Neuroprotective Effect Of Osthole In Ischemia-reperfusion Model

Fructus cnidii is fruit of Cnidium monnieri with a rich natural resource.The main pharmaco-components of cnidii are coumarins compounds which contain osthole(OST)as fundamental ingredient.Recent studies showed that osthole can play a role as phytoestrogens in vivo and has a potential contribution toward neuroprotection.Homerla protein can intervene the procedure of transporting and anchoring of metabotropic glutamate receptor(mGluR1a)to postsynaptic density(PSD),and it was cleared out that there were some neuroprotective effects of Homer1a.In this research,neuronal ischemia model by oxygen-glucose deprivation(OGD)in vitro and 4-vessel occlusion(4VO)model in vivo were employed to identify the neuroprotective effects of osthole and expression of Homer1a protein after ischemia-reperfusion injury to explore the osthole's effect upon Homerla to show the possible relationship between Homerla and the neuroprotection of osthole after the injury.PartⅠExpression of Homerla and neuroprotective effects of osthole in the neuronal ischemic model by oxygen and glucose deprivation in vitro Objective To identify the neuronal protective effects of osthole and the possible role of Homerla protein in this procedure.Methods The rat's high-density primary cultured cortical neurons basing on an improved method of cortex neuron culture in high-density were prepared seven days before the treatment of OGD.Primary cultured cortical neurons were identified with microscope and immunohistochemistry after 7 days in vitro cultivation.After assessment of purity of the neurons,the plates of neuron were divided into sham group(neurons avoided from the treatment of both OGD and OST), cnotrol group(neurons were exposed to OGD only)and OST groups(neurons were treated with different dose of osthole after that had been treated with OGD; OST0.01Mμ,OST0.1μM,OST1μM,OST10μM,OST100μM).Excepting the sham proup,oxygen ang glucose of the other groups were deprived for four hours and resupplied again.Immediately after the reperfusion,the OGD groups were treated with osthole 0.01μM,0.1μM,lμM,10μM and 10μM receptively. Twenty four hours after the treatment,neurons' expression of Caspase3 and Homerla of each of the groups were identified by means of Western Blot technique.Results All neurons showed multiform transparent cytoplasm with round or oblonga nucleuses and connected each other to form reticular structure; the neuron courting basing on the immunohistochemistry of anti-β-tubulin-Ⅲand NF200 indicated that purity of the cultured neurons was above 95%.The protein analysis showed that osthole could reduce the elevated level of Caspase3 protein induced by the OGD;the expression of Homerla was in inverse correlation in comparison with that of Caspase3.Conclusion The high-density primary cultured cortical neuron of rat was a kind of ideal test materials for OGD model and the following biochemical analysis.Decreased expression of Caspase3 and increased expression of Homer1a after the treatment of osthole in the Ischemia-reperfusion model of neurons indicated that the osthole can prevent neurons from the injury and the Homer1a protein might join the mechanism of osthole's neuroprotective effects.It was illuminated that Homerla could antagonize the functions of Homer1b/c which was relied on by mGluR1a for anchoring to PSD of neurons.In this study,the treatment effects of osthole were clarified and the pattern of Homerla expression after the treatment of osthole in the model of neuronal injury in vitro indicated that Homerla possibly played a role in the pathophysiological process after OGD injury of neurons,and a theoretical foundation was formed for the development of this new neuroprotective agent for the patient of brain ischemia.PartⅡNeuroprotective effects of osthole and expression of Homerla in the four-vessel occlusion model in vivoObjective To identify neuroprotective effects of osthole and expression of Homerla in the four-vessel occlusion model in vivo.Methods An improved method for four-vessel occlusion(4VO)model in rats was established.Fifty SD rats were divided into Sham group(animals avoided from the treatment of both 4VO and OST),control group(animals were exposed to 4VO only)and OST groups(animals were treated with different dose of osthole after that had been exposed to 4VO;OST5mg/kg,OST25mg/kg,OST125mg/kg).After having been anesthetized with chloral hydrate(400 mg/kg i.p.),the common carotid arteries(CCAs)and the beginning of the subclavical arteries(SCAs)of rats were separated and occluded for fifteen minutes,and then,the arteries were loosed to perform reperfusion one hour before the treatment of osthole excepting the rats in sham group avoiding the occlusion.The rats in OST groups were treated with osthole 5mg/kg,25mg/kg,125mg/kg receptively.The rats of sham and control groups were not treated with osthole.Twenty four hours later, neurological deficits were determined with the modified Garcia scoring system. After neurological deficits determination,the expression of Caspase3 and Homer1a of bilateral hippocampus were identified by using Western blot technique.Histopathology(hematoxylin eosin staining)was conducted and five images were randomly selected from each section for counting the number of survival cells at CA1 area of hippocampus.The neurological score was determined with the Kruskal-Wallis H,followed by the Mann-Whitney U with Bonferroni correction.The other values were analyzed by one-way ANOVA, followed by the LSD-t test.A P value of＜0.05 was considered statistically significant.Results This four-vessel occlusion model produced by the improved method is a safe,easy,reliable,stable,mini-invasive and quick procedure method for making bilateral hemispheric ischemia that can effectively decrease collateral circulations and lead to stable lesions in hippocampus and cortex. Both the histopathological changes and the neurological deficits scores of osthole groups are better than that of control group(P＜0.05)but lower than that of rats in sham group.Results of Western blot showed that osthole could reduce the expression of Caspase3 protein that was induced by the 4VO ischemia and reperfusion compared with that of the control group while the Homer1a was increased accompanying the increased dose of osthole.However, the expression of Caspase3 and Homer1a protein of the rats in group of osthole 125mg/kg showed an opposite result compared with that of the rats in the other treated groups indicated that the osthole of excess dose might play a negative role in the mechanism of brain ischemia.Conclusion The osthole can prevent neurons from the ischemia injury and the Homer1a protein might play a role in the mechanism of osthole's neuroprotective effects.It was illuminated that Homer1a can mitigate the excessive excitement of mGluR1a which relied on Homer1b/c to anchoring to PSD of neurons.In this study,the therapeutical effects of osthole were clarified and the pattern of Homer1a expression after the treatment of osthole in the model of ischemia-reperfusion neuronal injury in vivo and in vitro indicated that Homer1a possibly played a critical role in the pathophysiological process of ischemia-reperfusion injury of neurons,and a theoretical foundation was formed for the development of a new neuroprotective drug for the patients of brain ischemia.In conclusion, osthole as the natural coumarin derivative warrants further evaluation as a promising drug for brain ischemia patients,especially,if the results from this study can be confirmed in other experimental nervous system ischemia models in rodents and extrapolated to clinical settings finally.