| Background:Perinatal brain injury can occur because of a wide variety of events, including hypoxia-ischemia (HI), intrauterine infections, and cerebral hemorrhage. HI is the most common cause of perinatal brain injury. Moreover, Neonatal hypoxic-ischemic (HI) is a common cause of death and long-term neurological disability in newborns. The mechanisms underlying HI brain damage involve excitotoxicity, apoptosis, and inflammation. In this process, toxic intracellular accumulation of Ca2+ plays a prominent role in the damage. Previous studies have provided considerable evidence that glutamate receptors are the major pathway for Ca2+ influx in neurons following brain injury. Recently, transient receptor potential (TRP) channels have been shown to regulate Ca2+ homeostasis and to be involved in brain injury pathophysiology.Transient receptor potential vanilloid 1 (TRPV1, or the capsaicin receptor) is a Ca2+ permeable, nonspecific cation channel belonging to the TRP superfamily. TRPV1 is mainly expressed by nociceptors, the specialized subset of primary sensory neurons located in the dorsal root and trigeminal ganglia. TRPV1 is dedicated to the detection of noxious stimuli and is activated by vanilloid ligands such as capsaicin, noxious heat or acid. However, accumulating evidence suggests that TRPV1 is also expressed across the central nervous system (CNS), as has been suggested by many studies. In addition, TRPV1 can also be activated by endogenously generated compounds such as anandamide and lipoxygenase products, which accumulate during brain injury and it may aggravate the injury. Thus, TRPV1 seems to be a promising potential therapeutic target for drug development for brain injury.Vitexin is a c-glycosylated flavonoid that has been found in lots of medicinal plants. Vitexin has recently received increased attention because of its wide range of pharmacological effects. It has been reported to reduce nociceptive behavior by acting as an antagonist of TRPV1. Furthermore, the flavonoid cochinchinenin B shows an inhibitory effect on capsaicin-activated current in rat dorsal root ganglion neurons. Vitexin is therefore a potential pharmacological tool for studying the functions of TRPV1 in vitro and in vivo.Some studies have shown that HIF-la inhibition early after injury provides neuroprotective effects. Meanwhile, other study has proved that vitexin is an inhibitor of HIF-1a.Objective:(1) To identify the function of TRPV1 receptor in the neonatal hypoxia-ischemia brain damage on mouse via comparing the difference of cerebral infarction、 brain atrophy and animal behavior outcomes between newborn Trpvl gene knockout mice and wild type mice after hypoxia-ischemia brain damage.(2) To identify the treatment effect of vitexin in the neonatal hypoxia-ischemia brain damage on mouse by different experiment measures, furthermore, to explore the mechanism of pharmacological effect of vitexin and further clarify the function of TRPV1 receptor in the neonatal hypoxia-ischemia brain damage on mice.(3) To identify the neuroprotective effect of vitexin for other targets in the neonatal hypoxia-ischemia brain damage on rat.Methods:(1) With establishment of HI on postnatal 9 days C57BL/6 Trpvl gene knockout mice and wild type mice and combining TRPV1 receptor agonist capsaicin intervention treatment. TTC staining、Nissl staining and animal behavior are used to identify the function of TRPV1 receptor in the neonatal hypoxia-ischemia brain damage on mouse.(2) Different dosages of vitexin pretreatment on hypoxia-ischemia brain damage in 9-day-old C57BL/6 mice and TTC staining is used to determine the appropriate dose. Meanwhile, Nissl staining、TUNEL staining and animal behavior are also used to identify the neuroprotective effect of vitexin on HIBD.(3) Primary cortical neuronal cultures are prepared from the cerebral cortices of neonatal C57BL/6 mice (postnatal of 0-1) and cultured cortical neurons at 12 DIV are exposed to OGD. MTT assay、LDH release and PI staining are used to identify the neuroprotective effect of vitexin on the cultured cortical neurons.(4) The [Ca2+]i in OGD-treated primary cortical neurons is determined using the fluorescent Ca2+ indicator Fura-2-AM, and combining the whole cell patch clamp to identify the interaction of TRPV1 receptor with vitexin. Meanwhile, Western blot is used to verify the vitexin on expression of TRPV1 receptor.(5) Western blot is used to detect a batch of signaling pathway relative proteins (CaMKII/p-CaMKII and apoptotic proteins) after HI among different groups in vivo and in vitro.(6) Different dosages of vitexin treatment on hypoxia-ischemia brain damage in 7-day-old SD rats at different time. TTC staining is used to determine the appropriate dose and time. Nissl staining、brain water content measurement、 IgG staining、brain atrophy、foot-fault test and Morris water maze are used to to identify the neuroprotective effect of vitexin on HIBD.(7) To detect the expression of HIF-1 a and VEGF in different groups after HI via Western blot and ImmunohistochemistryResults:(1) TTC staining show that newborn trpv1gene knockout mice have lower infarct volume than wild-type mice, Moreover, TRPV1 receptor agonist capsaicin pretreatment on wild-type mice has shown the neuroprotective effect in a dose-dependent. But, this neuroprotection has gone if capsaicin post-treatment. Meanwhile, the same results are required in Nissl staining and animal behavior test.(2) Vitexin pretreatment significantly reduces the infarct volume compared to that in the HI group. The reduction of infarct volume is dose-dependent. Meanwhile, brain atrophy、TUNEL staining show that vitexin pretreatment resulted in a significant decrease in brain injury. Furthermore, vitexin improved neurobehavioral outcomes.(3) Vitexin reduces OGD-induced neuronal death and LDH release. Meanwhile, PI staining result also show that neuroprotective effects of vitexin.(4) The [Ca2+]i in OGD-treated primary cortical neurons is determined using the fluorescent Ca2+ indicator Fura-2-AM. Subjecting cortical neurons to OGD resulted in an increase in [Ca2+]i,However, vitexin reduces the increase. Vitexin quickly and reversibly inhibites the TRPV1 currents induced by capsaicin. But, western blot indicates that vitexin doesn’t changed TRPV1 receptor expression in the neuron after HI.(5) Western blot show that vitexin pretreatment also increases the Bcl-2-to-Bax protein ratio and decreased p-CaMKII/CaMKII, NF-κB and cleaved caspase-3 protein expression in brain damage area (cortex) after HI. Meanwhile, we get the same result in primary cortical neuron.(6) TTC staining result show that vitexin, administered 5 min after HI, was neuroprotective as seen by decreased infarct volume and it is a dose-dependent. This neuroprotection was removed when vitexin was administered 3 h after HI. Vitexin (best dose) ameliorated brain edema、BBB disruption、neuronal cell death、brain atrophy and animal behavior outcomes. HIF-la activator dimethyloxalylglycine (DMOG) increased the brain edema and BBB permeability compared to HI alone.(7) Western blot and Immunohistochemistry show that vitexin attenuated the increase in HIF-la and VEGF due to HI.Conclusions:(1) Our data has shown that newborn trpvl gene knockout mice decreased brain damage after HI. Moreover, TRPV1 receptor agonist capsaicin pretreatment may provide the neuroprotection against HI. It indicates that desensitizing inhibition of TRPV1 receptor may protects against hypoxia-ischemia Injury in the Neonatal Mouse Brain.(2) Vitexin pretreatment protects the brain from hypoxic-ischemic injury, improves neurobehavioral outcomes, promotes survival and inhibited pro-apoptotic signaling pathways in a mouse model of neonatal hypoxic-ischemic brain injury. The neuroprotection is likely mediated by the inhibition of the overactivation of TRPV1 receptor.(3) HIP-1a inhibition with vitexin provides both acute and long-term neuroprotection in the developing brain after neonatal HI injury via reducing infarct volume、protecting BBB integrity and brain edema. |
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