Oxidative Stress And Lysosomal Involved CDs-induced Hepatocyte Toxicity And Mechanism

With the rapid development of nanotechnology,nanomaterials have been widely used in materials,engineering,medicine and other fields.Quantum dots,as a new type of nanomaterial,have good fluorescence characteristics and are used in bioassays and bioimaging.Traditional quantum dots with a diameter of about 1-10 nm,are mainly consist of III-V or II-VI elements,because of their metal elements,the toxicity is generally large,which limits their application.As a new type of fluorescent quantum dot,Carbon dots not only have good fluorescence characteristics,but also have many advantages such as good water solubility,low toxicity,low cost,good biocompatibility,etc.,in vivo imaging,targeted diagnosis,drug carrier and other aspects have great application prospects.While carbon dots have shown great potential in medical applications,their biosafety evaluation has received much attention.In vivo studies have shown that CDs entering mice are mainly distributed in the liver,intestines and brain tissues,without causing significant liver damage.in vitro studies have shown that CDs can cause MC3T3-E1 cell survival rate reduced,dysfunctional,etc.,leading to toxic effects such as apoptosis.However,there are few studies on the safety and toxicity of CDs,and the toxicity characteristics and mechanism of toxicity are not clear,systematic research is still needed.Accordingly,this study selected KUP5 cells,AML12 cells and Hepa1-6 cells as in vitro toxicity research models.To study the toxic effects and mechanisms of CDs on three kinds of hepatocytes,and to explore the role of CDs in autophagy and apoptosis of three hepatocytes and the role of ROS in them.Further analysis of CDs-induced lysosomal damage and mechanism were performed.Provide scientific evidence for liver toxicity studies of CDs.The research results are as follows:(1)The CDs selected in this study were prepared by members of the cooperative research group of the School of Chemistry and Chemical Engineering.The maximum excitation and emission wavelength of CDs were 332 nm and 440 nm,the preservation of the primary heptazine-based unit and some oxygen-containing groups were functionalized on the surfaces of CDs,the particle diameter is 3.06±0.45 nm,the hydrated particle size in complete medium is 5.32±0.38 nm,and the zeta potential is-15.44 ± 0.56 mV.The water solubility and dispersibility of CDs are good and meet the experimental requirements.(2)After CDs were treated with KUP5 cells,AML12 cells and Hepa1-6 cells for 24 h,the survival rate of KUP5 cells and AML12 cells was significantly decreased,and the survival rate of Hepa1-6 cells was slightly decreased.The toxic effects of CDs on KUP5 cells and AML12 cells were significant,and the toxic effects on Hepa1-6 cells were not significant.There was a significant dose-effect relationship between cell viability and exposure dose.Subsequent ATP,LDH and cell morphology experiments were consistent with MTT results.(3)CDs induced the autophagy and apoptosis of KUP5 cells,AML12 cells and Hepa1-6 cells,and the excessive production of ROS.The expression of LC3-II and LC3-II/LC3-I were significantly increased in the three hepatocytes exposed to CDs for 12 h,indicating that autophagy has occurred.After 3-MA pretreatment,the survival rate of KUP5 cells and AML12 cells increased,indicating that the occurrence of autophagy aggravated CDs-induced cytotoxicity,and the survival rate of Hepa1-6 cells remained unchanged,the role of autophagy needs further analysis.When 3-MA pretreated cells,the expression level of Cleaved Caspase3 in the three hepatocytes was significantly lower than that in the CDs exposed group;Ac-DEVDCHO pretreated cells,the expression levels of LC3 II or LC3-II/LC3-I in the three hepatocytes were significantly decreased than that in the CDs exposed group,indicating that CDs-induced hepatocyte autophagy and apoptosis should be mutually constrained.After NAC(ROS scavenger)pretreated cells,the survival rates of all three hepatocytes increased,indicating that ROS production aggravated cytotoxicity.Compared with 400?g/ml group,the expression level of Cleaved Caspase3 in KUP5 cells was slightly increased in the NAC+400?g/ml CDs treatment group,indicating an increase in apoptotic rate,and the expression level of Cleaved Caspase3 in AML12 cells and Hepa1-6 cells was slightly decreased,indicating that the apoptosis rate decreased.The expression level of LC3-II in three hepatocytes increased slightly,but the relative expression levels of LC3-II/LC3-I in KUP5 and Hepa1-6 cells were significantly increased,in AML12 cells,the expression level of LC3-II/LC3-I was significantly decreased.(4)CDs treated three hepatocytes for 3 h had induced hepatocyte autophagy,and the uptake of CDs by three hepatocytes accumulated in lysosomes,causing lysosomal damage.In addition,CDs also induced the nuclear translocation of TFEB in three hepatocytes.CDsinduced TFEB nuclear translocation in KUP5 cells was mTOR-independent,while ERK pathway was not activated,CDs-induced TFEB nuclear translocation in AML12 cells and Hepa1-6 cells were involved in the mTOR and ERK pathways.Taken together,CDs have significant toxic effects on KUP5 cells,AML12 cells and Hepa1-6 cells,and are more cytotoxic to KUP5 cells and AML12 cells than Hepa1-6 cells.CDs induce oxidative stress,autophagy and apoptosis in three hepatocytes.Oxidative stress and apoptosis lead to increased hepatocyte mortality.Autophagy promotes the death of KUP5 cells and AML12 cells,but does not cause Hepa1-6 cells have an increased mortality rate.At the same time,CDs-induced lysosomal damage is involved in the induced autophagy process,which is involved in the mTOR and ERK pathways in three types of hepatocytes.By clarifying the role and regulation mechanism of reactive oxygen species and lysosomes in CDs-induced cell damage,this paper broadens the understanding of the comprehensive understanding of the toxicity of CDs and the further development of CDs toxicity.