The Role Of Carnosine In Brain Ischemic And Excitotoxic Injuries

Histamine is one of the most widely distributed neurotransmitter or neuromodulator in the central nervous system and controls a variety of neurobiological functions and behavioral responses including sleep-wake cycle,nociception,water consumption,food,motor activity and learning and memory.Histamine is involved in ischemic pathogenesis,and has a protective effect on the delayed neuronal death mediated by N-methyl-D-aspartate(NMDA) receptors.Postischemic loading with histidine,a precursor of histamine,decreases the amount of neuronal damage in the rat striatum.Histamine depletion withα-fluoromethylhistidine(α-FMH),an irreversible inhibitor of histidine decarboxylase(HDC),significantly increases the number of necrotic pyramidal cells in hippocampal CA1 region in rats subjected to cerebral ischemia.Histamine also protects against NMDA-induced necrosis in cultured cortical neurons.However,histamine itself cannot cross the blood-brain barrier and it is involved in brain inflammation.Therefore,specific histamine related compounds which have histamine-like effect but without the side-effect may have the potential clinical uses in preventing and treating cerebral ischemia.Carnosine(β-alanyl-L-histidine),first identified by Gulewitsch and Amiradzibi from Liebig's meat extract nearly a century ago and it occurs in innervated tissues including the animal and human brain and can easily enter the central nervous system from the periphery.There are many theories about its biological functions,such as anti-inflammatory agent,free radical scavenger,and protein glycosylation inhibitor,and may serve as a neurotransmitter in the olfactory bulb.However,so far,the physiological functions of carnosine in the brain remain obscure,and a unifying concept has not yet emerged.On the other hand,previous studies have suggested that carnosine can be metabolically transformed into histamine by carnosinase and histidine decarboxylase enzyme that exist in the brain and exerts its protective action through histamine H₁ receptor.Given the relationship between carnosine and histamine,carnosine is proposed as a new histaminergic drug that can replace histamine and avoid inflammation in clinical therapeutics.Therefore,our present studies concentrate on the effects of carnosine and the carnosine-histidine-histamine metabolic pathway on NMDA induced neurotoxicity in differentiated PC12 cells and focal cerebral ischemia in histidine decarboxylase knock-out(HDC-KO) mice.1 Neuroprotective effect of carnosine on NMDA-induced neurotoxicity in differentiated PC12 cellsPretreatment with carnosine increased the viability and decreased the number of apoptotic and necrotic cells in NMDA induced injury in differentiated PC12 cells measured by MTT and Hoechst 33342 and propidium iodide(PI) double staining assays. Carnosine also can reduce the extracellular glutamate level and increase HDC activity and the intracellular and extracellular contents of carnosine,histidine and histamine detected by high-performance liquid chromatography(HPLC).The protection by carnosine was reversed byα-fluoromethylhistidine,a selective and irreversible inhibitor of histidine decarboxylase(HDC).Pyrilamine and thioperamide,selective central histamine H₁ and H₃ antagonists also significantly reversed the protection of carnosine. Further,the inhibition of glutamate release by carnosine was reversed by thioperamide. Therefore,the protective mechanism of carnosine may not only involve the carnosine-histidine-histamine pathway,but also H1/H3 receptors and the effective inhibition of glutamate release.This study indicates that carnosine may be an endogenous protective factor and calls for its further study as a new antiexcitotoxic agent.2 Role of carnosine in permanent focal cerebral ischemia in HDC-KO mice Since camosine can protect against excitotoxicity in differentiated PC12 cells through a histaminergic pathway,in this study,we used HDC-KO and the corresponding wild type(WT) mice to elucidate whether histaminergic or other mechanisms are involved in.the effects of carnosine on permanent middle cerebral artery occlusion(pMCAO).The results showed that carnosine significantly improved neurological function and decreased infarct size both in HDC-KO and its WT mice,to the same extent.Carnosine decreased the glutamate levels,and preserved the expression of glutamate transporter-1(GLT-1) but not glutamate/aspartate transporter(GLAST) in astrocytes exposed to ischemia in vivo and in vitro.It suppressed the dissipation of mitochondrial membrane potentil(△Ψm) and generation of mitochondrial ROS induced by OGD in astrocytes.Furthermore,carnosine also decreased the mitochondrial ROS level and reversed the decrease of GLT-1 induced by rotenone,an inhibitor of mitochondrial complex I.Our study suggests that carnosine is neuroprotective in pMCAO in HDC-KO and WT mice and its mechanism of action may not be mediated by the histaminergic pathway,but by the effective regulation the expression of GLT-1 in astrocytes due to improving the moitochondrial function.In conclusion,our study demonstrates that there exists carnosine-histidine-histamine pathway in differentiated PC12 cells and it plays a protective roles in NMDA induced excitotoxicity.Carnosine exerts a neuroprotective effect on pMCAO induced injuries in HDC-KO and its WT mice and its action may not involve the histaminergic pathway,but through reducing glutamate excitotoxicity by reversing the GLT-1 decrease in astrocytes.