The Protective Effect Of Propolis Against Oxygen Glucose Deprivation/Reoxygenation In N2a Cells And Its Mechanisms Of Action

Bee Propolis was collected from the tree bud, which mixed pollen, secretion of bee tongue gland and wax gland. Propolis is rich in compounds, with a wide range of biological activities, Propolis had some antioxidant, anticancer and immunoregulatory activities,which could taken as a kind of good natural antioxidant and the antitumor activity material source. When the blood supply is restored, the aggravated damage of brain tissue is called the cerebral ischemia reperfusion injury. That presents severer nervous defects than earlier. The neurons survival rate was measured by colorimetric assay(CCK-8 kit). Leagene Giemsa staining was used to observe the morphology of apoptotic neurons. To investigate the effects of propolis against the cerebral ischemia-reperfusion injury induced by oxygen-glucose deprivation /reoxygenation(OGD/R) in mouse neuroblastoma N2 a cells.In this paper, the research topic from the National Natural Science Foundation project(31272509). The ethyl acetate extracts of 70% ethanol extract propolis was separated and purified systematically. Column chromatography was used to seperating ethyl acetate extracts. Then we got Fr.1, Fr.2, Fr.3, Fr.4, and Fr.3 showed the highest protective effect. Then HPLC were usd to seperating Fr.3, we got Caffeic acid phenethyl ester, Caffeic acid cinnamyl ester and Caffeic acid benzyl ester. Caffeic acid phenethyl ester, Caffeic acid cinnamyl ester and Caffeic acid benzyl ester were used to treated with N2 a cells injuryed by OGD/R. The results showed that Caffeic acid phenethyl ester and Caffeic acid cinnamyl ester showed significantly protective effect. Howere Caffeic acid benzyl ester showed no effect.Part of the mechanism of cerebral ischemia-reperfusion injury is cell oxidative damage caused by active oxygen free radicals and NO. We selected Caffeic acid cinnamyl ester to study on the mechanism of propolis effect. The contents of Catalase(CAT), Superoxide Dismutase(SOD1), Glutathione peroxidase(GPX1), NO and NOS in liver cells were determined by general methods. The results showed that after treated by Caffeic acid cinnamyl ester NO and NOS content in cells decreased significantly, the CAT, SOD1 and GPX1 activity were enhanced in cells, thereby reducing ischemia reperfusion injury induced oxidative damage to cells.HPLC was used to analyze the compounds of six propolis samples from Fusong, Benxi, Jianchang, Beipiao and wandasan. The six propolis samples were used to treated with N2 a cells injuryed by OGD/R to investigate the relationship between cerebral ischemia-reperfusion injury and the composition of propolis. This paper used the HPLC analysis method to get the pinocembrin and p-coumaric acid benzyl ester content. The results showed that dunhua propolis and Beipiao bee propolis showed the higher protective effect, followed by wandasan propolis, Benxi propolis, Fusong propolis. The Jianchang propolis showed the lower protective effect. A positive correlation between pinocembrin, p-coumaric acid benzyl ester content and protective effects was observed for all the samples. p-coumaric acid benzyl ester was used to treated with N2 a cells injuryed by OGD/R. The results showed that p-coumaric acid benzyl ester can protect the cerebral ischemia reperfusion injury remarkably.