Study on COX activity and its mRNA expression,and PGE2 release from cerebral microvascular endothelial cells stimulated by IL-1β |
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Authors: | GUO Jian-you HUO Hai-ru ZHAO Bao-sheng LIU Hong-bin LI Lan-fang GUO Shu-ying JIANG Ting-liang |
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Institution: | 1.Tang Center for Herbal Medicine Research, Institute of Materia Medica, China Academy of TCM, Beijing 100700, China; 2 Institute of Psychology, Chinese Academy of Sciences, Beijing 100101, China.E-mail:hrhuo@sohu.com |
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Abstract: | AIM: To study the cyclooxygenase(COX) activity and its mRNA expression, and PGE2 release from rats cerebral microvascular endothelial cells (rCEMC) stimulated by IL-1β(30 μg/L) at different times. METHODS: rCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor (Ⅷ factor, a marker for all endothelial cells) in cytoplasm of the cells. After rCEMC grew to confluency, they were stimulated with IL-1β for 0.5, 1, 2, 4, 8, 12 and 24 h, respectively. Activity of COX-1 and COX-2 in rCEMC and production of PGE2 in the conditioned media were detected by ELISA. COX-1 and COX-2 mRNA expressions were measured by real-time quantity PCR. The amplification product was tested by melting curve and identified by electrophoretic gel. RESULTS: ① Positive immunostaining for Ⅷ factor was present diffusely in the cytoplasm in more than 90% rCMEC. ② Compared to the cells without IL-1β stimulation, the production of PGE2 increased significantly (P<0.05) at 4 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ③ There was no significant difference on COX-1 activity between IL-1β group and non-IL-1β group. COX-2 activity increased significantly compared with those in non-IL-1β (P<0.05) at 8 h after rCEMC were incubated with IL-1β and reached the top level at 12 h (P<0.01), then declined thereafter at 24 h (P<0.05). ④ There was no significant difference on COX-1 mRNA expression between IL-1β group and non-IL-1β group. COX-2 mRNA was induced and became detectable at 1 h, and reached the top level at 4 h, then declined thereafter at 8 h and became undetectable by 12 h and 24 h after incubation with IL-1β. The melting curve showed there was no nonspecific amplification and electrophoretic gel showed the lengths of amplification products accorded with the predicted lengths. CONCLUSION: While rCEMC are stimulated by IL-1β, the excretion of PGE2 increases and reaches the top level at 12 h, which is related with its induction on COX-2 mRNA expression and COX-2 activity. |
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Keywords: | Interleukin-1 Brain Endothelial cells Prostaglandins E Cyclooxygenase-2 |
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