冠心康抗镉染毒致动脉硬化作用及机制研究

【作者】 詹杰；

【导师】 张静生；

【作者基本信息】 辽宁中医药大学 ， 中西医结合临床， 2011， 博士

【摘要】 镉(Cadmium,Cd)是工业上常用的一种二价重金属。自然界中天然存在的镉丰度并不高,但随着现代工业的发展,镉污染越来越成为威胁人类健康的重要危险因素[1]。镉主要通过消化道和呼吸道吸收,通过血液循环释放到全身组织器官中。一般认为镉的生物半减期是10～30年,因此具有很强的蓄积性。近年研究表明,镉中毒是引起动脉硬化、高血压病等心脑血管的原因之一,而西医治疗尚无可靠方法。中医虽没有镉中毒的记载,但在动脉硬化的防治中具有一定的优势,因此探索镉中毒诱发动脉硬化的中医病因病机和中医防治方法具有很好的现实意义和可能性。目的:1.通过体外培养HUVECs构建镉染毒损伤血管内皮细胞模型,对比观察冠心康和硫酸锌对镉染毒人脐静脉内皮细胞一氧化氮(NO)、细胞间黏附因子-1( ICAM-1)等血管内皮活性物质及细胞色素C(Cyt C)、细胞增殖率、细胞凋亡率等指标的变化,分析冠心康对镉染毒致血管内皮细胞损伤的保护机制;2.通过大鼠整体动物实验,对比观察冠心康和硫酸锌对镉染毒大鼠血清一氧化氮(NO)、细胞间黏附因子-1( ICAM-1)和主动脉病理及ICAM-1免疫组织化学的影响,分析冠心康对镉染毒致大鼠动脉硬化的保护作用,同时通过对比分析冠心康和硫酸锌对镉染毒大鼠心、肝、肾镉蓄积以及钙、锌含量变化的影响,分析冠心康对镉染毒大鼠抗镉蓄积和调节镉染毒大鼠钙锌稳态的保护作用的可能途径,为重金属中毒提供新的防治措施。材料与方法:实验一体外细胞培养实验60只健康Wister大鼠,随机分为6组,按药物血清制备通法制备药物血清。冠心康低、中、高浓度组按人的每日等效剂量的1O、2O及4O倍给药量折算出大鼠每日灌胃量,分别为0.8g/ml、1.6g/ml和3.2g/ml,按2 ml/(次·只)大鼠灌胃;正常对照组和模型组以生理盐水等量灌胃;西药组按人的每日等效剂量的1O倍,硫酸锌浓度1mg/ml,2 ml/(次·只)灌胃。1次/d,连续3 d。末次灌胃给药1 h后,腹主动脉采血,取含药血清。人脐静脉内皮细胞体外培养,随机分为空白组、模型组、补锌组,及冠心康低、中、高剂量组,分别以空白血清、1、5、10、30和60μM不同浓度镉(CdCl2)血清、不同浓度CdCl2及硫酸锌血清,以及不同浓度CdCl2和冠心康血清培养24h、48h、72h,荧光倒置显微镜下观察细胞表面及内部形态的变化,同时收集培养液,硝酸还原酶法测定一氧化氮(NO)浓度,免疫学酶联免疫吸附法测定细胞间黏附因子(ICAM-1)、细胞色素C(Cyt C),细胞生物学MTT法检测细胞增值率;流式细胞术(FCM)检测细胞凋亡率。实验二整体动物实验Wister雄性大鼠72只(250±50g),随机分6组,即空白组、模型组、补锌组、冠心康低、中、高剂量组。空白组饮水为正常自来水;模型组:饮水为0.05 mg/kg镉溶液,2周后改为正常饮水;补锌组造模同时,每天1mg/ml硫酸锌溶液2 ml灌胃;冠心康低、中、高剂量组造模同时,冠心康颗粒分别按1.5 g/kg/d、3 g/kg/d、6 g/kg/d,蒸馏水稀释至2 ml灌胃。试验期为60 d。观察大鼠存活率,试验结束后各组大鼠腹主动脉取血,分离血清,硝酸还原酶法测定一氧化氮(NO)含量,免疫学酶联免疫吸附法测定细胞间黏附因子(ICAM-1),主动脉取病理,,进行HE染色镜下观察,ICAM-1免疫组织化学检验,原子吸收法进行心、肝和肾中镉、锌、钙含量测定。结果:实验一体外细胞培养实验结果1.各组HUVECs细胞功能指标比较:模型组1μM、5μM、10μM、30μM和60μM各组HUVECs镉染毒24h后,一氧化氮(NO)分泌量均较正常组明显减少(P<0.01),48h后略有回升,随后明显下降,且随镉染毒浓度升高一氧化氮(NO)分泌减少,30μΜ和60μΜ镉组一氧化氮(NO)水平明显高于1μM、5μM、10μM组(P<0.05,P<0.01),与模型组相比,冠心康各剂量组和补锌组均可明显改善镉染毒后HUVECs的一氧化氮(NO)分泌浓度(P<0.01)。模型组各镉浓度组HUVECs镉染毒24h后,细胞间黏附因子-1( ICAM-1)分泌量较正常组明显增加(P<0.01),48h时ICAM-1分泌量继续升高(P<0.01),此后明显下降(P<0.01),但与空白组比较仍明显增高(P<0.01),不同染镉浓度对ICAM-1分泌量的影响有随镉染毒浓度升高而增加的趋势,且随染毒时间延长,增加趋势明显。与模型组比较,补锌与添加各剂量冠心康均可明显改善镉染毒后HUVECs的ICAM-1分泌量(P<0.01)。2.各组HUVECs细胞凋亡及细胞增殖指标比较:染镉后,镜下见模型组细胞出现较多凋亡细胞,以60μM组最明显,补锌组及冠心康各组细胞体积减小,但未见明显细胞死亡;ELLISA法测定细胞色素C(Cyt C)结果显示,各组细胞CytC分泌量均有不同程度升高,且与染镉浓度和染镉时间呈正相关,其中模型组细胞色素C(Cyt C)分泌量升高最明显(P<0.01),补锌组及冠心康有明显改善(P<0.05),并以冠心康中、高剂量组改善最明显;流式细胞术测定细胞凋亡率(AR)结果显示,模型组细胞凋亡率明显升高,且随染镉时间和浓度升高而升高(P<0.01),60μM模型组荧光染色细胞沉淀图片可见大量晚期凋亡细胞和死亡细胞存在,明显高于空白组和其他处理组(P<0.01)。补锌组、冠心康各剂量组与模型组比较AR明显降低(P<0.01),除冠心康高剂量-60μM染镉浓度组外,其余各处理组AR与空白组比较无统计学意义(P>0.05),各处理组组间比较无明显差异(P>0.05)。MTT法测定细胞增殖率(PR)结果显示,低剂量(30μM以下组)、短时间(24h)镉染毒可致HUVECs细胞增值率增高,大剂量(60μM组)、长时间(48h后)镉染毒各组均出现持续下降(P<0.01)。补锌组及冠心康各组PR有明显改善(P<0.01),且冠心康组优于补锌组(P<0.05)。实验二整体动物实验结果1.血清学检测结果:试验结束后,模型组与空白组比较大鼠血清一氧化氮(NO)水平明显降低(P<0.01),细胞间黏附因子-1( ICAM-1)水平明显升高(P<0.01);补锌组和冠心康低、中、高剂量组一氧化氮(NO)水平均明显高于模型组(P<0.01),ICAM-1水平较模型组均明显降低(P<0.01),其中冠心康高剂量组一氧化氮(NO)水平比空白组增高(P<0.05),ICAM-1水平高于空白组和其他处理组(P<0.01),冠心康中剂量组细胞间黏附因子-1( ICAM-1)水平比空白组比较无统计学意义(P>0.05)。2.病理、免疫组化结果:主动脉横断面HE染色,模型组可见内膜增厚,局部破裂、脱落,脂肪空泡,平滑肌细胞增生;补锌组和冠心康各组内膜较完整平坦,平滑肌细胞形态明显优于模型组。免疫组化结果显示,空白组主动脉内膜层及外膜层偶见细胞间黏附因子-1( ICAM-1)阳性表达,模型组各层均见较多ICAM-1阳性表达,主要存在于粥样斑块区,补锌组及冠心康各剂量组主动脉内膜及平滑肌层均可见少量ICAM-1阳性表达信号,但信号强度明显弱于模型组,且平滑肌细胞形态及排列明显优于空白对照组,尤以冠心康中剂量组改善明显。3.器官内镉蓄积量及钙、锌含量测定结果:模型组镉染毒后心、肝、肾中镉含量明显高于空白组(P<0.01),锌含量明显低于空白组(P<0.01),钙含量明显高于空白组(P<0.01);补锌组及冠心康各剂量组镉蓄积量明显减少(P<0.01),锌含量明显提高(P<0.01),与空白组无明显差异(P>0.05),且冠心康各剂量组中提供锌量明显低于补锌组(P<0.01),钙含量明显减少(P<0.01),其中冠心康低、中剂量组与空白组无明显差异(P>0.05)。结论:1.镉中毒可引起维持内皮细胞重要功能的一些关键性内稳定平衡失调,既出现血管内皮功能障碍,主要表现为血管舒张受损、细胞黏附因子增加、细胞凋亡相关因子释放,继而出现内皮细胞凋亡及内膜增生,机体出现动脉粥样硬化,说明镉染毒致动脉硬化与多个途径有关,它们之间是相互关联、相互作用的。2.以黄芪为君,栝楼、丹参为臣的冠心康可明显改善镉染毒造成的血管内皮损伤,增加血管内皮保护因子一氧化氮(NO)的释放,降低细胞黏附因子水平,抑制镉染毒造成的细胞内皮凋亡,多靶点共同发挥抗镉染毒致动脉粥样硬化作用;同时,冠心康对减少机体镉蓄积和维持锌、钙稳态亦有保护作用,为镉中毒的中医防治提供了新的途径。3.环境毒邪损失人体正气,造成气虚痰瘀可能是镉染毒诱发动脉粥样硬化的中医主要病机。更多还原

【Abstract】 Cadmium is a bivalent heavy metal used in industry. Although the concentration of it is relative low in natural environment, Cadmium has become an increasingly important environmental pollutant during the past century. It and its compounds have been extensively used in the smelting and electroplating industries, and in the manufacturing of batteries, dyes, paints, plastics, pesticide and fertilizes. Tobacco contains significant amounts of Cd and smoking is also one of the primary sources of Cd exposure in the general population. It currently ranks 7th on the United States Environmental Protection Agency’s priority list of hazardous substances, and Japan and China are the major countries of Cd exposure in the world.Exposure to Cd can result in a varity of adverse effects in humans and animals. With regard to the cardiovascular system, that has been associated with a wide varity of cardiovascular pathologies including atherosclerosis, hypertension and cardio- myopathy. At the same time, reliability and available therapies have not been established in modern medicine. Although the traditional Chinese Medicine(TCM) has not the recordation about Cd toxicology, the therapeutic benefits of TCM to cure atherosclerosis and to maintain health is there. Thus, studying on the Chinese medical pathology and treatment to Cd induced atherosclerosis is regarded with practical significance and probability.Purpose:1.Probe the protecting mechanism of GuangXinKang dissolvable powder to Cadmium exposure-induced functional impairment of the endothelium.2. Explore the protective effects of GuangXinKang dissolvable powder on the level of NO and ICAM-1 of serum, the pathological section and immunohistochemical staining of aorta, fetal rat and accumulating Cd of heart, liver and kidney and its impact of the concentration of Zn and Ca on the Cd exposured rat, so as to find an ideal pathway of protecting Cd-feed-rat.Material and Methods:1. Extrabody Cell Culture TestAfter fed 3 days adaptively, sixty rats are randomly divided into six groups by weight: the control group and the model group are fed city water, the low dose Guanxinkang group is fed Guanxinkang 0.8g/ml, the moderate dose group ia fed Guanxinkang 1.6g/ml, and the high dose group is fed Guanxinkang 3.2g/ml, the Zinc group is fed zinc sulfate 1mg/ml, 2ml each time orally, and 2 times per day. The rats are fed 3 days. Collect of blood serum from rat’s abdominal aorta and freeze at one hour after the rats take the last drug of 3 days.The HUVECs are randomly divided into six groups: the control group, the model group, the zinc group and the low, moderate and high dose Guanxinkang group. The control group does not add Cd, the other groups add 1, 5, 10, 30 and 60μmol/L Cd respectively, and cultured respectively with rat’s blank serum or serum of Zinc or different concentration of Guanxinkang for 24, 48, 72 hours. Respectively, the changes of cellular morphology were detected under the inverted fluorescence microscope, the NO concentration, the intercellular adhesion molecule-1(ICAM-1) expression , Cytochrome C(Cyt C), the proliferation rate (PR) and the apoptosis rate (AR) were observed.2. Animal Test72 adult male Wister rats(250±50g) were randomly divided into six groups of 12 animals each by weight: the control group, the model group, the zinc group and the low, moderate and high dose Guanxinkang group. The control group was fed city water; the model group taked orally Cd (0.05mg/kg) from water for 2 weeks before city water; the Zinc group also taked orally Cd (0.05mg/kg) from water for 2 weeks before city water, and was fed zinc sulfate 1mg/ml, 2ml each time orally everyday; the low, moderate and high dose Guanxinkang groups taked orally Cd (0.05mg/kg) from water for 2 weeks before city water ,meantime ,were respectively fed Guanxinkan1.5 g/kg/d、3 g/kg/d and 6 g/kg/d . The rats were fed 60 days and were observed survival rate. After the last treatment, blood were collected from rat’s abdominal aorta. The serum was obtained after centrifugation and used for NO and ICAM-1 measurements, the aorta was excised immediately for pathology and ICAM-1 immunohistochemistry test, the heart, liver and kidney were excised immediately for determination of Cd, Zn and Ca content by atomic absorption spectrometry(AAS).Results:1. Extrabody Cell Culture Test1.1 Comparison of HUVECs cell function index for each groupCompared with the control group, the exposure of HUVECs with Cd(1μM、5μM、10μM、30μM and 60μM) for 24h significantly(P<0.01)decreased concentration of NO and significantly(P<0.01)elevated protein level of ICAM-1 in model groups. The level of NO slight recovered and the level of ICAM-1 continue to rise at 48 hours, and then significant decreased in the level of of NO and ICAM-1 with the concentration of Cd-exposure, but the latter still significantly increased when compared with the control group. In contrast, upon administration of different dose Guanxinkang or Zn to Cd-treated HUVECs, significant(P<0.01)increases in NO and significant(P<0.01)decreases ICAM-1 expression were observed.1.2 Comparison of HUVECs cell apoptosis index and proliferation rate for each groupThe results showed that Cd exposure led to more apoptotic cells appeared under the inverted microscope in the model group, especially in 60μM Cd-treated model group, and less apoptotic cells appeared in the Zinc group and the different dose Guanxinkang groups, besides little small shape. Cd induces also the increase of the Cyt C level of each Cd-exposure groups, which increased with the concentration of Cd, and the most obvious increase was in the model group(P<0.01), whereas Zinc and Guanxinkang significantly caused inhibition of Cyt C oversecretion , which was distinctest in the middle and high dose Guanxinkang groups. FCM also showed that Cd induced increase of apoptotic rate(AR) , especially in 60μM Cd-treated model group. but Zinc and Guanxinkang inhibited the increase of AR. The AR of the Zinc group and Guanxinkang group, in addition to 60μM Cd-Guanxinkang group, had no significant difference compared with the control group(P>0.05).Interestingly, MTT showed Cd exposure inceased the PR of low concentrations of Cd ions (5-30μM)during short exposure time (24h) but continued to decline at higher concentrations(60μM)or longer exposured time(48-72h), which also showed the improvement in zinc group and Guanxinkang groups, and the latter was better than the former.2. Animal Test2.1 Comparison of Serological test resultsSubcutaneous administration of Cd showed a significant(P<0.01) reduction in NO with a significant(P<0.01) increase in ICAM-1 in rat plasma. In addition, the level of NO in the Zinc group and the different dose Guanxinkang group were significantly higher than the model group(P<0.01), and the level of ICAM-1 were significantly lower than the model group(P<0.01), meanwhile, the level of NO in the high dose Guanxinkang group were higher than the control group(P<0.05),and the level of ICAM-1 were higher than the control group and the other treatment groups(P<0.01),moreover, the level of ICAM-1 in the middle dose Guanxinkang group was similar with the control group(P>0.05).2.2 Comparison of pathological and immunohistochemical resultsThe pathological of aortic cross-section in HE staining showed intima got thickening, partially rupture or loss, with fat vacuoles under the endometrial, and vascular smooth muscle cell(VSMC) became proliferation. Immunohistochemical results showed that immunohistochemically visualized ICAM-1 occasionally expressed on aortic intima and adventitia of the control group, but expression increased significantly in the intima, the middle and outer membranes of aortic, espectly concentrated on the AS. Immunohistochemically visualized ICAM-1 also expressed on aortic intima and smooth muscle layer of the Zinc group and the different dose Guanxinkang group, but signal strength were significantly weaker than the model group, meanwhile, the shape and arrangement of smooth muscle cells was better than the model group, especially in the middle dose Guanxinkang group.2.3 Comparison of Cd, Zn and Ca assay results in organsThe concentrations of Cd and Ca in heart, liver and kidney of rats in Cd feed group were significantly higher than that of control group (P<0.01), with Zn concentration reversed (P<0.01). Compared to Cd feed group, Cd concentrations in heart, liver and kidney of rats in the Zn group and the different dose Guanxinkang group were significantly decreased(P<0.01). Likewise, Ca concentrations were significantly decreased either even though there were not differences between low and medium dose Guanxinkang groups. Meanwhile, the treatment of Cd-exposed HUVECs with different dose Guanxinkang significantly(P<0.01)elevated Zn concentration of the rat organ just as that of the Zinc group, with providing zinc significantly lower than the later(P<0.01).Conclusion:1. Cadmium induces some disorders of endothelial cells’stable and equilibrium which is key to maintain important features, that is vascular endothelial dysfunction, mainly as impaired vasodilation, increased cell adhesion molecules, release of apoptosis-related cytokine, followed by endothelial cells withered Apoptosis and intimal hyperplasia, and results in atherosclerosis. The results suggest that Cd exposure induced atherosclerosis through multiple pathways, among which are interrelated and interact.2. Guanxinkang, with basis Huangqi and associate Gualou and Danshen, can effectively improve endothelial dysfunction and prevent atherosclerosis by restoring NO concentration, down-regulating VCAM expression, and decreasing the apoptosis rate of vascular endothelial cell(VEC)induced by Cd. Meanwhile, Guanxinkang can also decrease the accumulation of Cd in body and maintain constant concentrations of Zn and Ca. The effects indicating a new pathway to protect and cure Cd toxicosis by TCM.3.The environmental toxic influences invade the body with the internal organs, and weaken the body’s protective qi by destroyed the relatively balanced state of qi, blood and body fluid, plus pathological products of disease outcome, such as phlegmn-humor and blood stasis. Which may be the etiology and pathogenesis of traditional Chinese medicine(TCM)about how Cd induce AS.更多还原