Endoplasmic Reticulum Stress And Autophagy Mediated CdTe QDs-induced Hepatocyte Toxicity And Mechanism

With the rapid development of nanotechnology,various nanomaterials have been widely used in various aspects of people's lives.Quantum Dots(QDs),as a novel nanomaterial with a diameter of about 2-20 nm,are mainly composed of III-V or II-VI elements,which have unique fluorescence characteristics.Most of them are Cadmium Telluride Quantum Dots(CdTe QDs),Cadmium selenide quantum dots,and novel cadmium-free quantum dots,among which CdTe QDs are the most common.Compared with traditional organic fluorescent dyes,CdTe QDs have the advantages of high fluorescence intensity,good stability,and great application prospects in living imaging,targeted diagnosis and treatment.At the same time,the biosafety evaluation is paid more and more attention.In vivo studies have shown that the liver is the main accumulation organ and can cause liver tissue damage after the CdTe QDs enter the organism.In vitro studies have shown that CdTe QDs can reduce the survival rate and mitochondrial damage of human hepatocellular carcinoma cells(HepG2 cells),resulting in oxidative stress and cell apoptosis.However,there are few studies on the hepatotoxicity of CdTe QDs,and the mechanism of toxicity is still not clear.There are many controversies about existing research conclusions derive from different quantum dots and cells were used.Therefore,the hepatotoxicity and mechanism of quantum dots need to be further explored.Based on the current research,this study selected HepG2 cells and human normal hepatocytes(L02 cells)as the extracorporal cell model to study the toxicity and toxicity mechanism of CdTe QDs on the two hepatocytes,and preliminarily explored the role of autophagic and endoplasmic retentive stress in CdTe QDs inducing two kinds of hepatocyte injury,and provided a basis for further study on the mechanisms of injury in low dose and early exposure.1.The CdTe QDs were synthesized in the aqueous phase in this study.The physicochemical properties such as the maximum excitation/emission wavelength,particle size,hydration particle size and Zeta potential of CdTe QDs were measured by fluorescence spectrophotometer,transmission electron microscope and Malvern laser particle size analyzer.The results showed that the maximum excitation and emission wavelength of the synthesized CdTe QDs were 300 nm and 559 nm,the particle diameter was 2.49 + 0.34 nm,the water particle diameter was 31.93 + 3.92 nm in the complete medium,as well as Zeta potential was-16.62 + 0.49 mV.CdTe QDs are well dispersed and meet the experimental requirements.2.The viability and apoptotic rate of HepG2 and L02 cells were detected by MTT assay and FITC/PI assay after 24 h exposure to CdTe QDs,respectively.The lactate dehydrogenase content in the supernatant by LDH kits,the cell morphological changes were observed under the microscope after 24 h exposure to CdTe QDs.The content of cadmium in cells was determined by graphite furnace.The results showed that the survival rate of the two hepatocytes decreased significantly(P<0.05),the apoptosis rate increased significantly(P<0.05),and the LDH content in the supernatant increased significantly(P<0.05)after 24 h exposure to CdTe QDs.Comparison between the two cells showed that CdTe QDs had a greater cytotoxic effect on L02 cells than did HepG2 cells.The observation of cell morphology showed that the morphology of the cells was abnormal after exposure,and the intercellular connections were destroyed,and the number of cells under the microscope was significantly reduced.The determination results of cadmium content showed that with the prolonged exposure time,the content of cadmium in cells gradually increased,and the content of cadmium in L02 cells was higher than that of HepG2 cells at the same time,which was consistent with the more serious damage of L02 cells.3.DCFH-DA method was used to detect the ROS content in hepatocytes after exposed to CdTe QDs for 24 h.At the same time,the intracellular fluorescence changes of ROS were observed under fluorescence microscope.Transmission electron microscopy was used to observe the change of endoplasmic reticulum after exposure to 25?M CdTe QDs for 12 h.Western blot was used to detect the expression level of endoplasmic reticulum stress marker proteins GRP78/Bip after VIxposure to CdTe QDs for 6h,12 h and 24 h,and the expression of PERK,ATF6 after VIxposure to CdTe QDs for 6h.The viability of 24 h cells exposed to CdTe QDs after pretreatment with ROS scavenger NAC,endoplasmic reticulum stress inhibitor 4-PBA and Salubrinal was measured by MTT assay.The results showed that a significant increase of ROS content in intracellular HepG2 and L02 cells exposed to CdTe QDs for 24 h,which causing oxidative stress,and the survival rate of both cells pretreated with ROS scavenger NAC was significantly increased,indicating that the oxidation caused by CdTe QDs should be promotes cell death.Western Blot results showed that CdTe QDs could induce the increase of Bip expression in HepG2 cells at 6 h after exposure,indicating that endoplasmic reticulum stress was induced in HepG2 cells,and endoplasmic reticulum stress was decreased at 12 h and 24 h after exposure.However,L02 cells were different.Bip only slightly increased at 6h,indicating that only a low level of endoplasmic reticulum stress was induced,and Bip expression was significantly increased at 12 h and 24h(P<0.05),which suggesting that the level of endoplasmic reticulum stress increased significantly in the later period of exposure.After pretreatment with endoplasmic reticulum stress inhibitors such as 4-PBA and Salubrinal,it was found that the survival rate of HepG2 cells was significantly reduced,indicating that endoplasmic reticulum stress exerts a protective effect on HepG2 cells,and the survival rate of L02 cells increases significantly.Endoplasmic reticulum stress contributes to the death of L02 cells,and this difference also explains to some extent the greater toxicity of CdTe QDs to L02 cells.4.Transmission electron microscopy was used to observe the production of autophagosomes after exposure to 25?M CdTe QDs for 12 h.The hepatocytes were transfected with mRFP-GFPLC3 double-stranded plasmid,and then CdTe QDs were exposed to both cells for different periods of time,observation of CdTe QDs induced autophagic flux in both cells by confocal laser microscope.Western blot was used to detect the expression level of autophagy marker proteins LC3 after exposured to CdTe QDs for 6h,12 h and 24 h.Cells were pretreated with autophagy inhibitor 3-ma and baflomycin A1,then exposed to CdTe QDs for 24 hours,the survival rate of cells was detected by MTT assay.The results showed that both HepG2 and L02 cells could be induced by CdTe QDs to induce autophagy.HepG2 cells could increase the expression of LC3-II in the early stage(6h)(P<0.05).The ratio of LC3-II/LC3-I was increased significantly,which indicates that HepG2 cells can produce obvious autophagy in the early stage of exposure.The LC3-II expression level decreases with the prolonged exposure time(12h and 24h),but it is still significantly higher than the control group showed that the level of autophagy was decreased at this time.The results of MTT showed that the survival rate of HepG2 cells increased significantly after pretreatment with 3-MA,indicating that autophagy in HepG2 cells exacerbated cytotoxicity induced by CdTe QDs.Pretreatment with baflomycin A1 inhibited the formation of autophagic lysosomes and caused the accumulation of autophagosomes,resulting in a significant decrease in the survival rate of both hepatocytes(P<0.05).The situation of L02 cells was different from that of HepG2 cells.In the early stage of exposure,only low-level autophagy appeared in the middle-high dose group,but autophagy increased significantly with prolonged exposure time.The L02 cells survived rate was slightly upregulated(P>0.05)after 3-MA pretreatment,indicating that the autophagy has little effect on the cytotoxicity induced by CdTe QDs,only slightly exacerbating the toxicity of CdTe QDs on L02 cells.In summary,CdTe QDs can have a significant toxic effect on HepG2 and L02 cells,and the toxicity of L02 cells is greater than that of HepG2 cells,mainly due to the more uptake of CdTe QDs by L02.CdTe QDs cause both cells oxidative stress and endoplasmic reticulum stress effect.Oxidative stress leads to an increase in hepatocyte mortality,and endoplasmic reticulum stress has a protective effect on HepG2 cells,and exacerbate the toxicity in L02 cells.CdTe QDs can induced autophagy in both cells,L02 cell autophagy level significantly increase later in HepG2 cells,and the generation of autophagy has intensified the toxic effects of HepG2 cells,smaller influence on L02 cell toxicity.