| 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. |
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