| BackgroundCarbon-based nanomaterials have been widely used because of their unique physical,chemical,and biological properties.Among these materials,graphene oxide(GO),as a two dimensional(2D)carbon nanosheet,has attracted considerable interest in recent years.GO possesses larger specific surface area,abundant hydrophilic groups and high stability in aqueous dispersion,which have enabled GO to be used in a growing number of applications in biomedical fields,such as drug carriers,cellular imaging,biosensors,and antibacterial agents.Simultaneously,it should be noted that the growing uses of GO have opened various opportunities for these nanomaterials to be released into the environment and the human body.Considering the unknown biological activity of these nanomaterials,it is urgently required to establish a paradigm for accurately evaluating their adverse effects in biological systems.After exposure,many reports have suggested that GO can transfer throughout the body,be retained in target organs over an extended period of time and further cause injurious responses.Additionally,in vitro data have also demonstrated the cellular toxicity of GO,including the excessive generation of oxidative stress,DNA damage,cell cycle arrest and apoptosis.The most widely investigated mechanisms of nanomaterial-induced toxicity are oxidative stress,inflammatory response and mitochondrial damage,but the underlying mechanisms are still largely obscure.Note that autophagy has been considered as a novel mechanism underlying GO-caused toxicity.It was reported that GO exposure might induce autophagic response or autophagic cell death both in vitro and in vivo.However,the detailed regulatory mechanism of autophagy in GO-induced toxic effect is still poorly defined.Appropriate regulation of autophagy is essential for human health,and autophagy dysfunctions may be related with various human pathologies.Furthermore,the proper acid environment and hydrolase activity inside lysosomes is crucial for intracellular content degradation.Under stimulus exposure,the dysfunctions of lysosomes may lead to incomplete autophagy and even initiate cell programmed death.In this study,we systematically explored the impact of GO on cell viability at different doses and time points.We further observed that GO could induce autophagy and the impairment of autophagic flux,which resulted in an elevated level of autoghagic substrate p62.Moreover,the relationship between p62 accumulation and caspase-9 mediated apoptosis was also determined using both chemical and genetic methods.Objective1.To observe the cytotoxicity of GO incubated with PC 12 cells and the important role of autophagy in the toxic effect of GO.2.To explore the relationships between the block of autophagic flux and lysosomal dysfunction.3.To explore the relationships between over accumulation of p62 proteins and apoptotic cell death in PC 12 cells.Materials and MethodsPart 1:Characterization of GO1)Scanning electron microscope(SEM)and atomic force microscope(AFM)were used to observe the primary particle size and morphology of GO.2)Fourier transform infrared(FTIR)spectroscopy and Raman spectroscopy were applied to analyze characteristic vibrations of GO.3)X-ray photoelectron spectroscopy(XPS)was used to observe the basic chemical bonds of GO.4)Dynamic Light Scattering was used to determine the hydrodynamic size and zeta potential of GO in distilled water(DW)and complete culture mediumPart 2:Cytotoxicity of GO and the interactions between GO and PC12 cells1)Preperation of GO suspension GO samples were dispersed in pure water firstly as the stock solution.Before administration,the suspension was diluted with complete culture medium and sonicated for 30 min using an ultrasonic processor.2)CCK-8 testPC12 cells were seeded in a 96-well plate and incubated with GO samples at concentrations of 5,10,20,30,40,50,and 60 pg/mL.After 6 h,12 h,and 24 h incubation,cell viability was respectively assessed by the cell counting kit-8(CCK-8)test.3)LDH testLeakage of LDH from the cells was conducted to examine the effect of GO on cell membrane permeability.Cells were seeded in 96-well plates and incubated with GO sulution at different concentrations.At indicated time points(6 h and 24 h),the cell culture medium collected from each well was centrifuged and measured at 490 nm on a microplate reader.4)The interactions between GO and PC 12 cellsa.Preparation of FITC-BSA-Conjugated GO GO samples and FITC-BSA solution were mixed at a mass ratio of 1:1 followed by incubation at 37? overnight with protection from light.Thereafter,the mixed solutions were centrifuged at 16,000 g and 4? for 30 min.Finally,the pellets were collected and resuspended with complete culture medium.b.Cellular adhesionCells were plated on a glass coverslip placed in a 12-well plate and incubated with 50?g/mL GO for 24 h.Cells were then fixed in 4%paraformaldehyde(PFA)for 20 min.Subsequently,the cell coverslips were dehydrated using ethanol,dried,and observed using SEM.c.Cellular uptakeCells were seeded in a 12-well plate and treated with FITC-BSA-GO for different time points.The cells were then fixed in 4%PFA and permeabilizated in Triton X-100,and then incubated with rhodaminephalloidin in the dark for 1 h.Finally,the cell nuclei were stained with DAPI.Representative images were captured using a fluorescent microscope.Quantitative analysis of fluorescence intensities was conducted by a flow cytometric(FACS).Part 3:GO-induced autphagy and involved signal pathways1)Inducement of autophagya.TEM evaluationCells were seeded in a 6-well plate and exposed to 50 ?g/mL GO.After 24 h,the cells were collected and fixed with 2.5%glutaraldehyde and embedded with epoxy resin.The ultrathin cell specimens(70 nm)were then stained with 1%lead citrate and 0.5%uranyl acetate and examined by a TEM.b.Fluorescence microscopy observationPC12 cells were first seeded in a 24-well plate and transfected with stubRFP-sensGFP-LC3 lentivirus.Cells stably expressing RFP-GFP-LC3 were treated with 50?g/mL GO.Rapamycin was added as the positive control.At the indicated time points(6 h and 24 h),the coverslips were fixed with 4%PFA and cell nuclei were labeled with DAPI.The intracellular distribution of LC3 puncta was observed by a fluorescent microscope.c.Cell lysis and protein extractionCells were treated with GO samples of 40,50,and 60 ?g/mL for 6 h and 24 h.For study of related signal pathways,740Y-P,SC79,and 3BDO were added for the duration of the stimulation.Then cell extracts were prepared using RIPA lysate buffer containing protease and phosphatase inhibitor at 4?.Cell pellets were analyzed for the protein concentrations using the BCA protein assay kit.d.Western blot analysisAn equal mass of protein was subjected to SDSPAGE and then transferred onto polyvinylidene fluoride(PVDF)membrane via semidry transfer.Membranes were blocked with 5%milk and washed with Tris-buffered saline in 0.1%Tween(TBST).Thereafter,the membranes were incubated with primary antibody at 4? overnight,and with horseradish peroxidaseconjugated(HRP)secondary antibody at room temperature for 1 h.Finally,protein bands were developed with chemiluminescence substrate and visualized on an enhanced chemiluminescence(ECL)detection system.Part 4:The relationship between impairment of autophagic flux and lysosomal dysfunction1)Lysosomal acidity assayCells were grown on glass coverslips in a 12-well plate and treated with GO for 24 h.LysoSensor Green DND-189 was added to the cells followed by 30 min of incubation at 37?.Cell coverslips were then washed in PBS for 2 times and visualized under a fluorescent microscope.The flow cytometry was used to quantitatively evaluate the intensity of FITC fluorescence.2)Lysosomal enzyme activity assayCells were treated with 40,50,and 60 ?g/mL of GO and washed with cold PBS.The cells were then lysed according to the manufacturer's instructions.Finally,the supernatant was collected and subjected to ACP and cathepsin B activity assay.3)Lysosomal membrane permeabilization assayCells were treated with different concentrations of GO for 24 h.Subsequently,the cells were incubated with anti-galectin-3 and anti-lysosomeassociated membrane protein-1(LAMP-1)antibodies at 4? overnight,washed,and labeled with the corresponding fluorescent secondary antibodies.Finally,the nuclei were stained with DAPI.LAMP-1-positive galectin-3 puncta were counted manually and analyzed by ImageJ software.Part 5:Cell apoptosis induced by over acuumulation of p62 protein and associated signal pathways1)Initiation of apoptosis and involved signal pathwaysa.Annexin V/FITC analysisCells were plated in a 6-well plate and exposed to GO 40,50,and 60 ?g/mL for 24 h.In the rescue experiment,rapamycin was added for the duration of the stimulation.Thereafter,the treated cells were collected and stained with FITC-conjugated Annexin V and propidium iodide(PI),and immediately analyzed with the flow cytometry.b.MMP measurementCells were grown in a 12-well plate and exposed to GO samples for 24 h.Subsequently,the cells were stained with JC-1 working solution for 20 min at 37?.The cells were then washed and instantly analyzed for red and green fluorescence with a flow cytometry.c.Nuclear stainingThe cells were treated with GO for 24 h.Additionally,CCCP was added as a positive control.After incubation,the cells were washed,fixed with 4%PFA and labeled with Hoechst 33258.Fluorescent images were acquired by a fluorescent microscope.d.Western Blot analysisSimilarly,cells were treated with GO for 24 h.At the end of the treatment,cell extracts were prepared using RIPA lysate buffer.An equal mass of protein was subjected to SDSPAGE and transferred onto PVDF membrane.The membranes were were incubated with primary antibody and HRP secondary antibody respectively.Finally,protein bands were visualized on an ECL detection system.2)The relationship between over accumulation of p62 and cell apoptosisa.LDH testSimilarly,cells were seeded in 96-well plates and treated with GO samples at 40,50,60?g/mL for 24 h.In the rescue group,rapamycin was added for the duration of the stimulation.Then cell culture medium collected from each well was centrifuged and measured at 490 nm on a microplate reader.b.Annexin V/FITC analysisCells were plated in a 6-well plate and exposed to GO for 24 h.In the rescue group,rapamycin was added for the duration of the stimulation.Thereafter,the treated cells were collected and stained with FITC-conjugated Annexin V and PI,and analyzed using a flow cytometry.c.Western Blot analysisSimilarly,cells were treated with GO for 24 h.In the rescue group,rapamycin was added for the duration of the stimulation.At the end of the treatment,cell extracts were prepared using RIPA lysate buffer.An equal mass of protein was subjected to SDSPAGE and transferred onto PVDF membrane.The membranes were were incubated with primary antibody and HRP secondary antibody respectively.Finally,protein bands were visualized on an ECL detection system.d.siRNA transfectionCells were seeded in a 12-well plate and transfected with siRNA duplexes with Lipofectamine 2000 reagent diluted in OptiMEM following instructions from the manufacturers.The western blot assay was used to examine knockdown efficiency after 48 h of transfection.e.Western Blot analysis on transfected cellsCells with the highest transfection efficiency were treated with GO for 24 h,and used for WB analysis.Statistical analysisGraphpad Prism(Graphpad,CA,USA)was used for statistical analysis.All data are expressed as mean± standard deviation.Comparisons between different groups were analysed using one-way analysis of variance(ANOVA)or Student's t test.All differences were considered significant whenP<0.05.Results1.SEM showed that the lateral size distribution for the GO nanosheets was mainly in the range of 500 to 800 nm.AFM revealed that the average thickness was approximately 1.0 nm.The FTIR spectra confirmed the characteristic vibrations of the GO materials and were consistent wit:h the observation of Raman spectroscopy,which detected the Raman shifts of D band(?1348 cm-1)and G band(?1602 cm-1).Moreover,the XPS spectra reflected the presence of basic chemical bonds in GO:C=O,C=C,C-O and O-C=O.The hydrodynamic diameter of GO samples in pure water was about 797.42 nm,and 439.14 nm in complete culture medium.Meanwhile,the zeta potential of GO in pure water was-47.9± 0.79 mV,and-9.82± 1.06 mV in cell culture medium.2.CCK-8 results showed that GO triggered cytotoxicity in a dose-and time-dependent manner.Treatment with GO could induce significant cell death(greater than 50%)at a dose of 60 ?g/ml after 6 h coincubation while causing the similar toxic effect at 40.g/mL after 24 h exposure.This finding was in consistent with the observation of LDH test.3.A growing amount of GO was taken up into the cells or absorbed on the plasma membrane as the incubation time increased from 1 h to 24 h.TEM found GO nanosheets adhered on the cell surface,absorbed in the cell invaginations,and finally internalized in the intracellular vesicles.Besides,a lot of autophagosome and swelling mitochondrial and lysosome were detected in the cytoplasm.The immunofluorescence images showed that GO caused significant accumulation of LC3-positive punctuate structures.Compared with the control and GO-treated cells,rapamycin induced more red puncta and significantly higher RFP/GFP ratio.Moreover,western blot analysis presented an increased LC3-II/LC3-I ratio and up-regulation of autophagy-related gene 5(Atg 5)and p62.4.The expression of PI3K,phosphorylated Akt and mTOR in treated cells significantly decreased in a dose-dependent manner compared with untreated cells.Simultaneously,the increased level of LC3-II/LC3-I was remarkably mitigated in the presence of pathway gene activators(740-YP,SC79 and 3BDO).5.The FITC fluorescence intensity decreased along with the GO concentration increasing from 40 ?g/mL to 60 ?g/mL.Flow cytometry further demonstrated that GO markedly weakened lysosome acidification in a dose-dependent manner.Besides,the activities of ACP and cathepsin B were significantly declined in a dose-dependent manner.Finally,GO was found to cause lysosomal membrane permeabilization by a maximum of 29%of the cell population.6.The Annexin V-FITC/PI based FACS assay demonstrated a clear induction of apoptosis in PC 12 cells in a dose-dependent manner.This finding was further confirmed by the MMP measurement.Western blot assay demonstrated that phosphorylation of caspase 9 and caspase 3 proteins were significantly increased after GO treatment for 24 h.Additionally,the expression level of Bax was markedly elevated while the level of Bcl-2 which suppressed apoptosis decreased dramatically.The presence of rapamycin presented a similar rescue effect in both early and total cell apoptosis.WB results found that evident LC3 turnover and increased level of Atg5.Simultaneously,the expression level of p62 decreased dramatically,along with the down-regulated levels of cleaved-caspase 3,cleaved caspase 9 and Bax proteins.The siRNA-mediated knockdown of p62 experiments found the same changing tendency as the rapamycin treatment.Conclusions1.GO triggered cytotoxicity in a dose-and time-dependent manner.Furthermore,GO induced-toxicity was more relevant to the nanomaterial concentration compared to the exposure duration.2.GO triggered an increased autophagic response via the inhibition of PI3K/Akt/mTOR pathways,and the impairment of autophagic flux in PC12 cells,which was determined to be associated with defects in lysosome degradation capability.3.The elevated level of p62 protein was functionally involved in cell apoptosis via the caspase-9 dependent pathway.Inhibition of excessive accumulation of autophagic substrate using chemical or genetic methods could attenuate GO induced apoptosis in PC12 cells. |
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