| Objective:In recent years,due to the rapid development of nanotechnology,nanomaterials with their unique physical and chemical properties such as thermal stability,corrosion resistance,high chemical stability,and good biocompatibility are gradually being used in the aviation industry,food packaging,and biomedical fields.Titanium dioxide nanoparticles（Ti O2NPs）,as one of the most commonly used synthetic materials,are expected to reach an annual production of 2.5 million tons by 2025,and the widespread use of Ti O2NP has also raised concerns about its safety.Cardiovascular diseases continue to be the leading cause of the burden of non-communicable diseases.According to the World Health Organization（WHO）,17.9million people die each year from cardiovascular disease,accounting for 31%of all deaths worldwide;of these,heart attacks and strokes can account for up to 85%;it is predicted that by 2030,about 23.3 million people will lose their lives each year due to cardiovascular disease.It is predicted that by 2030,approximately 23.3 million people will lose their lives each year due to cardiovascular disease.Thrombosis has been known as the main pathological basis for the development of many cardiovascular diseases,among which arterial thrombosis（AT）is a solid blood clot formed by abnormal coagulation of blood at the surface of the broken or repaired inner surface of blood vessels in the cardiovascular system due to multiple causes,including atherosclerosis（AS）and susceptibility gene polymorphism（SGP）are among the causes.Platelets have been reported to play an important role in hemostasis and thrombosis.The nuclear transcription factor E2-related factor 2（Nuclear factor erythroid-derived2-like 2,NEF2L2/NRF2）is involved in a variety of important pathway roles in the body,and it has been proposed that targeted activation of Nrf2 or downstream genes may prove to be a useful way to develop therapeutic approaches to against cardiovascular disease.In this study,we aimed to investigate whether titanium dioxide nanoparticles can cause arterial thrombosis and the role of Nrf2.Methods:1.In vivo mouse modeling:Mice were randomly divided into 4 groups:Nrf2+/+control group(Nrf2+/+,Control),Nrf2-/-control group(Nrf2-/-,Control),Nrf2+/+exposed group(Nrf2+/+,Ti O2NPs)and Nrf2-/-exposed group(Nrf2-/-,Ti O2NPs).The number of mice in each group ranged from 3-6 months.The mice were anesthetized with isoflurane by oral and intranasal inhalation and exposed by intravenous injection at a dose of body weight（g）×10/μL,with saline as the solvent control and Ti O2NP injection as the exposure group（corresponding to a dose of 25 mg/kg body weight）.After 1 h of injection,a model of common carotid artery thrombosis was performed,and the pathological tissues of mouse carotid arteries were collected.The blood was collected from the mice in commercial 1.5 m L EDTA anticoagulation centrifuge tubes,stored at 4℃for no more than 24 h.The blood indexes were measured using the 37℃saline method to detect the bleeding volume in the tail of mice.2.In vivo assays:platelet count and morphological indexes were measured by hematocrit analyzer,platelet aggregation rate was measured by resistance antibody,reactive oxygen species（ROS）levels in whole blood and platelets,P-selectin expression,phosphatidylserine（PS）exposure,microvesicle（MV）generation in platelets were measured by flow cytometry.The pathological indexes of the carotid artery tissues of mice were also measured using frozen sections and immunofluorescence staining.3.K562 cells were treated in vitro:human-derived leukemia cells were treated with IMDM basal medium,10%fetal bovine serum,and 1%anti-penicillin and anti-streptomycin antibacterial agents for approximately 72 h.When the cell density increased to 60%,the cells were exposed to titanium dioxide nanoparticles（concentrations of 0,10,25,50,100μg/m L for 6 h）.Cellular proteins were harvested and the expression of NRF2 protein was observed.Results:1.The particle size of Ti O2NPs used in this study ranged from 20 to 70 nm,with a mean particle size of 35.7 nm.In saline,the dynamic size distribution by intensity was around 100 nm for anatase titanium dioxide nanoparticles.2.The PI（Perfusion Index,PI）of Nrf2-deficient mice was elevated in the basal state,but was significantly reduced after Ti O2NPs exposure,and the results were statistically significant（P<0.05）.Thrombopathological histological sections showed increased platelet deposition in the thrombus tissue of Nrf2-deficient mice after Ti O2NPs exposure,suggesting that Nrf2-deficient mice are more sensitive to arterial thrombosis caused by Ti O2NPs exposure.3.When Ti O2NPs exposure was given,the aggregation rate of platelets increased;meanwhile,titanium dioxide nanoparticles caused an increase in platelet activation,and the difference was statistically significant（P<0.05）.4.When Ti O2NPs exposure was given,no significant changes were observed in the intraplatelet ROS levels,PS exposure,and MVs expression in mice,and platelets may not be affected in their activation and procoagulant activity through increased ROS production and PS exposure.5.When K562 cells were given Ti O2NPs exposure,there was a trend of increased NRF2 protein expression,and the difference was statistically significant（P<0.05）.Conclusions:Titanium dioxide nanoparticle exposure can induce arterial thrombosis by increasing platelet activation and aggregation in mice.Nrf2-deficient mice were more susceptible to arterial thrombosis caused by exposure to titanium dioxide nanoparticles. |
