| Plastic particles less than 5 mm in diameter are called microplastics(MPs),which are mainly derived from the decomposition of consumer plastic products.Polystyrene microplastics(PS-MPs)are a very common type of MPs.Microplastics are found in a wide range of environmental media and can enter the body through the digestive,respiratory and dermal tracts.Currently,microplastics are found in human feces,blood and even placenta.Microplastics with particle size less than 10μm can cross cell membranes,and have toxic effects on several tissues and organs of the body.Microplastics can induce oxidative damage and inflammatory responses,and have potential cardiovascular toxicity,but the mechanisms of cardiovascular toxicity have been less studied.In this study,we investigated the mechanism of the toxic effects of PS-MPs on the cardiovascular system by exposing human umbilical vein endothelial cells(HUVECs)to PS-MPs.Objective:To interpret the toxic effect of PS-MPs in HUVECs and the expression of mRNA and protein levels related with NLRP3/Caspase-1/GSMMD signal pathway,and to discuss the role of NLRP3-mediated pyroptosis caused by PS-MPs,as well as to provide scientific ideas for further elucidating the mechanism of PS-MPs cardiovascular toxicity.Methods:HUVECs were cultured using DMEM medium(containing 10%fetal bovine serum).HUVECs were exposure to 0.5μm PS-MPs in different doses for 48h,and the exposure dose was divided into 0,25,50,100 and 200μg/mL.HUVECs were transfected with NLRP3 siRNA,and the gene inhibition efficiency of NLRP3 was verified by Real Time PCR.The experiment was divided into five groups after NLRP3 gene inhibition for blank control group(0μg/mL PS-MPs),negative control group(siNC),gene inhibition group(siNLRP3),exposure group(100μg/mL PS-MPs)and silent exposure group(100μg/mL PS-MPs+siNLRP3).SEM and TEM were applied to determine the dispersion and surface characteristics of PS-MPs.Using CCK-8 to assay the survival rate of HUVECs.Cell apoptosis was assayed by FITC/PI staining,reactive oxygen species(ROS)content was assayed by flow cytometry and DCFH-DA.Colorimetry was used to assayed the amount of malondialdehyde(MDA),superoxide dismutase(SOD)and lactate dehydrogenase(LDH).Real Time PCR and Western Blot methods were used to detect the mRNA and protein expression levels of NLRP3,Caspase-1 and GSDMD those related to pyroptosis.The content of intracellular inflammatory factors IL-1β and IL-18 were assayed by ELISA kits.IBM SPSS 24.0 software was applied to statistical analysis,Graph Pad 8.0.1 for data analysis,and x ±s was employed to represent measurement data.If the data satisfied normality and homogeneity of variance,the index differences between 5 exposure groups were evaluated using one-way ANOVA(One Way ANOVA),and pairwise comparisons between exposure groups were used the LSD(Least Significant Difference)test.For index with nonnormal or uneven variance,rank sum test methods was used for bet between groups.The test level was set as α=0.05.Results:1.The survival rate of HUVECs decreased significantly in each dose group after exposure to PS-MPs compared with control group(P<0.05).2.Flow cytometry revealed that a significant increase in ROS content in all PSMPs exposure groups compared with the control group(P<0.05),with the highest intracellular ROS level in the 200μg/mL dose group.Immunofluorescence revealed that ROS levels increased with the increasing exposure dose of PS-MPs and were significantly higher than those in control group(P<0.05).Cellular MDA content was significantly increased in the 50μg/mL and 200μg/mL groups than those in the control group(P<0.05);cellular SOD content was significantly lower in the 50μg/mL,100μg/mL and 200μg/mL groups than those in the control group(P<0.05).3.Compared with control group,after 48 hours of PS-MPs exposure,the expression levels of NLRP3 mRNA and protein in HUVECs were significantly increased in the 50μg/mL,100μg/mL and 200μg/mL dose groups(P<0.05);the NLRP3 gene mRNA expression level in the 200μg/mL group was significantly higher than that in the 25μg/mL group(P<0.05),and the NLRP3 protein expression level was higher in the 50μg/mL group than in the 25μg/mL group(P<0.05).After 48 hours of PS-MPs exposure,the Caspase-1 mRNA expression levels in HUVECs were significantly increased in each dose group(P<0.05);the expression levels Caspase-1 mRNA and protein were significantly higher in the 100μg/mL and 200μg/mL groups than of other three groups(P<0.05).Compared with control group,the expression levels of caspase1 p20 protein were significantly increased in the 50μg/mL and 200μg/mL groups(P<0.05);the protein expression levels in the 200μg/mL group were significantly higher than that in the 25μg/mL group(P<0.05).Compared with the control group,GSDMD gene mRNA expression levels in HUVECs were significantly increased in the 50μg/mL,100μg/mL and 200μg/mL groups(P<0.05);the expression levels of GSDMD mRNA,GSDMD and GSDMD-N protein were significantly increased in the 100μg/mL and 200μg/mL groups than of the other three groups(P<0.05).4.After 48h exposure to PS-MPs,the content of IL-1β in HUVECs was significantly increased in the 100μg/mL and 200μg/mL groups than that of the other three groups(P<0.05).The content of IL-18 in the HUVECs in 25μg/mL,50μg/mL and 100μg/mL groups were significantly increased than that in the control group(P<0.05).5.After 48 hours of exposure to PS-MPs,the level of LDH were significantly increased in 100μg/mL and 200μg/mL groups than that of other three groups(P<0.05).The proportion of HUVECs late apoptosis increased as the contention of exposure dose increased.Except for control and 25μg/mL group,the other dose groups were significantly increased(P<0.05);the proportion of HUVECs late apoptosis in 100 and 200μg/mL groups were significantly increased than that in 25 and 50μg/mL groups(P<0.05).6.After inhibiting NLRP3 gene by siRNA,the mRNA and protein expression levels of NLRP3,Caspase-1 and GSDMD those related to pyroptosis were significant lower in PS-MPs+siNLRP3 group than PS-MPs exposure group(P<0.05);the content of IL-1β,IL-18 inflammatory factors and LDH in HUVECs were significantly decreased(P<0.05),and the percentage of HUVECs late apoptosis was also significantly decreased(P<0.05).Conclusions:1.PS-MPs exposure could alter the cell morphology,cause cell apoptosis,and decrease the survival rate of HUVECs.2.The exposure of PS-MPs elevated oxidative stress indices,resulting in higher ROS levels and reduced antioxidant enzymes levels in vivo.3.PS-MPs exposure activates NLRP3 inflammatory vesicles,inducing cellular membranes damage through the NLRP3/Caspase-1/GSDMD pyroptosis signaling pathway,leading to an inflammatory reaction.4.NLRP3 gene silencing inhibits NLRP3/Caspase-1/GSDMD pyroptosis signal pathway activation and reduces cell membrane damage and inflammatory reaction. |
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