Effect of intraperitoneal injection of cyclovirobuxine D on the expression of growth-associated protein-43 mRNA in hypertensive rats with cerebral ischemia reperfusion / 中国组织工程研究

ABSTRACT

BACKGROUND: Growth-associated protein-43, a kind of protein relatedto axonal growth, plays a key role in promoting neural development, axonalregeneration, synaptic growth and structural and functional reconstructionand so on. In the study, we find that cyclovirobuxine D can protect braininjury in rats with experimental cerebral ischemia reperfusion.OBJECTIVE: To observe the effect of cyclovirobuxine D on the expression of growth-associated protein-43 mRNA in hypertensive rats with cerebral ischemia reperfusion.DESIGN: A randomized controlled animal experiment.SETTING: Foshan Hospital of Traditional Chinese Medicine of Guangdong Province; Central Laboratory of Guangdong Hospital of Traditional Chinese Medicine.MATERIALS Cyclovirobuxine D is alkaloid monomer extracted from Chinese herb buxine. Cyclovirobuxine D powder with national protected traditional medicine number of ZYB20796057 was provided by Nanjing Xiaoyingyao Pharmaceutical Factory. Totally 120 two-to-three-month-old healthy male SD rats, of either gender, with body mass of 90 to 120 g, were used in this experiment.METHODS: This experiment was carried out in Foshan Hospital of Tra ditional Chinese Medicine of Guangdong Province and at the Central Laboratory of Guangdong Hospital of Traditional Chinese Medicine between June 2005 and March 2006. ①Stroke-prone-renovascular-hypertensive-rats models (RHRSP)were created by bilaterally narrowing the renal artery with silk loop clips. Totally 120 rats were randomly divided into blank group (n=20,renovascular hypertensive rats were given no treatments), sham operation group (n=20, rats were given only surgical trauma), model group (n=40, rats were given treatment of cerebral ischemia reperfusion) and cyclovirobuxine D-treated group (n=40, rats were given cyclovirobuxine D).② Unilateral occlusion of the middle cerebral artery ischemia reperfusion models were made with suture-occluded method. 6.48 mg/kg cyclovirobuxine D diluted by 1.5 mL normal saline was intraperitoneally injected into the rats of cyclovirobuxine D-treated group, twice per day; Normal saline was isochronously intraperitoneally injected into the rats of each subgroup of control group, 2 mL once, the method was the same as that of the cyclovirobuxine D-treated group; interval of injection time was 7 hours in each group. Rats in each group were executed on days 1, 7, 14 and 30after ischemia reperfusion. ③ Brain slice was prepared. The expression of growth-associated protein-43 mRNA of rats in each group was detected with in situ hybridization.MAIN OUTCOME MEASURES: ① The expression of growth-associated protein-43 mRNA around ischemia are following ischemia reperfusion for 2 hours. ②The expression of growth-associated protein-43 mRNA in hippocampus following ischemia reperfusion for 2 hours RESULTS: All the 120 rats entered the stage of result analysis. ① Immune in situ hybridization of growth-associated protein-43 mRNA In situhybridization showed that expression of growth-associated protein-43 mRNA and expression of growth-associated protein-43 mRNA could be detected respectively in the hippocampus and marginal area of hematoma after ischemia and reperfusion.②The expression of growth-associated protein-43RNA around haematoma following cerebral ischemia reperfusion group as not found in blank group and sham-operation group; The expression of growth-associated protein-43 mRNA was found in the marginal zones around haematoma of rats in the model group on the 1st day following ischemia reperfusion and it was significantly increased on the 7th day, gradually reduced on the 14th day and still expressed on the 30th day but less,with significant difference at each time point (P ＜ 0.01); Expression of growth-associated protein-43 mRNA around haematoma at each time point was more in the cyclovirobuxine D-treated group than in the model group ,with significant difference (P ＜ 0.05). ③There was no significant difference of the expression of growth-associated protein-43 mRNA in hippocampus of rats following cerebral ischemia reperfusion between blank group and sham-operation group; Expression of growth-associated protein-43 mRNA was found in hippocampus of rats in the model group on the 1st day after modeling, and it reached the peak on the 7th day, gradually decreased on the 14th day and significantly decreased on the 30th day, but significantly more than that of sham-operation group; The expression of growth-associated protein-43 mRNA in hippocampus was significantly more at each time point in cyclovirobuxine D-treated group than in model group, with significant difference (P ＜ 0.01).CONCLUSION: Cyclovirobuxine D up-regulates the expression of growthassociated protein-43 mRNA after reperfusion and promotes axonal regeneration of rats with experimental cerebral ischemia.