| Background: Chronic cerebral vasospasm (CVS) is the major cause of morbidity and mortality following subarachnoid hemorrhage (SAH) .The mechanism of CVS have last several decades and remained unclear up to now.It is indicated in experimental and clinical studies that pharmacological treatment aimed at persistent constriction of smooth muscle could meliorate CVS in some extent or in the early phase ,but couldn't take effects on severe CVS .It was observed in the arteries of patient and animals following SAH that there were necrosis, proliferation and trucking of muscle layer with inflammatory cells infiltrating. Studies also indicated that intracellular adhesion molecule-1 (ICAM-1),as a key factor in inflammtion was involved in the inflammtion of vessels following SAH, although the mechanism is unclear. Some scholars concluded that proliferative arteriopathy following inflammation ,rather than active constrictin of vascular smooth muscle cells (VSMC) produces CVS. Previous studies focused on the active constricting of VSMC and little attention have been paid to proliferation of VSMC. Animal model were commonly used in related studies, and the results were often differential from each other.Object : Artificial bloody cerebralspinal fluid(BCSF) was used as pathogeny on culturing microvessel endothelial cells (BmvECs) to mimik SAH status in vivo .We try to explore the mechanism of CVS and and effectiveness of EDS (ecdysterone) in treating CVS by observingpathologic changes , proliferative status , expression of CAM-1 on EC andproliferative effects of corresponding endothelial cells conditioned medium on VSMC.Methods::1. Pure BmvECs and VSMC were cultured in vitro , and the purity were confirmed by immunocytochemistry (ICC);2 . Using electronic microscope ,cell counting ,MTT assay, flow cytometric analysis to evaluate cytoxic effects of BCSF on EC .3. Immunocytochemistry and RT-PCR were used to observe the the expression of ICAM-1 protein and mRNA;4.Griess reaction and radioimmunoassay were used to determine the content of NO2- and ET-1. proliferative activity of corresponding conditioned medium on VSMC was tested by MTT assay , flow cytometric analysis and DNA synthesize. PCNA protein of VSMC was observed by Immunocytochemistry.Results:1. By MTT assay ,the longer BCSF were incubated , the more severe it injured the viability of EC. BCSF incubated for 7 days are suitable for study with obvious cytotoxic effects on EC.EDS had protective effects on these pathological changes;2. Following BCSF stimulating, immunocytochemistry reaction of ICAM-1 increased in BmvECs and HUVECs, and expression of ICAM-1 mRNA also increased in BmvECs.EDS could reduce the expression of ICAM-1 protein and mRNA;3. BCSF increased the secretion of ET-1 and decreased the secretion of NO,and conditioned medium could activate the DNA synthesize and proliferation of VSMC. EDS treatment tenuated the secretion disorder of EC and corresponding conditioned medium had lower proliferative activity on VSMC.Conclusion:1. Cytoxic effects of BCSF on BmvECs but not apoptosis changes of BmvECs were confirmed in this study.2. ICAM-1 expression in EC could be activate directly by BCSF ,although the mechanism is unclear.3. The proliferative effect of EC conditioned medium by BCSF may be based on the secretion disorder, which suggests there is possiblely VSMC proliferation following SAH;4. Our results show EDS have protective effects on EC in multiple aspects .It is possible to be a valid medicine in CVS therapy. |
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