| ObjectiveTo explore the effect and potential molecular mechanism on atherosclerosis(AS)of nanoiron sulfide(nFeS),and give new ideas and methods to treat AS.Methods1.Synthesis and characterization of nFeS:this experiment synthesized nFeS by hydrothermal method.The morphology and size of nFeS were observed by electron microscope;determination of crystallinity and material composition by X-Ray Diffraction(XRD);analyse of elements distribution by High Resolution Transmission Electron Microscopy(HRTEM)mapping.2.Vivo experiments:Six SPF grade 40-week-old C57BL/6 male mice and Twenty-four Apoe-/-male mice were selected for the pre-experiment,and the Apoe-/-mice were randomly divided into four groups,six mice in each group.Saline group and nFeS(12.5,25,50mg/kg)groups.Each group was given a high-fat diet(HFD)for 4 weeks.The study was administered by gavage.Saline was performed once a day,and nFeS groups mice were gavaged according to the design scheme.After 4 weeks of experiment,to euthanize mice and take tissue sampling.Firstly,observing the morphology of important organs and analyzing qualitative lesions of the H&E staining.To determine the gavage concentration of nFeS for the following experiments by Sudan Ⅳ staining of the aorta.Seventy-two 8-week-old SPF male Apoe-/-mice were selected for the formal experiment.They were randomly divided into six groups:Saline pre-treatment(pre)group,Atorvastatin(AT)(pre),nFeS(pre),Saline post-treatment(post),AT(post),nFeS(post).All groups were given HFD for 12 weeks,AT group was given AT(10mg/kg)by gavage and nFeS group was given nFeS(25mg/kg)by gavage,pre-treatment for the first 8 weeks of 12weeks,post-treatment for the last 8 weeks of 12weeks,once two days,recording mice body weight during the experiment weekly.After 12 weeks of experiment,to euthanize mice and take tissue sampling,liver and kidney function were tested by biochemical kit.The morphology of the vital organs was directly observed,and the tissue structural organic lesions were analyzed by H&E staining.The plaque pathomorphology was observed by Sudan Ⅳ staining,H&E staining and Masson staining.The positive area ratio and protein expression level of PCNA,IL-1β,TNF-α,4-HNE and Nrf2 in macrophage and vascular smooth muscle cells of the aortic arch were detected by immunofluorescent double staining and Western Blot.3.Vitro experiments:First,the effect of nFeS on vascular cells viability was detected by double staining of CCK-8 and live dead cells(proliferative activity and toxic activity).AS cell model was established with oxidized low density lipoprotein(ox-LDL).The experiments were divided into four groups:Control group,ox-LDL group,ox-LDL+nFeS(pre)group,ox-LDL+nFeS(post)group.Both the ox-LDL group and the ox-LDL+nFeS group were treated with ox-LDL(80μg/ml)stimulates cells.Through cell oil red o staining,it was verified that the effect of nFeS on lipid deposition in vascular cells.Through EdU,Western Blot,and q-PCR experiment,it was verified that the effect of nFeS on Vascular cells proliferation.The effect of nFeS on the migration of VSMCs cells was verified by scratch experiment.The Reactive Oxygen species(ROS),Western Blot and q-PCR experiment were used to assess the effect of nFeS on the level of inflammation and oxidative stress in RAW264.7 cells.Nrf2 expression in RAW264.7 cells was confirmed by siRNA knockdown.The experiment was randomized into four groups:ox-LDL group,ox-LDL+nFeS group,ox-LDL+siRNA group,ox-LDL+nFeS+siRNA group.Through the ROS,Western Blot and q-PCR experiment,it was verified that nFeS regulates the proliferation of Keap1/Nrf2/NQO1 signaling pathway,inhibiting RAW264.7 cells proliferation and reducing the level of inflammation and oxidative stress,which achieves the effect of preventing and treating AS.Results1.By electron microscopy,nFeS is roughly irregular sheet nanostructures.By XRD analysis,the specific components of the nFeS were determined.HRTEM mapping shows that Fe and S elements in nFeS are evenly distributed.2.Sudan Ⅳ staining and H&E staining results of pre-experiment showed that compared with the Saline group,the nFeS group(25mg/kg)had the smallest aortic plaque area,and the tissue morphology and structure of important organs in the nFeS group mice did not show significant changes.Compared with the Saline group,weekly body weight records showed that no obvious change in the body weight of Apoe-/-mice in the nFeS group;The biochemical kit test results showed that no obvious change in the liver and renal functions of mice in the nFeS group.The results of blood lipid testing showed that,compared with Saline group,the contents of TC,TG,LDL-C in serum of mice in nFeS group and AT group decreased,while the contents of HDL-C increased.However,the effect of the AT group was significantly greater than nFeS group.Sudan Ⅳ staining,H&E staining and Masson staining results of formal experiment showed that the atherosclerotic plaque in Saline group was serious and the lumen was narrow,the area of atherosclerotic plaque and necrotic core in AT group and nFeS group were less than Saline group,and the content of collagen in AT group and nFeS group were more than Saline group.Compared with AT group,the pathological improvement effect of the nFeS group is more significant,and the improvement effect of the nFeS(pre)group is more significant than nFeS(post)group.In macrophages,compared with Saline group,the results of immunofluorescence double staining and Western Blot showed that the proliferation,inflammation,and oxidative stress levels of macrophages in the nFeS group were significantly decreased,and the reduction effect of the nFeS(pre)group was obviously higher than the nFeS(post)group.In VSMCs cells,compared to the Saline group,the proliferation level in the nFeS group decreased.3.Within the visual field of cell oil red O staining,compared with ox-LDL group,no obvious decrease in the lipid deposition by VSMCs cells in the nFeS group.In the wound healing test,the healing degree of VSMCs cells in the nFeS group decreased.However,compared with ox-LDL group,lipid deposition by RAW264.7 cells in the nFeS group decreased,and the effect of the pre group was significantly greater than post group.CCK-8 and living dead cell double staining experiments showed that 0.5 mg/mL of nFeS was non-toxic to Vascular cells,and compared with ox-LDL group,the cell viability was inhibited.EdU,Western Blot and q-PCR experiment results showed that PCNA,IL-1β,TNF-α,4-HNE,Keap1,Nrf2 and NQO1 expression levels in RAW264.7 cells of ox-LDL group were greatly higher in the Control group.PCNA,IL-1β,TNF-α,4-HNE and Keapl expression levels in RAW264.7 cells of nFeS(pre)group was greatly lower than ox-LDL group.Nrf2 and NQO1 expression levels in RAW264.7 cells of nFeS(pre)group was greatly higher than ox-LDL group.PCNA,IL-1β,TNF-α,4-HNE and Keapl expression levels in RAW264.7 cells of ox-LDL+siRNA group was greatly higher than ox-LDL group.PCNA,IL-1β,TNF-α,4-HNE and Keapl expression levels in RAW264.7 cells of ox-LDL+nFeS+siRNA group was greatly lower than ox-LDL+siRNA group.Nrf2 and NQO1 expression levels in RAW264.7 cells of ox-LDL+siRNA group was greatly lower than ox-LDL group.Nrf2 and NQO1 expression levels in RAW264.7 cells of ox-LDL+nFeS+siRNA group was greatly higher than ox-LDL+siRNA group.The ROS experiment results showed that compared with the ox-LDL group,the red fluorescence intensity of the nFeS group decreased,and the pre group was weaker than the post group,the red fluorescence intensity enhancement in the ox-LDL+siRNA group.Compared with the ox-LDL+siRNA group,the red fluorescence intensity was decreased in the ox-LDL+nFeS+siRNA group.Conclusion1.nFeS significantly improved the AS plaque lesions in Apoe-/-mice.2.nFeS inhibited macrophages proliferation and reduced the levels of inflammatory and oxidative stress.3.nFeS may resist AS through the Keap1/Nrf2/NQO1 signaling pathway. |
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