Study On The Oxidative Damage Of Human Umbilical Vein Endothelial Cells Induced By PM2.5 And The Intervention Effect Of Resveratrol

Particulate air pollution, which is increasingly serious in recent years, has gradually become one of the most important risk factors for Chinese health. The harm of PM2.5 to human body is particularly serious. A large number of epidemiological studies found that PM2.5 seen as one of the most important environmental factors threatening human cardiovascular system is closely related to the incidence and mortality of cardiovascular disease in the population. The damage mechanism of the particles on the cardiovascular system has not been fully clarified while oxidative damage may be one of the significant ways to cause cardiovascular damage. RSV(Resveratrol) is a biologically active polyphenol compound as well as a natural antioxidant, and a number of studies have indicated that RSV has a cardiovascular protective effect. Nrf2(Nuclear factor-erythroid 2 related factor 2) is a vital nuclear transcription factor related to the intracellular antioxidant system. With promoting the Nrf2 m RNA synthesis and Nrf2 protein nuclear translocation Nrf2, RSV can increase the antioxidant level and reduce the oxidative damage in certain types of cells. There is less research on the oxidative damage of human umbilical vein endothelial cells induced by PM2.5 and the intervention effect of RSV and the role of Nrf2 in the intervention of RSV. Compared with the experiment in vivo, the experiment in vitro has the characteristics of great predictive ability, high sensitivity, low cost, no species difference. The experimental can be more accurate in the control of conditions in which PM2.5 exposure to the target cells.Objective: In view of the fact that the soluble components and some particles of PM2.5 can enter the blood circulation of the human body and cause toxic damage directly to the cardiovascular system, we used the method of experiment in vitro to study the oxidative damage of Wuhan’s PM2.5 to human umbilical vein endothelial cells(HUVEC) and the intervention effect of RSV so as to provide a new reference for the control of the internal exposure level and the prevention of cardiovascular damage by PM2.5.Methods: In the first part, we discussed the oxidative damage of HUVEC induced by PM2.5. Main indicators include basic cytotoxicity, reactive oxygen species(ROS) content, intracellular antioxidant enzymes superoxide dismutase(SOD) and glutathione peroxidase(GPX) activity and lipid and deoxyribonucleic acid(DNA) oxidative damage indicators. Different concentrations of PM2.5(0-250μg/cm2) was administrated to HUVEC. After 24 h exposure, the cell viability was measured by the CCK-8 method. Then, the HUVEC were exposed to 0, 1.95, 3.91, 7.81, 15.63, 31.25 μg/cm2 of PM2.5. The level of cellular reactive oxygen species(ROS) was detected with H2-DCFDA fluorescence probe after 1h and 3h exposure separately. After 24 h exposure, the activity of superoxide dismutase(SOD) and glutathione peroxidase(GPx), and malondialdehyde(MDA) content were detected by colorimetry, Comet tails rate was analysised by single cell gel electrophoresis assay in order to reflect the degree of DNA damage. 1m M or 2m M of N-acetyl cysteine(NAC) mixed with 31.25 μg/cm2 PM2.5 was separately administrated to HUVEC. After 3h exposure, the cell viability was measured by the CCK-8 method and ROS was observed by fluorescence microscope. After 12 h pretreatment with 0, 2.5, 5, 10μM of RSV, HUVEC were exposed to 0, 31.25 μg/cm2 of PM2.5, the cell viability was measured by the CCK-8 method. After 12 h pretreatment with 0, 10μM of RSV, HUVEC were exposed to 0, 31.25 μg/cm2 of PM2.5,In the second part, we discussed the intervention effect of resveratrol to the oxidative damage of HUVEC induced by PM2.5. After exposure of RSV(0-320μM), the inhibition rate of HUVEC was detected by CCK-8 method, the level of cellular ROS and MDA were detected. Western Blot was used to estimate the expression levels of Nrf2 in nuclear proteins and SOD-2(Manganese superoxide dismutase) in total proteins.Results: HUVEC viability decreased with the concentration of PM2.5 increased. Compared with the control, the cell viability significantly decreased in the group of greater than or equal to 7.81 g/cm2 PM2.5(P<0.05), and the IC50 is 156.94 μg/cm2. There was a large accumulation of ROS in HUVEC after 1h and 3h ecposure to PM2.5. After treatment with PM2.5 for 24 h, the activity of SOD and GPx decreased in a dose-dependent manner while the MDA level and comet tails rate increased in a dose-dependent manner. Compared with the control, the activity of GPx significantly decreased in the group of 15.63, 31.25 μg/cm2 PM2.5(P<0.05), the activity of SOD significantly decreased and the MDA level and comet tails rate significantly increased in the group of 7.81, 15.63, 31.25 μg/cm2 PM2.5(P<0.05). ROS is a metabolic substance produced in the human body in the process of sustaining life activity. In our study, the increased intracellular ROS production which was caused by the stimulation of PM2.5 led to the imbalance of oxidation and antioxidation system. Within a certain dose range of PM2.5, there was no significant change in cell viability and antioxidant capacity when cellular antioxidant levels were still in compensatory stage. With the concentration of PM2.5 increased, the production of ROS increased as well as the SOD and GPx activity decreased significantly, and that led to the aggravated oxidative damage to cells which was shown as the decrease of cell viability and the damage of lipid and DNA. As an antioxidant, NAC was able to clear the generated ROS. The treatment of 2m M NAC can significantly reduce the content of ROS and improve the survival rate of HUVEC which were exposed to PM2.5(P<0.05). It is further proved that ROS is an important factor for the cytotoxicity of PM2.5.RSV intervention showed that there is no significant cytotoxicity to HUVEC when the concentration of RSV was less than or equal to 20 m M. The pretreatment of 10 m M RSV can increase the survival rate and reduce the intracellular ROS and MDA production of the HUVEC significantly(P<0.05) which were exposed to 31.25 μg/cm2. Compared with the single PM2.5 treatment group, the content of Nrf2 nuclear protein and SOD-2 cell protein was increased in the cells of the RSV intervention group. RSV, a kind of natural antioxidant, has the cardiovascular protective effects, such as anti atherosclerosis, anti thrombosis, prevention of coronary heart disease and antihyperlipemia, etc. A number of studies have indicated that the Nrf2 pathway plays an important role in the regulation of the oxidation and antioxidation in vascular endothelial cells. The results in our study suggested that the RSV intervention can reduce oxidative damage induced by PM2.5 in HUVEC, which might be one of the ways that RSV protected vascular endothelial cells. At the same time, RSV can also regulate the expression of nuclear protein of Nrf2 and the cell total protein of SOD-2, townstream protein of Nrf2. This reveals that the mechanism of the antioxidant function may be related to the regulation of Nrf2/SOD-2 pathway by RSV, but it still need to be further studied.Conclusion: Oxidative damage may be one of the pathways of vascular endothelial cell injury induced by PM2.5. With the exposure dose of PM2.5 increased, the imbalance of the redox state of the cells increased, which resulted in the oxidative damage of lipids and DNA in HUVEC. The intervention of RSV can reduce oxidative damage induced by PM2.5 in cells, and the mechanism may be related to the regulation of Nrf2 and SOD-2 by RSV, which still need to be further studied.