Protective Effects Of Novel Twin Compounds Containing Tetramethylpyrazine And Carnitine Substructures In Experimental Ischemic Stroke

Background and ObjectiveAlthough studies have seen dramatic advances in the pathogenesis of stroke,the delivery of stroke therapies is still a great challenge.One of the major reasons is that current therapies specifically target a single pathogenic factor,therefore,it is very important to develop new therapeutic agent candidates with multiple therapeutic targets in the vicious cascade of stroke.In this study,on the basis of the potential neuroprotective effects of 2-hydroxymethyl-3,5,6-trimethylpyrazine(HTMP)and the drug delivery property of carnitine esters,we designed and synthesized a series of novel twin compounds based on structural combination principles.We screened the protective effects of synthesized compounds by the viability of PC 12 cells under OGD condition in vitro and in experimental animal ischemic stroke model,and further explored the potential mechanisms of their neuroprotective effects though the pathways of inflammatory responses,NADPH oxidase(NOX2)-mediated oxidative stress,the mitochondrial structure and function together with energy metabolism,which are proved to be closely associated with stroke.Methods and Results1 Primary screening of twin compounds containing HTMP and carnitine substructures in OGD-induced cell deathWe first evaluated the protective effects of HTMP-carnitine ester twin compounds in PC 12 cells under OGD condition using CCK8 assay,the concentration for 50%of maximal effect(EC50)was calculated by plotting the dependence between drug concentration and percentage of protection on OGD-induced cell death.Among the ten candidate compounds,the EC50 of four of the candidates(LR134,137,140,143)was less than 100nM and the compound LR134(with the minimum EC50)was selected for in vivo dose and time screening study.2 Both LR134 and LR143 protected against cerebral ischemia reperfusion(I/R)injury in rats.Transient middle cerebral artery occlusion(MCAO)was induced in male Sprague-Dawley rats.We first evaluated the effects of different doses and time treatment of compound LR134 in rats subjected to MCAO.Stroke outcome was assessed at 24 hours after reperfusion using a 5-tiered neurological scoring system and 2,3,5-triphenyltetrazolium chloride(TTC)staining method.The results showed that compared with vehicle-treated I/R group,the neurobehavioral manifestations,infarct volume and edema index were significantly reduced in LR134 administrated group at 30 minutes before MCAO,together with the pharmacokinetic results that after administration,the peak time of compound LR134 in the rats’ brain is about 30 minutes.As for dose screening,2.5 and 5mg/kg LR134 treated groups show significant neuroprotection as compared with vehicle group.Therefore,we chose the minimal tested protective molar doses(2.5mg/kg of LR134,6.82μmol/kg)and 30 minutes before MCAO for further comparison of other candidate compounds.At the equal molar dose(6.82 μmol/kg),we found that besides compound LR134,pretreatment of compound LR143 also exhibited significantly protective effects on cerebral ischemia/reperfusion injury,while compounds LR137 and LR140 had no significant protection effects at the same dose level.However,the precursors of the compounds,L-carnitine and HTMP had no significant protective effects on cerebral I/R injury at the same dose.To further assess the neuroprotective effects of these two candidate compounds in ischemia-evoked neuronal injury,HE and Nissl staining was used to examine morphological features and the surviving cells of neurons in cerebral cortex,TUNEL staining was used to assess the apoptotic like neurons.We found that I/R induced severe neuron necrosis,a significantly reduction of Nissl substance indicating loss of surviving neurons and an increased number of TUNEL-positive apoptosis-like cells.However,administration of LR134 and LR143 significantly ameliorated neuronal injury.3 The mechanism of LR134 and LR143 protected against cerebral ischemia reperfusion(I/R)injury in rats.Chemokines and cytokines were measured with real time RT-PCR.Macrophages(CD68)and neutrophils(Ly6B)expression were observed by immunohistochemistry.Cerebral I/R markedly enhanced the production of pro-inflammatory mediators,which was significantly attenuated by LR134 and LR143.We further measured O2·-production by Electron Spin Resonance(ESR)and the expression of NOX2 by WB and real time RT-PCR,the result showed that the O2·-level was markedly increased in the brain after MCAO,which was in consistent with the up-expression of NOX2(gp91phox)in the brain.The results from co-immunofluorescence staining for NOX2 and a neuronal marker NeuN indicated the upregulation of NOX2 in neurons.Pretreatment of LR134 and LR143 markedly decreased O2·-production and neuronal NOX2 expression induced by I/R injury.The total ATP content in the brain tissue and cells was determined by using a luciferin-luciferase assay kit.We found that pretreatment of compounds LR134 and LR143 significantly improved energy metabolism in experimental ischemic stroke.Since the AMP-activated protein kinase(AMPK)is a critical energy sensor in all types of cells,we then measured the activation of AMPK presenting by the ratio of phosphorylation form of AMPK on Thy 172 to total AMPK.It was found that LR134 and LR143 remarkably reduced AMPK activity.Considering of the importance of mitochondrial dysfunction in the ischemic stroke,we then evaluated the effects of LR134 and LR143 on both structure and function of brain mitochondria.The ultra-structural analysis revealed the presence of abnormal-irregularly swelling mitochondria with shortening and disintegrating cristae,even vacuole in ischemic brains.Pretreatment with LR134 and LR143 improved structure mitochondria as evidenced by the presence of regularly-rounded with increased number of elongated cristae.Mitochondria and cytoplasm were fractionated using a mitochondria isolation,the release of cytochrome C from mitochondria into the cytoplasm was also observed to detect the mitochondrial function.Western blot results showed the elevated levels of cytochrome C in cytosol and concomitantly lower levels in mitochondria of ischemic brain tissues,indicating that this apoptogenic factor was released from mitochondria to cytoplasm.Moreover,LR134 and LR143 inhibited the release of cytochrome C into the cytoplasm.Similar results were also found in OGD/R injured PC12 cells.Conclusion and innovationConclusionIn summary,our study provides direct evidence showing the novel compounds containing HTMP and carnitine substructures had neuroprotection with multiple therapeutic targets,suggesting that modulation of these chemical structures may be an innovative therapeutic strategy for treating patients with stroke.InnovationTwin compounds containing HTMP and carnitine substructures used in this study are a series of novel compounds.It was the first time that the neoroprotective effects of synthesized compounds were screened by using both in vitro and in vivo cerebral ischemia/reperfusion injury models.It was the first time that the potential mechanisms of the neoroprotective effects of candidate compounds were explored.