The Role Of CircRNA Regulation Of Nrf2 Signaling Pathway In PM_2.5-induced Oxidative Stress In Human Umbilical Vein Endotheilial Cells

ObjectiveFine particulate matter(PM_2.5)is an important productive poison and air pollutant.Although the primary route of PM_2.5 entry into the body is through the respiratory system,the greatest risk of PM_2.5 exposure is the development of cardiovascular disease.A large number of domestic and foreign studies have shown that oxidative stress is an important pathological basis for PM_2.5-induced cardiovascular disease,and nuclear factor E2-related factor 2（Nrf2）is believed to play a key role in cellular antioxidant defense mechanisms.In recent years,with the development of high-throughput sequencing technology,a large number of non-codingRNAs,such as circularRNAs have been increasingly recognized for their functions in regulating cardiovascular diseases.At present,there is not enough research on circRNA in the mechanism of PM_2.5-induced cardiovascular disease.In order to give full play to the role of circRNA as an ideal target for diagnosis of biomarkers,treatment and intervention in the future,this study will investigate the effects of PM_2.5 toxicity on oxidative stress,Nrf2signaling pathway,and circRNA expression profile in human umbilical vein endothelial cells（HUVEC）cells,and explore the role of circRNA in PM_2.5-induced HUVEC cells oxidative stress and Nrf2 signaling,to enrich the toxic mechanism of PM_2.5 and provide a scientific basis for the prevention and treatment of cardiovascular diseases caused by PM_2.5.Methods1.PM_2.5 in the atmosphere was collected and prepared into PM_2.5 mother liquor for the preparation of different concentrations of venom;human umbilical vein endothelial cells（HUVEC）were taken for in vitro experiments;CCK-8 method was used to detect 0,25,50,75,100,150,200μg/m L PM_2.5poisoning solution for 24h and 48h HUVEC cell survival rate;HUVEC cells were treated with 0,25,50 and 75μg/m L PM_2.5venom for 24h,and the WST-8method,NADPH method and TAB method were used to detect superoxide dismutase（SOD）activity,glutathione peroxidase（GPX）activity and Malondialdehyde（MDA）content;RT-q PCR and Western Blot methods were used to detect the mRNA and protein expression levels of nuclear factor E2-related factor 2（Nrf2）,heme oxygenase-1（HO-1）,and quinone oxidoreductase1（NQO1）.HUVEC cells were treated with 75μg/m L PM_2.5 venom for 24 h,differential circRNAs related to oxidative stress and Nrf2 signaling pathway were screened by circRNA sequencing and bioinformatics analysis technology,and verified by RT-q PCR experiments.2.RNase R digestion experiments were used to verify the circular structures of hsa＿circ＿0004983 and hsa＿circ＿0087960;the levels of hsa＿circ＿0004983 and hsa＿circ＿0087960 in HUVEC cells were knocked down by cell transfection technology andRNA interference technology,and the optimal interfering sequence siRNA was determined by RT-q PCR;after knocking down the hsa＿circ＿0004983 and hsa＿circ＿0087960,HUVEC cells was exposed to 75μg/m L PM_2.5 for 24 h,in order to detect SOD,GPX activity and MDA content,as well as mRNA and protein expression levels of Nrf2,HO-1,NQO1 in HUVEC cells.Results1.Effects of PM_2.5 exposure on HUVEC cell survival rate,oxidative stress level,Nrf2 signaling pathway expression and circRNA expression profile.1.1 Cell survival rate:With the increase of PM_2.5 infection concentration,HUVEC cell survival rate decreased,and half of the inhibitory effect concentrations（IC50）of cells infected for 24 hours were between 100 and 150μg/m L,and IC50 infected for 48 h was between 75 and 100μg/m L.1.2 Oxidative stress levels:SOD activity decreased significantly（all P<0.05）in the 25,50 and 75μg/m L PM_2.5 infected groups,GPX viability decreased significantly in the 75μg/m L infected group（P<0.05）,and MDA content in the 50 and 75μg/m L infected groups increased significantly（all P<0.05）.1.3 Nrf2 signaling pathway expression:mRNA and protein expression levels of Nrf2 in the 50 and 75μg/m L PM_2.5 infected groups were significantly increased（all P<0.05）,the expression levels of mRNA in the 50 and 75μg/m L contaminated groups were significantly increased（both P<0.05）,and the protein expression levels of HO-1 in the 25,50 and 75μg/m L infectious groups were significantly increased（all P<0.05）,and the expression levels of mRNA in the 25,50 and 75μg/m L infectious groups were significantly increased（all P<0.05）,and 25,50 and 75μg/m L were significantly increased.The mRNA and protein expression levels of NQO1 in the m L infected group were significantly increased（both P<0.05）.1.4 CircRNA expression profile and circRNA screening,verification:In the 0 and 75μg/m L PM_2.5 infected groups,there were 7210 upregulations（fold change≥2,q values≤0.001）,6936 downregulations（fold change≤-2,q values≤0.001）;5 upregulated circRNAs:hsa＿circ＿0004983,hsa＿circ＿0018982,hsa＿circ＿0023249,hsa＿circ＿0000199,and hsa＿circ＿0087960 are linked to antioxidant activity and the Nrf2 signaling pathway.The RT-q PCR verification of the sequencing results showed that the transcription level of 5 circRNAs in the HUVEC cells of the infected group was increased compared with the control group,and the trend of change was consistent with the sequencing results.When the PM_2.5 concentration≥25μg/m L,the expression levels of hsa＿circ＿0004983,hsa＿circ＿0087960 and hsa＿circ＿0000199 were significantly increased（all P<0.05）,and the correlation analysis results showed that the correlation coefficients between hsa＿circ＿0004983 and hsa＿circ＿0087960 and PM_2.5concentrations were larger（r=0.988,r=0.947,P<0.05）。2.Effects of knockdown hsa＿circ＿0004983 and hsa＿circ＿0087960 on PM_2.5-induced HUVEC cellular oxidative stress and Nrf2 signaling pathway expression2.1 Cyclic structure verification:The results ofRNase R digestion experiments show that hsa＿circ＿0004983 and hsa＿circ＿0087960 are not easily digested byRNase R（both P>0.05）.2.2 The effect of hsa＿circ＿0004983 on the level of oxidative stress:Compared with the negative transfection group（NC group）,the activities of SOD and GPX in the positive transfection group（siRNA1 group）were significantly increased,and the MDA content was significantly decreased（all P<0.05）,the SOD and GPX activities of the NC+PM_2.5 group were significantly reduced,and the MDA content was significantly increased（all P<0.05）;compared with the siRNA1+PM_2.5 group,the SOD and GPX activities of the siRNA1 group were significantly higher,and the MDA content was lower（all P<0.05）,the SOD and GPX activities of the NC+PM_2.5 group were significantly lower,and the MDA content was significantly higher（all P<0.05）;there was no statistically significant difference between the siRNA1+PM_2.5 group and the NC group in SOD and GPX activities,as well as the MDA content（all P>0.05）;Compared with the siRNA1 group,the NC+PM_2.5 group had significantly lower GPX and SOD activities and higher MDA content（all P<0.05）.2.3 The effect of hsa＿circ＿0004983 on Nrf2 signaling pathway:Compared with the NC group,the mRNA and protein expression levels of Nrf2 in the siRNA1 group did not change significantly（both P>0.05）,and the mRNA and protein expression levels of Nrf2 in the PM_2.5 treatment group(NC+PM_2.5 group and siRNA1+PM_2.5 group)were significantly increased（all P<0.05）,and the mRNA expression level of Nrf2 in the siRNA1+PM_2.5 group was significantly higher than that in the siRNA1 group（P<0.05）.Compared with the NC group,the mRNA and protein expression levels of HO-1 in the siRNA1 group and PM_2.5 treatment group(NC+PM_2.5group and siRNA1+PM_2.5 group)were significantly increased（all P<0.05）.Compared with siRNA1+PM_2.5 group,the mRNA and protein levels of HO-1 in siRNA1group and NC+PM_2.5 group were significantly lower（both P<0.05）;The mRNA level was significantly lower（all P<0.05）.Compared with the NC group,the mRNA level of NQO1 in the siRNA1group was decreased（P>0.05）,and the protein level was significantly increased（P<0.05）;the mRNA and protein levels of NQO1 in the PM_2.5treatment groups(NC+PM_2.5 group and siRNA1+PM_2.5 group)were significantly increased（all P<0.05）;compared with the siRNA1+PM_2.5group,the mRNA and protein levels of NQO1 in the siRNA1 group and the protein level and the protein level of NQO1 in the NC+PM_2.5 group were significantly lower（all P<0.05）;compared with the siRNA1 group,the mRNA level of NQO1 in the NC+PM_2.5 group was significantly higher（P<0.05）.2.4 The effect of hsa＿circ＿0087960 on the level of oxidative stress:Compared with the NC group,the activities of SOD and GPX in the siRNA2 group were significantly increased,and the content of MDA was significantly decreased（all P<0.05）;the activities of SOD and GPX in the NC+PM_2.5 group were significantly decreased,and the content of MDA was significantly increased（all P<0.05）.The activities of SOD and GPX in the siRNA2+PM_2.5 group were slightly higher,and the content of MDA was slightly lower（all P>0.05）;compared with siRNA2+PM_2.5 group,the activities of SOD and GPX in the siRNA2 group were higher,and the content of MDA was lower（GPX activity:P>0.05,SOD activity and MDA content both P<0.05）,SOD and GPX activities were significantly lower in NC+PM_2.5 group,and MDA content was significantly higher（all P<0.05）;compared with the siRNA2group,activities of SOD and GPX in the NC+PM_2.5 group were significantly lower,and the MDA content was higher（all P<0.05）.2.5 The effect of hsa＿circ＿0087960 on Nrf2 signaling pathway:Compared with the NC group,the mRNA expression levels of Nrf2 in the siRNA2 group did not change significantly（P>0.05）,while the mRNA expression levels of HO-1 and NQO1 increased significantly（both P<0.05）,the mRNA levels of Nrf2,HO-1 and NQO1 in the PM_2.5 treatment group(NC+PM_2.5 and siRNA2+PM_2.5 group)were significantly increased（all P<0.05）;compared with siRNA2+PM_2.5 group,the mRNA levels of Nrf2,HO-1 and NQO1 in the siRNA2 group and the NC+PM_2.5 were significantly lower（all P<0.05）;compared with the siRNA2 group,the mRNA expression level of Nrf2in the NC+PM_2.5 group were significantly higher（P<0.05）,and the mRNA expression level of HO-1 was significantly lower（P<0.05）.Compared with the NC group,the protein expression levels of Nrf2,HO-1 and NQO1 in the siRNA2 group did not increase significantly（all P>0.05）,the protein expression levels of Nrf2,HO-1 and NQO1 in the PM_2.5 treatment group(NC+PM_2.5 group and siRNA2+PM_2.5 group)were significantly higher（all P<0.05）;compared with the siRNA2+PM_2.5 group,the protein expression levels of Nrf2,HO-1 and NQO1 in siRNA2 group were significantly lower（all P<0.05）,and were slightly higher in the NC+PM_2.5 group（all P>0.05）.Conclusion1.PM_2.5 can inhibit HUVEC cell activity,induce oxidative stress,activate the Nrf2 signaling pathway,and alter the circRNA expression profile.2.Low expression of hsa＿circ＿0004983 and hsa＿circ＿0087960 can inhibit the oxifativec cell oxidative stress caused by PM_2.5 exposure by promoting the expression of the Nrf2 signaling pathway and its downstream HO-1 and NQO13.hsa＿circ＿0004983 and hsa＿circ＿0087960 play a role in promoting oxidative stress,and have the potential to become a new target for the prevention and treatment of cardiovascular diseases.