| Objective To explore the influence of hyperhomocysteinemia (Hhcy) and asymmetric dimethylarginine (ADMA) on the structure and function of rats’cerebral vascular. On this basis to investigate the change of Hey, ADMA, oxidized low-density lipoprotein (OX-LDL), dimethylarginine dimethylaminohydrolase (DDAH) levels in rats and evaluate the interventional effects after giving the probucol,folic acid, and vitamin B12.Methods Seventy male SD rats were randomly divided into seven groups. Each group had ten rats:normal control (A) group, Hhcy model (B) group, low-dose-probucol (C) group, middle-dose-probucol (D) group, high-dose-probucol (E) group, folic-acid-vitamin B12(F) group, folic-acid-vitamin B12and middle-dose-probucol (G) group. Group A was fed on plain feedstuff for twelve weeks and from the beginning of ninth week added water for four weeks. Other groups were fed on homomethionin feedstuff for twelve weeks.And then from the beginning of ninth week water, probucol(340、510、680mg/kg/d), folic acid and vitamin B12,folic acid and vitamin B12and probucol were added respectively. The plasma concentrateons of the Hey, ADMA and OX-LDL were assayed at the beginning,the end of eighth and twelfth week. The DDAH2expression in brain homogenate was measured at the end of twelfth week. Hey and ADMA were measured by high-performance liquid chromatography (HPLC)method. OX-LDL was measured by enzyme linked immunosorbent assay (ELISA) method. DDAH2expression in the brain was measured by Werstern blot method. The changes of rats’ cerebral vascular and cerebral cortex were performed with electron microscope at the end of twelfth week.Results1、There were no statistically differences (P>0.05) in seven groups of rats about the levels of Hey, ADMA, OX-LDL at the beginning of the experiment. When the rats had been fed on homomethionin feedstuff for eight weeks, the Hhcy had been induced successfully, the level of Hcy in blood plasma was increased from6.39±1.51μmol/l to60.42±11.92μmol/l in group B. Meanwhile the levels of ADMA, OX-LDL were higher than those of the same period of group A (P<0.05).2、The Hcy level in group B was significantly higher than that of group A at the end of eighth week and twelfth week (P<0.01). At the end of twelfth week, the levels of Hcy in groups C、D、E were all lower than that of group B (P<0.05), but were significantly higher than that of group A (P<0.01), as well as groups F and G (P<0.05). The level of Hcy in group F was significantly lower than that of group B(P<0.01), but still higher than that of groups A and G(P<0.05).The level of Hcy in group G had no statistically significant differences compared with that of group A(P>0.05).3、The ADMA level in group B was significantly higher than that of group A at the end of eighth week and twelfth week (P<0.01). At the end of twelfth week, the levels of ADMA in groups C、D、E、F were all lower than that of group B(P<0.05), but were significantly higher than that of group A and G(P<0.05), and the ADMA levels had no statistically significant differences among the four groups (P>0.05), the level of ADMA in group G had no statistically significant differences compared with that of group A(P>0.05).4、The OX-LDL level in group B was significantly higher than that of group A at the end of eighth week and twelfth week (P<0.01). At the end of twelfth week, the levels of OX-LDL in groups C、D、E、F were all lower than that of group B(P<0.05), but were significantly higher than that of group Aand G(P<0.05), and the OX-LDL levels had no statistically significant differences among the four groups (P>0.05), the level of OX-LDL in group G had no statistically significant differences compared with that of group A(P>0.05).5、The levels of Hcy, ADMA and OX-LDL in groups C, D, E, Fat the end of twelfth week were lower than that of the end of eighth week, but higher than that of the beginning of the experiment(P<0.05). The levels of Hcy, ADMA and OX-LDL in group G at the end of twelfth week were lower than that of the end of eighth week(P<0.05) while had no statistically significant differences compared with that of the beginning of the experiment(P>0.05).The levels of Hcy, ADMA and OX-LDL in group A at the end of twelfth week had no statistically significant differences compared with that of the beginning of the experiment and the end of eighth week(P >0.05). The levels of Hcy, ADMA and OX-LDL in group B at the end of twelfth week were higher than those at the beginning of the experiment and the end of eighth week(P<0.05).6、Pearson-correlation analysis showed:Plasma levels of ADMA were positively correlated with Hcy, OX-LDL (P<0.01). Plasma levels of Hcy were positively correlated with that of ADMA, OX-LDL(P<0.01).7、The DDAH2expression in brain homogenate of group B was lower than that of the other six groups at the end of twelfth week(P<0.05).The DDAH2expression in groups C、D、E、F were all higher than that of group B, but were lower than that of group A and G(P<0.05). The DDAH2expression in group G had no statistically significant differences compared with group A(P>0.05).8、On electron microscope, we observed that in group B, the cerebrovascular endothelial cell membranes and cytoplast were degraded and vague. The endothelial cell was swelling and disrupted. Some cell debris which were disrupted and falled off almost blocked the lumen. Perivascular brain tissue was swollen. The perivascular neurons damaged and collapsed. Most cell organelles were degraded and the neurons were lost. In groups C, D, E and F the damages were reduced than group B. We observed that part of the artery endothelial cells were still well. The vascular intima inclined to be more thicker. Some cell organelles could be recognized. In cerebral cortex the small vessel was partially damaged, nearby neurons and the nerve blanket were mild swelling, the glial cells baseboard also mild swelled and so on. The ultrastructure of group G was the best one in intervention groups. We observed that the artery was open. The thickness of vascular intima significantly reduced. The vessel wall was partially not clear. The cell organelles could still be recognized. Small blood vessels in the brain tissue were open, extravascular without swelling. The neurons and nerve blanket structure were basically normal, synaptic structure was clear.Conclusion The hyperhomocysteinemia model could be induced successfully by homomethionin feedstuff in rats, as well as the levels of ADMA and OX-LDL higher, and the expression of DDAH2lower, which demonstrate that cerebral vascular struction damage and endothelial dysfunction are prevalence in rats. ADMA levels are positively correlated with Hcy levels, and both of them are also positively correlated with OX-LDL. The present study demonstrate that the three of them interact with each other, accelerating oxidative stress, forming vicious cycle, and leading to cerebral vascular endothelial injury and dysfunction. With supplementation of folic acid,vitamin B12and probucol the levels of Hcy,ADMA and OX-LDL are decreased significantly, in the meanwhile the expression of DDAH2is increased, and sequentially inhibition of oxidative stress and improvement of the endothelial function, which is propitious to alleviate the damage of cerebral vascular and tissue. And the effect is more significant by the three kinds of drug combination. |
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