The Role Of Fractalkine In Atherosclerosis And The Effect Of Aspirin Intervention

Partr 1 Expression of FKN in HUVEC and the effect of aspirin interventionObjective:To investigate the effect of aspirin intervention on fractalkine expression in human umbilical vein endothelial cells(HUVEC) stimulated by tumor necrosis factor-α.Methods:The well-growing HUVEC were randomly assigned to the following 8 groups:①control group:only HUVEC;②TNF-αstimulated group: HUVEC+TNFα;③NS-398 intervention group:HUVEC+ NS-398 (0.5μmol/L)+TNFα;④PDTC intervention group:HUVEC+ PDTC (40μmol/L)+TNFα;⑤0.02mmol/L aspirin intervention group: HUVEC+0.02mmol/L aspirin+TNFα;⑥0.2mmol/L aspirin intervention group:HUVEC+0.2mmol/L aspirin+TNFα;⑦1mmol/L aspirin intervention group:HUVEC+1mmol/L aspirin+TNFα;⑧5mmol/L aspirin intervention group:HUVEC+5mmol/L aspirin+TNFα.The latter 6 groups of HUVEC were incubated with the corresponding intervention medicine in RPMI1640 culture medium for 24 hours.After washing, RPMI1640 was supplied to the HUVEC in control group while RPMI1640 containing 4ng/mlTNFαwas supplied to the HUVEC in the rest 7 groups and HUVEC incubation continued for another 24 hours.Then total RNA in HUVEC was extracted.Reverse transcriptional polymerase chain reaction(RT-PCR)were applied to measure the FKN,COX-1,COX-2 and NF-kappaB p65 mRNA expression in HUVEC in different groups.Membrane protein and cytoplasmic protein were extracted and western blot was applied to measure the FKN,COX-1,COX-2 and NF-kappaB p65 protein expression in HUVEC in different groups.One-way ANOVA was used to analyzed the relative grey intensity of FKN,COX-1,COX-2 and NF-kappaB p65 mRNA and protein expression.Results:1.RT-PCR showed that electrophoresis band intensity of FKN, COX-1,COX-2 and NF-kappaB p65 mRNA was different among the 8 groups.The results of one-way ANOVA were as follows:①The difference of FKN mRNA expression was obvious among 8 HUVEC groups (P＜0.001).4ng/ml TNFαsignificantly increased the FKN expression in HUVEC when compared with control group(P＜0.001).FKN mRNA expression stimulated by TNFαwere decreased after 0.02mmol/L, 0.2mmol/L,1mmol/L,5mmol/L aspirin intervention in a dosagedependent manner(P＜0.05 or P＜0.001).FKN mRNA expression stimulated by TNFαwere also decreased by 40μmol/L PDTC intervention(P＜0.001).0.5μmol/L NS-398 intervention had no effect on FKN mRNA expression stimulated by TNFα.②The difference of COX-1 mRNA expression was obvious among HUVEC groups(P＜0.001). 4ng/ml TNFαdid not increase the COX-1 expression in HUVEC when compared with control group.Compared with TNFα-stimulated group, COX-1 mRNA expression in HUVEC intervened by 0.2mmol/L,1mmol/L,5mmol/L aspirin was significant decreased in a dosage-dependent manner(P＜0.05).While 0.02mmol/L aspirin, 40μmol/L PDTC and 0.5μmol/L NS-398 showed no influence on COX-1 mRNA expression.③The difference of COX-2 mRNA expression was obvious among HUVEC groups(P＜0.001).4ng/ml TNFαsignificantly increased the COX-2 expression in HUVEC when compared with control group(P＜0.001).COX-2 mRNA expression stimulated by TNFαwere decreased after 0.2mmol/L,1mmol/L,5mmol/L aspirin intervention in a dosage-dependent manner(P＜0.05).0.02mmol/L aspirin and 40μmol/L PDTC had no effect on COX-2 mRNA expression stimulated by TNFα.. 0.5μmol/L NS-398 obviously decreased COX-2 mRNA expression stimulated by TNFα.(P＜0.001).④NF-kappaB p65 mRNA expression in control group was not different from that of TNFα-stimulated group. 0.02mmol/L,0.2mmol/L,1mmol/L,5mmol/L aspirin intervention decreased the NF-kappaBp65 mRNA expression in HUVEC(P＜0.05 or P＜0.001).40μmol/L PDTC aspirin intervention also decreased the NF-kappaB p65 mRNA expression(P＜0.001).0.5μmol/L NS-398 intervention showed no effect on NF-kappaB p65 mRNA expression.2.Western blot showed band intensity of FKN,COX-1,COX-2 and NF-kappaB p65 protein was different among the 8 groups.The results of one-way ANOVA were as follows:①The difference of FKN protein expression was obvious among 8 HUVEC groups(P＜0.001).4ng/ml TNFαsignificantly increased the FKN expression in HUVEC when compared with control group(P＜0.001).FKN protein expression stimulated by TNFαwere decreased after 0.2mmol/L,1mmol/L,5mmol/L aspirin intervention in a dosage-dependent manner(P＜0.01 or P＜0.001). FKN protein expression stimulated by TNFαwere also decreased by 40μmol/L PDTC intervention(P＜0.001).0.02mmol/L aspirin and 0.5μmol/L NS-398 intervention had no effect on FKN protein expression stimulated by TNFα.②The difference of COX-1 protein expression were not significant among the 8 HUVEC groups(P＞0.05).③The difference of COX-2 protein expression was obvious among HUVEC groups (P＜0.001).4ng/ml TNFαsignificantly increased the COX-2 expression in HUVEC when compared with control group(P＜0.001).COX-2 protein expression stimulated by TNFαwere decreased after 0.02mmol/L,0.2mmol/L,1mmol/L,5mmol/L aspirin intervention in a dosage- dependent manner(P＜0.05 or P＜0.001).0.5μmol/L NS-398 and 40μmol/L PDTC intervention also significantly decreased the COX-2 protein level stimulated by TNFα(P＜0.001).④NF-kappaB p65 protein expression in control group was not different from that of TNFα-stimulated group.0.2mmol/L,1mmol/L,5mmol/L aspirin intervention decreased the NF-kappaB p65 protein expression in HUVEC(P＜0.05 or P＜0.01).40μmol/L PDTC aspirin intervention also decreased the NF-kappaB p65 protein expression(P＜0.001).0.02mmol/L or 0.5μmol/L NS-398 intervention showed no effect on NF-kappaB p65 protein expression.Conclusions:①TNFα(4ng/ml)induces fractalkine mRNA and protein expression in human umbilical vein endothelial cells through activation of NF-kappaB.②Aspirin inhibits TNFα-induced fractalkine mRNA and protein expression in a dosage-dependent manner.The effect may relate to the inhibition of NF-kappaB p65,COX-1 and COX-2 activity,or NF-kappaB p65 expression by aspirin.③TNFα(4ng/ml)induces COX-2 mRNA and protein expression in human umbilical vein endothelial cells through activation of NF-kappaB.Upregulation of COX-2 is not related to TNFα-induced fractalkine expression.Aspirin inhibits TNFα-induced COX-2 expression in a dosage-dependent manner. Part 2 Expression of FKN in atherosclerosis plaque of ApoE^-/-mouse and the effect of aspirin interventionObjective:To investigate the effect of aspirin intervention on aorta atherosclerotic plaque in ApoE gene knockout mice and the related mechanism.Methods:Twenty male 7-week ApoE gene knockout mice were fed with normal chow and observed for 1 weeks.Then they were randomly allocated into 3 groups:①control group with mice on normal chow and placebo(n=6);②high-fat-diet group with mice on a high fat diet and placebo(n=7);③aspirin intervention group with mice on a high fat diet and 58mg/kg/day aspirin(n=7).After 12 weeks of experiment,the mice were executed and blood were collected to measure serum total cholesterol and high-sensitivity C reactive protein.The proximal segment of thoracic aorta was saved to observe the atherosclerotic lesion under light microscopy as well as to check the fractalkine,COX-1,COX-2 and NF-kappaB p65 expression by immunohistochemistry methods.The distal segment was saved and total RNA was extracted.The fractalkine,COX-1,COX-2 and NF-kappaB p65 mRNA expression were detected by reverse transcriptional polymerase chain reaction(RT-PCR).Results:1.Comparison of mice body weight in each group showed no significant difference either before experiment or after 12 weeks of experiment.After experiment the serum total cholesterol was different among the mice in 3 groups(P＜0.01).Post hoc analysis showed that serum total cholesterol level in high-fat-diet group was much higher than that in control group with mice on normal chow(P＜0.01),while there was no difference between high-fat-diet group and aspirin intervention group.The C reactive protein level was not obviously different among the 3 groups(P＞0.05).Correlation analysis showed no relationship between serum total cholesterol and C reactive protein level(P＞0.05)2.Observation under light microscopy showed thickness of endothelium and focal artherosclerosis lesion formation in aorta of mice in control group.In mice of high-fat-diet group,the aorta atherosclerotic plaque was very obvious made the lumen narrowed.There was lipids,foam cells macrophage and smooth muscle cell infiltration.The elastic fiber became thicker and part of endothelium was not intact.There was atherosclerotic lesion in aorta of mice in aspirin intervention group.But the lesion was less severe than that of high-fat-diet group.No obvious abnormality was detected in normal control group.3.Pathology image analysis showed that lesion to lumen size ratio and PICAO of aorta plaque among the 3 groups were obviously different (P＜0.05),while the difference of plaque maximal thickness and average thickness were not significantly different among the 3 groups (P＞0.05).The four analysis criteria(lesion to lumen size ratio,PICAO,maximal thickness and average thickness)in normal control group were obviously different from those in high-fat-diet group (all P＜0.05),while in aspirin intervention group,only PICAO was significantly decreased when compared with mice in high-fat-diet group (P＜0.01)4.Immunohistochemistry analysis showed that there was positive expression of NF-kappaB p65,COX-2 and fractalkine in aorta atherosclerotic lesion and the positive expression was the most obvious in mice of high-fat-diet group.The positive expression of COX-1 was not obviously different among the three groups.5.RT-PCR analysis showed the difference of FKN,COX-1,COX-2, NF-kappaB p65 mRNA expression was obvious among 3 groups (ANOVA:P＜0.001).FKN mRNA expression in both control group and aspirin-intervention groups were decreased when compared with that in high-fat-diet group(P＜0.05 or P＜0.001).There was no difference of COX-1 mRNA expression between control group and aspirin-intervention groups(P＞0.05),but COX-1 mRNA expression in aspirin-intervention group was obviously decreased compared with high-fat-diet group(P＜0.001).COX-2 mRNA expression in both control group and aspirin-intervention groups were decreased when compared with that in high-fat-diet group(P＜0.001).NF-kappaB p65 mRNA expression in both control group and aspirin-intervention groups were decreased when compared with that in high-fat-diet group(P＜0.05 or P＜0.001)Conclusions:①Severity of atherosclerotic lesion in ApoE gene knockout mice was improved by aspirin intervention.This effect may attribute to the inhibiton of NF-kappaB p65,COX-1 and COX-2 by aspirin and had no relation to serum cholesterol.②There was fractalkine chemokine expression in aorta of ApoE gene knockout mice.Fractalkine expression in high-fat-diet group was downregulated by aspirin intervention through the inhibition of NF-kappaB p65 activation.③The great lessen of plaque lesion and serum cholesterol in control group indicated that diet control is helpful to slow down the progression of atherosclerosis in ApoE gene knockout mice.④C reactive protein is not valuable to evaluate the atherosclerosis in ApoE gene knockout mice.