Effects Of Carbon Dots On Osteoblast Differentiation Of MG63 Cells By Golgi Stress Response

BackgroundIn recent years,bone diseases caused by various causes such as tumors,trauma,infection and heredity have been paid more and more attention.Traditional treatment methods include surgical removal of diseased bone tissue,and autologous bone transplantation,allogeneic bone transplantation and tissue engineered bone,operative treatment combined with gene therapy and physical therapy can accelerate bone tissue growth and promote bone remodeling.In the past few years,with the development of nanotechnology,more and more nanomaterials have been used to treat bone diseases.Ascorbic acid has the potential to promote osteoblast differentiation.Carbon dots always retain some of the physical and chemical properties of their carbon sources.Ascorbic acid-polyethyleneimine carbon dots,a new kind。of nanoparticle,made from ascorbic acid and polyethyleneimine.However,it is not clear whether it can promote osteoblast differentiation.In order to provide new ideas for the treatment of bone diseases,it is crucial to elucidate the role of AA-PEI carbon dots in osteoblasts differentiation.In the study of nanomaterials,it is found that nanomaterials can cause cellular oxidative stress and subcellular organelle level stress after being taken up by cells,the latter mainly include endoplasmic reticulum stress response and mitochondria stress response,both of them participate in the regulation of osteoblast differentiation.Golgi is an important subcellular organelle structure and distributed in animal and plant cells widely,which is involved in protein reprocessing,transport,sorting and glycosylation modification.But,it is unclear whether nanomaterials can induce Golgi stress.Golgi phosphoprotein 3(GOLPH3),a kind of marker protein for Golgi stress,provides an essential function in Golgi morphology,vesicle trafficking,mitochondrial function regulation,and which can regulate Golgi apparatus function by changing the localization of the enzyme on the Golgi membrane and participating in the glycosylation modification of the proteins.All in all,we suggest that GOLPH3 is involved in the osteoblast differentiation process.Therefore,,it is very important to elucidate whether the uptake of nanomaterials can also induce Golgi stress response through GOLPH3,and the effect of Golgi stress response on osteoblast differentiation.ObjectiveExplore the effect of AA-PEI carbon dots on Golgi stress.Determine the effect of Golgi stress induced by AA-PEI carbon dots on osteoblast differentiation of human osteosarcoma cell line MG63,to provide new theoretical basis for the treatment of maxillofacial bone diseases.Materials and methodAA-PEI carbon dots are synthesized by microwave method.Observe the imaging of carbon dots in cells by inverted fluorescence microscope.Detect the effect of carbon dots on cell proliferation by MTT and apoptosis by flow cytometry.Silence GOLPH3 gene and detect the effect of carbon dots and GOLPH3 on ROS levels by flow cytometry.Observe the effect of carbon dots on Golgi structure by immunofluorescence staining.Extract mRNA of MG63 cells by TRIzol reagent with different time and different concentration of carbon dots,and detect the expression of GOLPH3 gene by RT-PCR.Osteogenic induction of MG63 for 3 days and 7 days,and detect the expression of GOLPH3 gene and osteogenic differentiation-related genes(RUNX2,OCN,ALP)by RT-PCR;after 14 days of osteogenic induction,detect mineralization ability of carbon dot and GOLPH3 gene silencing by alizarin red staining.ResultsThe carbon dots stimulates green fluorescence under blue excitation source;when the carbon dots concentration is lower than 40 mg·L-1,the cell proliferation rate is greater than 75%after treatment with carbon dots for 24h,48h,and 72h(P<0.05);the carbon dots cause a certain degree of apoptosis rate and an increase of intracellular ROS levels(P<0.05);carbon dots promote the dispersion of GOLPH3 antibody-labeled Golgi in the cytoplasm;RT-PCR results show that the expression of GOLPH3 gene increase with the increase of concentration and the prolongation of reaction time of carbon dots in cells;induce osteoblast differentiation of MG63 cells,carbon dots inhibit the expression of on osteogenic differentiation-related genes(RUNX2,OCN,ALP)and Golgi stress-related gene GOLPH3 obviously,GOLPH3 gene inhibits the expression of R UNX2 and OCN,but promote the expression of ALP(P<0.05);alizarin red staining results show that CDs and GOLPH3 gene silencing can reduce the mineralization level of cells.Conclusion1.The carbon dots have photoluminescence property;the carbon dots have low toxicity and good biosafety.2.The carbon dots induce oxidative stress,time-dependent and concentration-dependent Golgi stress response and cause intracellular dispersion of the Golgi structure.3.Carbon dots inhibit osteogenic differentiation and mineralization of MG63 cells,and silence GOLPH3 gene will also inhibit mineralization.