| Objective: To fully investigate the regulatory role of pterostilbene on the nuclear related Nrf2 in atherosclerotic diseases and its correlation with the TLR-4/MyD88NF-?B signaling pathway,and to explore the pathological mechanism of pterostilbene in improving atherosclerotic diseases.Materials and Methods:Sixty randomly selected adult SD rats were made into rat atherosclerosis injury model based on aortic endothelial injury method.These rats were randomly divided into the following four groups: Control group(intraperitoneally injected with normal saline).Nrf2-p group(intraperitoneally injected with Nrf2 antagonist);Pterostilbene group(intraperitoneally injected with pterostilbene solution);Pterostilbene+ Nrf2 group(intraperitoneally injected with pterostilbene+ Nrf2 agonist).These rats were fed for 4 weeks,and then were executed for following analysis.Morphological analysis: HE staining was used to analyze the cell morphology of rats in each group.Immunohistochemical staining and immunofluorescence staining were used to analyze the positive expressions of Nrf2,Survivin,Bax and Bcl-2 in the rat cells of each group.Concentrations of TNF-?,IL-6and il-1? in rat tissues were detected by ELISA.Cell proliferation and apoptosis:cck-8 detected the proliferation trend of cells in each group;Annexin v-fitc /PI double staining was used to detect the apoptosis rate of rats in each group.Mitochondrial index analysis: mitochondrial membrane potential analysis and ATP synthesis ability quantitative analysis;ROS analysis;The activity expressions of Nrf2,TLR-4/MyD88/NF-B signaling key proteins,caspase-3,caspase-8 and caspase-9 were detected by the dual-luciferase assay system.TEM analysis of apoptotic corpuscle in rat cells;MRNA analysis of Bax,Bcl-2,Survivin and Nrf2 proteins in rat cells was detected by RT-PCR.Western-blot analysis of expression levels of key proteins inBax,Bcl-2,Survivin,Nrf2,TLR-4 /MyD88/NF-?B signaling pathways in rat cells.Results: Weight changes of the rats in each group: within 2 weeks,the weight gain of the rats in each group was relatively low,and the rats showed a relatively slow growth pattern.The growth trend of the rats in each group was significantly different between 3 and 6 weeks after the operation,and the comparison trend was Nrf2-p<Control<Pterostilbene<Pterostilbene+Nrf2,P>0.05,the data difference was not statistically significant.Serum correlation index analysis: serum lipid level analysis showed the similar changes in TC,TG and LDL-2 concentrations: Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,HDL-C data analysis showed the opposite trend,P<0.05.Analysis of antioxidant stress capacity: MDA analysis: the comparison trend was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,while SOD level showed the opposite trend.Compared with each group,P<0.05,the data difference had statistical significance.Analysis of aortic endothelial function: the concentration of NO and 6-keto-pgf showed the same trend,the comparison trend was Nrf2-p<Control<Pterostilbene<Pterostilbene+Nrf2,and the expression concentration of 6-keto-pgf showed the opposite trend.Histopathological analysis: HE staining: in control group and Nrf2 antagonist group,the boundaries between the middle membrane and the outer membrane were unclear,and the nucleus showed shrinkage,accompanied by a certain degree of vacuolization in the cell,and thrombus fragments appeared in the cell lumen.After the addition of pterostilbene and Nrf2 agonist,the structure of cell artery wall was gradually improved and its texture clarity became better.Immunohistochemical staining and immunofluorescence chemical detection: Bax proteins: the comparison trend of data in each group was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p;Nrf2 ?Survivin? Bcl-2: the comparison trend of data in each group was Nrf2-p<Control<Pterostilbene<Pterostilbene+Nrf2;Inflammatory status analysis: ELISA: TNF-?,IL-6 and IL-1? were expressed in the concentration of each group and the comparison trend was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p The comparison resulted in P-value<0.05,and the difference was statistically significant.Lipid precipitation accumulation: in the control group and the Nrf2 antagonist group,lipid droplets in the aorta were significantly observed,and lipid precipitation accumulated in different degrees.With the increase of Nrf2 expression in the cells,lipid precipitation accumulation in the cells decreased,and the comparative trend of lipid precipitation accumulation in the rats in each group was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p.Cell proliferation and apoptosis: cell proliferation trend: in the data analysis at 0h after transfection,the data in each group showed the same trend,with P > 0.05,and the data difference was not statistically significant.At 24 h,36 h and 48 h after transfection,the comparison trend of the data in each group was Nrf2-p<Control<Pterostilbene<Pterostilbene+Nrf2,and the comparison between the data in each group was P<0.05,indicating statistically significant difference.Annexin v-fitc/PI double staining method was used to detect apoptosis: the comparison trend of apoptosis of rat aortic cells in each group was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,P<0.05,the data difference was statistically significant.Mitochondrial index analysis: mitochondrial microfilament distribution and membrane potential: the comparison of the density and intensity of microfilament distribution around the mitochondria of rat aortic cells in each group was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,With the increase of Nrf2 expression in cells and the intervention effect of pterostilbene,the mitochondria in cells changed from red fluorescence high energy state to green fluorescence low energy state.Membrane potential and ATP synthesis capacity: the comparison trendof cells in each group was Nrf2-p<Control<Pterostilbene<Pterostilbene+Nrf2,and the statistical data P values were 0.030,0.023,0.034 and 0.045,respectively.The data difference was statistically significant.ROS level analysis: the expression trend of rat aortic endothelial cells in each group was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,and the data of each group were compared,P<0.05,the difference was statistically significant.Dual-luciferase detection system: Nrf2,tlr-4 /MyD88/NF-B signaling pathway key proteins: Nrf2: the comparison trend of data in each group was Nrf2 Key proteins of tlr-4 /MyD88/NF-B signaling pathway: the expression trend of data in each group was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,and the difference was statistically significant(P<0.05).Activity expression of caspase-3,caspase-8 and caspase-9 proteins: the concentration of caspase-3 and caspase-9,the key protease of mitochondrial apoptosis pathway,changed to a certain extent.Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,P<0.05,the difference was statistically significant.the variation of caspase-8 protein concentration was small.TEM analysis: the number and distribution of apoptotic corpuscule in rat aortic cells in each group were compared as Pterostilbene+Nrf2< Pterostilbene<Control<Nrf2-p;RT-PCRanalysis:The expression trend of Nrf2 group was Nrf2 Survivin: the expression trend was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p;Bax and Bcl-2: the expression trend of Bax was Nrf2 The expression trend of bcl-2 cells was Nrf2-p <Control<Pterostilbene<Pterostilbene+Nrf2;Western-blot analysis: The expression trend of Nrf2 group was Nrf2 Survivin:the expression trend was Pterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p;Bax and Bcl-2: the expression trend of Bax was Nrf2 The expression trend of bcl-2 cells was Nrf2-p <Control<Pterostilbene<Pterostilbene+Nrf2;TLR-4 ? My88 ? NF-?B : the expression trend of data in each group wasPterostilbene+Nrf2<Pterostilbene<Control<Nrf2-p,and the comparison between data in each group was P<0.05,with statistically significant difference.Conclusions:(1)The rat atherosclerosis model in each group was successfully established,and there was no death in the rats;Nrf2 plays a protective role on aortic cells in the development of atherosclerosis in rats,mainly manifested in the following aspects: atherosclerosis was improved,the antioxidant capacity of aortic endothelial cells was increased,and the cell recovery was gradually enhanced.(2)In the pathological process of rat atherosclerosis,pterostilbene can target Nrf2 concentration expression and regulate tlr-4 /MyD88/NF-?B cell signaling pathway to regulate the proliferation and apoptosis of aortic cells.With the intervention of pterostilbene,the intracellular Nrf2 expression concentration increased,which effectively reduced the expression concentration of TLR-4/MyD88/NF-?B cell signaling pathway key proteins TLR-4,MyD88,NF-?B,and finally effectively inhibited the inflammatory state of rat aortic endothelial cells,promoted the proliferation trend of vascular endothelial cells,and inhibited the trend of apoptosis.(3)Pterostilbene regulates the apoptosis of rat atherosclerosis cells mainly through mitochondrial pathway.It inhibits TLR-4 /MyD88/NF-?B cell signaling pathway through targeted regulation of Nrf2,effectively enhances the energy state of vascular endothelial cells mitochondria,and inhibits the apoptosis process of vascular endothelial cells.In addition,the changes of Caspase family kinases were fully combined to reduce the expression concentration of Bax protein and increase the expression concentration of Bcl-2 protein,ultimately inhibiting the process of cell apoptosis and promoting the recovery of atherosclerotic diseases. |
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