Protection Of Piceid On Ischeimic Reperfusion Injury In Rats

OBJECTIVES The model of acute reperfusion injury after cerebral ischemia in rats was established by unilateral middle cerebral artery(MCA) occlusion and bilateral carotid arteries (CCA) occlusion, to investigate the effects and the protective mechanisms of Piceid on cerebral ischemia-reperfusion (I/R) injury in rats.METHODS The rats were divided into 6 groups: model group, sham-operate group, pentoxifylline (PTX) group and 3 Piceid groups. 1h after the ischemia occurred, the saline, Piceid (7.5,15,30mg/kg) and PTX (16.5mg/kg) were administered via sublingual vena to the rats respectively, and the ischemic condition lasted for an hour with subsequent reperfusion of 24 hours.Then, cerebral tissues were taken for measurement of lactic acid (LD), malondialdehyde (MDA) content, monoamine oxidase (MAO) activity, the level of superoxide dismutase (SOD) by the special devised experiment kits respectively. The H₂O content was measured by wet-dry method. The level of endothelin-1 (ET-1) and the expression of endothelial NOS (eNOS) were estimated by radio-immunity method and immunohistochemistry technique. At the same time, some of the cerebral tissues were stained with hematoxylin-eosin (HE) and 2, 3, 5-triphenyltetrazolium chloride (TTC) to observe the change of ultrastructural and the infarct sizes of the cerebraltissues.6 New Zealand rabbits were used, blood was obtained by cardiac puncture, platelet rich plasma (PRP) was prepared and was divided into 7 groups: control-group, NS-controlgroupand5Piceid(0.01> 0^ 2.68> 10.72 ^ lOQamol/L) groups. Except the control one, all groups were treated with saline of Piceid to inhibit the platelet aggregation.The cerebral blood flow (CBF), cerebrovascular resistance (CVR) and mean blood pressure (MBP) was employed to investigate the effects of Piceid in anaesthetic dogs, ligated left carotid external artery. Electro magnetic flowmeter was used to measure the CBF; pressure transmitter was used to investigate the change of MBP.In the experiments in vitro, IL-16 was successfully used to produce the intercellular adhesion molecule-1 (ICAM-1) damage model of hypoxia. And then, the effect of Piceid on inhibit ICAM-1 from damage rat cerebral micrangium endothelial cell (RCMEC) was determined by the ELISA method.RESULTS The experiments in vivo showed that CBCA and MCAO methods could successfully produce I/R model in adult rats, which not only induced secondary edema in the local injured cerebral, but also brought about significant changes in morphology of injured cerebral and statistically meaningful disturbances of energetic metabolic system, production of free oxygen radical, increase of peroxidation. Therefore, the present I/R models could be used for the study of pharmacodynamics of Piceid on I/R.Piceid obviously inhibited the secondary cerebral edema with the most remarkable suppressing rate. Meantime, the ultrastructural findings suggested that Piceid caused profound cerebral damage, which could be protected of improved by injection of Piceid. All rats in sham group showed normal ultrastructure with intact axons, neurons such as nucleus. In model group, similar ultrastructural morphological abnormities were seen in all rats at 24h after I/R, including cellular edema, empty appearance of the axons, such as edematous, which was not seen in Sham group. Compared with sham group and 3 Piceid groups, cerebral water content was muchhigher in model group(P<0.01). Piceid-treated, like or even better than PTX-treated, soundly maintained the ultrastructure of the injured cerebral in a relatively good appearance.Piceid significantly suppressed LD production and MAO activity, and at the same time, Piceid obviously prevented reduction of SOD activity, reduced MDA production and inhibited ET-1 content in the injured cerebral tissue in comparison with the I/R model at the dose of 7.5, 15 and 30mg/kg, with the most remarkable effects appearing at 24h after I/R.Compared with sham group and 3 Piceid groups, the infarct sizes of the cerebral were much larger in model group, expression of eNOS was not observed, but in another groups, eNOS immumoreactivity in neurons of ischemic territory was up regulated. And also, Piceid could prevent platelets from inhibit the releasing reactions(/><0.01). There is a relationship between dose and efficacy of Piceid.The femoral artery was cannulated for blood pressure measurement. Record the values of CVR, MBP and CBF for each location before occlusion (baseline), and 30minutes after induction of ischemia (occlusion). Occlusion in dogs resulted in significantly less of decrease of CVR and MBP in the dogs. And at the time, Piceid obviously prevented reduction of CBF.Similarly, the experiments in vitro demonstrated that IL-16-induced, The expression of ICAM-1 was markedly increased. The expression of ICAM-1 was confirmed in REMEC by ELISA, which showed ICAM-1 expression to be increased. Piceid could directly and effectively protect the RCMEC from these damages through inhibiting the expression of ICAM-1.CONCLUSIONS Piceid effectively repressed tissue edema, removed oxygen free radical; blocked lipid peroxidation and improved energetic metabolic system to raise the expression of eNOS so as to further ameliorate the light microenvironment, which were involved in the protective influences of Piceid on the cerebral structure, cell morphology, and functional repair and even neuron regeneration after cerebral trauma. The effects of Piceid were the same as or greater than those of PTX. All the resultssuggested that Piceid has potent therapeutic effects in I/R via an oxygen free radical-Ca2+-NO pathway.