| ObjectiveGraphene is a new type of carbon nanomaterials,the thickness of only one carbon atom,is the world’s thinnest nano-materials.Graphene composed of carbon atoms to sp2 hybrid orbit composed of hexagonal two-dimensional structure,each atom has a strong force,the structure is very stable,with high hardness,good stretch and other characteristics,in physics,Medicine and many other disciplines have a good application prospects.Graphene oxide(GO)is a derivative of graphene after oxidation,and has more hydrophilic and biocompatibility than graphene.Researchers have extensively discussed their effects on drug delivery,tumor Treatment,optical imaging and biosensors.With the extensive application of GO materials,its biosafety issues are also of concern.The liver is an important detoxification organ in the human body and an important target organ for the toxic effects of nanomaterials.Therefore,the aim of this study is to find a biomarker that can be used to evaluate the acute inflammatory response of nanomaterials liver,To investigate the effect of different diameter GO on the acute inflammation of the liver after exposure to the tail vein and to explore the mechanism of GO,and to provide experimental support and theoretical basis for the safe application of GO in the field of biomedicine.MethodsIL-6-Fluc reporter gene was targeted to the liver by hydrodynamic gene delivery technology.Bioluminescence imaging was used to detect the luciferase expression in mouse liver after inflammatory stimulation.Besides,the relevance between the light intensity and IL-6 level was also intensively investigated.The liver IL-6 was used as a marker to indicate acute inflammation of liver in large,medium and small scale GO,and the visualized mouse model was used to monitor the real-time dynamic(large diameter of 500-1000 nm,marked as Large,abbreviated as L),medium(50-500 nm in diameter,labeled as Middle,abbreviated as M),small(diameter of 50-200 nm,marked as Small,abbreviated as S)three sizes of GO nanomaterials by tail vein injection The activation of IL-6 in the liver of mice with different sizes was analyzed from the oxidative damage induced by Reactive Oxygen Species(ROS)and the macrophage-induced macrophage polarization in mice.And the relationship between the activation of IL-6 and the NF-κB signaling pathway was investigated by visualizing the mouse model of NF-κB signaling pathway constructed by this group.ResultsThe pIL-6-Fluc was successfully delivered to the liver with a transfection efficiency of about 20% in the hepatic parenchymal cells.The hydrodynamic gene delivery could cause a transient liver injury but could return to the normal in 5-7 days.The expression of pIL-6-Fluc could be induced by LPS treatment with an about 46.80±13.35 fold increase at the peak value,which was significantly higher than that detected by ELISA(4.09±0.96 fold).PDTC,the NF-κB signaling pathway inhibitor,significantly suppressed the expression of pIL-6-Fluc induced by LPS,which was in consistent with the signaling pathway where LPS activated the IL-6 expression.Using this mouse model,the tail vein S,M,L three sizes GO,The fluorescence signal was detected in the mouse liver after 3 hours of treatment,and the peak intensity of the fluorescence signal was gradually decreased after about 9 hours,and the fluorescence signal intensity decreased gradually after about 3 hours.Disappeared after 48 hours.We found that the activation of IL-6 signal in a dose-dependent and size-dependent manner,that is,the higher the concentration of GO injection,the stronger the activation of IL-6 in the liver,the stronger the GO size,the stronger the activation of IL-6 in the liver.At the same time,the greater the GO size of the liver caused by ROS and NF-κB signaling pathway activation stronger,ROS inhibitors and NF-κB signaling pathway inhibitors can inhibit liver IL-6 activation,GO activation of mouse liver IL-6 may ROS → NF-κB → IL-6 pathway.In vitro mechanism studies have shown that L-GO is more adherent to the cell membrane of macrophages and acts directly on the surface of the cell membrane.The TRL-4 receptor transduces the signal into the cell and promotes the polarization of macrophages to M1,Secretion of a large number of inflammatory factors,which mediate the inflammatory response.ConclusionsAn IL-6 reporter mouse model was constructed in this study by hydrodynamic gene transfection,allowing noninvasively monitoring IL-6 activation specifically in hepatic tissues.The reporter model was capable of monitoring the IL-6 activation with sensitivity higher than that of ELISA.The results showed that large-scale GO could induce more oxidative stress in the liver,and then activate the IL-6 expression in the mouse liver through the NF-κB signaling pathway.At the same time,the in vitro cell experiments showed that the large size GO would be more The surface of the phage,through the TRL-4 receptor to transduce the signal to promote its polarization to the M1,and secretion of proinflammatory cytokines to activate the expression of IL-6 in the liver.Through the above two aspects,GO in the size and dose-dependent way to activate the mouse liver IL-6,induced liver inflammatory response.SignificanceIn the basic medical research,the transcription and expression of IL-6 in the liver were measured by RT-PCR,immunohistochemistry and ELISA.However,these traditional monitoring methods can measure the expression level of IL-6,but not only cumbersome to operate the experimental animals at different time points,but also can not obtain continuous observation data on the same experimental animal.In this study,we constructed a real-time,dynamic,noninvasive and highly sensitive mouse model for monitoring the expression of IL-6 in situ.The effect of different concentrations of GO on the activation of IL-6 in liver was evaluated by the model.The results showed that the small size of GO had a low degree of hepatic inflammatory response,Biomedical field of safe application to provide experimental support and theoretical basis. |
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