Research On The Mechanisms Of Polybrominated Diphenyl Ethers Induces Protein Oxidative Damage And Promotes Lipid Accumulation To Aggravate The Progression Of Atherosclerosis

Polybrominated diphenyl ethers(PBDEs)are a kind of persistent organic pollutants(POPs),usually used as brominated flame retardant additives.They widely distribute in dust,water and air in high-risk areas.Because of their high lipophilicity and long halflife,PBDEs would expand toxicity through environmental migration and environmental enrichment,thereby endangering human health.After entering the human body,PBDEs would be metabolized into hydroxylated metabolite(OH-PBDEs)by P450 enzymes in liver,and further converted into quinone-type metabolite(PBDEQ)under the effect of peroxidase.Because of its electrophilic properties,PBDEQ is easy to undergo Michaeladdition reactions with nucleophile reagents such as DNA and proteins in organisms.Due to the instability of quinone type structure in vivo,PBDEQ could generate abundant reactive oxygen species(ROS).Previously,study has shown that PBDEQ exhibits higher biological activity and higher toxicity than the precursor.Therefore,a comprehensive exploration of the toxic effects induced by PBDEQ and the exploration of cellular defense system resistance are conducive to profound understanding of the toxic mechanisms of PBDEs and their metabolites.Part Ⅰ: This part aims to investigate the mechanism of PBDEQ-induced intracellular protein oxidative damage triggers ubiquitin-proteasome and autophagy-lysosomal system activation in LO2 cells.First,we analyzed the physicochemical properties of PBDEQ according to its chemical structure,and detected its cytotoxicity by CCK-8 assay,showing that PBDEQ induced LO2 cells death in a dose-dependent manner.Next,we investigated the toxic effects to proteins.The results showed that PBDEQ decreased the amount of reductive protein-SH and increased protein carbonyls groups in cells,which indicated that PBDEQ could cause protein oxidative modification.Next,PROTEOSTAT(?) aggresome dye was used to capture protein aggregates,the similar result was confirmed by transmission electron microscope.In addition,we observed the structural changes of endoplasmic reticulum(ER),lysosome and other organelles,which confirmed that PBDEQ could trigger the cellular defense system mechanism.Subsequently,the expression of ER stress-related proteins,such as GRP78 and ATF4,was detected by Western blot,demonstrating that PBDEQ activated ER stress.To further explore how ER stress regulated the damaged proteins,the ER activator Tm and inhibitor4-PBA was used to further observe the effect of ER stress on protein damage.By evaluating the content of protein-SH,protein carbonyls and the number of protein aggregates,we found that the activator TM had little effect on the regulation of protein damage,but the inhibitor 4-PBA could significantly aggravate the oxidative damage of proteins,indicating that ER stress played a protective role in the process of PBDEQinduced protein damage,but the ability was limited.Researches reported that most of the damaged proteins are removed by degradation systems.Here,we evaluated the effect of the proteasome system and the autophagy lysosome,indicating that both degradation systems could be activated by PBDEQ and work together in the clearance of damaged proteins.Once inhibition of them would increase the toxicity of PBDEQ.Finally,we found that PBDEQ-induced protein oxidative damage was related to oxidative stress,moreover,antioxidant N-acetylcysteine(NAC)protected proteins against oxidative damage.Part Ⅱ: This part aims to explore the effect of PBDEQ on the progression of atherosclerosis.we found that PBDEQ could induce lipid accumulation,activate NLRP3 inflammasome and pyroptosis through CD36 receptor in J774 A.1 macrophages.First,we found that PBDEQ exposure induced lipid accumulation in oxidized low-density lipid(Ox-LDL)treated J774 A.1 macrophages by detecting the levels of total cholesterol and other indicators.Furthermore,the formation of lipid droplets was also observed by oil red O staining.Then,we found that PBDEQ induced intracellular lipid accumulation by promoting lipid intake and reducing cholesterol exclusion.Additionally,CD36 played a major role in regulation lipid homeostasis.Further research showed that PBDEQ could activate NLRP3 inflammasomes and induce pyrolysis,which were confirmed by PI,LDH and other experiments.Subsequently,we found that silencing CD36 with small interfering RNAs effectively attenudes these effects,suggesting that lipid accumulation was related to NLRP3 inflammasomes and pyroptosis.Finally,we found that PBDEQ induced lipids accumulation and activated pyroptosis was ROS-dependent.In conclusion,the two-part experiments above investigated the toxicity mechanism of PBDEQ.The first part mainly revealed the mechanism of protein oxidative damage induced by PBDEQ and explored the cellular defense system,which would be helpful to expand our understanding of the toxic effects of PBDEQ and provide new ideas for its prevention and treatment.The second part mainly focused on the effects of PBDEQ exposure on health and disease.We aimed to demonstrate the pyroptosis and NLRP3 inflammasome activation induced by PBDEQ in J774 A.1 macrophages,and we also evaluated the lipid accumulation and foam cell formation to further confirm the involvement of CD36 during these processes,which offer a better understanding of the cardiovascular toxicity of PBDEQ.The above two parts further complement the underlying toxicity of PBDEs and their metabolites.