Protection Of Histamine H3Receotor Antagonists Against Cerebral Ischemic Injury And The Involved Mechanisms

Ischemic stroke is characterized by the sudden loss of blood circulation to an area of the brain, resulting in a corresponding loss of neurologic function, and it is one of the three leading causes of death worldwide. Few therapies are effective other than tissue plasminogen activator. Despite the fact that many neuroprotective agents, have been developed in attempts to cure ischemic stroke, the results are unimpressive for a variety of reasons. Therefore, more effort is needed to elucidate the ischemic mechanisms and further explore new neuroprotective drugs.Histamine is an endogenous neurotransmitter in brains. Histaminergic neurons locate in the tuberomammillary nucleus of the posterior hypothalamus, and their fibers are widely distributed throughout the brain. To date, four subtypes of receptors have been identified:H1, H2, H3and H4, of which H1-H3receptors are found in brains. Histamine is known to regulate numerous neurological functions, such as learning and memory, sleep disorders, epilepsy. Up to now, reports indicated that histamine in the brain is closely related to ischemia. The histamine concentration in the brain increases significantly during ischemia period. More research indicated that a-FMH, a histamine synthesis blocker, aggravates the later period of ischemic injury. Histidine, from which histamine is synthetized, alleviated the transient ischemia induced infarct volume. Our earlier research also indicated that exogenous administration of histamine ameliorates the neuronal excitotoxicity induced by NMDA. Taken together, it indicates that histamine and its related drugs are protective against ischemia. However, owing to the permeability of exogenous histamine is not high enough to pass through the blood-brain barrier, the clinical application of histamine is obstructive.The histamine H3receptor (H3R) was first identified in1983as a presynaptic autoreceptor that inhibits histamine release from histaminergic neurons via negative feedback. Increasing attention has been paid to the role of H3R in brain disorders such as narcolepsy, Alzheimer’s disease, Parkinson’s disease, schizophrenia and attention deficit hyperactivity disorder. Our previous report indicated that H3R antagonist is protective against100μM NMDA-induced neurotoxicity However, Mariottini et al. showed that H3R agonist protects neurons against100μM NMDA-induced neurotoxicity. Therefore, the effect of H3R in the excitotoxicity is still unclear. Therefore, the present study was designed to investigate the role of H3R in I/R injury both in vivo and in vitro, and to explore the underlying mechanisms, providing a new target for clinical ischemic therapy.Protection of Histamine H3Receptor Antagonists against Cerebral Ischemic Injury and the Involved MechanismsAt present, the role of histamine H3R in cerebral ischemia is not fully elucidated. It was shown that OGD/R significantly increased H3R expression, and H3R transfection increased vulnerability of HEK293cells to serum deprivation. H3R antagonist thioperamide protected against OGD/R injury. The protection by thioperamide was significantly reversed by the selective H3R agonist immepip, suggesting that H3R may exaggerate ischemic injury. In vivo study indicated that three types of H3R antagonists (thioperamide, clobenpropit and A331440) protected against I/R-induced focal brain ischemic infarct and neurological deficient. H3R deletion ameliorated ischemic brain injury and alleviated apoptosis. More importantly, the protections of thioperamide was absent in H3R-/-mice. Further study indicated that the protective mechanism by thioperamide was non-histaminergic. Here we found that neither the HIR antagonist pyrilamine nor the H2R antagonist cimetidine compromised the protection by thioperamide. Both a-FMH, a selective HDC inhibitor that decreases histamine synthesis, and HDC knockout failed to reverse the protection by thioperamide. Therefore, the results suggested that histamine H3R antagonism was protective against ischemic injury and the involved mechanism was non-histaminergic.The present work found that LiCl (GSK-3β inhibitor) completely reversed the protection of thioperamide, indicating that activation of GSK-3β was necessary for the protection by H3R antagonism. Interestingly, the activation of GSK-3β by H3R inhibition subsequently inhibited mTOR activity, as revealed by decreased phosphorylation of P70S6K. We also found that both H3R inhibition and knockout induced an enhancement of autophagy in I/R. In addition, inhibition of autophagy by3-MA, Atg7gene knock down and Atg5gene knock out all reversed the protection by thioperamide against I/R injury. The interaction of H3R with CLIC4increased in OGD/R, and H3R inhibition by thioperamide disturbed the H3R-CLIC4interaction which was reversed by the H3R agonist immepip. The peptide Tat-H3RCT414-436, which competitively blocked the CLIC4-H3R interaction by binding with CLIC4conferred protection to an extent similar to H3R blockade in OGD/R on neurons. In addition, Tat-H3RCT414436inhibited Akt, activated GSK3β,inhibited mTOR/P70S6K signaling and ultimately upregulated the autophagy level in the OGD/R period. Furthermore, siRNA for CLIC4also exhibited protection through mTOR inhibiting induced autophagy against OGD/R injury. Therefore, these data strongly suggested that H3R blockade disturbs CLIC4-H3R binding and then activates autophagy to protect cells against I/R injury.