The Influence Of Propolis Flavonoids On Regulatory T Cells Atherosclerosis

Previous studies have shown that propolis has the exact anti-atherosclerosisfunction, and many detailed studies had been done to explore its mechanism ofthis action, but it is still unclear. It was known that CD4⁺CD25⁺regulatory Tcells could inhibit atherosclerosis, and if propolis play the role of anti-atheros-clerosis throuth adjusting the quantity and/or function of CD4⁺CD25⁺regulatoryT cells. In this study, experimental animal models were used to explore the in-fluence of propolis on the CD4⁺CD25⁺regulatory T cells and atherosclerosis.Objective（1）To further validate the relationship between propolis flavonoids and li-pids, NO of NOS, the ET of VEGF and other indicators;（2） To further verify theanti-atherosclerotic effect of propolis;（3）To explore whether propolis flavo-noids play an anti-atherosclerotic role through adjusting the number and/orfunction of CD4⁺CD25⁺regulating T cells.The extraction way of propolis flavonoids was ultrasound-helped alcoholmethod. The purification way of propolis flavonoids was silica gel column chro-matography method. Spectrophotometer method was used to evaluate the puritof propolis flavonoids is.40ApoE-/-mice of age of7-8weeks were used toestablish the model of atherosclerosis. These mice were randomly divided into4groups, and each group included10mice. The four groups were:①high-fatgroup（HL group）:the mice were raised with high-fat feeds;②normal controlgroup（NC group）:the mice were raised with normal feeds;③fat+propolis flavo-noids group （HP group）: the mice were raised with normal feeds and treated withpropolis flavonoids：300mg.kg^-1.d^-1;（4） propolis flavone （PF group）: treatednormal feeds added by propolis flavonoids：300mg.kg^-1.d^-1. mice were treated for14weeks by the way of intragastric administration. The serum samples werecollected from angular vein. The concentration of triglyceride （TG）,cholesterol（TC）, high density lipoprotein（HDL）,low density lipoprotein（LDL） and very lowdensity lipoprotein （VLDL） were measured using automatic biochemistry ana-lyzer. Nitric acid reduction enzymic method was used to analyze the concen-tration of nitric oxide （NO） and nitric oxide synthase （NOS）. The concentrationof endothelin （ET） and vascular endothelial growth factor （VEGF） were evalu-ated by the way of Enzyme-Linked Immunosorbent Assay （ELISA） method. Theaorta was collectedand dyed with Oil Red O to measure the area of atheros-clerotic plaque. The spleen of mice was collected and splenetic cells were ob-tained. After splenetic cell culure for24h,the proportion of CD4⁺CD25⁺Foxp³⁺regulatory T cells in CD4+T cells was detected by the way of flow cytometry.The mRNA was extracted from splenetic cells then the expression of Foxp3m-RNA was evaluated using RT-PCR.Result（1）The concentration of TG in HL group, HP group, NC group and PF groupdecreased one by one: HL group16.04±1.41mmol/L, HP group13.97±1.34mmol/L, NC group10.00±1.51mmol/L, PF group6.92±1.04mmol/L. TGconcentration in PF group, NC group, HP group were significantly higher thanthat in HL group, and ANOVA analysis showed that the differences were statis-tically significant （P <0.05for all）.Similarly, the concentration of LDL in HL group, HP group, NC group andthe PF group decreased one by one: HL group11.05±1.81mmol/L, HP group,8.82±0.91mmol/L;NC group,7.38±1.04mmol/L, PF group6.16±0.96mmol/L.ANOVA analysis showed that LDL concentration of PF group, NC group, HPgroup was significantly higher than that in HL group （P <0.05for all）.（2） The concentration of NO in HL group, HP group, NC group and PFgroup decreased one by one:87.40±6.56umol/L;80.38±6.75umol/L80.18±7.13umol/L;71.6±6.13umol/L. The NO concentration in PF group, NC group,and HP group was significantly higher than that in HL group, ANOVA analysisshowed that the differences were statistically significant （P <0.05for all）.（3） The concentration of TNOS in PF group was significantly lower thanthat in HL group （20.14±5.99U/mL vs45.48±7.38U/mL）,HP group （40.32±5.91U/mL）, and NC group （36.70±5.70U/mL）（P <0.05for all）. The concentration of iNOS in PF group （15.06±3.34U/mL） was signifi-cantly lower than that in other three groups: HL group （39.11±5.25）, HP group（34.49±2.80U/mL）, NC group （31.66±2.73U/mL）（P <0.05for all）.concentration of TNOS and iNOS in HP group was significantly lower thanthat in NC group （P <0.05for both）. There was no statistically difference in con-centration of TNOS and iNOS between PF group and NC group （P>0.05）.（4） ANOVA analysis showed that the concentration of ET in PF group wassignificantly lower than that in HL group （57.04±6.20ng/L vs82.19±6.85ng/L）,and also lower than NC group （73.42±6.21ng/L） and HP group （78.42±5.81ng/L）（P <0.05for all）..The ET concentration in PF group was signifi-cantlylower than that in NC group （P <0.05）.But there was no significant diffe-rencein ET concentration between HP group and NC group （P>0.05）.（5） Oil Red O staining showed that the atherosclerotic plaque area of aortain HL group was significant larger than that in NC group and HP group （P<0.05）.There was no significant difference of plaque area between the HP group and NCgroup （P>0.05）. The atherosclerosis plaque area of PF group was significantlylower than that in NC group （P <0.05）（6） The concentration of VEGF in atherosclerotic plaques is an importantsymbol of plaque stability. The concentration of VEGF in HL group （155.62±23.96ng/L）was significantly higher than that in NC group （123.28±16.83ng/L）and HP group （135.85±15.65ng/L）（P <0.05for both）. The concentration ofVEGF in PF group （78.90±13.13ng/L） was significantly lower than those inother grops（P <0.05for all）.（7） ANOVA analysis showed that the ratio of CD4⁺CD25⁺Foxp³⁺Tregs inPF group （5.549±0.77%） was significantly higher than those in HL group （2.79±0.67%）,HP group （4.31±0.69%）, and NC group （5.49±0.91%）（P <0.05for all）.T ratio of CD4⁺CD25⁺Foxp³⁺Tregs in HP group was significantly lowerthanthat in NC group （P <0.05）. No statistical difference ws found between PFgroup and NC group （P>0.05）.（8） ANOVA analysis showed that the Foxp3mRNA expression in PF groupwas significantly higher than that in HL group （1.50±0.66copys/ul vs0.77±0.37copys/ul）,and higher than that in HP group （1.38±0.23copys/ul） and NC group（1.43±0.13copys/ul）（P <0.05）. There was no significant difference between HPgroup and NC group （P>0.05）. Conclusion（1） The propolis flavonoids could reduce the concentration of TG,TC,LDLand VLDL in ApoE-/-mice.It also could elevate concentration of NO, and lowerthe concentration of iNOS and ET.（2） The propolis flavonoids could reduce the level of VEGF in serum andatherosclerotic plaque and could inhibit the development of artery atheroscle-rosis in ApoE-/-mice.（3） The propolis flavonoids could increase the ratio of CD4⁺CD25⁺Foxp³⁺Tregs and the expression of Foxp3mRNA in ApoE-/-mice, suggesting that thepropolis flavonoids play an anti-atherosclerotic role through CD4⁺CD25⁺Fox-p³⁺Tregs.