Effect Of Yishenhuoxue Capsule On Regulating Immune Response And Plaque Stability In Atherosclerosis

Part 1 Effect of Yishenhuoxue capsule on regulating immune response and plaque stability in apolipoprotein E-knockout miceBackgroundAtherosclerosis(AS) is a common disease which done great harm to human health.Multitude studies had indicated that both humoral and cellular immunity participate the processes of AS.So regulating the immune response will become an important method in treatment of AS.Cell differentiation antigen 40(CD40) and CD40 ligand(CD40L) is a pair of transmembrane glycoprotein which belongs to tumor necrosis factor receptor(TNFR) and tumor necrosis factor(TNF) superfamilies.It has been shown that the costimulatory signal delivered by CD40-CD40L interactions participate the immune inflammatory response caused by oxLDL.CD40-CD40L interactions can induce endothelial cells(ECs),vascular smooth muscle cells(VSMCs) and macrophage(MΦ) expressing adhesion molecules such as vascular adhesion molecule-1(VCAM-1), intercellular adhesion molecule-1(ICAM-1),E selectin and chemotatic factors such as interleukin-8(IL-8),monocyte inflammatory protein-1(MIP-1) and monecyte chemoattractant protein-1(MCP-1),et al.These adhesion molecules and chemotatic factors can promote the leukocyte dipping into the vascular wall and the migration and proliferation of VSMCs in intima.In addition,CD40-CD40L interactions can promote ECs,VSMCs and MΦexpressing matrix metalloproteinase and tissue factor, thus can cause plaque unstability,rupture and thrombopoiesis.Yishenhuoxue Capsule(YC),an extract of traditional Chinese medicine,has been used as an antiatherosclerotic agent for years in clinic.Previous studies have shown that YC can lower atherosclerotic index,down-regulate the expression of inflammatory factors caused by high hyperhomocysteinemia,but the effect of YC on modulating the immune response and plaque stability has not been clarified.In this study,we observed the effects of YC on serum levels of lipid and oxLDL and the expression of CD40,matrix metalloproteinase-9(MMP-9) and VCAM-1 in atherosclerotic plaques and on the plaque stability in vivo.The study coule provide scientific evidence for the YC on treatment of AS in clinic.AimTo investigate the possible mechanisms of YC on modulating immune response in atherosclerosis and on plaque stability through observing the plasma oxLDL level, changes of the morphology and the ultrastructure and detecting the variation of CD40, VCAM-1 and MMP-9 mRNA and protein expression and the composition of plaques.MethodsMail 45 apolipoprotein E-knockout(apoE^-/-) mice at 8 weeks of age were fed a high fat high cholesterol diet including 15%fat and 0.25%cholesterol.Mail 9 C57BL/6J mice at 8 weeks of age were fed a normal chow diet.At 20 weeks of age, apoE^-/- mice were randomly divided into five groups,the ApoE-KO group,the apoE^-/- +YSHXH group(YSHXH group),the ApoE^-/-+ YSHXL group(YSHXL group),the apoE^-/- + total panax notoginsenoside group(TPNS group) and the apoE^-/-+ simvastain group(Simvastain group)(n=9 for each group).C57BL/6J mice were the normal control group(Control group).Mice in apoE^-/-+YSHXH and apoE^-/-+YSHXL group were given YC at 2500 mg/kg,500 mg/kg,respectively.Mice in TPNS group were given TPNS at 60 mg/kg.And mice in Simvastain group were given Simvastain at 3.3 mg/kg.All drugs were given orally,once a day for 12 weeks.Mice in ApoE-KO and control groups were given 0.2 ml distilled water,once a day for 12 weeks.After administered for 12 weeks,all mice were sacrificed.1 ml blood was obtained from each mouse by removing eyeball.Serum levels of total cholesterol(TC),low density lipoprotein cholesterol(LDL-C) and trigliyceride(TG) was detected by oxidation enzyme method.Serum oxLDL level was mas determined by enzyme linked immunosorbent assay(ELISA).The aorta was perfused with sterile phosphatebuffered saline(PBS) by cardiac puncture.The aortic arch about 1mm was fixed in 3% glutaraldehyde to make transmission electron microscope sample,and the surplus aortic arch and the root of aorta was fixed in 4%paraformaldehyde overnight for histology and immunohistochemistry.The thoracic aorta was rapidly frozen in liquid nitrogen for later detection of CD40,VCAM-1,MMP-9 mRNA expression.Results1.Comparison of serum lipid concentrationThe serum TC,TG and LDL-C concentration was significantly higher in apoE^-/- group than that in Comal group(P<0.001).Lipid levels in YSHXH,Simvastain and TPNS group were significantly lower compared with that in apoE^-/- group(P<0.001). The TC,LDL-C levels were no difference between YSHXL and apoE^-/- group mice(P >0.05),while the TG concentration in YSHXL group was lower than that in apoE^-/- group(P<0.05).High dose YC was superior to low dose YC in lowering serum lipid level(P<0.001).2.Comparison of serum oxLDL concentrationThe serum oxLDL concentration in apoE^-/- group was significantly higher than that in Control group(P<0.001).OxLDL levels in drugs treated groups were lower than that in apoE^-/- group(P<0.001 or P<0.01).There were significant difference between YSHXH and YSHXL group(P<0.001).3.Effect of YC on areas of atherosclerotic lesion in apoE^-/- miceA normal aorta wall was observed in Control group mice.Atherosclerotic lesions were observed in all apoE^-/- mice and the lumens were narrowed in a different degree. The ratio of lesion area to vessel area in apoE^-/- group was higher than that in YSHXH, TPNS and Simvastain group(32.54±5.13%vs 14.16±5.18%,12.04±5.02%,14.43±4.29%,P<0.05 or P<0.01).No difference between YSHXL and apoE^-/- group (32.54±5.13%vs 26.22±4.74%,P>0.05).4.Effect of YC on plaque composition in apoE^-/- miceCompared with the apoE^-/- group,MΦand lipid infiltration in plaques were lower and VSMCs in plaques were higher in the YSHXH,Simvastain and TPNS group(P<0.001).Lipid infiltration in the YSHXL group was also lower than the apoE^-/- group(P<0.05),but the level of MΦand VSMCs in plaques of the two groups was no significant difference(P>0.05).These indicated that YC could promote plaque stability,and high dose YC was superior to low dose YC.5.Ultrastructural observation of the ECs in aorta In the Control group,the ECs exhibited characteristics of monolayer pavement cells with a good succession,the cell membrane was intacted and kytoplasm was uniformity.Morphous of mitochondria is normal.The membrana elastica interna was succession and uniformity.In the apoE^-/- group,the ECs had obviously engorgement and the succession between ECs was interruption.ECs amotic phenomenon was observed and nuclei heterochromatin obviously increased.The mitochondria obviously swelled with the ridge swelling and dissolved.The membrana elastica interna was uneven and interrupted and VSMCs migrated into intima from the gap. However,these changes were ameliorated when treated with YC,Simvastain and TPNS.The nuclei had slight heterochromatin.The numbers of mitochondria with slight increasing,a consecutive membrana elastica intema was observed.6.Comparison of expression of CD40,VCAM-1 and MMP-9 protein in aorta wall(1) Comparison of expression of CD40 protein:The expression of CD40 protein in the apoE^-/- group was higher than that in the Control group(P<0.001).The expression of CD40 protein was significantly decreased after treatment with YC, simvastain and TPNS(P<0.001).The expression of CD40 protein was decreased significantly in the Simvastain group,the high dose YC group compared with that in the low dose group(P<0.05).(2) Comparison of expression of VCAM-1 protein:The expression of VCAM-1 protein in the apoE^-/- group was higher than that in the Control group(P<0.001). After treatment with YC,simvastain and TPNS,the expression of VCAM-1 protein in each group was decreased in a different degree compared with the apoE^-/- group(P< 0.01 or P<0.05).The expression of VCAM-1 protein was decreased significantly in the YSHXH group compared with that in the YSHXL group(P<0.05).(3) Comparison of expression of MMP-9 protein:The expression of MMP-9 protein in the apoE^-/- group was higher than that in the Control group(P<0.001).The expression of VCAM-1 protein in drugs treated groups was significantly decreased compared with the apoE^-/- group(P<0.001 or P<0.01 or P<0.05).The expression of VCAM-1 protein was decreased significantly in the YSHXH group,the Simvastain group and the TPNS group compared with that in the YSHXL group(P<0.01 or P< 0.05).7.Comparison of expression of CD40,VCAM-1 and MMP-9 mRNA in aorta wall(1) Comparison of expression of CD40 mRNA:The expression of CD40 mRNA in the apoE^-/- group was higher than that in the normal group(P<0.001). After treatment with YC,simvastain and TPNS,CD40 mRNA was decreased in a different degree compared with the apoE^-/- group,and among those treatment groups, the Simvastain group had the lowest CD40 mRNA expression(P<0.001),next was the TPNS group and the YSHXH group.The expression of CD40 mRNA in the YSHXH group,the Simvastain group and the TPNS group were lower than that in the YSHXL group(all P<0.001).(2) Comparison expression of VCAM-1 mRNA:The expression of VCAM-1 mRNA in the apoE^-/- group was higher than that in the normal group(P<0.001). VCAM-1 mRNA was decreased in a different degree in drugs treatment groups compared with the apoE^-/- group.The expression of VCAM-1 mRNA in the YSHXH group and the Simvastain group were lower than that in the TPNS group and YSHXL group(P<0.001).(3) Comparison expression of MMP-9 mRNA:The expression of MMP-9 mRNA in the apoE^-/- group was higher than that in the normal group(P<0.001).VCAM-1 mRNA was decreased significantly in drugs treatment groups compared with the apoE^-/- group(P<0.001).The expression of VCAM-1 mRNA in the YSHXH group, the TPNS group and the Simvastain group were lower than that in the YSHXL group (P<0.001).Conclusion1.YC could decrease blood lipid level in apoE^-/- mice.2.YC could decrease serum oxLDL concentration in apoE^-/- mice. 3.YC could down-regulated the mRNA and protein expression of CD40,MMP-9 and VCAM-1.4.YC could regulate immune response in AS,inhibit the development of AS and prevent transformation from stable plaque to unstable plaque.The mechanisms were possibly associated with the suppressive effect of YC on blood levels of lipid and oxLDL and CD40,MMP-9 and VCAM-1 expression in aortas of apoE^-/- mice. Part 2 Effect of Yishenhuoxue extraction on CD40,MMP-9 and VCAM-1 expression in human umbilical vein endothelial cells induced by oxLDLAimTo observe the Yishenhuoxue extraction on the CD40,MMP-9 and VCAM-1 expression in human umbilical vein endothelial cells(HUVECs) induced by oxLDL. Investigate the effect of Yishenhuoxue extraction on atherosclerosis in vitro.MethodsThe Yishenhuoxue extraction was prepared by decocting with distilled water and precipitating with ethanol.The HUVECs were obtained by perfusing trypsinase liquid in umbilical cord and cultured in vitro.The 3-5 generation cells were used in the study. Cells were divided into 7 groups,the normal group,the oxLDL stimulated group (stimulate group),the oxLDL+low dose Yishenhuoxue extraction group(low dose group),the oxLDL+middle dose Yishenhuoxue extraction group(middle dose group), the oxLDL+high dose Yishenhuoxue extraction group(high dose group),the oxLDL +simvastain group(simvastain group) and the oxLDL+total panax notoginsenosidum group(TPNS group).Cells in stimulate group were admoved oxLDL at 50μg/ml into cell culture fluid and cultured for 24 h.Cells in low dose group,middle dose group and high dose group were pretreated with Yishenhuoxue extraction at 100μg/ml,500μg/ml and 1 mg/ml,respectively for 24 h,after 24 h, cells were given oxLDL at 50μg/ml and cultured for another 24 h.Cells in simvastain group and TPNS group were pretreated with simvastain at 10μmol/l and TPNS at 100μg/ml for 24 h,after 24 h,cells were given oxLDL at 50μg/ml and cultured for another 24 h.The expression of VCAM-1 mRNA was detected by real time RT-PCR, the expression of CD40,MMP-9 protein was detected by western blot.All experimental data analyzed by statistics.Results1.Comparison of VCAM-1 mRNA:The expression of VCAM-1 mRNA in stimulate group was significantly higher than that in the normal group(P<0.001). The VCAM-1 mRNA expression was significantly lower in lose dose group,middle dose group,high dose group,simvastain group and TPNS group compared with that in stimulate group(P<0.05 or P<0.001).Among those five groups,the simvastain group had the lowest expression of VCAM-1 mRNA,next was the high dose group and TPNS group.The VCAM-1 mRNA was significantly lower in simvastain group and high dose group compared with low dose group(P<0.05).2.Comparison of CD40 protein:The expression of CD40 protein in stimulate group was significantly higher than that in the normal group(P<0.001).HUVECs treated with Yishenhuoxue extraction,simvastain and TPNS for 24 hours indicated a decrease of CD40 protein expression.CD40 protein was the lowest in simvastain and high dose group,high dose Yishenhuoxue extraction and middle dose Yishenhuoxue extraction was superior to low dose Yishenhuoxue extraction(P<0.05 or P<0.01).3.Comparison of MMP-9 protein:The expression of MMP-9 protein in stimulate group was significantly higher than that in the normal group(P<0.001).The MMP-9 protein expression was significantly lower in lose dose group,middle dose group, high dose group,simvastain group and TPNS group compared with that in stimulate group(P<0.01 or P<0.001).The MMP-9 protein was significantly lower in high dose group and simvastain group compared with that in middle dose group,TPNS group and low dose group(P<0.05,P<0.01 or P<0.001).Conclusion1.oxLDL could induce the expression of CD40,VCAM-1 and MMP-9 in HUVECs at a high level.2.Pretreatment with Yishenhuoxue extraction,simvastain and TPNS could down-regnlate the expression of CD40,VCAM-1 and MMP-9 in HUVECs induced by oxLDL. 3.The expression of CD40,VCAM-1 and MMP-9 in HUVECs had a descending tendency along with the increasing concentration of Yishenhuoxue extraction.It indicated that the effect of Yishenhuoxue extraction on modulating immune inflammatory factors in HUVECs induced by oxLDL was in a dose dependent manner.