| Objiective:With protein chip technology, to investigate the protective effect of vascular endothelial cells(VECs) of Jiawei Danshen Yin and the antiatherosclerotic mechanism by analyzing related proteins and cytokines of AS in atherosclerosis model of ApoE knockout (ApoE-/-) mice induced on high-fat diet.And then to validate by further clinical trials.Methods:1.507-8-week-old male ApoE-/-mice induced on high-fat diet for12weeks to establish AS model,then were randomly divided into the low-dose, high-dose Jiawei Danshen Yin group, simvastatin plus aspirin control group and model group. Another12age and strain matched male C57BL/6J mice as the blank group, fed with normal diet. To observe the changes of serum lipid, Nitric oxide(NO), endothelin(ET), high sensitivity C-reactive protein(hs-CRP), vascular endothelial growth factor(VEGF) and matrix metalloproteinases9(MMP-9) after8weeks treatment.The pathological morphology of aortic plaques and ultrastructure of endothelial cells in aortic plaques were also observed under the light microscope and electron microscope respectively.2. First,the AS related proteins and cytokines in lysate of aortic plaques were determinated by using Ray Biotech QAM-CAA-4000,which could Quantitative measure200kinds of mouse cytokines.Then the maximum value of signal and fold change and the AS related cytokines such as Pro-MMP-9, bFGF, VEGF were selected as the screening targets, further Verified By immunoblot technique (Western blot, WB) and real-time PCR technique (RT-PCR)3.60patients with primary hyperlipidemia plus carotid atherosclerotic plaques were randomly divided into treatment group(Ultramicro-Jiawei Danshen Yin) and control group(simvastatin plus aspirin) to observe the changes of serum lipid, hs-CRP,VEGF, MMP-9, carotid intima-media thickness(IMT), carotid intimal plaque and the adverse reactions.The treatment lasted for6months.Results:1. The levels of serum TC,TG,LDL-C were obviously increased,and the level of HDL-C decreased significantly in model group(P<0.05or P<0.01).High-fat diet could induce ApoE"’’mice to develope severe hyperlipidemia and to promote early atherosclerotic lesion formation. Jiawei Danshen Yin could decrease the levels of serum TC,LDL-C,hs-CRP,ET and increase the level of HDL-C, NO, Lowing the levels of VEGF,MMP-9(P<0.05or P<0.01). Besides lipids and hs-CRP,the therapeutic effects with a dose-dependent were better than the low-dose group(P<0.05). Compared with the control group,high dose group was superior to decrease the level of ET,but there was no obvious difference to hs-CRP,NO,VEGF and MMP-9(P>0.05). Jiawei Danshen Yin could improve the aortic plaques and endothelial cell ultrastructure and function.2. Model group vs Jiawei Danshen Yin treatment group,200kinds of proteins microarray results show that,a total of51kinds of protein had found with significant differences. Up-regulated proteins were39kinds, including Pro-MMP-9,IL-2Ra,MIP-3b,VEGF,TremL1,C5a,MMP-3,Lipocalin-2,IL-17F,E GF,OPN,CD27,IL-6,bFGF,RAGE,IL-7Ra,CD36,VEGFR1,LOX-1,MMP-2,I-TA C,IL-1b,Fcg RIIB, TCK-1,PDGF-AA,DLL4,CD48,IL-5,TNFR1,TCA-3,LIX,B7-1,MCP-1,Os teoactivin,TGFb1,IGFBP-3,BLC,JAM-A.And down-regulated proteins were12kinds such as CD40L, BTC,FetuinA,TROY,TACI,Dkk-1, Dkk-1, SDF-1a, OPG,MIG,Meteorin,sFRP-3,IL-2Ra,4-1BB. The maximum value and fold change proteins were bFGF(36,549), Pro-MMP-9(fold change=13.665).3. Jiawei Danshen Yin could downregulating protein and gene expression of VEGF,MMP-9,bFGF in lysate of aortic plaques(P<0.05or P<0.01).Antibody protein chip and WB,RT-PCR had a similar results. There was no significant difference in high-dose Jiawei Danshen Yin group and model group about protein expression of VEGF,MMP-9,bFGF(P>0.05),but the therapeutic effects were better than ligh-dose group(P<0.05). Chinese medicine groups were dose-dependent of the gene expression of MMP-9,but without significant difference of the gene expression of VEGF,bFGF to all treatment groups.4. The levels of lipid, hs-CRP,VEGF,MMP-9,carotid IMT value and plaque area were obviously improved in both groups After treatment(P<0.05or P<0.01). Ultramicro-Jiawei Danshen Yin group was better than control group in reducing the levels of TC, APOB,VEGF,MMP-9and carotid IMT,elevating the level of HDLC,and decreasing the plaque area. The difference has statistical significance(P<0.05).But Both groups showed similar efficacy in hs-CRP. There was no adverse reaction and side effect during the treatment in both groups.Conclusion:1. High-fat diet could induce ApoE-/-mice to establish a stable AS model, it was the ideal model for the study of AS.2. Bye protein microarray we found that51kinds of proteins were significantly different,and Up-regulated proteins were39kinds, down-regulated proteins were12kinds.If choosed the fold change was greater than1.5, less than0.66for different factors,it would contain more inflammatory cytokines.It show that the complexity of AS,and inflammation was an important mechanism for leading to endothelial cell damage, dysfunction, and plaque progression in AS, immunomodulatory was also involved.NF-κB signal pathway was an important signaling pathway related to inflammation regulation.So,we suspect that TLR4/NF-κB signal pathway,MAPKs signal pathway maybe involved in AS. but it need to make further studies.3. Jiawei Danshen Yin could regulate lipid, reduce the levels of hs-CRP,ET,lower the levels of serum VEGF,MMP-9and downregulate the protein and gene expression of VEGF,MMP-9,bFGF in lysate of aortic plaques in ApoE-/-mice, increase the level of serum NO, reduce inflammation injury, protect the endothelial cells, improve endothelial cell ultrastructure and function in aortic of ApoE-/-mice, stabilize plaques, delay AS progression. Comprehensive effect was superior to routine western medicine treatment,and the therapeutic effects had some correlation with Drug dosage. Antibody protein chip and WB,RT-PCR had a similar results.4. Ultramicro-Jiawei Danshen Yin is effective for primary hyperlipidemia plus carotid atherosclerotic plaques without obvious adverse reaction and side effect. It’s more effective than routine western medicine treatment,and It’s mechanismof of anti-atherosclerosis was correlated with the function of regulating lipid, reducing the level of hs-CRP and anti-inflammation, lowering level of serum VEGF,MMP-9, stabilizing plaques, decreasing IMT value,carotid intimal plaque area. |
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