Active Ingredient Of Rhizoma Acori Graminei Against Neuronal Apoptosis Through CaMKⅡPathway

ObjectiveCa2+/calmodulin dependent protein kinases is the receptor proteinases of Ca2+, activated CaM is aroused path way by the Ca2+/CaM dependence proteinase. CaMKⅡmainly be expressed to the nervous tissue. It has an indivisibility relation with neuronal apoptosis.The Chinese herbal medicine Rhizoma acori graminei is the root-stock of southern a Acorus tatarinowii Schott, has the efficacy of nourishing the brain. There some researches detected that, the composition of Rhizoma acori graminei to nourishing the brain and protect neuronal apoptosis is mainly of theβ-asarone and the Eugenol. Theβ-asarone and the Eugenol can reduce the hippocampus neuronal apoptosis. The member mechanism is not clear.Our previous studies show thatβ-asarone and the Eugenol can raise expression of CaMKⅡpath way.To further study, this experiment inquires if the active ingredient of Rhizoma acori graminei against neuronal apoptosis through CaMKⅡpath way or not.MethodsExperiment one, this Experiment have two parts. Firstly,β-asarone and the Eugenol affect neuronal apoptosis of original generation hippocampus nerve cell, which induced by A-β. Develop original generation hippocampus nerve cell of the hippocampus tissue of foetus rat. On the sixth day, use the A-βfunction carries on a cell model establishment at the cell, giveβ-asarone and the Eugenol make used for the cell after 24 hours. Then western-blot is applied to observe expression 3 days later. Lastly, Applied CaMKⅡinhibitor to stop CaMKⅡpath way to observe the effection of the the active ingredient of Rhizoma acori graminei against neuronal apoptosis through CaMKⅡpathway. Use the CaMKⅡinhibitor at the original generation hippocampus nerve cell, inquire into Active ingredient of Rhizoma acori graminei Against neuronal apoptosis through CaMKⅡpathway.Experiment two, EndoFectinTM-Plus reagent transiently transfected CaMKⅡexpression plasmid and the CaMKⅡgene silencing to the PC12 cells. Observe the the active ingredient of Rhizoma acori graminei against neuronal apoptosis through CaMKⅡpath way. Develop the PC12 cells at the same times. PC12 cells stub come from the adrenal gland chromaffin cell of big rat oncocyte, it grows quickly, stability and does't easily take place automatic conversion. It can build up 2 times body cells that can spread generation to fasten, have the characteristic of more typical sacral neuronal endocrine cell. It usually be used to a condition outside the body under research neuronal cell hurt the function mechanism and drug is to the ideal cell of the protection function of neuronal cell. To further study, in this study, EndoFectinTM-Plus reagent transiently transfected CaMKⅡexpression plasmid, extraction of PC12 transfected cell protein, using Western blotting observations ofβ-asarone on differentiation of CaMKⅡetc., and apoptosis pathway-related protein expression. The same transfection, the CaMKⅡgene silencing. using Western blotting observations ofβ-asarone on differentiation of CaMKⅡetc., and apoptosis pathway-related protein expression.Experiment three, the effection of Rhizoma acori graminei reduce the hippocampus neuronal apoptosis of cerebral ischemia skin layer neuronal cells through CaMKⅡpathway. Build up cerebral ischemia model of the rat.All animals were randomly divided into three groups:group, mode group,β-asarone treatment group and Eugenol treatment group. Ten days later carry on infusing to note to get material and take a tapetum to divide syrup, carry on a Western blotting examination.ResultsExperiment one The western-blotting illustrated that the expressing of CaMK2a, CREB1, p-CREB, BCL-2 in theβ-asarone treatment group and Eugenol treatment group were higher than that of the model group. The expressing of Caspase3 inβ-asarone treatment group and Eugenol treatment group were less than that of the model group. Western blotting showed that the expression ofCREB, P-CREB in the inhibitor group were significantly less than the non-inhibitor group.Experiment two CaMKⅡexpression plasmid was transfected, Western blotting showed that CaMK2a, CREB, P- CREB, BCL-2 expression were significantly higher than that of empty vector group.The expressing of theβ-asarone treatment group was higher than CaMKIIgroup. CaMKII gene silencing, Western blotting showed that the expression ofCREB, P- CREB were significantly less than the empty vector group.Experiment three The western-blotting illustrated that the expressing of CREB1, BCL-2 in theβ-asarone treatment group and Eugenol treatment group were higher than model group. The expressing of Bax, Caspase3 inβ-asarone treatment group and Eugenol treatment group were less than model group.ConclusionCaMKⅡpathway is important to the neuronal apoptosis.β-asarone and the Eugenol can protect the neuronal cell from the damage by adjust the expression of controling the gene of apoptosis. These results indicate that active ingredient of Rhizoma acori graminei against neuronal apoptosis through CaMKⅡpathway. It can raise the expressing of CaMKⅡ, CREB1, BCL-2 and decreased the expressing of Bax, Caspase3 to protect the neuronal cell from apoptosis.