author at: Teaching Center for Preventive Medicine, School of Public Health, Anhui Medical University, Meishan Road 81, Hefei, Anhui, China. E-mail addresses: [email protected] (L. Wen), [email protected] (J. Cao). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.386 Received 6 May 2017; Received in revised form 26 October 2017; Accepted 27 October 2017 Available online 28 October 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved.
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the oil surface (Supplementary Fig. S2). The filter paper was renewed every 2 h. Then the filter paper with fumes attached was processed separately with 50 ml acetone for 24 h in Soxhlet extractor. The extracts were dried by rotary evaporation at 40 °C, and then further evaporated to yellow viscous solution. Dimethyl sulfoxide (DMSO) was used to dilute the solution to 200 mg/ml. The solution was transferred to brown glass vials, sealed, and stored at −80 °C until use. For the experiments in this study, the stoke solution was diluted to 12.5, 25, 50, 100, and 200 μg/ml with the cell culture medium. The compositions of the COFsderived PM2.5 have already been measured and reported by our group [1].
kitchen in China (Supplementary Table S1), which was confirmed by another recent study [4]. COFs-derived PM2.5 could especially affect the women in China, as they are generally the ones cooking food for the families. While for the pregnant women, such exposure could possibly affect the fetus. Previous studies in our group have demonstrated that maternal exposure of COFs-derived PM2.5 during pregnancy could lead to low birth weight (LBW) in mice (Supplementary Fig. S1). However, the underlying mechanisms are still unclear. Umbilicus is the only link between the mother and fetus during pregnancy, and is responsible for the exchange of oxygen, nutrients, and waste materials between fetus and mother [5]. Veras et al. found that ambient PM2.5 could induce oxidative stress and alter endothelin receptor expression to affect the umbilical cord blood vessels, and therefore contribute to the decreased fetal weight [6]. Peyter et al.[7] also demonstrated that the umbilical cord diameter was significantly lower in growth-restricted newborns than appropriate for gestational age term newborns; in addition, the relaxation of the umbilical vein induced by nitric oxide was significantly attenuated in growth-restricted neonates than controls. Previous in vitro [8] and in vivo [9] studies in our group have further confirmed that COFs-derived PM2.5 exposure could evidently affect the umbilical cord blood vessels. These findings suggest that PM2.5 exposure could increase the risk of endothelial dysfunction of the umbilical cord blood vessels, and possibly increase the risk of decreased fetal weight. Previous studies have shown that COFs-derived PM2.5 can induce oxidative damage not only to type II alveolar epithelial cells in vitro [1], but also to umbilical cord blood vessels in vitro and vivo [8,9]. Oxidative stress can cause severe damages to DNA, RNA, and proteins, and thus trigger autophagy and apoptosis [10]. Autophagy enables eukaryotic cells to capture cytoplasmic components for degradation within lysosomes. Although baseline autophagy is important for the maintenance of normal cellular homeostasis, abnormal autophagy contributes to many physiological and pathological processes, including morphogenesis, cancer, neurodegenerative disorders, and infectious diseases [11,12]. Therefore, we hypothesize that the elevated ROS production induced by the exposure of COFs-derived PM2.5 could disturb the balance of autophagy in umbilical vein endothelial cells, thus consequently impair the functions of umbilical veins. PI3K/AKT/mTOR pathway plays an important role in the modulation of cell autophagy [13]. Mechanistic target of rapamycin (mTOR) is one of the major modulators of autophagy that can be regulated by various signaling pathways [14]. For instance, several studies have demonstrated that the PI3K/AKT/mTOR signaling pathway participates in the regulation of autophagy induced by PM2.5 exposure [15,16]. However, the effects of COFs-derived PM2.5 exposure on autophagy of human umbilical vein endothelial cells (HUVECs), as well as the role of the PI3K/AKT/mTOR signaling pathway in this process have not been investigated to date. Here we investigated the effects of COFs-derived PM2.5 on autophagy of HUVECs and the role of the ROS-AKT-mTOR axis in this event. The findings of this study could provide experimental evidence of the adverse effects of COFs-derived PM2.5 on the functions of umbilical cord.
2.2. Cell culture This study was approved by the Ethics Committee of Anhui Medical University. HUVECs were kindly provided by the School of Life Sciences, University of Science and Technology of China. The cells were cultured with DMEM culture medium (supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was changed every day. Cells were used in the following experiments after the culture reached 80% confluence. 2.3. Cell viability assay Cytotoxicity of COFs-derived PM2.5 was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In brief, HUVECs were incubated with 0 (control, containing 1‰ DMSO), 12.5, 25, 50, 100, and 200 μg/ml of COFs-derived PM2.5 for 12, 24, and 36 h in 96-well culture plate, respectively. To confirm the effects of ROS on cell viability, N-acetyl-L-cysteine (NAC), a ROS inhibitor, was used along with different concentrations of COFs-PM2.5 for 24 h. Then the culture medium was carefully discarded, 200 μl MTT solution (prepared with the culture medium at a ratio of 9:1) was added to each well, and the plate was incubated for another 4 h at 37 °C. The formazan crystals were dissolved with 150ul of DMSO, and the absorbance in each well was read at 490 nm using ELx800 microplate reader (Bio-TEK, USA). 2.4. ROS measurement To determine the ROS levels in the cells, the HUVECs were loaded with the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFHDA). The HUVECs were seeded into 6-well plates with 5.0× 105 cells/ well. Before the following treatment, the cells were synchronization for 24 h by serum starvation. According to the results of the MTT assay, the cells were treated with different concentrations (0, 25, 50, 100 μg/ml) of COF-derived PM2.5, 100 μg/ml COF-derived PM2.5+10 mmol/L NAC, and 10 mmol/L NAC for 12, 24, and 36 h, respectively. The fluorescent probe DCFH-DA was added to each well with the final concentration of 10 μM and incubated at 37 °C for 30 min in dark. The plate was washed three times with PBS, trpsinized, re-suspended, and immediately subjected to fluorospectrophotometer analysis and fluorescence microscopy, at the excitation and emission wavelength of 488 and 525 nm, respectively. To further determine the mitochondrial reactive oxygen species in HUVECs, the cells were treated with COFsderived PM2.5, with or without 10 mmol/L NAC, for 24 h, and then stained with MitoSOX Red dye according to manufacturer's instructions. The pictures were taken with a fluorescence microscopy and analyzed with Image-Pro Plus 6.0 software. All these experiments were performed in triplication.
2. Materials and methods 2.1. Collection of COFs-derived PM2.5 To generate and collect the COFs-derived PM2.5 in the laboratory, 200 ml peanut oil (5S pressing first-class peanut oil; Luhua, Luhua Group, Shandong, China) was poured into an iron pot, and heated to smoke point (280 ± 10 °C) by an electric heater to generate cooking oil fumes. The fumes were collected with filter paper connected to a total suspended particulates sampler (XY-2200, Qaingdao Xuyu Environmental Co., Ltd, Qingdao, China), which is designed to collect different sizes of particulate matters (including PM2.5), at 50 cm above
2.5. Observing autophagosome with TEM Transmission electron microscope (TEM) was adopted to observe autophagosomes in the HUVECs. In brief, HUVECs were treated with 0 and 100 μg/ml COFs-derived PM2.5 for 24 h, respectively. Cells treated 453
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Fig. 1. Effects of COF-derived PM2.5 on viability of HUVECs. A, HUVECs were treated with different concentrations (0, 12.5, 25, 50, 100, and 200 μg/ml) of COFs-derived PM2.5 for 12, 24, and 36 h, and then the cell viability was assessed by MTT assay; B, HUVECs were treated with NAC and different concentrations of COFs-derived PM2.5 for 24 h. ** P < 0.01, *** P < 0.001, comparing with the corresponding control group; #P < 0.05, ##P < 0.01, comparing with the corresponding COFs-derived PM2.5 treatment group.
for 3 h at room temperature. After washing with TBST, the membrane was incubated with corresponding secondary antibodies for 2 h at room temperature, and then extensively washed with TBST. The proteins were visualized by using enhanced chemiluminescence's kit (Thermo Scientific, Rockford, USA).
with serum-free rapamycin (50 nM) were used as the positive control. In addition, cells were also treated with 100 μg/ml COFs-derived PM2.5+10 mmol/L NAC to demonstrate the effects of ROS on cell autophagy. After the treatment, the cells were collected and fixed, and sent to the Electron Microscopy Room of the Provincial Hospital, Anhui Medical University for sample preparing and image analysis.
2.9. Statistical analysis 2.6. Immunofluorescent assay SPSS 17.0 software was used for the statistical analysis. All the data were described with means and standard divisions. One-way analysis of variances (ANOVA) and post-hoc Bonferroni test was used for the comparisons among different groups. P < 0.05 was considered statistically significant.
HUVECs were grown on coverslips, and treated with 0 and 100 μg/ ml COFs-derived PM2.5, 100 μg/ml COFs-derived PM2.5+10 mmol/L NAC, and 50 nM rapamycin for 24 h, respectively. Then the cells were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.4% Triton X-100 for 15 min, and blocked (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min. The expression of LC3 and beclin1 was detected using primary antibodies against LC3-I/II (Cell Signling Technology) and beclin1 (Cell Signling Technology) respectively, at 4 °C overnight. The coverslips were then washed with PBS, and incubated with corresponding secondary antibodies at 37 °C for 1 h in dark. The nuclei of the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min in dark. Immunofluorescent images were obtained by laser scanning confocal microscope (Leica SP5, Germany).
3. Results 3.1. Effects of COFs-derived PM2.5 on viability of HUVECs The viability of the HUVECs treated with different concentrations of COFs-derived PM2.5 was evaluated by MTT assay. As shown in Fig. 1A, the HUVECs viability reduced dose-dependently after the COFs-derived PM2.5 treatment. When treated with 50, 100, and 200 μg/ml COFs-derived PM2.5 for 12, 24, and 36 h, the cell viability were significant lower than in the control group (P < 0.01). In addition, the cell viability also decreased significantly with the increase of exposure time when the COFs-derived PM2.5 doses were relatively high (100 and 200 μg/ml). However, the cell viability was effectively rescued by the administration of NAC (Fig. 1B).
2.7. Apoptosis assay In order to investigate the effects of autophagy on apoptosis in the HUVECs treated by COFs-derived PM2.5, the cells were treated with 100 μg/ml COFs, 100 μg/ml COFs + 10 mmol/L NAC, or 100 μg/ml COFs + 5 mM 3-MA for 24 h, respectively. Then apoptosis of the cells was measured by flow cytometry using annexin V-FITC/propidium iodide (PI) staining.
3.2. Effects of COFs-derived PM2.5 on ROS levels in HUVECs We further investigated the cellular ROS levels after the COFs-derived PM2.5 treatments. As shown in Fig. 2A, the green fluorescence (indicating ROS production) in the HUVECs increased greatly after treated with 100 μg/ml of COFs-derived PM2.5, which peaked at 12 h and then started to decrease with time. However, co-incubation of the cells with COFs-derived PM2.5 plus NAC effectively inhibited the green fluorescence. The quantitative analysis with fluorescence spectrophotometer confirmed that treating with COFs-derived PM2.5 for 12 and 24 h dose-dependently increased the ROS level in the cells. The fluorescence intensity in the cells treated with 100 μg/ml of COFs-derived PM2.5 was significantly higher than in the control group (P < 0.01). In addition, the fluorescence intensity also decreased with the increase of exposure time (Fig. 2B). Consistent with the cellular ROS levels, the mitochondrial ROS levels also increased with the concentrations of COFs-derived PM2.5. The mitochondrial ROS level, as shown by the intensity of MitoSOX Red staining, in the HUVECs treated by 100 μg/ml COFs-derived PM2.5 was significantly higher than in the control group, while the co-treatment by
2.8. Western blot analysis Expressions of LC3-I/II, beclin 1, PI3K/p-PI3K, AKT/p-AKT, and mTOR/p-mTOR were quantified by western blot. HUVECs were treated with different concentrations (0, 25, 50, and 100 μg/ml) of COFs-derived PM2.5 for 24 h to explore the dose-response effects. In addition, the cells were also treated with 10 mmol/L NAC, 100 μg/ml COFs-derived PM2.5 + 10 mmol/L NAC, 100 μg/ml COFs-derived PM2.5 + 5 mM 3-MA, and 50 nM serum-free rapamycin, respectively, for 24 h to explore the role of ROS in autophagy. After the treatment, cells were harvested, lysed on ice, and centrifuged at 4 °C, 14,000 rpm for 15 min. Protein concentration was determined by Bicinchoninic acid assay. Equal amount of protein was dissolved in loading buffer, boiled for 10 min, resolved by SDS-PAGE, and then transferred onto nitro-cellulose membranes. The membranes were blocked with 5% non-fat dry milk in Tris-Buffered-Saline with Tween (TBST) overnight at 4 °C, and then incubated with appropriate primary antibodies in blocking buffer 454
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A
B
D
C
100 μg/ml PM2.5
NAC + 100 μg/ml PM2.5
Fig. 2. Effects of COFs-derived PM2.5 on generation of reactive oxygen species (ROS) in HUVECs. A) Typical images of ROS level in HUVECs detected by fluorescence microscopy; B) Fluorescence intensity in the cells were measured by fluorescence spectrophotometer; C) Typical images of mitochondrial ROS level in cells treated by 100 μg/ml COFs-derived PM2.5 with or without NAC; and D) Fluorescence intensity in the cells by MitoSOX red staining. ** P < 0.01, ***P < 0.001, comparing with the corresponding control group; #P < 0.05, ##P < 0.01, comparing with the corresponding COFs-derived PM2.5 treatment group.
3.4. Apoptosis of the HUVECs
NAC significantly reduced the mitochondrial ROS level (Fig. 2C and 2D).
We further assessed the effects of COFs-derived PM2.5 on HUVECs apoptosis to explore the cell death mode. To our surprise, we found that the rate of cell apoptosis was generally low in all the treatment group, although the rate in the cells treated with 100 μg/ml COFs-derived PM2.5 was significantly higher than in the control group (P < 0.01). The application of NAC slightly reduced the apoptosis rate, while the application of 3-MA slightly increased the apoptosis rate, although the difference with the 100 μg/ml COFs-derived PM2.5 group was not statistically significant (P > 0.05). (Fig. 7)
3.3. Autophagy of HUVECs induced by COFs-derived PM2.5 For the cells in the control group, TEM showed that the mitochondria were normal, mitochondrial cristae were clearly, while no autophagosome was detected (Fig. 3A). In contrast, the exposure of HUVECs to 100 μg/ml of COFs-derived PM2.5 or 50 nM of rapamycin evidently increased the number of autophagosomes in the cytoplasm (Fig. 3B and C). However, co-incubation of the cells with NAC evidently reduced the number of autophagosomes induced by COFs-derived PM2.5 treatment. The structures of mitochondria were also clear and normal in the cells co-incubated with NAC (Fig. 3D). The expression of LC3 (including LC3-I and LC3-II) and beclin1, two most common autophagic markers, was also evaluated to reflect the autophagy. As shown in Fig. 4, immunofluorescent assay showed that COFs-derived PM2.5 or rapamycin evidently increased LC3 puncta and beclin1 expression in HUVECs. The LC3 proteins were mainly displayed as dot-like structures distributed within the cytoplasm or localizing in the perinuclear regions (Fig. 4A). While beclin1 proteins mainly aggregated around the nucleus (Fig. 4B). Co-incubation of the cells with NAC and COFs-derived PM2.5 evidently reduced the levels of LC3 and beclin1 proteins. Quantitative analysis showed that treating with COFs-derived PM2.5 dose-dependently increased the LC3-II level (Fig. 5A and B), leading to the increased ratio of LC3-II/LC3-I (Figs. 5A and 5C). Consistently, beclin1 expression also increased after the treatment of COFs-derived PM2.5 (Figs. 5A and 5D). In addition, co-incubation of the cells with either 3-methyladenine (3-MA, an autophagy inhibitor) or NAC significantly reduced the expression of beclin 1 and ratio of LC3-II/LC3-I induced by COFs-derived PM2.5 (Fig. 6).
3.5. Role of ROS-AKT-mTOR axis in HUVECs autophagy induced by COFsderived PM2.5 After treated with different concentrations of COFs-derived PM2.5 (0, 25, 50, and 100 μg/ml), western blot showed that the expressions of phosphorylated PI3K, AKT, and mTOR proteins (p-PI3K, p-AKT, and pmTOR) all tended to decrease with the increase of COFs-derived PM2.5 concentrations. In addition, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR all decreased significantly (P < 0.05) in dose-dependent manners (Fig. 8A-C). However, co-incubation of the cells with NAC effectively rescued the effects of COFs-derived PM2.5 (P < 0.05) (Fig. 8D-F). 4. Discussion In the present study, HUVECs were treated with different concentrations of COFs-derived PM2.5, and the findings revealed that COFsderived PM2.5 could dose-dependently reduce the cellular viability and increase ROS level. In addition, COFs-derived PM2.5 also effectively induced autophagy in the cells via ROS-AKT-mTOR axis, which could 455
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Fig. 3. Typical images of autophagosome in HUVECs by transmission electron microscopy (×30 k). A) Control group; B) HUVECs treated with 100 μg/ml of COF-derived PM2.5 for 24 h; C) HUVECs treated with serum-free rapamycin (50 nM) for 24 h; and D) HUVECs treated with 100 μg/ml of COF-derived PM2.5 and 10 mmol/L NAC for 24 h. Arrow shows autophagosome, arrow head shows mitochondria.
increased the viability of the cells treated by 100 and 200 μg/ml of COFs-derived PM2.5. Interestingly, although the viability of the cells decreased with the time of incubation with COFs-derived PM2.5, the ROS level in the HUVECs treated with 100 μg/ml of COFs-derived PM2.5 for 12 h was the highest, which decreased with time gradually. We suspected that the appeared ROS level decreased could be partly resulted from the increased cell death, which reduced the cell counts, and thus resulted in lower DCF fluorescence. In addition, there could be an adaptation process in the survived cells after stimulated by COFs-derived PM2.5. In a previous study, Deng et al.[10] treated A549 cells with different concentrations of ambient particulate matters, and found that the ROS level peaked at 4 h of treatment, but decreased to normal level at 24 h of treatment, which was in agreement with our findings. In light of the previous findings that ROS is one of the mechanisms participated in the functional and morphological changes of human umbilical vein caused by COFs-derived PM2.5[9], our findings suggested that the exposure of COFs-derived PM2.5 could help forming an oxidative environment, which in turn damages the umbilical tissues. COFs-derived PM2.5 is a complex mixture containing a lot of compounds including PAHs. In a recent study, 300 ng/ml PAHs were used to treat HepG2 cells for 24 h, which did not significantly affect the cell viability [18]. Another study used 25, 50, and 100 μg/ml of PM2.5
be rescued by the co-treatment of NAC. To our knowledge, this is the first study investigating the effects and mechanisms of COFs-derived PM2.5 on autophagy of HUVECs. HUVEC is a layer of endothelial cells covering the luminal surface of the umbilical blood vessels, which not only serves as an interface between circulating blood and surrounding tissues, but also contributes to the resistance against thrombosis and mediation of immune responses. Endothelial dysfunction can cause abnormalities in vascular vasomotor function, and therefore plays an important role in various diseases [17]. Previous studies in our group have shown that COFs-derived PM2.5 exposure could cause LWB in mice (Supplementary Fig. S1), while oxidative stress in umbilical cord blood vessel cells could play an important role [8,9]. The findings of this study confirmed that COFs-derived PM2.5 exposure could dose-dependently increase the cellular ROS levels in the HUVECs. The treatment of COFs-derived PM2.5 also significantly reduced the cell viability, and 100 μg/ml was the concentration of COFs-derived PM2.5 with the most evident responses, which is in parallel with the increase of ROS levels, indicating that the ROS generation induced by COFs-derived PM2.5 exposure could be one of the mechanisms participated in the reduced cell viability. In addition, ROS seemed to be one of the major causes of the decreased cell viability in this study, as the co-treatment of NAC significantly
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Fig. 4. Typical images show the distribution of LC3 and beclin1 proteins in HUVECs. After HUVECs were treated, immunofluorescent assay was used to display LC3 and beclin 1 proteins in the cells. A) LC3; and B) Beclin 1.
on ROS generation and cell death were comparable. Oxidative stress is an important mechanism participated in the cytotoxic effects of PM2.5[22], which can trigger cell autophagy and apoptosis [10]. Autophagy is a programmed cell death that enables eukaryotic cells to capture cytoplasmic components for degradation within lysosomes, which contributes to many physiological and pathological processes, including morphogenesis, cancer, neurodegenerative disorders, and infectious diseases [11,12]. Autophagy is characterized by the conversion of LC3-I to the PE-conjugated LC3-II form that accompanied by beclin 1 disappearance [23]. LC3 has been considered the only credible marker of the autophagosome in mammalian cells [24]. In this study, immunofluorescent assay showed that the level of LC3 and beclin1 both increased significantly in HUVECs treated with COFs-derived PM2.5, which was confirmed by the results of western blot. In addition, TCM examination also showed that the treatment of COFs-derived PM2.5 or rapamycin, which was used as the positive control of autophagy in this study, greatly increased the number of autophagosomes in the cells. These findings demonstrated that COFsderived PM2.5 treatment could effectively induce autophagy in HUVECs. In addition, the TCM examination of this study also showed
solvent-extractable organics to treat A549 cells for different time, and found the cell viability decreased with the treatment time; the viability of the cells decreased by over 20% at 24 h treatment [19]. However, 100 μg/ml COFs-derived PM2.5 means about 15.643 ng/ml PAHs [1], which is greatly lower than the concentrations used in the abovementioned studies, significantly reduced the viability of HUVECs in this study. We speculated that the difference between the two studies could be as follows: 1) the cell types were different, which could respond different to the treatment; 2) in addition to PAHs, COFs-derived PM2.5 consisted of some other compounds (such as metals), which could increase the toxic effects; and 3) COFs-derived PM2.5 could also results in mechanical effects on the cells, and thus increase the toxic effects. In contrast, two other studies found that 50 μM BaP could significantly increase the cellular ROS levels (by about 40%)[20,21] and reduce the viability of cells (by about 20–30%)[20]. According to our previous findings [1], 100 μg/ml COFs-derived PM2.5 contains about 0.0028 μg/ ml BaP. However, the other compounds in COFs-derived PM2.5, such as metals, pyrene, benzo(a)anthracene, benzo(ghi)pyrene, and fluoranthene could also affect the cytotoxicity. Thus although the level of BaP in the COFs-derived PM2.5 was much lower in this study, the effects
Fig. 5. Expression of LC3 and beclin1 proteins in HUVECs treated with different concentration (0, 25, 50, and 100 μg/ml) of COFs-derived PM2.5. A) Western blot of LC3 and beclin 1 protein expression in HUVECs treated with different concentrations of COFs-derived PM2.5; B) level of LC3-II; C) Ratio of LC3-II/LC3-I; and D) Level of beclin 1. *P < 0.05, **P < 0.01, comparing with the control group.
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Fig. 6. 3-MA and NAC rescued the effects of COFs-derived PM2.5 on the expression of LC3-I/II and beclin1 proteins in HUVECs treated with 100 μg/ml COFs-derived PM2.5. A1-A3) LC3 and beclin 1 expression in HUVECs treated with COFs-derived PM2.5 and/or 3-MA; B1-B3) LC3 and beclin 1 expression in HUVECs treated with COFs-derived PM2.5 and/or NAC. ** P < 0.01, comparing with the control group; #P < 0.05, ##P < 0.01, comparing with the PM2.5 group.
the dominant role over apoptosis and necrosis in the HUVECs upon COFs-derived PM2.5 treatment are to be further investigated. Previous studies have demonstrated that exogenous stimuli including malnutrition, hypoxia, and stress could inhibit the activity of PI3K, which in turn down-regulate the activation of downstream Akt, inhibit cell proliferation, induce cell apoptosis and autophagy, and finally promote cell death [27,28]. This study found that treating HUVECs with COFs-derived PM2.5 significantly inhibited the phosphorylation of PI3K, AKT, and mTOR, in dose-dependent manners, suggesting that COFs-derived PM2.5 exposure could inhibit the activation of PI3K-AKT-mTOR signaling pathway and thus up-regulate autophagy in HUVECs. In agreement with our findings, previous studies have shown that ambient PM2.5 exposure could induce autophagy of human bronchial epithelial cells and macrophages, in which processes the inhibition of PI3K-AKT-mTOR signaling pathway plays an important role [15,16]. Our study also showed that co-incubation of the cells with NAC effectively rescued the inhibitory effects of COFs-derived PM2.5 on phosphoylation of PI3K, AKT, and mTOR, indicating that ROS participated in the COFs-derived PM2.5-induced autophagy of HUVECs mediated by PI3K-Akt-mTOR signaling pathway. In a previous study, Cui et al.[29] treated A549 cells with an anti-tumor drug, and found that this drug could inhibit tumor growth by inducing cell autophagy and apoptosis via ROS mediated PI3K-AKT-mTOR signaling pathway, which was consistent with our findings.
Fig. 7. Apoptosis rate of the HUVECs upon treatment with different concentrations of COFs-derived PM2.5 with or without NAC/3-MA. **P < 0.01, comparing with the control group.
evident morphological changes of mitochondria in the cells. Therefore, we speculate that the changes of mitochondria could result in increased ROS level in the cells, which then participate in the processes of autophagy [25]. MitoSOX Red staining in this study showed that the ROS levels in the mitochondria of the HUVECs treated with 100 μg/ml of COFs-derived PM2.5 was significantly higher than in the control group, which was in accordance with the increase of the cellular ROS level. These results suggested that mitochondrial dysfunction was one of the major sources of the ROS in these cells. However, The co-incubation of the cells with NAC, not only reduced the mitochondrial ROS level, but effectively reduced the levels of LC3 and beclin 1, as well as the ratio of LC3-II/LC3-I. Similarly, co-incubation of the cells with COFs-derived PM2.5 and 3-MA, an autophagy inhibitor, also effectively reduced the levels of LC3 and beclin 1, as well as the ratio of LC3-II/LC3-I. Interestingly, the apoptosis rate of the cells remained mild across the treatment, while the co-incubation with 3-MA slightly increased the apoptosis rate. These findings implied that in addition to causing cell death, the autophagy in these cells also played another role, namely cell survival [26]. Taken together, these findings demonstrated that ROS plays an essential role in autophagy induced by COFs-derived PM2.5 exposure. However, the detailed mechanisms of how autophagy played
5. Conclusions In summary, this is the first study demonstrate that COFs-derived PM2.5 induces autophagy of HUVECs via ROS- AKT-mTOR axis, which could lead to endothelial damages of umbilical vein. However, there are still several limitations in this study. First, the findings of this study could not be directly applied in population studies, as the defense system of the whole body could not be included in this study. Second, COFs-derived PM2.5 was collected from only peanut oil in this study; although peanut oil is commonly used in China, it could not loyally represent the overall situation in China. Third, the HUVECs were directly exposed to COFs-derived PM2.5 in this study, which could not loyally reflect the effects of COFs-derived PM2.5 after absorbed through inhalation. Finally, HUVECs, an immortal cell line, were used in this study to investigate the effects of COFs-derived PM2.5 on cell 458
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Fig. 8. Effects of ROS-AKT-mTOR axis in HUVECs treated with COFs-derived PM2.5. A-C) COFs-derived PM2.5 affects the expression of proteins in the PI3K-AKT-mTOR signaling pathway. D-F) NAC rescues the effects of COFs-derived PM2.5 on the expression of the proteins in the PI3K-AKTmTOR signaling pathway. *P < 0.05, ** P < 0.01, comparing with the control group; #P < 0.05, ## P < 0.01, comparing with the PM2.5 group.
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autophagy, which could at least bias the results. Therefore, it is important to note that extrapolation of these findings to in vivo conditions remains to be established. Acknowledgments We sincerely thank the School of Life Sciences, University of Science and Technology of China for kindly providing HUVECs, thank the Electron Microscopy Room of the Provincial Hospital, Anhui Medical University for the help in TEM. Funding This work was supported by the National Natural Science Foundation of China (Grand Number: 31430028), National Basic Research Programme of China (Grand Number: 2013CB933900), the Natural Science Foundation of Anhui Province (Grand Number: 1708085MH220), the Project Foundation for the Young Talents in Colleges of Anhui Province (Grand Number: gxyq2017003), and the Grant for Scientific Research of BSKY from Anhui Medical University (Grand Number: XJ201621). Competing interests The authors declare that they have no competing interests. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.10. 386. References [1] Y. Liu, Y.Y. Chen, J.Y. Cao, F.B. Tao, X.X. Zhu, C.J. Yao, D.J. Chen, Z. Che, Q.H. Zhao, L.P. Wen, Oxidative stress, apoptosis, and cell cycle arrest are induced in primary fetal alveolar type II epithelial cells exposed to fine particulate matter from cooking oil fumes, Environ. Sci. Pollut. Res. Int. 22 (2015) 9728–9741. [2] K. Ge, The transition of Chinese dietary guidelines and food guide pagoda, Asia Pac. J. Clin. Nutr. 20 (2011) 439–446. [3] T. Dearth-Wesley, P. Gordon-Larsen, L.S. Adair, A.M. Siega-Riz, B. Zhang, B.M. Popkin, Less traditional diets in Chinese mothers and children are similarly linked to socioeconomic and cohort factors but vary with increasing child age, J. Nutr. 141 (2011) 1705–1711. [4] S. Li, J. Gao, Y. He, L. Cao, A. Li, S. Mo, Y. Chen, Y. Cao, Determination of time- and size-dependent fine particle emission with varied oil heating in an experimental kitchen, J. Environ. Sci. 51 (2017) 157–164. [5] N. Shen, W. Zhang, G. Li, Impact of isolated single umbilical artery on pregnancy outcome and delivery in full-term births, J. Obstet. Gynaecol. Res. 42 (2016) 399–403. [6] M.M. Veras, R.M. Guimaraes-Silva, E.G. Caldini, P.H. Saldiva, M. Dolhnikoff, T.M. Mayhew, The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: a quantitative morphological and immunohistochemical study, Reprod. Toxicol. 34 (2012) 598–606. [7] A.C. Peyter, F. Delhaes, D. Baud, Y. Vial, G. Diaceri, S. Menetrey, P. Hohlfeld, J.F. Tolsa, Intrauterine growth restriction is associated with structural alterations in human umbilical cord and decreased nitric oxide-induced relaxation of umbilical vein, Placenta 35 (2014) 891–899.
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Original article
ROS-AKT-mTOR axis mediates autophagy of human umbilical vein endothelial cells induced by cooking oil fumes-derived fine particulate matters in vitro
MARK
Rui Dinga,1, Chao Zhanga,1, Xiaoxia Zhub,1, Han Chenga, Furong Zhua, Yachun Xua, Ying Liuc, ⁎ ⁎⁎ Longping Wend, , Jiyu Caoa,e, a
Department of Occupational and Environmental Health, School of Public Health, Anhui Medical University, Hefei, Anhui, China Department of Chronic Disease Control and Prevention, Shanghai Putuo District Center for Disease Control and Prevention, Shanghai, China Department of Maternal, Child and Adolescent Health, School of Public Health, Anhui Medical University, Hefei, Anhui, China d School of Medicine, South China University of Technology of China, Guangzhou, Guangdong, China e Teaching Center for Preventive Medicine, School of Public Health, Anhui Medical University, Hefei, Anhui, China b c
A R T I C L E I N F O
A B S T R A C T
Keywords: Cooking oil fumes PM2.5 Reactive oxygen species Autophagy Human umbilical vein endothelial cells
Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) exposure can induce oxidative stress and cytotoxic effects. Here we investigated the role of ROS-AKT-mTOR axis in COFs-derived PM2.5-induced autophagy in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with different concentrations of COFs-derived PM2.5, together with or without N-acetyl-L-cysteine (NAC, a radical scavenger) or 3-methyladenine (3-MA, an autophagy inhibitor). Cell viability was assessed with MTT assay, and ROS level was measured with DCFH-DA assay after the treatment. Transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes, while immunofluorescent assay and western blot were used to assess the expression of LC3-I/II and beclin 1. Proteins involved in the PI3K-AKT-mTOR signaling pathway were measured with western blot. The results showed that the treatment of COFs-derived PM2.5 dose-dependently reduced the viability of HUVECs and increased the ROS levels in the cells. Both immunofluorescent assay and western blot showed that treatment with COFs-derived PM2.5 significantly increased LC3-II and beclin 1 levels, as well as the ratio of LC3-II/LC3-I, which could be rescued by the co-incubation with NAC or 3-MA. TEM also confirmed the increased formation of autophagosomes in the cells treated with COFs-derived PM2.5, while co-treatment with NAC evidently decreased autophagosomes formation. In addition, western blot also showed that the phosphorylation of PI3K, AKT, and mTOR all decreased by the treatment of COFs-derived PM2.5, which was effectively rescued by the co-treatment with NAC. These findings demonstrate ROS-AKT-mTOR axis plays a critical role in HUVECs autophagy induced by COFs-derived PM2.5.
1. Introduction Cooking oil fumes-derived PM2.5 (COFs-derived PM2.5) is an important source of indoor air pollution, especially in East Asian countries such as China. In contrast to ambient PM2.5, COFs-derived PM2.5 contains more noxious components, such as polycyclic aromatic hydrocarbons, and could persistently exist in the microenvironment at
relatively high concentration [1]. Although the China Food-Based Dietary Guidelines (FBDGs, 2008) issued by the Ministry of Health recommended that the daily intake of fats and oils should be 25–30 g per reference man [2], the actual consumption in China is much higher [3], thus the contribution of COFs-PM2.5 to indoor air pollution in Chinese families is substantial. In a previous survey, we found that the COFsderived PM2.5 level was as high as 1.679–7.023 mg/m3 in domestic
Abbreviations: PM2.5, Fine particulate matter of < 2.5 µm in aerodynamic diameter; COFs, Cooking oil fumes; LBW, Low birth weight; ROS, Reactive oxygen species; PI3K, Phosphatidylinositol 3-kinase; mTOR, Mechanistic target of rapamycin; HUVECs, Human umbilical vein endothelial cells; DMSO, Dimethyl sulfoxide; MTT, 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide; DCFH-DA, 2’,7’-dichlorofluorescin diacetate; NAC, N-acetyl-L-cysteine; TEM, Transmission electron microscopy; DAPI, 4',6-diamidino-2-phenylindole; PI, Propidium iodide; 3-MA, 3-methyladenine; TBST, Tris-Buffered-Saline with Tween; PAHs, Polycyclic aromatic hydrocarbons; BaP, Benzo(α)pyrene; ANOVA, Analysis of variances ⁎ Corresponding author. ⁎⁎ Corresponding author at: Teaching Center for Preventive Medicine, School of Public Health, Anhui Medical University, Meishan Road 81, Hefei, Anhui, China. E-mail addresses: [email protected] (L. Wen), [email protected] (J. Cao). 1 These authors contributed equally to this study. http://dx.doi.org/10.1016/j.freeradbiomed.2017.10.386 Received 6 May 2017; Received in revised form 26 October 2017; Accepted 27 October 2017 Available online 28 October 2017 0891-5849/ © 2017 Elsevier Inc. All rights reserved.
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the oil surface (Supplementary Fig. S2). The filter paper was renewed every 2 h. Then the filter paper with fumes attached was processed separately with 50 ml acetone for 24 h in Soxhlet extractor. The extracts were dried by rotary evaporation at 40 °C, and then further evaporated to yellow viscous solution. Dimethyl sulfoxide (DMSO) was used to dilute the solution to 200 mg/ml. The solution was transferred to brown glass vials, sealed, and stored at −80 °C until use. For the experiments in this study, the stoke solution was diluted to 12.5, 25, 50, 100, and 200 μg/ml with the cell culture medium. The compositions of the COFsderived PM2.5 have already been measured and reported by our group [1].
kitchen in China (Supplementary Table S1), which was confirmed by another recent study [4]. COFs-derived PM2.5 could especially affect the women in China, as they are generally the ones cooking food for the families. While for the pregnant women, such exposure could possibly affect the fetus. Previous studies in our group have demonstrated that maternal exposure of COFs-derived PM2.5 during pregnancy could lead to low birth weight (LBW) in mice (Supplementary Fig. S1). However, the underlying mechanisms are still unclear. Umbilicus is the only link between the mother and fetus during pregnancy, and is responsible for the exchange of oxygen, nutrients, and waste materials between fetus and mother [5]. Veras et al. found that ambient PM2.5 could induce oxidative stress and alter endothelin receptor expression to affect the umbilical cord blood vessels, and therefore contribute to the decreased fetal weight [6]. Peyter et al.[7] also demonstrated that the umbilical cord diameter was significantly lower in growth-restricted newborns than appropriate for gestational age term newborns; in addition, the relaxation of the umbilical vein induced by nitric oxide was significantly attenuated in growth-restricted neonates than controls. Previous in vitro [8] and in vivo [9] studies in our group have further confirmed that COFs-derived PM2.5 exposure could evidently affect the umbilical cord blood vessels. These findings suggest that PM2.5 exposure could increase the risk of endothelial dysfunction of the umbilical cord blood vessels, and possibly increase the risk of decreased fetal weight. Previous studies have shown that COFs-derived PM2.5 can induce oxidative damage not only to type II alveolar epithelial cells in vitro [1], but also to umbilical cord blood vessels in vitro and vivo [8,9]. Oxidative stress can cause severe damages to DNA, RNA, and proteins, and thus trigger autophagy and apoptosis [10]. Autophagy enables eukaryotic cells to capture cytoplasmic components for degradation within lysosomes. Although baseline autophagy is important for the maintenance of normal cellular homeostasis, abnormal autophagy contributes to many physiological and pathological processes, including morphogenesis, cancer, neurodegenerative disorders, and infectious diseases [11,12]. Therefore, we hypothesize that the elevated ROS production induced by the exposure of COFs-derived PM2.5 could disturb the balance of autophagy in umbilical vein endothelial cells, thus consequently impair the functions of umbilical veins. PI3K/AKT/mTOR pathway plays an important role in the modulation of cell autophagy [13]. Mechanistic target of rapamycin (mTOR) is one of the major modulators of autophagy that can be regulated by various signaling pathways [14]. For instance, several studies have demonstrated that the PI3K/AKT/mTOR signaling pathway participates in the regulation of autophagy induced by PM2.5 exposure [15,16]. However, the effects of COFs-derived PM2.5 exposure on autophagy of human umbilical vein endothelial cells (HUVECs), as well as the role of the PI3K/AKT/mTOR signaling pathway in this process have not been investigated to date. Here we investigated the effects of COFs-derived PM2.5 on autophagy of HUVECs and the role of the ROS-AKT-mTOR axis in this event. The findings of this study could provide experimental evidence of the adverse effects of COFs-derived PM2.5 on the functions of umbilical cord.
2.2. Cell culture This study was approved by the Ethics Committee of Anhui Medical University. HUVECs were kindly provided by the School of Life Sciences, University of Science and Technology of China. The cells were cultured with DMEM culture medium (supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin) at 37 °C in a humidified atmosphere with 5% CO2. The culture medium was changed every day. Cells were used in the following experiments after the culture reached 80% confluence. 2.3. Cell viability assay Cytotoxicity of COFs-derived PM2.5 was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. In brief, HUVECs were incubated with 0 (control, containing 1‰ DMSO), 12.5, 25, 50, 100, and 200 μg/ml of COFs-derived PM2.5 for 12, 24, and 36 h in 96-well culture plate, respectively. To confirm the effects of ROS on cell viability, N-acetyl-L-cysteine (NAC), a ROS inhibitor, was used along with different concentrations of COFs-PM2.5 for 24 h. Then the culture medium was carefully discarded, 200 μl MTT solution (prepared with the culture medium at a ratio of 9:1) was added to each well, and the plate was incubated for another 4 h at 37 °C. The formazan crystals were dissolved with 150ul of DMSO, and the absorbance in each well was read at 490 nm using ELx800 microplate reader (Bio-TEK, USA). 2.4. ROS measurement To determine the ROS levels in the cells, the HUVECs were loaded with the fluorescent probe 2’,7’-dichlorofluorescin diacetate (DCFHDA). The HUVECs were seeded into 6-well plates with 5.0× 105 cells/ well. Before the following treatment, the cells were synchronization for 24 h by serum starvation. According to the results of the MTT assay, the cells were treated with different concentrations (0, 25, 50, 100 μg/ml) of COF-derived PM2.5, 100 μg/ml COF-derived PM2.5+10 mmol/L NAC, and 10 mmol/L NAC for 12, 24, and 36 h, respectively. The fluorescent probe DCFH-DA was added to each well with the final concentration of 10 μM and incubated at 37 °C for 30 min in dark. The plate was washed three times with PBS, trpsinized, re-suspended, and immediately subjected to fluorospectrophotometer analysis and fluorescence microscopy, at the excitation and emission wavelength of 488 and 525 nm, respectively. To further determine the mitochondrial reactive oxygen species in HUVECs, the cells were treated with COFsderived PM2.5, with or without 10 mmol/L NAC, for 24 h, and then stained with MitoSOX Red dye according to manufacturer's instructions. The pictures were taken with a fluorescence microscopy and analyzed with Image-Pro Plus 6.0 software. All these experiments were performed in triplication.
2. Materials and methods 2.1. Collection of COFs-derived PM2.5 To generate and collect the COFs-derived PM2.5 in the laboratory, 200 ml peanut oil (5S pressing first-class peanut oil; Luhua, Luhua Group, Shandong, China) was poured into an iron pot, and heated to smoke point (280 ± 10 °C) by an electric heater to generate cooking oil fumes. The fumes were collected with filter paper connected to a total suspended particulates sampler (XY-2200, Qaingdao Xuyu Environmental Co., Ltd, Qingdao, China), which is designed to collect different sizes of particulate matters (including PM2.5), at 50 cm above
2.5. Observing autophagosome with TEM Transmission electron microscope (TEM) was adopted to observe autophagosomes in the HUVECs. In brief, HUVECs were treated with 0 and 100 μg/ml COFs-derived PM2.5 for 24 h, respectively. Cells treated 453
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Fig. 1. Effects of COF-derived PM2.5 on viability of HUVECs. A, HUVECs were treated with different concentrations (0, 12.5, 25, 50, 100, and 200 μg/ml) of COFs-derived PM2.5 for 12, 24, and 36 h, and then the cell viability was assessed by MTT assay; B, HUVECs were treated with NAC and different concentrations of COFs-derived PM2.5 for 24 h. ** P < 0.01, *** P < 0.001, comparing with the corresponding control group; #P < 0.05, ##P < 0.01, comparing with the corresponding COFs-derived PM2.5 treatment group.
for 3 h at room temperature. After washing with TBST, the membrane was incubated with corresponding secondary antibodies for 2 h at room temperature, and then extensively washed with TBST. The proteins were visualized by using enhanced chemiluminescence's kit (Thermo Scientific, Rockford, USA).
with serum-free rapamycin (50 nM) were used as the positive control. In addition, cells were also treated with 100 μg/ml COFs-derived PM2.5+10 mmol/L NAC to demonstrate the effects of ROS on cell autophagy. After the treatment, the cells were collected and fixed, and sent to the Electron Microscopy Room of the Provincial Hospital, Anhui Medical University for sample preparing and image analysis.
2.9. Statistical analysis 2.6. Immunofluorescent assay SPSS 17.0 software was used for the statistical analysis. All the data were described with means and standard divisions. One-way analysis of variances (ANOVA) and post-hoc Bonferroni test was used for the comparisons among different groups. P < 0.05 was considered statistically significant.
HUVECs were grown on coverslips, and treated with 0 and 100 μg/ ml COFs-derived PM2.5, 100 μg/ml COFs-derived PM2.5+10 mmol/L NAC, and 50 nM rapamycin for 24 h, respectively. Then the cells were fixed in 4% paraformaldehyde for 30 min, permeabilized in 0.4% Triton X-100 for 15 min, and blocked (Beyotime Institute of Biotechnology, Shanghai, China) for 30 min. The expression of LC3 and beclin1 was detected using primary antibodies against LC3-I/II (Cell Signling Technology) and beclin1 (Cell Signling Technology) respectively, at 4 °C overnight. The coverslips were then washed with PBS, and incubated with corresponding secondary antibodies at 37 °C for 1 h in dark. The nuclei of the cells were stained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min in dark. Immunofluorescent images were obtained by laser scanning confocal microscope (Leica SP5, Germany).
3. Results 3.1. Effects of COFs-derived PM2.5 on viability of HUVECs The viability of the HUVECs treated with different concentrations of COFs-derived PM2.5 was evaluated by MTT assay. As shown in Fig. 1A, the HUVECs viability reduced dose-dependently after the COFs-derived PM2.5 treatment. When treated with 50, 100, and 200 μg/ml COFs-derived PM2.5 for 12, 24, and 36 h, the cell viability were significant lower than in the control group (P < 0.01). In addition, the cell viability also decreased significantly with the increase of exposure time when the COFs-derived PM2.5 doses were relatively high (100 and 200 μg/ml). However, the cell viability was effectively rescued by the administration of NAC (Fig. 1B).
2.7. Apoptosis assay In order to investigate the effects of autophagy on apoptosis in the HUVECs treated by COFs-derived PM2.5, the cells were treated with 100 μg/ml COFs, 100 μg/ml COFs + 10 mmol/L NAC, or 100 μg/ml COFs + 5 mM 3-MA for 24 h, respectively. Then apoptosis of the cells was measured by flow cytometry using annexin V-FITC/propidium iodide (PI) staining.
3.2. Effects of COFs-derived PM2.5 on ROS levels in HUVECs We further investigated the cellular ROS levels after the COFs-derived PM2.5 treatments. As shown in Fig. 2A, the green fluorescence (indicating ROS production) in the HUVECs increased greatly after treated with 100 μg/ml of COFs-derived PM2.5, which peaked at 12 h and then started to decrease with time. However, co-incubation of the cells with COFs-derived PM2.5 plus NAC effectively inhibited the green fluorescence. The quantitative analysis with fluorescence spectrophotometer confirmed that treating with COFs-derived PM2.5 for 12 and 24 h dose-dependently increased the ROS level in the cells. The fluorescence intensity in the cells treated with 100 μg/ml of COFs-derived PM2.5 was significantly higher than in the control group (P < 0.01). In addition, the fluorescence intensity also decreased with the increase of exposure time (Fig. 2B). Consistent with the cellular ROS levels, the mitochondrial ROS levels also increased with the concentrations of COFs-derived PM2.5. The mitochondrial ROS level, as shown by the intensity of MitoSOX Red staining, in the HUVECs treated by 100 μg/ml COFs-derived PM2.5 was significantly higher than in the control group, while the co-treatment by
2.8. Western blot analysis Expressions of LC3-I/II, beclin 1, PI3K/p-PI3K, AKT/p-AKT, and mTOR/p-mTOR were quantified by western blot. HUVECs were treated with different concentrations (0, 25, 50, and 100 μg/ml) of COFs-derived PM2.5 for 24 h to explore the dose-response effects. In addition, the cells were also treated with 10 mmol/L NAC, 100 μg/ml COFs-derived PM2.5 + 10 mmol/L NAC, 100 μg/ml COFs-derived PM2.5 + 5 mM 3-MA, and 50 nM serum-free rapamycin, respectively, for 24 h to explore the role of ROS in autophagy. After the treatment, cells were harvested, lysed on ice, and centrifuged at 4 °C, 14,000 rpm for 15 min. Protein concentration was determined by Bicinchoninic acid assay. Equal amount of protein was dissolved in loading buffer, boiled for 10 min, resolved by SDS-PAGE, and then transferred onto nitro-cellulose membranes. The membranes were blocked with 5% non-fat dry milk in Tris-Buffered-Saline with Tween (TBST) overnight at 4 °C, and then incubated with appropriate primary antibodies in blocking buffer 454
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A
B
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100 μg/ml PM2.5
NAC + 100 μg/ml PM2.5
Fig. 2. Effects of COFs-derived PM2.5 on generation of reactive oxygen species (ROS) in HUVECs. A) Typical images of ROS level in HUVECs detected by fluorescence microscopy; B) Fluorescence intensity in the cells were measured by fluorescence spectrophotometer; C) Typical images of mitochondrial ROS level in cells treated by 100 μg/ml COFs-derived PM2.5 with or without NAC; and D) Fluorescence intensity in the cells by MitoSOX red staining. ** P < 0.01, ***P < 0.001, comparing with the corresponding control group; #P < 0.05, ##P < 0.01, comparing with the corresponding COFs-derived PM2.5 treatment group.
3.4. Apoptosis of the HUVECs
NAC significantly reduced the mitochondrial ROS level (Fig. 2C and 2D).
We further assessed the effects of COFs-derived PM2.5 on HUVECs apoptosis to explore the cell death mode. To our surprise, we found that the rate of cell apoptosis was generally low in all the treatment group, although the rate in the cells treated with 100 μg/ml COFs-derived PM2.5 was significantly higher than in the control group (P < 0.01). The application of NAC slightly reduced the apoptosis rate, while the application of 3-MA slightly increased the apoptosis rate, although the difference with the 100 μg/ml COFs-derived PM2.5 group was not statistically significant (P > 0.05). (Fig. 7)
3.3. Autophagy of HUVECs induced by COFs-derived PM2.5 For the cells in the control group, TEM showed that the mitochondria were normal, mitochondrial cristae were clearly, while no autophagosome was detected (Fig. 3A). In contrast, the exposure of HUVECs to 100 μg/ml of COFs-derived PM2.5 or 50 nM of rapamycin evidently increased the number of autophagosomes in the cytoplasm (Fig. 3B and C). However, co-incubation of the cells with NAC evidently reduced the number of autophagosomes induced by COFs-derived PM2.5 treatment. The structures of mitochondria were also clear and normal in the cells co-incubated with NAC (Fig. 3D). The expression of LC3 (including LC3-I and LC3-II) and beclin1, two most common autophagic markers, was also evaluated to reflect the autophagy. As shown in Fig. 4, immunofluorescent assay showed that COFs-derived PM2.5 or rapamycin evidently increased LC3 puncta and beclin1 expression in HUVECs. The LC3 proteins were mainly displayed as dot-like structures distributed within the cytoplasm or localizing in the perinuclear regions (Fig. 4A). While beclin1 proteins mainly aggregated around the nucleus (Fig. 4B). Co-incubation of the cells with NAC and COFs-derived PM2.5 evidently reduced the levels of LC3 and beclin1 proteins. Quantitative analysis showed that treating with COFs-derived PM2.5 dose-dependently increased the LC3-II level (Fig. 5A and B), leading to the increased ratio of LC3-II/LC3-I (Figs. 5A and 5C). Consistently, beclin1 expression also increased after the treatment of COFs-derived PM2.5 (Figs. 5A and 5D). In addition, co-incubation of the cells with either 3-methyladenine (3-MA, an autophagy inhibitor) or NAC significantly reduced the expression of beclin 1 and ratio of LC3-II/LC3-I induced by COFs-derived PM2.5 (Fig. 6).
3.5. Role of ROS-AKT-mTOR axis in HUVECs autophagy induced by COFsderived PM2.5 After treated with different concentrations of COFs-derived PM2.5 (0, 25, 50, and 100 μg/ml), western blot showed that the expressions of phosphorylated PI3K, AKT, and mTOR proteins (p-PI3K, p-AKT, and pmTOR) all tended to decrease with the increase of COFs-derived PM2.5 concentrations. In addition, the ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR all decreased significantly (P < 0.05) in dose-dependent manners (Fig. 8A-C). However, co-incubation of the cells with NAC effectively rescued the effects of COFs-derived PM2.5 (P < 0.05) (Fig. 8D-F). 4. Discussion In the present study, HUVECs were treated with different concentrations of COFs-derived PM2.5, and the findings revealed that COFsderived PM2.5 could dose-dependently reduce the cellular viability and increase ROS level. In addition, COFs-derived PM2.5 also effectively induced autophagy in the cells via ROS-AKT-mTOR axis, which could 455
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Fig. 3. Typical images of autophagosome in HUVECs by transmission electron microscopy (×30 k). A) Control group; B) HUVECs treated with 100 μg/ml of COF-derived PM2.5 for 24 h; C) HUVECs treated with serum-free rapamycin (50 nM) for 24 h; and D) HUVECs treated with 100 μg/ml of COF-derived PM2.5 and 10 mmol/L NAC for 24 h. Arrow shows autophagosome, arrow head shows mitochondria.
increased the viability of the cells treated by 100 and 200 μg/ml of COFs-derived PM2.5. Interestingly, although the viability of the cells decreased with the time of incubation with COFs-derived PM2.5, the ROS level in the HUVECs treated with 100 μg/ml of COFs-derived PM2.5 for 12 h was the highest, which decreased with time gradually. We suspected that the appeared ROS level decreased could be partly resulted from the increased cell death, which reduced the cell counts, and thus resulted in lower DCF fluorescence. In addition, there could be an adaptation process in the survived cells after stimulated by COFs-derived PM2.5. In a previous study, Deng et al.[10] treated A549 cells with different concentrations of ambient particulate matters, and found that the ROS level peaked at 4 h of treatment, but decreased to normal level at 24 h of treatment, which was in agreement with our findings. In light of the previous findings that ROS is one of the mechanisms participated in the functional and morphological changes of human umbilical vein caused by COFs-derived PM2.5[9], our findings suggested that the exposure of COFs-derived PM2.5 could help forming an oxidative environment, which in turn damages the umbilical tissues. COFs-derived PM2.5 is a complex mixture containing a lot of compounds including PAHs. In a recent study, 300 ng/ml PAHs were used to treat HepG2 cells for 24 h, which did not significantly affect the cell viability [18]. Another study used 25, 50, and 100 μg/ml of PM2.5
be rescued by the co-treatment of NAC. To our knowledge, this is the first study investigating the effects and mechanisms of COFs-derived PM2.5 on autophagy of HUVECs. HUVEC is a layer of endothelial cells covering the luminal surface of the umbilical blood vessels, which not only serves as an interface between circulating blood and surrounding tissues, but also contributes to the resistance against thrombosis and mediation of immune responses. Endothelial dysfunction can cause abnormalities in vascular vasomotor function, and therefore plays an important role in various diseases [17]. Previous studies in our group have shown that COFs-derived PM2.5 exposure could cause LWB in mice (Supplementary Fig. S1), while oxidative stress in umbilical cord blood vessel cells could play an important role [8,9]. The findings of this study confirmed that COFs-derived PM2.5 exposure could dose-dependently increase the cellular ROS levels in the HUVECs. The treatment of COFs-derived PM2.5 also significantly reduced the cell viability, and 100 μg/ml was the concentration of COFs-derived PM2.5 with the most evident responses, which is in parallel with the increase of ROS levels, indicating that the ROS generation induced by COFs-derived PM2.5 exposure could be one of the mechanisms participated in the reduced cell viability. In addition, ROS seemed to be one of the major causes of the decreased cell viability in this study, as the co-treatment of NAC significantly
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Fig. 4. Typical images show the distribution of LC3 and beclin1 proteins in HUVECs. After HUVECs were treated, immunofluorescent assay was used to display LC3 and beclin 1 proteins in the cells. A) LC3; and B) Beclin 1.
on ROS generation and cell death were comparable. Oxidative stress is an important mechanism participated in the cytotoxic effects of PM2.5[22], which can trigger cell autophagy and apoptosis [10]. Autophagy is a programmed cell death that enables eukaryotic cells to capture cytoplasmic components for degradation within lysosomes, which contributes to many physiological and pathological processes, including morphogenesis, cancer, neurodegenerative disorders, and infectious diseases [11,12]. Autophagy is characterized by the conversion of LC3-I to the PE-conjugated LC3-II form that accompanied by beclin 1 disappearance [23]. LC3 has been considered the only credible marker of the autophagosome in mammalian cells [24]. In this study, immunofluorescent assay showed that the level of LC3 and beclin1 both increased significantly in HUVECs treated with COFs-derived PM2.5, which was confirmed by the results of western blot. In addition, TCM examination also showed that the treatment of COFs-derived PM2.5 or rapamycin, which was used as the positive control of autophagy in this study, greatly increased the number of autophagosomes in the cells. These findings demonstrated that COFsderived PM2.5 treatment could effectively induce autophagy in HUVECs. In addition, the TCM examination of this study also showed
solvent-extractable organics to treat A549 cells for different time, and found the cell viability decreased with the treatment time; the viability of the cells decreased by over 20% at 24 h treatment [19]. However, 100 μg/ml COFs-derived PM2.5 means about 15.643 ng/ml PAHs [1], which is greatly lower than the concentrations used in the abovementioned studies, significantly reduced the viability of HUVECs in this study. We speculated that the difference between the two studies could be as follows: 1) the cell types were different, which could respond different to the treatment; 2) in addition to PAHs, COFs-derived PM2.5 consisted of some other compounds (such as metals), which could increase the toxic effects; and 3) COFs-derived PM2.5 could also results in mechanical effects on the cells, and thus increase the toxic effects. In contrast, two other studies found that 50 μM BaP could significantly increase the cellular ROS levels (by about 40%)[20,21] and reduce the viability of cells (by about 20–30%)[20]. According to our previous findings [1], 100 μg/ml COFs-derived PM2.5 contains about 0.0028 μg/ ml BaP. However, the other compounds in COFs-derived PM2.5, such as metals, pyrene, benzo(a)anthracene, benzo(ghi)pyrene, and fluoranthene could also affect the cytotoxicity. Thus although the level of BaP in the COFs-derived PM2.5 was much lower in this study, the effects
Fig. 5. Expression of LC3 and beclin1 proteins in HUVECs treated with different concentration (0, 25, 50, and 100 μg/ml) of COFs-derived PM2.5. A) Western blot of LC3 and beclin 1 protein expression in HUVECs treated with different concentrations of COFs-derived PM2.5; B) level of LC3-II; C) Ratio of LC3-II/LC3-I; and D) Level of beclin 1. *P < 0.05, **P < 0.01, comparing with the control group.
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Fig. 6. 3-MA and NAC rescued the effects of COFs-derived PM2.5 on the expression of LC3-I/II and beclin1 proteins in HUVECs treated with 100 μg/ml COFs-derived PM2.5. A1-A3) LC3 and beclin 1 expression in HUVECs treated with COFs-derived PM2.5 and/or 3-MA; B1-B3) LC3 and beclin 1 expression in HUVECs treated with COFs-derived PM2.5 and/or NAC. ** P < 0.01, comparing with the control group; #P < 0.05, ##P < 0.01, comparing with the PM2.5 group.
the dominant role over apoptosis and necrosis in the HUVECs upon COFs-derived PM2.5 treatment are to be further investigated. Previous studies have demonstrated that exogenous stimuli including malnutrition, hypoxia, and stress could inhibit the activity of PI3K, which in turn down-regulate the activation of downstream Akt, inhibit cell proliferation, induce cell apoptosis and autophagy, and finally promote cell death [27,28]. This study found that treating HUVECs with COFs-derived PM2.5 significantly inhibited the phosphorylation of PI3K, AKT, and mTOR, in dose-dependent manners, suggesting that COFs-derived PM2.5 exposure could inhibit the activation of PI3K-AKT-mTOR signaling pathway and thus up-regulate autophagy in HUVECs. In agreement with our findings, previous studies have shown that ambient PM2.5 exposure could induce autophagy of human bronchial epithelial cells and macrophages, in which processes the inhibition of PI3K-AKT-mTOR signaling pathway plays an important role [15,16]. Our study also showed that co-incubation of the cells with NAC effectively rescued the inhibitory effects of COFs-derived PM2.5 on phosphoylation of PI3K, AKT, and mTOR, indicating that ROS participated in the COFs-derived PM2.5-induced autophagy of HUVECs mediated by PI3K-Akt-mTOR signaling pathway. In a previous study, Cui et al.[29] treated A549 cells with an anti-tumor drug, and found that this drug could inhibit tumor growth by inducing cell autophagy and apoptosis via ROS mediated PI3K-AKT-mTOR signaling pathway, which was consistent with our findings.
Fig. 7. Apoptosis rate of the HUVECs upon treatment with different concentrations of COFs-derived PM2.5 with or without NAC/3-MA. **P < 0.01, comparing with the control group.
evident morphological changes of mitochondria in the cells. Therefore, we speculate that the changes of mitochondria could result in increased ROS level in the cells, which then participate in the processes of autophagy [25]. MitoSOX Red staining in this study showed that the ROS levels in the mitochondria of the HUVECs treated with 100 μg/ml of COFs-derived PM2.5 was significantly higher than in the control group, which was in accordance with the increase of the cellular ROS level. These results suggested that mitochondrial dysfunction was one of the major sources of the ROS in these cells. However, The co-incubation of the cells with NAC, not only reduced the mitochondrial ROS level, but effectively reduced the levels of LC3 and beclin 1, as well as the ratio of LC3-II/LC3-I. Similarly, co-incubation of the cells with COFs-derived PM2.5 and 3-MA, an autophagy inhibitor, also effectively reduced the levels of LC3 and beclin 1, as well as the ratio of LC3-II/LC3-I. Interestingly, the apoptosis rate of the cells remained mild across the treatment, while the co-incubation with 3-MA slightly increased the apoptosis rate. These findings implied that in addition to causing cell death, the autophagy in these cells also played another role, namely cell survival [26]. Taken together, these findings demonstrated that ROS plays an essential role in autophagy induced by COFs-derived PM2.5 exposure. However, the detailed mechanisms of how autophagy played
5. Conclusions In summary, this is the first study demonstrate that COFs-derived PM2.5 induces autophagy of HUVECs via ROS- AKT-mTOR axis, which could lead to endothelial damages of umbilical vein. However, there are still several limitations in this study. First, the findings of this study could not be directly applied in population studies, as the defense system of the whole body could not be included in this study. Second, COFs-derived PM2.5 was collected from only peanut oil in this study; although peanut oil is commonly used in China, it could not loyally represent the overall situation in China. Third, the HUVECs were directly exposed to COFs-derived PM2.5 in this study, which could not loyally reflect the effects of COFs-derived PM2.5 after absorbed through inhalation. Finally, HUVECs, an immortal cell line, were used in this study to investigate the effects of COFs-derived PM2.5 on cell 458
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Fig. 8. Effects of ROS-AKT-mTOR axis in HUVECs treated with COFs-derived PM2.5. A-C) COFs-derived PM2.5 affects the expression of proteins in the PI3K-AKT-mTOR signaling pathway. D-F) NAC rescues the effects of COFs-derived PM2.5 on the expression of the proteins in the PI3K-AKTmTOR signaling pathway. *P < 0.05, ** P < 0.01, comparing with the control group; #P < 0.05, ## P < 0.01, comparing with the PM2.5 group.
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autophagy, which could at least bias the results. Therefore, it is important to note that extrapolation of these findings to in vivo conditions remains to be established. Acknowledgments We sincerely thank the School of Life Sciences, University of Science and Technology of China for kindly providing HUVECs, thank the Electron Microscopy Room of the Provincial Hospital, Anhui Medical University for the help in TEM. Funding This work was supported by the National Natural Science Foundation of China (Grand Number: 31430028), National Basic Research Programme of China (Grand Number: 2013CB933900), the Natural Science Foundation of Anhui Province (Grand Number: 1708085MH220), the Project Foundation for the Young Talents in Colleges of Anhui Province (Grand Number: gxyq2017003), and the Grant for Scientific Research of BSKY from Anhui Medical University (Grand Number: XJ201621). Competing interests The authors declare that they have no competing interests. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.freeradbiomed.2017.10. 386. References [1] Y. Liu, Y.Y. Chen, J.Y. Cao, F.B. Tao, X.X. Zhu, C.J. Yao, D.J. Chen, Z. Che, Q.H. Zhao, L.P. Wen, Oxidative stress, apoptosis, and cell cycle arrest are induced in primary fetal alveolar type II epithelial cells exposed to fine particulate matter from cooking oil fumes, Environ. Sci. Pollut. Res. Int. 22 (2015) 9728–9741. [2] K. Ge, The transition of Chinese dietary guidelines and food guide pagoda, Asia Pac. J. Clin. Nutr. 20 (2011) 439–446. [3] T. Dearth-Wesley, P. Gordon-Larsen, L.S. Adair, A.M. Siega-Riz, B. Zhang, B.M. Popkin, Less traditional diets in Chinese mothers and children are similarly linked to socioeconomic and cohort factors but vary with increasing child age, J. Nutr. 141 (2011) 1705–1711. [4] S. Li, J. Gao, Y. He, L. Cao, A. Li, S. Mo, Y. Chen, Y. Cao, Determination of time- and size-dependent fine particle emission with varied oil heating in an experimental kitchen, J. Environ. Sci. 51 (2017) 157–164. [5] N. Shen, W. Zhang, G. Li, Impact of isolated single umbilical artery on pregnancy outcome and delivery in full-term births, J. Obstet. Gynaecol. Res. 42 (2016) 399–403. [6] M.M. Veras, R.M. Guimaraes-Silva, E.G. Caldini, P.H. Saldiva, M. Dolhnikoff, T.M. Mayhew, The effects of particulate ambient air pollution on the murine umbilical cord and its vessels: a quantitative morphological and immunohistochemical study, Reprod. Toxicol. 34 (2012) 598–606. [7] A.C. Peyter, F. Delhaes, D. Baud, Y. Vial, G. Diaceri, S. Menetrey, P. Hohlfeld, J.F. Tolsa, Intrauterine growth restriction is associated with structural alterations in human umbilical cord and decreased nitric oxide-induced relaxation of umbilical vein, Placenta 35 (2014) 891–899.
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