The Study Of Platinum Nanoparticles-caused Endothelial Cell Damage By Disrupting VE-cadherin

Objective:Nanotechnology is a new subject with great development prospect in recent years.With the rapid development of nanomaterial science,nanobiology and nanomedicine,the chances of human contact with or even ingestion of nanomaterials are greatly increased.Platinum nanoparticles are one of the most commonly used materials in the field of nanomaterials due to their excellent catalytic performance and good biocompatibility.Platinum nanoparticles are widely used in cosmetics,supplements,food additives,etc.,which increases the possibility of human exposure to platinum nanoparticles.Platinum nanoparticles may have multiple biological effects,inducing different cellular responses in vitro,causing multiple organ damage in vivo in mice.However,there is seldom research on the toxicity of platinum nanoparticles on the endothelium.Platinum nanoparticles may enter blood vessels through various ways and affect the endothelium.Therefore,we studied the toxicity and potential mechanism of Pt NP-5(5 nm in diameter)and Pt NP-70(70 nm in diameter)on the endothelium.Methods:1.Platinum nanoparticles with particle sizes of 5 nm and 70 nm were selected for the study.2.Primary human umbilical vein endothelial cells(HUVECs)were cultured.The cells were thawed and resuscitated for subculture.3.Transmission electron microscopy(TEM)was used to observe endocytosis of Pt NPs in HUVECs.4.The effects of different concentrations of Pt NPs on the proliferation of HUVECs were detected by CCK8 kit.5.To detect the effects of Pt NPs on the level of lactate dehydrogenase(LDH)in HUVECs.6.Transwell was used to detect the effects of different concentrations of Pt NPs on the migration of HUVECs.7.The effect of Pt NPs on the tube formation capacity of HUVECs was observed by tube formation test.8.Flow cytometry was used to detect the effects of Pt NPs on reactive oxygen species(ROS)in HUVECs.9.Western blot was used to detect the expressions of VE-cadherin after Pt NPs incubated with HUVECs.10.The effect of Pt NPs on the cell-cell connection of HUVECs was observed by immunofluorescence staining,and the addition of ROS scavenger(NAC)could reduce the damage of Pt NPs-induced endothelial adhesion connection.11.Three samples of untreated cells,Pt NP-5 cells(40 μg/ m L)and PNP-70 cells(40μg/m L)were prepared for RNA-SEQ analysis to screen the differential genes.Results:1.Uptake of Pt NPs by HUVECs: Endocytosis of Pt NP-5 and Pt NP-70(both 20μg/m L)was observed by HUVECs 24 h after exposure to Pt NPs.Both sizes of Pt NPs were mainly located in HUVECs vesicles.2.Cytotoxicity test: Both sizes of Pt NPs reduced cell viability and increased LDH release in a concentration-dependent manner.These results indicated that Pt NPs reduced the activity of HUVECs and damaged the integrity of HUVECs membrane,and Pt NP-5was more cytotoxic than Pt NP-70.3.Migration and tube formation experiments: The results showed that both sizes of Pt NPs could significantly inhibit the migration and the tube formation of HUVECs in a dose and size dependent manner,compared with the control group.The inhibitory effect of Pt NP-5 was more obvious than that of Pt NP-70.4.Detection of cellular ROS: both Pt NP-5 and Pt NP-70 induced significantly higher intracellular ROS levels in a dose-dependent manner,compared with the control group.Pt NP-5 was more potent to stimulated ROS production compared to Pt NP-70.5.Expression of VE-cadherin: Western blot results showed that compared with the control group,Pt NPs decreased the expression of VE-cadherin in HUVECs in treatment groups,and the inhibitory effect of Pt NP-5 on VE-cadherin expression was greater than that of Pt NP-70.6.Immunofluorescence staining: Confocal results showed that Pt NPs disrupted the integration of VE-cadherin and induced the rearrangement of F-actin in HUVECs.The addition of NAC(ROS scavenger)attenuated the Pt NPs-induced disruption of VEcadherin continuity.7.RNA-seq: There were a large number of differentially expressed genes between the control group and the Pt NP-treated group according to the bioinformatics analysis.Pt NP-5 has greater influence on the expression of HUVECs gene compared to Pt NP-70.Conclusion:We found that Pt NPs are cytotoxic,and can reduce the activity of HUVECs and destroy cell membranes.After incubating with Pt NPs,HUVECs produced reactive oxygen species,and the increase of ROS is considered to be the main reason for the damage of HUVECs by Pt NPs.In addition,the addition of NAC has also confirmed that the production of ROS was the main reason for the endothelial barrier damage induced by platinum nanoparticles.We also concluded that Pt NPs can reduce the expression of VE-cadherin,disrupt VE-cadherin integration,and thus damage the endothelial barrier.Meanwhile,cytoskeleton protein F-actin was also studied,which proved that the rearrangement of F-actin was involved in the destruction of endothelial barrier induced by platinum nanoparticles.Finally,transcriptome sequencing of the HUVECs showed that platinum nanoparticles could alter the expression of genes related to the endothelial barrier.