*Contributed equally
Graphene is a two-dimensional structured material with a hexagonal honeycomb lattice composed of carbon atoms. The biological effects of graphene oxide (GO) have been extensively investigated, as it has been widely used in biological research due to its increased hydrophilicity/biocompatibility. However, the exact mechanisms underlying GO-associated lung toxicity have not yet been fully elucidated. The aim of the present study was to determine the role of GO in lung injury induction, as well as its involvement in oxidative stress, inflammation and autophagy. The results revealed that lower concentrations of GO (5 and 10 mg/kg) did not cause significant lung injury, but the administration of GO at higher concentrations (50 and 100 mg/kg) induced lung edema, and increased lung permeability and histopathological lung changes. High GO concentrations also induced oxidative injury and inflammatory reactions in the lung, demonstrated by increased levels of oxidative products [malondialdehyde(MDA) and 8-hydroxydeoxyguanosine (8-OHdG)] and inflammatory factors (TNF-α, IL-6, IL-1β and IL-8). The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CLQ) inhibited autophagy in the lung and attenuated GO-induced lung injury, as demonstrated by a reduced lung wet-to-dry weight ratio, lower levels of protein in the bronchoalveolar lavage fluid, and a reduced lung injury score. Furthermore, 3-MA and CLQ significantly reduced the levels of MDA, 8-OHdG and inflammatory factors in lung tissue, suggesting that autophagy also mediates the development of oxidative injury and inflammation in the lung. Finally, autophagy was directly inhibited in BEAS-2B cells by short hairpin RNA-mediated autophagy protein 5 (ATG5) knockdown, which were then treated with GO. Cell viability, as well as the extent of injury (indicated by lactate dehydrogenase level) and oxidative stress were determined. The results revealed that ATG5 knockdown-induced autophagic inhibition significantly decreased cellular injury and oxidative stress, suggesting that autophagy induction is a key event that leads to lung injury during exposure to GO. In conclusion, the findings of the present study indicated that GO causes lung injury in a dose-dependent manner by inducing autophagy.
Graphene is a two-dimensional structured material with a hexagonal honeycomb lattice composed of carbon atoms, and is currently the thinnest and most widely used non-metallic nanomaterial (
Previous animal studies have demonstrated that graphene can enter the body through tracheal instillation, inhalation, intravenous injection, intraperitoneal injection and oral administration. Graphene penetrates the blood-air, blood-brain and blood-placenta barriers, and subsequently accumulates in the lung, liver and spleen, resulting in acute or chronic injury (
Previous studies on the toxic effects of graphene have primarily focused on mitochondrial damage, DNA damage, the inflammatory response, apoptosis and oxidative stress (
Therefore, the aim of the present study was to determine the role of GO in lung injury induction, as well as its involvement in oxidative stress, inflammation and autophagy in a rat model, in the hope that the findings may help elucidate the mechanisms underlying GO-induced lung injury.
Male Sprague-Dawley rats (age, 10 weeks; weight, 250-300 g) were obtained from the Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine (Shanghai, China). The animals were housed in the animal center of Xinhua Hospital at a constant temperature of 25±2˚C, a relative humidity of 41%, and on a 12:12 h light/dark cycle. All animals had free access to food and water. The experiment was conducted according to the principles of the Bioethics Committee of Shanghai Jiaotong University School of Medicine for the care and use of laboratory animals (no. AS-20183265), as well as the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996). The humane endpoints used to identify the adverse effects of surgery/treatments were as follows: i) Weight loss of 20-25%; ii) inability or extreme reluctance to stand, persisting for 24 h; iii) depression coupled with body temperature <37˚C; iv) infection involving any organ system failing to respond to antibiotic therapy within 48 h and accompanied by systemic signs of illness; and v) signs of severe organ system dysfunction.
To determine the effects of GO on lung injury, rats were randomly assigned to five groups (n=12 per group) as follows: i) Control; ii) GO (5 mg/kg); iii) GO (10 mg/kg); iv) GO (50 mg/kg); and v) GO (100 mg/kg). Rats in the control group were fed a normal diet and received no special treatment. Rats in the GO groups received 5, 10, 50 and 100 mg/kg GO injections, respectively. GO was injected into the tail vein once a day for 7 consecutive days. There were no significant differences in age or weight among the groups. Following treatment, the lung wet-to-dry (W/D) weight ratio, levels of protein in the bronchoalveolar lavage fluid (BALF), lung injury scores, oxidative stress, and levels of autophagy-related proteins and inflammatory factors in the lung tissue were determined.
Furthermore, to evaluate the involvement of autophagy in the pathology of GO-induced lung injury, the rats were randomly assigned to the following six groups (n=12): i) Control; ii) vehicle; iii) GO50; iv) GO50 + vehicle; v) GO50 + 3-methyladenine (3-MA); and vi) GO50 + chloroquine (CLQ). Rats in the control group were fed a normal diet and received no special treatment. Rats in the vehicle group were also fed a normal diet, and received saline (the same volume as was used for GO administration) via the tail vein once a day for 7 days. Rats in the GO50 group received 50 mg/kg GO injections via the tail vein once a day for 7 days; those in the GO50 + vehicle group received 50 mg/kg GO injected via the tail vein, and the same volume of saline intraperitoneally for 7 days. The rats in the GO50 + 3-MA group received 50 mg/kg GO injected via the tail vein, and 15 mg/kg 3-MA (Sigma-Aldrich; Merck KGaA) intraperitoneally once a day for 7 days. Finally, the rats in the GO50 + CLQ group were administered 50 mg/kg GO injected via the tail vein, and 20 mg/kg CLQ intraperitoneally (Sigma-Aldrich; Merck KGaA), once a day for 7 days. The levels of autophagy-related proteins, lung W/D weight ratio, protein levels in the BALF, lung injury scores, and levels of oxidative stress and inflammatory factors in lung tissue were then determined.
To confirm the role of autophagy in GO-induced lung cell injury, autophagy was specifically inhibited in BEAS-2B cells by short hairpin (sh) RNA-mediated autophagy protein 5 (ATG5) knockdown. The cells were subsequently treated with GO as previously described (
Following treatment with GO, the rats were euthanized with an overdose of pentobarbital (200 mg/kg via i.p. injection), and the breathing and heartbeat were checked to verify rat death before opening the thoracic cavity to expose the lungs. The right middle lobe of the lung was removed and weighed to obtain the wet weight. The lungs were then dried in an oven at 60˚C for 3 days to obtain the dry weight, and the lung W/D weight ratio was calculated to evaluate lung edema. To collect BALF, the left lung was lavaged three times with saline (5 ml, 4˚C). The collected lavage fluid was centrifuged at 1,200 x g for 10 min at 4˚C, and the total protein levels were measured using a BCA protein assay kit (Beyotime Institute of Biotechnology).
After the rats were euthanized, the lung tissue was harvested and stained with hematoxylin and eosin (H&E). Three specimens were randomly selected from each rat, and five fields for each section were analyzed under a microscope (magnification, x200) by two independent pathologists who were blinded to the experimental groupings. The staining scores were calculated according to the following variables: Alveolar congestion, hemorrhage, infiltration or aggregation of neutrophils in the airspace, and hyaline membrane formation. The lung injury scores ranged between 0 and 4 as follows: i) 0, no injury; ii) 1, <25% lung involvement; iii) 2, 25-50% lung involvement; iv) 3, 50-75% lung involvement; and v) 4, >75% lung involvement.
Autophagy was inhibited by ATG5 knockdown using shRNA, as previously described by Domagala
BEAS-2B cells were harvested with trypsin and re-suspended in culture medium. The cell suspension was adjusted to a concentration of 5x104 cell/ml, and 100 µl was added to each well of a 96-well plate. The cells were maintained in a CO2 incubator for 12 h at 37˚C, after which time 50 µg/ml GO was added to each well. After a further 24 h, the culture medium was discarded and the cells were harvested by gentle centrifugation; 10 µl MTT solution (5 mg/ml, 0.5% MTT; Beyotime Institute of Biotechnology) was then added to each well and the cells were incubated for another 4 h at 37˚C. To terminate the reaction, 150 µl dimethyl sulfoxide (Sigma-Aldrich; Merck KGaA) was added to each well, and the 96-well plate was placed on a shaking platform for 10 min to fully dissolve the formazan crystals. The absorbance of each well was measured at OD490 nm and the cell survival rate was calculated.
The release of LDH was measured using the method described by Zhang
The levels of oxidative products (MDA, 8-OHdG and protein carbonyl) in the lung homogenates were detected using the respective kits according to the manufacturers' instructions. The 8-OHdG ELISA kit was purchased from Cusabio Technology, Ltd. (cat. no. CSB-E10526r), and the MDA (cat. no. A003-1-2) and protein carbonyl (cat. no. A087-1-2) kits were purchased from Nanjing Jiancheng Bioengineering Institute.
The expression levels of TNF-α (cat. no. E-EL-R2856c), IL-6 (cat. no. E-EL-R0015c), IL-1β (cat. no. E-EL-R0012c) and IL-8 (cat. no. SEKR-0071-96T) (all from Beijing Solarbio Science & Technology Co., Ltd.) in the lung tissue were measured using commercial ELISA kits (Elabscience, Inc.) according to the manufacturers' protocols. The cytokine levels were determined using a spectral scanning plate reader (Varioskan; Thermo Fisher Scientific, Inc.).
Briefly, the right lung was harvested from each rat and placed in ice-cold homogenization buffer (100 mmol/l NaCl, 50 mmol/l Tris base, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA and 1% Triton X-100; pH 7.5), and then homogenized in a 15-ml glass homogenizer. The homogenate was centrifuged at 1,500 x g for 10 min at 4˚C to collect the supernatant. The protein concentration was measured using a BCA protein assay kit (Beyotime Institute of Biotechnology). The proteins (50 µg/per lane) were separated by 10% SDS-PAGE and transferred to PVDF membranes, which were then incubated with a mixture of tris-buffered saline (TBS), Tween-20 (0.1%) and non-fat milk for 1 h at 37˚C. The membranes were incubated with the following primary antibodies overnight at 4˚C in Universal Antibody Dilution Buffer (Santa Cruz Biotechnology, Inc.): Anti-p-mTOR (cat. no. sc-293133), anti-mTOR (cat. no. sc-517464), anti-LC3B-I (cat. no. sc-398822), anti-LC3B-II (cat. no. sc-271625), anti-GAPDH (cat. no. sc-365062), anti-p62 (cat. no. sc-48402), anti-ATG5 (cat. no. sc-133158) and anti-beclin-1 (cat. no. sc-48341) (all 1:1,000; all from Santa Cruz Biotechnology, Inc.). The membranes were washed three times with TBST, and then incubated with secondary antibody (cat. no. sc-516102; 1:5,000; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The protein bands were visualized using the enhanced chemiluminescence method with an Electro-Chemi-Luminescence Substrate Kit (Rahn AG) and quantified using Quantity One software (v4.6.6; Bio-Rad Laboratories Inc.). The bar graphs show the average values, and the error bars are quantified from the optical densities of six bands (experimental repeats) per group.
Statistical analysis was conducted with SPSS software version 19 (IBM Corp.) using one-way ANOVA. The data fulfilled ANOVA assumptions, such as normality and homoscedasticity. Dunnett's post hoc test was performed following ANOVA for comparing experimental groups with the control group only; Bonferroni's post hoc test was performed following ANOVA for comparing the data between experimental groups. P<0.05 was considered to indicate a statistically significant difference.
Changes in the lung W/D weight ratio, the amount of protein in the BALF, histopathological appearance and lung injury score are presented in
Changes in oxidative products (MDA, protein carbonyl and 8-OHdG) and inflammatory factors (TNF-α, IL-6, IL-1β and IL-8) in the lung were then assessed. As shown in
The expression levels of autophagic proteins (mTOR, LC3B-I/II, p62 and beclin-1) were then detected in lung tissues; representative western blot images are displayed in
The expression levels of autophagy-related proteins (mTOR, LC3B-I/II, p62 and beclin-1) in rat lung tissues after treatment with the autophagy inhibitors 3-MA and CLQ are shown in
To confirm the role of autophagy in the development of GO-induced lung injury, the effects of 3-MA and CLQ on the severity of GO-induced lung injury were assessed. First, treatment with the vehicle control did not induce lung injury. The lung W/D weight ratio and amount of protein in the BALF were significantly decreased, but not completely restored by 3-MA or CLQ (
To confirm the role of autophagy in the development of GO-induced oxidative stress and inflammation in lung tissue, the effects of 3-MA and CLQ on the production of MDA, 8-OHdG and inflammatory factors were investigated. First, treatment with vehicle did not induce oxidative stress or inflammation. The levels of MDA and 8-OHdG were significantly decreased, but not completely restored by 3-MA and CLQ (
To confirm the role of autophagy in GO-induced lung cell injury, autophagy was specifically inhibited in BEAS-2B cells via shRNA-mediated ATG5 knockdown, and the changes in cell viability, LDH levels and oxidative stress were determined. Treatment with shNTC or shATG5 alone did not significantly affect cell viability, or the levels of LDH and oxidative stress. The protein expression of ATG5 following shRNA transfection is shown in
Due to their unique physical and chemical properties, graphene nanomaterials have been widely used in various fields, such as those surrounding energy and the environment. As a result, the environmental behavior and biological toxicity of graphene have been attracting increasing attention. GO may promote acute inflammatory reactions and chronic injury by interfering with the normal physiological functions of important organ systems. The results of various
The GO-induced production of excess reactive oxygen species (ROS) is associated with acute lung injury, inflammatory response, cell apoptosis and DNA damage (
Intratracheal infusion and intravenous injection of high doses of graphene nanomaterials cause significant inflammatory reactions in animals, such as inflammatory cell infiltration, pulmonary edema and granuloma formation (
The number of reports on GO-induced autophagy has increased in recent years. In the present study, the expression levels of autophagy-related proteins (mTOR, LC3B-I/II, p62 and beclin-1) were assessed to confirm the role of autophagy in rat GO-induced lung injury, following treatment with different concentrations of GO. The results revealed that GO at 50 and 100 mg/kg significantly increased the level of mTOR phosphorylation and beclin-1 expression, but decreased the LC3B-I/II ratio and the expression of p62. These data directly confirm that higher concentrations of GO can induce autophagy in the lung in an mTOR-dependent manner. To elucidate the role of autophagy in GO-induced lung injury, rats were then treated with 50 mg/kg GO and the autophagy inhibitors 3-MA and CLQ, and the levels of autophagy-related proteins and the severity of GO-induced lung injury were assessed. Autophagy was also specifically inhibited in BEAS-2B cells via shRNA-mediated ATG5 knockdown, and changes in cell viability, LDH release and oxidative stress were determined. The results revealed that 3-MA and CLQ inhibited autophagy in the lung and attenuated GO-induced lung injury. 3-MA and CLQ also significantly reduced the production of MDA, 8-OHdG and inflammatory factors in the lung tissue, suggesting that autophagy may mediate the development of oxidative injury and inflammation in the lung. Furthermore, cell viability was significantly increased by shATG5 compared with the control shRNA. By contrast, LDH release in the shATG5 group was significantly lower compared with that in the shNTC group. Furthermore, the levels of both MDA and 8-OHdG were significantly decreased by shATG5 treatment.
There is a close association between oxidative stress and autophagy. ROS can induce autophagy via various associated signaling pathway proteins (
In conclusion, the results of the present study confirmed that the intravenous administration of GO induces lung injury in a dose-dependent manner, as demonstrated by lung edema and increased lung vascular permeability, histopathological changes, oxidative injury and inflammation. GO also significantly induced autophagy in lung cells, while autophagy inhibitors attenuated GO-induced lung injury. These results suggest that GO promotes autophagy-induced lung injury.
Not applicable.
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
LZ conducted lung W/D weight ratio measurement and BALF collection, histopathological examination, MTT assays and wrote the manuscript; SO conducted shRNA-mediated ATG5 knockdown and LDH measurements; HZ conducted the measurement of oxidative products; MQ conducted western blotting; YD conducted inflammatory cytokine ELISAs; SW collected and analyzed the data; YW interpreted the results; JO designed the study, financially supported the study and revised the manuscript. All authors read and approved the final version of the manuscript.
The experiment was conducted according to the principles of the Bioethics Committee of Shanghai Jiaotong University School of Medicine for the care and use of laboratory animals (no. AS-20183265), as well as the Guide for the Care and Use of Laboratory Animals (NIH publication no. 85-23, revised 1996).
Not applicable.
The authors declare that they have no competing interests.
Changes in lung edema, histopathological appearance and vascular permeability. (A) Lung W/D weight ratio. (B) Protein levels in the BALF. (C) Histopathological appearance (magnification, x200). (D) Lung injury scores. Data are expressed as the mean ± SEM; n=12. #P<0.05 vs. control. Dunnett's post hoc test following ANOVA was used to compare experimental groups with the control group. W/D, wet-to-dry; BALF, bronchoalveolar lavage fluid; GO, graphene oxide.
Changes in the levels of oxidative products and inflammatory factors in the lung. Expression levels of (A) MDA, (B) protein carbonyl, (C) 8-OHdG, (D) TNF-α, (E) IL-6, (F) IL-1β and (G) IL-8. Data are expressed as the mean ± SEM; n=12. #P<0.05 vs. control. Dunnett's post hoc test following ANOVA was used to compare experimental groups with the control group. MDA, malondialdehyde; 8-OHdG, 8-hydroxy-2'-deoxyguanosine.
Changes in autophagic protein levels in the lung tissue. (A) Representative western blot images of p-mTOR, mTOR, LC3B-I and LC3B-II. (B) Representative western blot images of p62 and beclin-1. Ratios of (C) p-mTOR/mTOR and (D) LC3B-I/II expression. Expression levels of (E) p62 and (F) beclin-1. Data are expressed as the mean ± SEM; n=12. #P<0.05 vs. control. Dunnett's post hoc test following ANOVA was used to compare experimental groups with the control group. GO, graphene oxide.
Effects of autophagy inhibitors on autophagic proteins in lung tissue. (A) Representative western blot images of p-mTOR, mTOR, LC3B-I and LC3B-II. (B) Representative western blot images of p62 and beclin-1. Ratios of (C) p-mTOR/mTOR and (D) LC3B-I/II expression. Expression levels of (E) p62 and (F) beclin-1. Data are expressed as the mean ± SEM; n=12. *P<0.05 vs. GO50 + vehicle (the same dose of saline). Bonferroni's post hoc test was performed following ANOVA to compare the data between experimental groups. 3-MA, 3-methyladenine; CLQ, chloroquine; GO, graphene oxide.
Effects of GO on lung edema, histopathological appearance and vascular permeability. (A) Lung W/D weight ratio. (B) Protein levels in the BALF. (C) Histopathological appearance (magnification, x200). (D) Lung injury scores. Data are expressed as the mean ± SEM; n=12. #P<0.05 vs. vehicle (the same dose of saline). *P<0.05 vs. GO50 + vehicle (the same dose of saline). N.S., not significant. Bonferroni's post hoc test was performed following ANOVA for comparing the data between experimental groups. W/D, wet-to-dry; BALF, bronchoalveolar lavage fluid; 3-MA, 3-methyladenine; CLQ, chloroquine; GO, graphene oxide.
Effects of autophagy inhibitors on oxidative products and inflammatory factors in the lung. Expression levels of (A) MDA, (B) 8-OHdG, (C) TNF-α, (D) IL-6, (E) IL-1β and (F) IL-8. Data are expressed as the mean ± SEM; n=12. #P<0.05 vs. vehicle (the same dose of saline); *P<0.05 vs. GO50 + vehicle (same dose of saline). N.S., not significant. Bonferroni's post hoc test was performed following ANOVA to compare the data between experimental groups. MDA, malondialdehyde; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; 3-MA, 3-methyladenine; CLQ, chloroquine; GO, graphene oxide.
Effects of ATG5 knockdown on GO-induced injury and oxidative stress in BEAS-2B cells. (A) Protein expression of ATG5. (B) Cell viability. (C) Relative LDH release. Levels of (D) 8-OHdG, (E) MDA and (F) protein carbonyl. Data are expressed as the mean ± SEM; n=12. #P<0.05 vs. control; *P<0.05 vs. GO + shNTC. N.S., not significant. Bonferroni's post hoc test was performed following ANOVA to compare the data between experimental groups. ATG5, autophagy protein 5; GO, graphene oxide; LDH, lactate dehydrogenase; 8-OHdG, 8-hydroxy-2'-deoxyguanosine; MDA, malondialdehyde.