Glaucocalyxin A Reduces Neonatal Hypoxic-Ischemic Brain Injury In Rats

Neonatal hypoxic–ischemic brain damage (HIBD) means an injury induced byperinatal asphyxia.It continues to be a major contributor of mortality and life-long neurologicalimpairments in infants and children. Neonatal hypoxic ischemic encephalopathy (HIE)affects1–3per1000term births in the developed world, which has higher mortality andis the leading cause of neurological sequelae. There are1.6to2.2million infantssuffering asphyxia each year in China. About400,000suffer severe brain damage,epilepsy, mental retardation, and different degrees of disability. Neonatal braindevelopment is very critical. Patients with HIE, need positive, timely andcomprehensive treatment and are adhered to the convalescent treatment and earlyintervention. Currently, cerebral metabolism activator, salvia, naloxone and all kinds ofnutritional factors areused to cure HIE. Some studies indicated that the extract from roots of C. sinicawas of significant tolerance to ischemia and hypoxia. Our article is dedicated to theprotective effect of the study of Chinese herbs on neonatal rats with HIBD.Many phytochemical studies on this plant have revealed that the ethanol extract ofRabdosia japonica (Burm. F.) Hara var. glaucocalyx (Maxim.) Hara can improvemicrocirculation function and has platelet aggregation function, then to reducemyocardial ischemia-reperfusion injury and protect the heart. It exhibits variousbiological activities such as inhibition platelet aggregation and apoptosis. In view of theefficacy of glaucocalyxin A (GLA), we propose that GLA protect against HIBD innewborn rats. So we establish the HIBD animal models in neonatal rats to simulate HIE,observing the protective effect of glaucocalyxin A and research its mechanism. I hopeour study can provide experimental evidence about the treatment of hypoxic-ischemicencephalopathy of GLA in the future. Methods:1．Extract and Isolate glaucocalyxin A.Glaucocalyxin A (GLA) is a biologically active ent-kauranoid diterpenoid isolatedfrom Rabdosia japonicavar.2. Establish the HIBD animal models.HIBD was induced in postnatal day7(P7) rat pups by unilateral carotid ligation and2.5hours of hypoxia. One hundred and sixty-three seventy-seven P7rat pups wereused in this study and randomly divided into the following groups: sham-operated (n=50), HI group (n=50), HI groupstreated with GLA (n=55).3. Brain atrophy was assessed at4weeks after HI. All animals were anesthetized anddecapitated, and the brains were removed. Brain tissue loss was expressed as themass ratio of the ipsilateral hemisphere compared with the contralateral hemisphere.4. At4weeks after HI, a battery of behavioral test–Morris Water Maze Test wasperformed. Morris Water Maze Test has been used to examine exploratory learningand working memory by hippocampus dysfunction.5. As studied previously,2,3,5-triphenyltetrazolium chloride monohydrate (TTC;Sigma) staining was performed to determine the infarct volume. At48hours afterHI, brains were removed and sliced into1-mm sections. Slices were incubated for ina0.1%solution of TTC at37C for30min in the dark and then fixed in10%buffered formaldehyde solution at room temperature. The infarct area in each brainslice was determined with a computerized image analysis system (AlphaEase ImageAnalysis Software V3.1.2).6. The animals were killed at24hours after HI, western blotting was performed.Underlying mechanism study showed that GLA treatment significantly preservedAkt phosphorylation, decreased ERK phosphorylation and cleaved caspase-3levels.Results:1. By observing the brain development and neurological behavior results after HIsurgery, it is proved tnat HIBD animal model is successful.2. At4weeks after HI, extensive atrophy of ipsilateral brain tissue was observed. Therewas no significant atrophy in the sham-operated group. However, severe atrophywas observed in the HI group（n=20，p<0.001）. So we know that hypoxia and ischemia affect the brain development.3. Morris Water Maze Test results: the HI group showed a significantly longer escapelatency and a lower frequency of original platform crossing compared with thesham-operated group（n=15，p<0.001），. We have the conclusion that hypoxia andischemia can lead to a long-term deficit of behavioral functions in neonatal rats.4. Compared with the vehicle group, the infarct volume in the GLA500μg/kg groupand the GLA1mg/kg groupwas significant smalle（rn=10，p<0.01）, while the GLA50μg/kg treatment had no protection on infarct volum（en=10，p<0.01）. In this study,we concluded that GLA500μg/kg and GLA500μg/kg can reduce the infarct volumeafter HI.5. The signaling pathway underlies GLA-induced neuroprotection in the neonatal HImodel:HI injury caused significant increase of cleaved caspase-3compared with thecontrol group and GLA treatment decreased the level of cleaved caspase-3（n=10，p<0.01）.The phosphorylation of Akt significantly increased after GLA treatment comparedwith vehicle group（n=10，p<0.01）. And The phosphorylation of ERK levelsdrastically decreased compared with vehicle group (n=10，p<0.01）.Conclusion:We concluded that GLA provides protection against hypoxic–ischemic brain injuryby inhibiting apoptosis in neonatal rats. The signal pathways include PI3K/Akt pathwayand MAPK signaling pathway.