Comparative Research On Mechanism Of Neuroprotective Effect And Influence Of Blood-brain Barrier Between L-borneolum And Borneolum Syntheticum

Objective:To compare the mechanism of neuroprotective effect and influence of blood-brain barrier between L-borneolum and borneolum syntheticum. It provides experimental basis to explain the scientific connotation that borneol with the effect of inducing resuscitation and restoring consciousness can be distributed in brain for flavour of pungent.Methods:In this study, mechanisms of neuroprotective effect and influence of blood-brain barrier were explained from whole-cell-molecular level.①The mouse models of cerebral anoxia and ischemia were established by sodium nitrite, decollation, intravenous injection of magnesium chloride and the occlusion of bilateral common carotid arteries with vague nerves. The survival times were detected to observe the influence of the mouse models after drugs.②The focal middle cerebral artery occlusion (MCAO) model was made by suture-occluded method. The rectal temperature of rats was observed on preoperative and reperfusion4h,22h. Rates of cerebral edema were examined after22h reperfusion.③Borneol in mice brain was determined by GC, and compare borneol transmittance in mice brain between L-borneolum and borneolum syntheticum after intragastric administration.④superoxide dismutase(SOD) activity and the content of malondialdehyde(MDA) both in serum and ischemia side brain tissue, the level of nitrogen monoxidum (NO) and nitricoxide synthase (tNOs and iNOs) activity both in serum and ischemia side brain tissue, calcium ion(Ca2+) content and ATPase(T-ATPase, Na+-K+-ATPase, Ca2+-Mg2+-ATPase) activity in ischemia side brain tissue were measured by spectrophotometry.⑤The expression of MDR1mRNA and P-GP in ischemia side brain tissue were measured by real-time PCR and western-blot.⑥the ultra micro structure of blood brain barrier of ischemia hemisphere side in fronto-parietal cortex region was observed by transmission electron microscope.⑦Growth of Hypoxic ischemic PCl2(rat pheochromocytoma cell) was measured by MTT, and Calcium ion (Ca2+) content in cell was measured by spectrophotometryResult:L-borneolum and borneolum syntheticum showed the ability of anti-cerebral ischemia and hypoxia against brain ischemia and hypoxia: ①Compared with the model group, opening times of L-borneolum (200,133.33,66.67mg/kg) and borneolum syntheticum(200mg/kg) increased significantly (P<0.05,P<0.01).;Compared with the control group, survival time of the mouse which were established by sodium nitrite, decollation, intravenous injection of magnesium chloride and the occlusion of bilateral common carotid arteries with vague nerves in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) increased significantly (P<0.05,P<0.01).②Compared with the model group, increases of rectal temperature were inhibited significantly in groups of L-borneolum (200mg/kg) after reperfusion4h(P<0.05). Compared with the model group, increases of rectal temperature were inhibited significantly in groups of L-borneolum (200,133.33mg/kg) and borneolum syntheticum (200mg/kg) after reperfusion22h (P <0.01, P<0.05); Compared with the model group, rates of cerebral edema in groups of L-borneolum(200,133.33mg/kg) and borneolum syntheticum(200mg/kg,133.33mg/kg) were reduced significantly (P<0.05,P<0.01).Mechanism of neuroprotective effect and influence of blood-brain barrier:①After oral administration of L-borneolum, borneol transmittance was0.174%at5min, reached the maxima of0.225%at15min, and then reduced gradually. After oral administration of borneolum syntheticum, borneol transmittance was0.112%at5min, reached the maxima of0.190%at20min, and then reduced rapidly.②Compared with the model group, content of malondialdehyde(MDA) both in serum and ischemia side brain tissue in groups of L-borneolum(200mg/kg) and bomeolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01),and superoxide dismutase(SOD) activity in serum and ischemia side brain tissue increased extremely significantly (P<0.01).③Compared with the model group, the content of nitrogen monoxidum(NO) and the activity of nitricoxide synthase(tNOs and iNOs) both in serum and ischemia side brain tissue in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01).④Compared with the model group, content of calcium ion(Ca2+) and activities of T-ATPase,Na+-K+-ATPase and Ca2+-Mg2+-ATPase in ischemia side brain tissue in groups of L-borneolum(200mg/kg) and borneolum syntheticum(200mg/kg) decreased significantly (P<0.05,P<0.01).⑤Compared with the model group, P-GP protein expression levels and MDR1mRNA gene expression level in ischemia side brain tissue in groups of L-borneolum and borneolum syntheticum (200mg/kg) decreased extremely significantly (P<0.01).⑥Structure of blood-brain barrier in modle group was damaged compared with sham-operated groups, which exhibited tight junction between endothelial cells being opened, swelling of mitochondria and rough endoplasmic reticulum expansion.Damage of structure of blood-brain barrier in groups of L-borneolum and borneolum syntheticum was improved to different extent.⑦Compared with the model group, OD values in Hypoxia-ischemia PC12in groups of L-borneolum (1.6、0.8、0.4μg/100μL) and borneolum syntheticum (1.6%0.8%0.4μg/100μL) increased extremely significantly (P<0.01).content of calcium ion (Ca2+) in Hypoxia-ischemia PC12in groups of L-borneolum(1.6、0.8、0.4μg/100μL) and borneolum syntheticum(200mg/kg)(1.6、0.8、0.4μg/100μL) decreased extremely significantly (P<0.01).Compared with the groups of borneolum syntheticum in same dosage, content of calcium ion (Ca2+) in groups of L-borneolum decreased extremely significantly (P<0.01).Conclusion:1. Neuroprotective effect:L-borneolum and borneolum syntheticum have protective effect obviously on cerebral ischemia and anoxia. It may be the pharmacodynamics basis for explaining borneol can induce resuscitationthe and restore consciousness2. Mechanism:①L-borneolum and borneolum syntheticum can distribute in the brain tissue quickly and stabilize BBB structure and reduce the damage of tight junction.②They can affect lipid peroxidation. It manifested as increasing the activity of SOD and decreasing the content of MDA in serum and tissue.③They can affect NO pathway. It manifested as decreasing the content of NO and decreasing activities of NOs (tNOs and iNOs) in serum and tissue.④They can affect Ca2+pathway. It manifested as decreasing the content of Ca2+in cell and tissue. Also they can decreasing the activities of T-ATPase,Na+-K+-ATPase and Ca2+-Mg2+-ATPase.⑤They can affect P-GP pathway. It manifested as decreasing MDR1gene expression levels and P-GP protein expression level. There are multiple links and multiple paths in mechanisms of neuroprotective effectThrough comprehensive analysis, it can be seen that neuroprotective effect of L-borneolum and borneolum syntheticum may be most closely with the P-GP pathway, lipid oxidation reaction and NO pathway. It is associated with Ca2+pathway secondly. It may be the main biological mechanism of borneol that show the property of "pungent-float-heart" and the effect of inducing resuscitation and restoring consciousness3. Comparison between L-borneolum and borneolum syntheticum:Based on the statistical results of the intensity integral rules, L-borneolum showed a superior trend than borneolum syntheticum in neuroprotective effect and mechanism of influence of blood-brain barrier. Tip:to select L-borneolum properly when cerebrovascular disease.