| Homocysteine ( Hcy ) is recognized as an independent risk factor for atherosclerosis (AS) and thrombogenesis. Epidemiological evidence has gradually accumulated implicating Hcy in the pathophysiology of many neurological disorders. Recent in vitro and in vivo studies have demonstrated that endothelial injury was the initial event in the pathogenesis of arteriosclerosis. The cellular mechanisms of hyperhomocysteinemia (HHcy)-induced endothelial injury and dysfunction include oxidative stress, endoplasmic reticulum stress, and dysfunction of endothelium-dependent vascular tone, coagulation and inflammation. It has been reported that vitamin B12, B6 and folic acid supplementation could lower the serum Hcy level, which may have important implications for reducing stroke rates. However, it is not clear whether these treatments could reverse the pathological processes of arteriosclerosis and relieve or eliminate any symptoms associated with it, since Hcy has already activated the pathway that leads to the impairment of endothelial cells. It is also important to investigate the mechanisms by which Hcy induces endothelial dysfunction during and after atherosclerotic lesion in those people with prolonged HHcy. In order to address the above questions, we studied the toxic effects of Hcy in rat brain microvascular endothelial cells (BMEC) in vitro and it's potential mechanisms. Methods: (1) To establish rat brain microvascular endothelial cells culture in vitro. (2) To examine the dose effects of Hcy on BMEC growth inhibition by MTT assay. (3) The changes of Bcl-2 and Bax mRNA level were measured by RT-PCR. (4) The changes of ICAM-1 expression were studied by immunocytochemistry and flow cytometry (FCM). (5) The changes of NF-κB expression were assessed by immunocytochemistry and Western blot. (6) To analyze the association between the expression of NF-κB and Bcl-2, Bax, and ICAM-1.Results:1. Rat brain microvascular endothelial cells (BMEC) culture has been established in vitro.2. Treatment of BMEC with 4μmol/L copper plus Hcy in the range of 0.2 to 2 mmol/L reduced cell viability in a dose-dependent manner, whereas 0.2-2 mmol/L Hcy or 4μmol/L copper by itself had no effect.3. After 0-18 h of Hcy plus copper treatment, the Bcl-2 and Bax mRNA level in BMEC was decreased and increased respectively in a time-dependent manner.4. Expression of ICAM-1 in BMEC was increased in the presence of 1.0 mmol/L Hcy .Compared with the 0 h group, the ICAM-1 of 12 h and 18 h groups were significantly increased(p<0.05;p<0.01), whereas the 6 h group was not significantly(p>0.05).5. Hcy stimulated the nuclear transfer of NF-κB, which was detected as early as 6h after Hcy treatment, peaked at 12h and then decreased gradually back to normal level.In summary, our studies confirmed the toxic effects of Hcy in BMEC, suggesting that HHcy could inhibit the growth of the BMEC by inducing apoptosis. And the decrease in cell viability of HHcy was in a dose– dependent manner. Our work demonstrated that homocysteine-induced Bcl-2 , Bax and ICAM -1 expression variation is mediated via the activation of NF-κB in BMEC. There is little analogic report in domestic now. As the result of the ICAM -1 expression mediated by NF-κB activation signal during the homocysteine-induced endothelial inflammatory injury, the functon of the endothelial cells was damaged. In addition, we speculate that homocysteine-induced Bcl-2 , Bax gene expression may be associated with the BMCE apoptosis .the BMEC can represent the tissue specificity of brain vessels, so as to build up the basis for the evaluation of the relationship between HHcy and some nervous system diseases. Moreover, the data forthcoming from these settings will suggest new avenues to slow down the pathological process of arteriosclerosis and block the activation of chemokines, such as NF-κB, upon endothelial injury, and provide new insights for the prevention of ischemic cerebrovascular diseases.
|
PDF Full Text Request