MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW



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MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
09-30-2021, 07:59 PM
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09-30-2021, 07:59 PM
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RE: MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
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09-30-2021, 08:04 PM
Post: #3
RE: MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
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.
Introduction

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 (1). Graphene has unique electrical and mechanical properties, a large specific surface area and potential biocompatibility. As a result, it is widely used in materials, electronics, energy, optics and biomedical fields, such as cell imaging, drug delivery and biosensing (2-5). With the large-scale production and application of graphene, research into its biological toxicity has been attracting increasing attention. A large number of studies have confirmed that the biotoxicity of graphene nanomaterials depends on their individual physicochemical properties (including size, morphology and functional groups), concentration, route of biological ingestion and the organs involved (6-9). Different forms of graphene and their derivatives display different physical/chemical properties and biological toxicities. Graphene may be categorized as single-layer graphene, few-layer graphene, graphene oxide (GO), reduced graphene oxide (rGO) and graphene nanoribbons. Among these subtypes, the biological effects of GO have received the most attention, as it has been more widely used in biological research due to its higher hydrophilicity/biocompatibility.

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 (10-13). Different organs exhibit different levels of graphene nanomaterial accumulation and clearance. The accumulation of GO in the lungs increases with increasing injection dose and particle size (14). In the lung, graphene is engulfed by alveolar macrophages and excreted in the sputum via mucosal cilia. Furthermore, 28 days after tracheal instillation, 46.2% of graphene layers may be excreted in the feces (15). As graphene can directly act on the respiratory system, research has mainly focused on graphene-induced damage to this system. It was previously reported that, in humans, the majority of inhaled graphene nanoparticles pass through the upper respiratory tract and are deposited in the lungs. As such, the deposition rate of graphene nanoparticles in the respiratory tract is ~4% (16). Therefore, lung injury is the primary symptom of graphene-induced toxicity, and mice exposed to GO nanomaterials reportedly experienced acute injury and chronic fibrosis of the lung (8).

Previous studies on the toxic effects of graphene have primarily focused on mitochondrial damage, DNA damage, the inflammatory response, apoptosis and oxidative stress (17-19). GO-associated lung injury may be relieved with the antioxidant drug dexamethasone, indicating that GO may cause pulmonary toxicity through oxidative stress (8). Autophagy is a process through which cells degrade proteins, damaged organelles and foreign matter through the lysosomal degradation pathway. Active autophagy usually results in increased phosphorylation of mTOR and beclin-1 expression, and a decreased ratio of LC3B-I/II and p62 expression levels. Therefore, the expression levels of phosphorylated mTOR, the ratio of LC3B-I/II, and the expression of p62 and beclin-1, are commonly used indicators of this process. Autophagy is regulated by multiple intracellular molecules and plays a key role in the homeostatic maintenance of cells. Autophagy may be involved in cellular defense, although excessive uncontrolled autophagy may also result in tissue damage. A 2012 study demonstrated that GO triggers Toll-like receptor-mediated autophagy in RAW264.7 mouse macrophages (20). Chen et al (21) observed that GO also triggered autologous effects in CT26 cells through Toll-like receptors. They also determined that co-administration of GO and cisplatin promoted the nuclear localization of cisplatin and LC3 protein while triggering autophagy, thereby altering the original LC3 pathway in the early stages of autophagy and enhancing the antitumor effects of cisplatin (22). However, the role of autophagy and its association with oxidative stress and inflammation in the development of GO-induced lung injury has not been extensively investigated.

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.

Materials and methods

Animals and study design

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 (23). BEAS-2B cells were seeded into 6-well plates (Beyotime Institute of Biotechnology) at a concentration of 1x105 cells/ml, and then exposed to 50 µg/ml GO for 24 h. Cell viability was assessed using the MTT method. The concentration of lactate dehydrogenase (LDH) in the culture media was determined using an LDH assay kit (cat. no. C0016; Beyotime Institute of Biotechnology) and the levels of oxidative stress indictors [malondialdehyde (MDA, cat. no. S0131S; Beyotime Institute of Biotechnology), 8-hydroxy-2'-deoxyguanosine (8-OHdG; cat. no. ab201734; Abcam) and protein carbonyl (cat. no. ab235631; Abcam)] were measured using the corresponding kits as per the manufacturers' protocols.

Lung W/D weight ratio measurement and BALF collection

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 no g for 10 min at 4˚C, and the total protein levels were measured using a BCA protein assay kit (Beyotime Institute of Biotechnology).

Histopathological examination

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.

ShRNA-mediated ATG5 knockdown

Autophagy was inhibited by ATG5 knockdown using shRNA, as previously described by Domagala et al (24). Briefly, BEAS-2B cells were infected with lentiviral particles (Sigma-Aldrich; Merck KGaA) encoding ATG5-specific shRNA (MISSION shRNA TRCN0000151474; shATG5 group) or a scrambled (non-targeting) shRNA plasmid (SHC002V; shNTC group). After transduction, 2 µg/ml puromycin was added to the culture medium.

MTT assay

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.

LDH measurement

The release of LDH was measured using the method described by Zhang et al (25). First, BEAS-2B cells were harvested as mentioned above, and the cell suspension was adjusted to 5x104/ml, 500 µl of which was added to each well of a 6-well plate. The cells were cultured in a CO2 incubator for 12 h at 37˚C, after which time 50 µg/ml GO was added to each well for a further 24-h incubation period (37˚C). The supernatants were then harvested and LDH level was measured using an LDH assay kit (Nanjing Jiancheng Bioengineering Institute). The supernatants were incubated at 37˚C for 15 min, and 2,4-dinitrophenylhydrazine was added for a further 15 min. Finally, 0.4 M NaOH was added to each well and the absorbance at 450 nm was measured using a microplate reader (Bio-Rad 680; Bio-Rad Laboratories, Inc.).

Measurement of oxidative products

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.

Inflammatory cytokine ELISA

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.).


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09-30-2021, 08:05 PM
Post: #4
RE: MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
Hello!!!!

-------------------------------
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.

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09-30-2021, 08:07 PM
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RE: MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
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10-01-2021, 05:09 PM
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RE: MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
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10-05-2021, 08:03 PM
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Pin it
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10-08-2021, 06:24 PM
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RE: MUST HEAR: GRAPHENE OXIDE - NWO HORROR SHOW
heis
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Absolute Crap Crap Reasonable Nice Amazing
 


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