The Screening Method Of Cerebral Nerves Protective Composition From TCM Based On "Calcium Signal Pathway-blood Brain Barrier"

Objective:Choose the traditional Chinese medicine(TCM) effectively used to treat cerebral ischemia,and study the neuroprotective effect, regulation of calcium signal pathway and blood-brain barrier (BBB) permeability of the ingredients from TCM, reveal the feasibility to establish a screening method of cerebral nerves protective composition based on "calcium signal pathway-blood brain barrier"Methods:1.To cultivate rat cerebral cortical neurons cell in vitro and determine the survival rate by MTT, the neuroprotection active ingredients were screened.2.To establish the model of rat cortical neurons cell injury caused by glutamate and KCl, the change of fluorescence intensity detected by Laser Scanning Confocal Microscopy(LSCM), active ingredients with regulation of calcium signal pathway were screened.3. To co-culture brain microvascular endothelial cells (BMVEC) and astrocytes cells (AC) in vitro, blood-brain barrier model (BBBM) was established with the active components analysised by HPLC.4.To establish the model of middle cerebral artery occlusion(MCAO) by suture embolism in rats, Through the observation of ethology and mensuration of cerebral infarction proportion, SOD, MDA, GSH-Px and NOS in rats'blood serum, evaluated the effect of resisting the ischemical reperfusion injury of active ingredients.Results:1. The samples, such as erigeron injection, could promote the survival of cortex nerve cell in vitro, so did scutellarin, caffeic acid,3,5-di-O-caffeoyl quinic acid, aetherolea from Chuanxiong,chuanxiongzine,ferulaic acid, aetherolea from Shichangpu and asaricin.2. The samples, such as erigeron injection,, could resist cortical neurons cell injury caused by Glutamate and KCl, so did 3,5-di-O-caffeoyl quinic acid, Chuanxiong aetherolea,chuanxiongzine,ferulaic acid and shichangpu aetherolea.Both scutellarin and asaricin could only resist cortical neurons cell injury caused by glutamate.3. The blood-brain barrier model in vitro was successful. erigeron injection, 3,5-di-O-caffeoyl quinic acid, aetherolea from Chuanxiong, chuanxiongzine, ferulaic acid, aetherolea from Shichangpu and asaricin could penetrate BBB, but scutellarin didn't,the products of the structural modification of which, scutellarin ethyl ester, meanwhile, has neuroprotective effect and regulation of calcium signal pathway.4.3,5-di-O-caffeoyl quinic acid and aetherolea from Chuanxiong Chuanxiong could enhance the activities of SOD, GSH-Px,NOS and decrease the content of MDA in rats to resist the damages caused by ischemical reperfusion injury5. Aggregate analysis indicated that the neuroprotective effect of erigeron injection is related to regulation of calcium signal pathway,so did 3,5-di-O-caffeoyl quinic acid, aetherolea from Chuanxiong, chuanxiongzine, aetherolea from Shichangpu and asaricin. Furthermore, the ability to penetrate BBB of active ingredients is an important factor to resist cerebral ischemia.Conclusions:All the results above showed can elementarily attest to that establishing a screening method of cerebral nerves protective composition from TCM based on "calcium signal pathway-blood brain barrier"is feasible, and offered a new research idea for development of original new drug to treat cerebral ischemia.