Protective Effect And Mechanism Of Salidroside Analogue Against Cerebral Ischemia Injury

Objective:Studies have shown that salidroside,a traditional Tibetan medicine,is difficult to cross the blood brain barrier and penetrate into the brain due to its large molecular polarity and small fat-soluble character.Therefore,we have synthesized the salidroside analogue(SaIA-4g)through structure modification.The present study was aimed to analyze the distribution of SalA-4g in various organs of rats and its pharmacokinetic model,and explore the protective effects and mechanism of SalA-4g against oxygen and glucose deprivation(OGD)-induced injury in primary hippocampal neurons and middle cerebral artery occlusion(MCAO)-induced injury in rats.The research will provide an important experimental basis for the development of SalA-4g as a potential neuroprotective agent used in clinical treatment of ischemic cerebrovascular disease.Methods:1.Rat plasma and organs were collected at different times,and the content of SalA-4g was detected in plasma and homogenate carried out by high-performance liquid chromatography(HPLC)after tail vein injection of SalA-4g.The plasma concentration-time curve and the pharmacokinetic parameters fitted by DAS 2.1.1 software were analyzed to explore the dynamic regulation of SalA-4g metabolic process and the distribution of SalA-4g in various organs of rats.2.The MCAO model in rats was established by a monofilament nylon suture.The modified neurological severity score(mNSS),the cerebral infarction volume,and the TUNEL assay were applied to evaluate the protective effects of SalA-4g on ischemic injury in MCAO rats;18F-FDG PET/CT was applied to evaluate the effects of SalA-4g on cerebral glucose metabolism in MCAO rats;Western blot analysis was performed to examine the effects of SalA-4g on the protein expression levels of GLUT3 in MCAO rats.3.Embryonic 17-18 d SD rats hippocampal neurons were cultured under normal conditions,and then an OGD injury model was established in primary hippocampal neurons.MTT assay and Hoechst staining were used to identify the protective effects of SalA-4g against OGD-induced injury in primary hippocampal neurons;2-NBDG and immunofluorescence staining were used to examine the effect of SalA-4g on the glucose uptake and the expression levels of GLUT3 in OGD injured hippocampal neurons;pathway analysis was performed to examine whether the PKA/CREB/GLUT3 signaling was involved in the enhancement of glucose uptake by SalA-4g and the neuroprotection of SalA-4g in OGD-injured hippocampal neurons.Results:1.The linear range of SalA-4g was 0.1～200 μg/ml(r2=0.9996),The lowest limit of quantification of SalA-4g was 0.1 μg/ml.The pharmacokinetic parameters of SalA-4g were as follows:the half life time and apparent volume of distribution of SalA-4g were 0.657 h and 7.376 L/kg,respectively.In vivo,SalA-4g was cleared mainly through hepatic metabolism and renal excretion.The distribution of SalA-4g was relatively small in the lung,heart,spleen and brain,but the removal in these organs was slow.2.SalA-4g had a significant protective effect on OGD-injured hippocampal neurons.SalA-4g could inhibit the OGD-induced cell viability decrease and cell apoptosis in a dose-effect manner,with the most obvious changes at the concentration of 200 μM.Importantly,200 μM SalA-4g was significantly better than 200 μM salidroside for inhibition of cell viability loss and apoptosis.3.SalA-4g had a significant protective effect on ischemia injury in MCAO rats.SalA-4g could decrease the mNSS scores and the infract volume,attenuate cell apoptosis in the cortex and the hippocampal CA1 region.The effects of SalA-4g exhibited a dose-dependent pattern,and the reducing effects of 50 mg/kg SalA-4g were significant.Moreover,50 mg/kg SalA-4g was significantly better than 50 mg/kg salidroside in terms of the reducing effects.4.SalA-4g could significantly improve the glucose uptake as well as the expression levels of GLUT3 in OGD-injured hippocampal neurons in a dose-effect manner,with the most obvious changes at the concentration of 200 μM.However,there was little effect of salidroside on GLUT3 expression.5.SalA-4g could significantly increase the glucose metabolism,improve the metabolic recovery in the ischemic hemisphere,and improve the expression levels of GLUT3 in the cortex and hippocampus in MCAO rats.The effects of SalA-4g exhibited a dose-dependent pattern,with the most obvious changes at the dose of 50 mg/kg.However,there was little effect of salidroside on GLUT3 expression in the ischemic brain.6.SalA-4g could significantly increase the expression levels of p-CREB and PKA RⅡ.The induction of neuronal GLUT3、the enhancement of glucose uptake and the neuroprotection induced by SalA-4g were alleviated by H-89(PKA inhibitor)in OGD injured hippocampal neurons.Conclusions:1.Salidroside analogue(SalA-4g)had increased hydrophobicity and lipophilicity through structure modification.SalA-4g was more likely to cross the blood brain barrier and penetrate into the brain to exert its neuroprotective effects,as compared to salidroside.2.SalA-4g had a significant protective effect on OGD-injured hippocampal neurons and ischemia injury in MCAO rats.3.The neuroprotective effects of SalA-4g were associated with enhanced glucose uptake in ischemic brain.SalA-4g exerted neuroprotective roles possibly through the PKA/CREB/GLUT3-mediated glucose uptake.