【作者】 刘桂林；

【导师】 张继东；

【作者基本信息】 山东大学 ， 中西医结合临床， 2009， 博士

【摘要】 第一部分益肾活血胶囊对载脂蛋白E基因敲除小鼠主动脉粥样硬化的免疫调节作用及斑块稳定性的影响研究背景动脉粥样硬化（Atherosclerosis,AS）是严重危害人类健康的常见病,其发病率有逐年增加的趋势。众多研究表明,体液免疫和细胞免疫参与了AS形成的全过程。而且,免疫介导和炎症反应在AS的发生机制和冠心病心血管突发事件的发生中起重要作用。因此免疫调节和抗炎治疗将有望成为防治AS的重要手段之一。白细胞分化抗原40（Cell differentiation antigen 40,CD40）与其配体（CD40 ligand,CD40L,又称CD154）是一对互补跨膜糖蛋白,分别属于肿瘤坏死因子（Tumornecrosis factor,TNF）受体和TNF超家族成员,是免疫系统细胞间信息传递的主要介质,其相互作用显著影响AS相关细胞的功能,并且与斑块的发生发展及斑块的稳定性密切相关。研究发现CD40通路很可能是动脉粥样硬化发生发展过程中免疫炎症调节的上层机制。致AS抗原如氧化修饰的低密度脂蛋白（Oxidized lowdensity lipoprotein,oxLDL）,热休克蛋白60（Heat shock protein 60,HSP60）等可促进CD40/CD40L的激活与表达,CD40L与CD40结合,可诱导内皮细胞（Endothelialcells,ECs）、血管平滑肌细胞（Vascular smooth muscle cells,VSMCs）,巨噬细胞（Macrophage,MФ）等表达产生血管细胞粘附分子-1（Vascular adhesion molecule-1,VCAM-1）、细胞间黏附分子-1（Intercellular adhesion molecule-1,ICAM-1）和E-选择素（E selectin）及化学趋化因子白介素-8（Interleukin-8,IL-8）、单核细胞炎性蛋白-1和单核细胞趋化蛋白-1（Monocyte chemoattractant protein-1,MCP-1）等,促进白细胞进入血管壁及平滑肌细胞向内膜的迁移与增殖。而且,CD40/CD40L相互作用还可诱导ECs、VSMCs、MΦ表达和释放基质金属蛋白酶和组织因子,促进斑块不稳定、破裂及局部血栓形成。益肾活血胶囊（Yishenhuoxue Capsule,YC）是根据导师的经验方研制而成,是临床治疗AS的有效的中药复方制剂。我们以前的研究表明其能够降低动脉粥样硬化指数,降低高同型半胱氨酸血症引起的炎症因子的高表达。但是其对CD40信号系统及斑块稳定性的影响尚不明了,本研究从体内试验探讨了益肾活血胶囊对动脉粥样硬化过程中免疫炎症因子oxLDL、CD40、基质金属蛋白酶-9（Matrixmetalloproteinase-9,MMP-9）、VCAM-1的调节作用及对动脉粥样硬化斑块稳定性的影响,以期为该药及益肾活血法治疗AS提供科学依据。目的观察益肾活血胶囊对载脂蛋白E基因敲除(Apolipoprotein E-knockout,apoE^-/-)小鼠血脂,血清oxLDL,主动脉壁的组织学及超微结构、血管壁免疫炎症因子CD40、VCAM-1和MMP-9水平以及斑块内部构成（如脂质,胶原纤维,MΦ,SMCs的含量等）的变化,探讨益肾活血胶囊对动脉粥样硬化的免疫调节作用及对动脉粥样硬化斑块稳定性的影响。方法8周龄雄性apoE^-/-小鼠45只,同种属C57BL/6J小鼠9只。apoE^-/-小鼠给予高脂饮食喂养（15%脂肪,0.25%胆固醇,84.75%基础饲料）,C57BL/6J小鼠给予普通饲料喂养,于高脂喂养12周末,将apoE^-/-小鼠随机分为模型组、益肾活血胶囊小剂量组（小剂量组）、益肾活血胶囊大剂量组（大剂量组）、辛伐他汀对照组（辛伐他汀组）和三七总皂苷对照组（三七总皂苷组）,每组9只。C57BL/6J小鼠为正常组,共6组。小剂量组和大剂量组分别给予YC 500 mg/kg,2500 mg/kg灌胃,1次/d;辛伐他汀组给予辛伐他汀3.3 mg/kg灌胃,1次/d;三七总皂苷组给予三七总皂苷60 mg/kg灌胃,1次/d;灌胃时间为12周,模型组和正常组给予生理盐水0.2 ml/只,1次/d,时间12周。于试验24周末,处死各组试验鼠,摘眼球取血1 ml,氧化酶法测定各实验组动物血清总胆固醇（Total cholesterol,TC）、低密度脂蛋白胆固醇（Low density lipoprotein cholesterol,LDL-C）、甘油三脂（Triglyceride,TG）浓度,酶联免疫吸附试验（Enzyme linked immunosorbent assay,ELISA）测定动物血清中oxLDL含量。用磷酸盐缓冲液（Phosphate buffer saline,PBS）从左心室逆行灌注主动脉,自主动脉根部起至胸主动脉离断主动脉,主动脉弓部约1 mm迅速投入3%戊二醛中制备透射电镜标本,余主动脉根部及主动脉弓投入4%多聚甲醛中固定,制备冰冻切片,分别行HE染色,天狼星红染色,油红O染色及SMCs、MΦ和免疫炎症因子CD40、VCAM-1、MMP-9免疫组化染色。胸主动脉迅速投入液氮中。采用实时定量RT-PCR技术检测胸主动脉CD40、VCAM-1和MMP-9的mRNA表达。采用SPSS 11.5统计软件进行统计分析。结果1.小鼠血脂测定与正常对照组相比,模型组小鼠血清TC、TG、LDL-C浓度明显升高（P均＜0.001）;大剂量组、辛伐他汀组及三七总皂苷组小鼠血脂浓度与模型组比较均有明显程度下降,其中三七总皂苷组与大剂量组下降最为显著（P均＜0.001）,其次为辛伐他汀组;小剂量组与模型组相比,除TG外,TC、LDL-C浓度与模型组相比差异无显著性（P均＞0.05）;小剂量组和大剂量组相比具有非常显著性差异（P均＜0.001）。2.小鼠血清oxLDL含量比较与正常对照组相比,模型组小鼠血清oxLDL含量明显升高（P＜0.001）;各个药物处理组小鼠oxLDL含量与模型组比较均有明显程度下降,其中大剂量组oxLDL含量下降最为显著（P＜0.001）,其次为辛伐他汀组,三七总皂苷组和小剂量组（P＜0.001,或P＜0.01）。3.apoE^-/-小鼠AS病变面积检测正常组主动脉壁光滑,完整,无AS斑块形成。apoE^-/-小鼠均见AS斑块形成,管腔呈不同程度的狭窄。斑块面积/管腔面积分别是32.54±5.13%（模型组）,26.22±4.74%（小剂量组）,14.16±5.18%（大剂量组）,14.43±4.29%（辛伐他汀组）,12.04±5.02%（三七总皂苷组）。与模型组相比,各用药组斑块面积/管腔面积比值有不同程度降低,其中大剂量组,辛伐他汀组,三七总皂苷组与小剂量组相比差异有统计学意义（P＜0.05或P＜0.001）。4.apoE^-/-小鼠AS斑块内部构成的变化大剂量组,辛伐他汀组和三七总皂苷组斑块内MΦ浸润及脂质含量低于模型组（P均＜0.001）,同时斑块内VSMCs含量高于模型组（P均＜0.001）。小剂量组与模型组相比,斑块内脂质含量降低（P＜0.05）,MΦ浸润水平及VSMCs含量经统计无显著差异（P＞0.05）。大剂量组与小剂量组相比,差异有显著性（P＜0.001,或P＜0.01）。大剂量组与辛伐他汀组,三七总皂苷组之间差异无显著性（P＞0.05）。5.主动脉内皮细胞超微结构改变正常组血管EC为单层扁平细胞,连续性好,胞膜平整,胞浆均匀,紧贴内弹性膜,线粒体形态结构正常。内弹性膜连续,厚度均匀。模型组EC明显肿胀,连续性中断,有明显的内皮细胞脱落现象,胞核内异染色质明显增多,边集于核膜下,线粒体明显肿胀,脊肿胀、溶解。内弹性膜有中断,厚度不均匀,可见VSMC自缺口伸入内膜。各药物处理组与模型组相比,EC形态有不同程度的改善,连续性较好,胞核形态结构基本正常,线粒体数目略增多,形态结构基本正常。内弹性膜连续,厚度均匀。6.主动脉CD40、VCAM-1、MMP-9蛋白表达水平比较（1）CD40蛋白表达比较:正常主动脉CD40表达很少,模型组小鼠血管壁CD40蛋白表达显著升高（P＜0.001）;各药物处理组CD40蛋白表达与模型组相比,有非常显著性差异（P＜0.001）;辛伐他汀组和大剂量组CD40蛋白表达明显低于小剂量组（P＜0.05）。（2）VCAM-1蛋白表达比较:与正常对照组相比,模型组小鼠血管壁VCAM-1蛋白表达显著升高（P＜0.001）;与模型组相比,各药物处理组VCAM-1蛋白表达均明显下降（P＜0.01或P＜0.05）;大剂量组与小剂量组比较,差异有统计学意义（P＜0.05）。（3）MMP-9蛋白表达比较:与正常对照组相比,模型组小鼠血管壁MMP-9蛋白表达显著升高（P＜0.001）;与模型组相比,各药物处理组MMP-9蛋白表达均明显下降（P＜0.001或P＜0.01或P＜0.05）;大剂量组、辛伐他汀组、三七总皂苷组MMP-9蛋白表达较小剂量组明显下降（P＜0.01或P＜0.05）。7.主动脉CD40、VCAM-1、MMP-9 mRNA表达水平比较（1）CD40 mRNA表达比较:与正常对照组相比,模型组小鼠血管壁CD40mRNA表达显著升高（P＜0.001）;各药物处理组CD40 mRNA表达较模型组均有不同程度下降,其中辛伐他汀组下降最为显著（P＜0.001）,其次为三七总皂苷组和大剂量组（P＜0.001）;小剂量组与大剂量组、辛伐他汀组、三七总皂苷组比较有显著差异（P＜0.001）。（2）VCAM-1 mRNA表达比较:与正常对照组相比,模型组小鼠血管壁VCAM-1mRNA表达显著升高（P＜0.001）;与模型组相比,各药物处理组VCAM-1mRNA表达均显著下降（P＜0.05或P＜0.001）;大剂量组、辛伐他汀组VCAM-1表达低于三七总皂苷组和小剂量组（P＜0.001）。（3）MMP-9 mRNA表达比较:与正常对照组相比,模型组小鼠血管壁MMP-9mRNA表达明显升高（P＜0.001）;与模型组相比,各药物处理组MMP-9 mRNA表达均显著下降（P＜0.001）;大剂量组、辛伐他汀组、三七总皂苷组与小剂量组比较,差异具有统计学意义（P＜0.001）。结论1.益肾活血胶囊能够降低apoE^-/-小鼠血脂水平。2.益肾活血胶囊能够降低apoE^-/-小鼠血清oxLDL含量。3.益肾活血胶囊抑制了免疫炎症因子CD40、MMP-9、VCAM-1的mRNA和蛋白表达。4.益肾活血胶囊对AS具有免疫调节作用,能够抑制AS的进展,抑制AS斑块由稳定向不稳定发展。其机制与降低血脂,下调oxLDL及CD40、MMP-9、VCAM-1的表达有关。第二部分益肾活血提取液对oxLDL诱导的人脐静脉内皮细胞CD40、MMP-9及VCAM-1表达的影响目的观察益肾活血提取液对oxLDL诱导的人脐静脉内皮细胞（Human umbilicalvein endothelial cells,HUVECs）CD40、MMP-9及VCAM-1表达的影响,从体外试验探讨益肾活血胶囊抗AS的机制。方法采用水煎醇沉法制备益肾活血提取液。采用胰蛋白酶液灌注消化法分离获取HUVECs并进行体外培养,取3-5代细胞进行试验。试验共分7组,空白组不予oxLDL刺激及药物处理;oxLDL刺激组（刺激组）细胞培养液中加入oxLDL（终浓度为50μg/ml）培养24 h;oxLDL+益肾活血提取液小剂量组（剂量组）、oxLDL+益肾活血提取液中剂量组（中剂量组）、oxLDL+益肾活血提取液大剂量组（大剂量组）细胞培养液中加入益肾活血提取液（终浓度分别为100μg/ml,500μg/ml,1mg/ml）培养24 h,oxLDL+辛伐他汀组（辛伐他汀组）细胞培养液中加入辛伐他汀（终浓度为10μmol/1）培养24 h,oxLDL+三七总皂苷组（三七总皂苷组）细胞培养液中加入三七总皂苷（终浓度为100μg/ml）培养24 h,后5组于药物培养24 h后加入oxLDL（终浓度为50μg/ml）继续培养24 h进行检测。采用实时定量RT-PCR检测VCAM-1的mRNA表达,免疫印迹法（Western blot）检测CD40、MMP-9的蛋白表达。试验重复3次,实验数据采用统计学方法进行分析。结果1.VCAM-1 mRNA表达水平的比较。与空白组相比,刺激组VCAM-1 mRNA表达明显增加（P＜0.001）。与刺激组相比,各药物处理组VCAM-1 mRNA表达均显著下降（P＜0.05或P＜0.001）。其中辛伐他汀组下降最为显著,其次是大剂量组、三七总皂苷组。辛伐他汀组、大剂量组与小剂量组相比,差异有统计学意义（P＜0.05）。2.CD40蛋白表达水平的比较。与空白组相比,刺激组CD40蛋白表达明显增加（P＜0.001）。与刺激组相比,除小剂量组外余各药物处理组CD40蛋白表达均显著下降（P＜0.05或P＜0.01）。其中辛伐他汀组和大剂量组下降最为显著,其次是中剂量组和三七总皂苷组。3.MMP-9蛋白表达水平的比较。与空白组相比,刺激组MMP-9的蛋白表达显著增高（P＜0.001）。与刺激组相比,各药物处理组MMP-9的蛋白表达均显著下降（P＜0.01或P＜0.001）。大剂量组、辛伐他汀组与中剂量组、三七总皂苷组和小剂量组比较,差异有统计学意义（P＜0.05,P＜0.01或P＜0.001）。结论1.oxLDL可诱导HUVECs高表达VCAM-1、CD40和MMP-9。2.给予益肾活血提取液、辛伐他汀及三七总皂苷预处理后,再给予oxLDL刺激,VCAM-1的mRNA表达降低,CD40、MMP-9的蛋白表达下降。3.随着益肾活血提取液浓度的增加,VCAM-1、CD40和MMP-9的表达呈下降趋势。提示益肾活血提取液对oxLDL诱导的免疫炎症因子的调节作用在一定范围内具有剂量依赖性。更多还原

【Abstract】 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.更多还原