益气化痰活血法对AS粘附分子的影响及其与血脂异常证候的相关性

【作者】 郭杨志；

【导师】 杨惠民；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2014， 博士

【摘要】 目的：1通过观察调脂通脉颗粒对载脂蛋白E基因敲除(ApoE-/-)小鼠血脂水平及血清粘附分子sICAM-1、sVCAM-1的影响,对小鼠主动脉弓AS斑块的影响,及对小鼠主动脉弓NF-k B、ICAM-1和LFA-1表达的影响,探讨调脂通脉颗粒调节血脂及干预AS的可能作用机制,为益气化痰活血法指导血脂异常及AS的治疗提供实验依据。2通过观察血脂异常患者中医证候类型与血脂水平、血清粘附分子的关系,研究血脂异常患者证候分布规律,并初步探讨将TC、TG、HDL-C、s ICAM-1. sVCAM-1等血清检测指标作为血脂异常中医辨证依据的可行性,为血脂异常的中医辨证论治提供新思路。方法：1动物实验部分将40只6周龄ApoE-/-雄性小鼠随机分为模型组、血脂康组、调脂通脉颗粒低、中、高剂量组,每组8只,予高脂饮食结合药物饲养；另取8只相同遗传背景的C57BL/6雄性小鼠作为对照组,予普通饮食饲养。各组小鼠饲养12周后处死,血清学检测血脂、sICAM-1、sVCAM-1变化；主动脉弓根部石蜡切片苏木素伊红(HE)染色及马松(Masson)染色观察组织形态学变化；另取主动脉弓根部石蜡切片,行免疫组织化学染色法观察NF-k B、ICAM-1及LFA-1表达。收集数据后采用统计学方法分析,观察调脂通脉颗粒对以上指标的影响。2临床研究部分筛选120例于北京中医药大学东直门医院及北京市房山区大安山乡社区卫生服务中心门诊就诊的患者,根据相应纳入、排除标准收集血脂异常患者,签署知情同意书,采集患者一般资料并填写用于证候研究的临床调查量表,留取患者静脉血,血清学检测血脂、sICAM-1及sVCAM-1水平,另收集20例正常人作为对照组,研究内容同血脂异常患者。收集数据后进行统计分析观察血脂异常患者与正常人各项指标的差异；观察血脂异常不同证候类型间各项指标的差异；探讨将以上检测指标作为中医辨证依据的可行性。结果：1血脂检测表明,模型组、血脂康组、调脂通脉颗粒各组小鼠血清TC、LDL-C. HDL-C水平高于正常对照组；模型组、调脂通脉颗粒低剂量组TG水平高于正常对照组；调脂通脉颗粒中、高剂量组、血脂康组TG水平低于模型组。2ELISA法检测表明,调脂通脉颗粒各组、血脂康组、模型组小鼠血清sICAM-1、 sVCAM-1水平高于正常对照组；调脂通脉颗粒高剂量组血清sICAM-1、sVCAM-1水平低于模型组。3HE染色、Masson染色可见,对照组主动脉内膜光滑未见粥样斑块形成；模型组主动脉内膜增厚伴易损斑块形成；血脂康组及调脂通脉颗粒高剂量组内膜增厚伴稳定斑块形成。4免疫组化染色表明,模型组、血脂康组、调脂通脉颗粒各组主动脉弓NF-k B、ICAM-1及LFA-1表达高于正常对照组。调脂通脉颗粒高剂量组主动脉弓NF-k B、ICAM-1及LFA-1表达低于模型组。5临床研究发现,血脂异常患者证候分布比例由高到低依次为：肝肾阴虚组(40.83%)>阴虚阳亢组(25.83%)>痰浊阻滞组(20.83%)>脾肾阳虚组(8.33%)>气滞血瘀组(4.17%)。6各证候类型的血清TC水平由高到低依次为：气滞血瘀组>肝肾阴虚组>痰浊阻滞组>阴虚阳亢组>脾肾阳虚组>正常对照组。肝肾阴虚组、痰浊阻滞组、阴虚阳亢组及气滞血瘀组血清TC水平高于正常对照组。气滞血瘀组TC水平明显高于肝肾阴虚组及阴虚阳亢组。7各证候类型的血清TG水平由高到低依次为：痰浊阻滞组>阴虚阳亢组>肝肾阴虚组>脾肾阳虚组>气滞血瘀组>正常对照组。痰浊阻滞组及阴虚阳亢组TG水平高于正常对照组。痰浊阻滞组血清TG水平明显高于阴虚阳亢组。8各证候类型的血清HDL-C水平由低到高依次为肝肾阴虚组<正常对照组<阴虚阳亢组<脾肾阳虚组<痰浊阻滞组<气滞血瘀组,组间比较无差异。9血脂异常患者血清sICAM-1水平高于正常人,各证候类型血清sICAM-1水平由高到低依次为：阴虚阳亢组>肝肾阴虚组>脾肾阳虚组>痰浊阻滞组>气滞血瘀组>正常对照组。各证候组的血清sICAM-1水平均较正常对照组升高,但各证候组间无差异。10血脂异常患者血清sVCAM-1水平高于正常人,各证候类型血清sVCAM-1水平由高到低依次为：肝肾阴虚组>痰浊阻滞组>阴虚阳亢组>脾肾阳虚组>气滞血瘀组>正常对照组。各证候组的血清sVCAM-1水平均较正常对照组升高,但各证候组间无差异。结论：1以益气化痰活血法为指导的调脂通脉颗粒可在一定程度上缓解AS病理学改变,这可能与其降低血清TG、sICAM-1及sVCAM-1水平,抑制NF-kB. ICAM-1及LFA-1在斑块处的表达有关,抑制粘附分子及相关炎性因子表达可能是调脂通脉颗粒防治AS的作用机制之一。2肝肾阴虚证、阴虚阳亢证及痰浊阻滞证是血脂异常患者的重要证候类型,脾肾阳虚证出现较少,气滞血瘀证比例最少。3血脂异常患者的血清TC水平升高与气滞血瘀证密切相关；血清TG水平升高与痰浊阻滞证联系紧密；可考虑将其作为相应证候类型的辨证依据。4血脂异常患者的血清sICAM-1及sVCAM-1水平明显高于正常人,提示血脂异常早期阶段就已发生与AS相关的血管炎性反应。但本研究中未观察到其差异与不同证候类型存在相关性,暂不考虑将其作为辨证依据。更多还原

【Abstract】 Objective:To study the effect of "Tiaozhitongmai" granules on serum lipid,atherosclerosis plaque and NF-κ B,ICAM-1and LFA-1’s expressions in the aortic arch of ApoE-/-Mice. Provided experimental foundation for the method of reinforcing Qi as well as removing phlegm and stagnant blood to treat blood lipid abnormality and AS. To study the distribution rule of TCM syndromes of dysl ipidemia, and the relationship between that and serum level of TC, TG, HDL-C, sICAM-1and sVCAM-1.Methods:In the part of animal research, Six week-old male C57BL/6mice and ApoE-/-mice were divided into control group, model group, Xuezhikang group and "Tiaozhitongmai" granule group (low-,medium-, high-dosage) There were8mice in each group. They were fed by chow diet combined with medicine for12weeks. Changes in serum lipid, sICAM-1and sVCAM-1, as well as that of atherosclerosis and expressions of NF-k B, ICAM-1, and LFA-1in aortic arch were monitored by automatic biochemical serum analysis machine, ELISA, HE staining, and Immunohistochemical staining respectively. In the part of clinical research, we selected120patients with dyslipidemia according inclusion and exclusion criteria. Collecting their general information and finishing clinical research form after signed informed consent form. Then use automatic biochemical serum analysis machine and ELISA to monitor patients’serum level of lipid, sICAM-1and sVCAM-1respectively. Use statistic approach to analyze the relationship between patients’medical indexes of a test and TCM syndromes.Results:1By comparison to the control group, the serum levels of TC, LDL-C, and HDL-C have significantly increased in ApoE-/-mice (P<0.05), so was the serum levels of TG in the modeling group and in "Tiaozhitongmai" granule low-dosage group (P<0.05).2Compared with control group, serum level of sICAM-1and sVCAM-1significantly increased in ApoE-/-mice(P<0.05), and compared with model group, serum level of sICAM-1and sVCAM-1significantly decreased (P <0.05)3The HE staining showed that there were no atherosclerosis plaques formed in control group, yet vulnerable atherosclerosis plaques was formed in model group, and that stable atherosclerosis plaques were also formed in "Xuezhikang" group and "Tiaozhitongmai" granule high-dosage group. 4In comparison to the control group, NF-κ B、ICAM-1and LFA-1expression significantly increased in ApoE-/-mice (P<0.05), while, expressions of NF-κ B、ICAM-1and LFA-1decreased (P<0.05) in "Tiaozhitongmai" granule high-dosage group comparing to the modeling group.5In clinical research, we found the distribution rule of TCM syndrome was:Ganshenyinxu (40.83%)> Yinxuyangkang (25.83%)> Tanzhuozuzhi (20.83%)>Pishenyangxu (8.33%)> Qizhixueyu (4.17%)6The syndrome type of TC level from high to low in the order was: Qizixueyu> Ganshenyinxu> Tanzhuozuzhi> Yinxuyangkang> Pishenyangxu>normal group. Syndrom of Ganshenyinxu, Tanzhuozuzhi, Yinxuyangkang and Qizhixueyu had higher TC level than normal group. Syndorm of Qizhixueyu had higher TC level than that of Ganshenyinxu and Yinxuyangkang.7The syndrome type of TG level from high to low in the order was: Tanzhuozuzhi> Yinxuyangkang> Ganshenyinxu> Pishenyangxu> Qizhixueyu>normal group. Syndrom of Tanzhuozuzhi and Yinxuyangkang had higher TG level than normal group. Syndorm of Tanzhuozuzhi had higher TG level than that of Yinxuyangkang.8The syndrome type of HDL-C level from low to high in the order was: Ganshenyinx<normal group<Yinxuyangkang<Pishenyangxu<Tanzhuozuzhi <Qizhixueyu. There was no significant difference between six groups.9The patients with dyslipidemia had higher serum sICAM-1level than normal group. The syndrome type of sICAM-1level from high to low in the order was:Yinxuyangkang> Ganshenyinxu> Pishenyangxu> Tanzhuozuzhi> Qizhixueyu> normal group. There was no significant difference between six groups.10The patients with dyslipidemia had higher serum sVCAM-1level than normal group. The syndrome type of sVCAM-1level from high to low in the order was:Ganshenyinxu> Tanzhuozuzhi> Yinxuyangkang> Pishenyangxu> Qizhixueyu> normal group. There was no significant difference between six groups.Conclusion:1"Tiaozhitongmai" granule appeared to be effective in decreasing the serum level of TG, sICAM-1and sVCAM-1, as well as the expressions of NF-κ B, ICAM-1, and LFA-1in the aortic arch of ApoE-/-mice.2The syndrome of Ganshenyinxu, Yinxuyangkang and Tanzhuozuzhi is one of the important syndrome types in patients with dyslipidemia, Pishenyangxu appear less, the lowest proportion is Qizhixueyu.3Dyslipidemia in patients with higher TC is closely related to the syndrome of Qizhixueyu, elevated TG and the syndrome of Tanzhuozuzhi connected, may identify those two syndrome based on serum TC and TG level.4The serum sICAM-1and sVCAM-1level of patients with dyslipidemia were significantly higher than normal people, that means the inflammatory reaction connected with AS had already happened in the early stage of dyslipidemia. But there was no correlation between serum adhesion molecular level and different syndrome types.更多还原