【作者】 吴同启；

【导师】 顾宁；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2011， 博士

【摘要】 目的：1.观察宁心痛颗粒(由黄芪、川芎、葛根、毛冬青、细辛组成)对动脉粥样硬化易损斑块的稳定作用；2.探讨宁心痛颗粒稳定易损斑块的机制。方法：42只新西兰大白兔随机分为A、B1、B2、B3和B4组,A组6只,给予普通饲料喂养。B1、B2、B3和B4组每组9只,给予高脂饲料喂养,两周后采用球囊损伤腹主动脉,第10周末于球囊损伤部位转染野生型p53基因,12周末处死,处死前48h、24h分别予两次药物(中国斑点蝰蛇毒和组胺)触发建立兔动脉粥样硬化易损斑块模型。第3周起B1、B2、B3组分别予宁心痛颗粒高剂量(宁心痛颗粒2g-kg-1d-1)、宁心痛颗粒常规剂量(宁心通颗粒lg-kg-1·d-1)、血脂康胶囊60mg·kg-1·d-1)干预,B4组作为模型组,不予药物干预。各组分别于实验开始时和处死前取空腹血检测总胆固醇(TCH)、甘油三酯(TG)、低密度脂蛋白胆固醇(LDL-C)、高密度脂蛋白胆固醇(HDL-C)水平,计算LDL-C/HDL-C、TG/HDL-C及TCH/HDL-C值；各组分别于实验开始时和药物触发前、药物触发后(处死前)取空腹血检测血浆纤维蛋白原(Fib)水平,并用酶联免疫吸附法(ELISA)检测血清超敏C反应蛋白(hs-CRP).白介素6(IL-6)、基质金属蛋白酶1(MMP-1)、基质金属蛋白酶抑制物1(TIMP-1)、细胞间黏附分子1 (ICAM-1)、血管细胞黏附分子1 (VCAM-1)水平。各组均以普通病理观察斑块形态,测量纤维帽厚度(FCT)及内膜-中膜厚度(IMT),并计算FCT/IMT比值；透射电镜观察斑块局部的超微结构；用免疫组化(IHC)观察斑块核因子κB (NF-κB)、单核细胞趋化因子1 (MCP-1)、MMP-1、TIMP-1、ICAM-1、VCAM-1的蛋白表达情况。采用蛋白印迹技术(Western Blot)检测斑块局部NF-κB、MCP-1、MMP-1、TIMP-1的蛋白表达水平。采用实时荧光定量PCR技术(real-time PCR)检测斑块局部核因子KB抑制蛋白(IκB)、MCP-1、MMP-1、TIMP-1、VCAM-1的mRNA表达量。采用原位末端脱氧核苷酸转移酶介导的缺口末端标记法(TUNEL)观察斑块局部平滑肌细胞凋亡情况。结果：1.使用新西兰大白兔,通过高脂喂养加球囊损伤,外源性p53基因转染,中国斑点蝰蛇毒(CRVV)和组胺触发,成功地建立了动脉粥样硬化易损斑块的动物模型。2.B1组、B2组、B3组实验兔斑块IMT低于B4组,差异有统计学意义(P<0.05)；B1组、B2组、B3组斑块的FCT及FCT/IMT比值均高于B4组,差异有统计学意义(P<0.05)；B1组斑块IMT、FCT及FCT/IMT比值与B3组比较差异无统计学意义(P>0.05)；B1组、B3组FCT/IMT比值高于B2组,差异有统计学意义(P<0.05)。3.透射电镜显示B1、B2、B3、B4组实验兔斑块局部细胞超微结构无明显差异。4.高脂饲料喂养12周后,B1、B2、B3和B4组实验兔TCH、TG、LDL-C和HDL-C水平较A组明显升高,差异有统计学意义(P<0.05)；B1组TCH、TG、LDL-C水平低于于B4组,差异有统计学意义(P<0.05)；B3组TCH、LDL-C、HDL-C水平低于于B4组,差异有统计学意义(P<0.05)；B1组、B2组、B3组LDL-C/HDL-C、TG/HDL-C及TCH/HDL-C比值与B4组比较无明显差异(P>0.05)；B1组TCH、TG、LDL-C、HDL-C水平与B3组相当,差异无统计学意义(P>0.05)；B1组TCH水平低于B2组,差异有统计学意义(P<0.05)。5.触发前、触发后(处死前)B1、B2、B3、B4组实验兔血浆纤维蛋白原水平较入组时升高,差异有统计学意义(P<0.05)；B1组、B2组、B3组触发前和触发后血浆纤维蛋白原水平均稍低于B4组相同时间点纤维蛋白原水平,差异无统计学意义(P>0.05);三者组间比较,差异亦无统计学意义(P>0.05)。6.触发前及触发后,B1、B2、B3、B4组实验兔血清hs-CRP、IL-6、MMP-1、TIMP-1、ICAM-1、VCAM-1水平均较基线升高,差异有统计学意义(P<0.05)。药物触发后B1、B3组血清hs-CRP、IL-6、MMP-1、TIMP-1、ICAM-1、VCAM-1水平低于B4组,差异有统计学意义(P<0.05);B1、B3组间比较,差异无统计学意义(P>0.05)；触发后B1组hs-CRP、IL-6、TIMP-1水平低于于B2组,差异有统计学意义(P<0.05)。7.IHC显示Bl、B2、B3、B4组实验兔斑块局部NF-κB、MCP-1、MMP-1、TIMP-1、ICAM-1、VCAM-1的蛋白表达水平较A组正常血管明显升高,差异有统计学意义(P<0.05)；B1组、B3组斑块局部NF-κB、MCP-1、MMP-1、ICAM-1、VCAM-1的蛋白表达水平低于B4组,差异有统计学意义(P<0.05)；B1组斑块局部TIMP-1的蛋白表达水平低于B4组,差异有统计学意义(P<0.05)；B1组斑块局部NF-κB、MCP-1、MMP-1、TIMP-1、ICAM-1、VCAM-1的蛋白表达水平与B3组比较差异无统计学意义(P>0.05)；B1组斑块局部NF-κB、ICAM-1的蛋白表达水平低于B2组,差异有统计学意义(P<0.05)。8. Westem Blot显示B1、B2、B3、B4组实验兔斑块组织NF-κB、MCP-1、MMP-1、TIMP-1的蛋白表达水平均高于A组,差异有统计学意义(P<0.05)；B1组、B2组、B3组斑块组织NF-κB、MCP-1、MMP-1、TIMP-1的蛋白表达水平均低于B4组,差异有统计学意义(P<0.05)；B1组斑块组织NF-κB、MCP-1、MMP-1、TIMP-1的蛋白表达水平与B3组水平相当,差异无统计学意义(P>0.05)；B1组斑块组织NF-κB、MCP-1、MMP-1、TIMP-1的蛋白表达水平低于B2组,差异有统计学意义(P<0.05)。9. Real-time PCR显示B4组实验兔斑块组织IκB、MCP-1、VCAM-1、MMP-1、TIMP-1的nRNA表达水平高于A组,差异有统计学意义(P<0.05)；B1组、B3组斑块组织IκB、MCP-1、MMP-1、TIMP-1的mRNA表达水平低于于B4组,差异有统计学意义(P<0.05),B3组斑块组织VCAM-1的mRNA表达水平低于B4组,差异有统计学意义(P<0.05),B1组斑块组织VCAM-1的mRNA表达水平与B4组比较差异无统计学意义(P>0.05)；B1组斑块组织IκB, MCP-1、MMP-1、TIMP-1的mRNA表达水平与B3组比较差异无统计学意义(P>0.05)；B1组斑块组织IκB、MMP-1的mRNA表达水平低于B2组,差异有统计学意义(P<0.05)。10. TUNEL显示B1组、B2组、B3组实验兔斑块局部平滑肌细胞凋亡率均低于B4组,差异有统计学意义(P<0.05)；B1组凋亡率稍低于B3组,但差异无统计学意义(P>0.05)。结论：宁心痛颗粒对模型兔动脉粥样硬化易损斑块具有一定的稳定作用。宁心痛颗粒能够通过调节脂质代谢,影响相关的细胞因子、黏附分子、基质金属蛋白酶等炎性介质表达,抑制炎症反应,抑制VSMC凋亡,从而降低AS斑块IMT,增加FCT及FCT/IMT比值,达到稳定模型兔动脉粥样硬化易损斑块的目的。更多还原

【Abstract】 Mechanism study for effects of Ningxintong granule on atherosclerosis vulnerable plaques in rabbit modelObjective:To investigate the effect of Ningxintong granule (NXTG, a self-made drug composed from Chinese natural medicine of Radix Astragali, Radix Puerariae, Rhizoma Chuanxiong, Radix Ilecis Pubescens and Herba Asari, each pack contains 1Og of compound) on stabilizing the vulnerable plaques and address the mechanisms of therapeutic effects of NXTG on stabilization of vulnerable plaques.Methods:42 of healthy male new Zealand white rabbits were randomized into the group A (n=6), and the group B (n=36), rabbits in group B were divided into four subgroups randomly: group B1 was treated with high dose of NXTG (n=9,2g-kg-1·-d-1), B2 was treated with normal dose of NXTG (n=9, 1g-kg-1·d-1), group B3 was administered with Xuezhikang (n=9, 60mg-kg-1·d-1) and group B4 was as baseline control group (n=9). Rabbits in group A were fed with standard chow. Rabbits in group B were fed with the high cholesterol diet for 2 weeks, then underwent the ballon-induced abdominal aortic wall injury. At 3rd week, first of drug intervention was added during the process. Rabbits in group B were still fed by the cholesterol-rich diet. At the end of the 8th week, recombinant adenovirus, which carrying the p53 gene was injected through a catheter into the aortic segments rich in plaques in group B. After two weeks, all of the remaining rabbits in group B underwent pharmacological triggering with injection of Chinese Russell’s viper venom (CRVV) and histamine. At the beginning of the experiment and 12th week, blood samples were collected for measuring the lipid profile. The Fibrinogen level on grouping, pre-pharmacological triggering and post-pharmacological triggering, the concentrations of hs-CRP, IL6, MMP-1, TIMP-1, ICAM-1, VCAM-1 on grouping, pre-pharmacological triggering and post-pharmacological triggering were measured using the ELISA method.The abdominal aorta were processed and examined by HE staining. Using transmission microscope to observe the ultra structure of the AS segments. The levels of NF-κB, MCP-1, MMP-1, TIMP-1, ICAM-1 and VCAM-1 were determined by means of immunohistochemistry. The protein expressions of NF-κB, MCP-1, MMP-1 and TIMP-1 in the lesions were examined using Western Blot analysis The mRNA expressions of IκB, MCP-1, VCAM-1, MMP-1 and TIMP-1 in the abdominal arterial atherosclerosis lesions were measured using the real-time fluorescence quantitative PCR. SMC apoptosis was detected by TUNEL assay.Results:1. Establishment of vulnerable plaques animal model:Rabbits were treated with high cholesterol diet followed by balloon-induced abdominal aortic wall injury, then, tranfected the human wild-type p53 gene in the location of plaques to build the vulnerable plaques animal model.2. Compared with Group B4, the fibrous cap thickness(FCT) in medication groups (group B1, B2 and B3) was significantly thicker and the intima-media thickness(IMT) was significantly decreased (P<0.05). The ratio of FCT and IMT was significantly different between Group B4 and treatment groups (P<0.05). There were no significant difference in the parameter such as FCT, IMT, ratio of FCT and IMT between groupB1 and group B3. The ratio of FCT and IMT was significantly lower in group B2 than group B1 (P<0.05).3. Transmission microscopy showed that there was no significant difference in ultra structure of the AS segments among four model groups.4. Lipid profile:After 12 weeks of high lipid diet, the serum lipid levels (TCH, TG,LDL-C level and HDL-C)in group B1, B2, B3 and B4 significantly increased (P<0.05). Compared with group B4, the levels of TCH, TG and LDL-C in group B1 were decreased significantly(P<0.05), Compared with the group B4, the levels of TCH, LDL-C and HDL-C in group B3 were decreased significantly(P<0.05), There is no significant differences in TCH, TG, LDL-C and HDL-C between group B1 and B3.5. Fibrinogen level:Compared with the beginning of the experiment, the level of fibrinogen in group B1, B2, B3 and B4 was increased significantly before pharmacological triggering and after pharmacological triggering. There was no significant difference among these four groups.6. Compared with the beginning of the experiment, the concentrations of hs-CRP, IL-6, MMP-1, TIMP-1, ICAM-1 and VCAM-1 in group B1, B2, B3 B4 was increased significantly before pharmacological triggering and after pharmacological triggering (P<0.05). Compared with group B4,the concentrations of hs-CRP, IL-6, MMP-1, TIMP-1, ICAM-1 and VCAM-1 in group B1, B3 were decreased significantly after pharmacological triggering (P<0.05), but there were no significant difference between group B1 and B3. The concentrations of hs-CRP, IL-6 and TIMP-1 was significantly lower in group B1 than in group B2 (P<0.05).7. Immunohistochemistry analysis showed that expressions of NF-κB, MCP-1, MMP-1, TIMP-1, ICAM-1 and VCAM-1 were found in the atherosclerotic plaques. Compared with group B4, the expressions of NF-κB, MCP-1, MMP-1, ICAM-1 and VCAM-1 detected by immunohistochemistry in the group B1 and B3 were much lower (P<0.05), but there was no significant difference between group B1 and group B3. Compared with group B4, TIMP-1 positive cells was lower in group B1 (P<0.05). The expressions of NF-κB and ICAM-1 was significantly lower in group B1 than in group B2 (P＜0.05).8. Western Blot analysis showed, that the protein expressions of NF-κB, MCP-1, MMP-land TIMP-1 were detected in the atherosclerosis lesions. Compared with group B4, the protein expressions of NF-κB, MCP-1, MMP-land TIMP-1 in group B1, B2 and B3 were much lower in the atherosclerosis lesions (P<0.05), but there was no significant difference between group B1 and group B3. The expressions of NF-κB, MCP-1, MMP-land TIMP-1 in the group B2 were higher compared with group Bl (P<0.05).9. The expressions of IκB, MCP-1, MMP-land TIMP-1 mRNA in Group B4 were significantly higher than Group A (P<0.05).Compared with group B4, the mRNA expressions of IκB, MCP-1, MMP-land TIMP-1 in group B1 and B3 were significantly lower in the atherosclerosis lesions (P<0.05), but there was no significant difference between group B1 and group B3. The mRNA expression of VCAM-1 was significantly lower in group B3 than group B4(P<0.05), but there was no significant difference of VCAM-1 mRNA expression between group B1 and group B4. The mRNA expressions of MMP-1, and IκB in the group B2 were higher compared with group B1(P<0.05).10. TUNEL analysis showed the positive cells in four groups. Compared with group B4, positive cells in group B1, B2 and B3 were significantly different(P<0.05). The positive cells in group B1 was slightly lower than group B3, but there was no significant difference between the two groups.Conclusion:NXTG has effects of anti-inflammation, decreasing the level of lipid and leading to stabilize the vulnerable plaques.更多还原