| Objective: Stroke, the second leading cause of death worldwide, leads to long-term severe disability in patients. Despite ongoing advances in stroke research, the high morbidity and disability associated with cerebral infarction remain to be adequately addressed. Because of the narrow and restricted time window of thrombolysis, neuroprotection is an alternative or adjunct approach, used to preserve the function of the ischemic penumbra in the acute ischemic phase. Present therapeutic strategies are focused on the administration of drugs that have anti-inflammatory and anti-free radical properties, or that reduce calcium overload, among others. More recent studies have highlighted the importance of neurovascular regeneration and neural functional recovery in the regenerative stage. Kaempferol, belongs to the common flavonoids, mainly comes from rhizome of Kaempferia galanga, and widely exists in a variety of fruits, vegetables and drinks. Because of its multiple efficacy such as anti-cancer, anti-inflammatory, antioxidant, antibacterial, antiviral and so on,kaempferol arouse extensive interesting. Existing experiments has proved the neuroprotection of kaempferol in the acute phase of cerebral ischemia through anti-inflammatory and antioxidant action, but whether it can promote the recovery of neural function after stroke and the underlying molecular mechanisms are still dimness.Generation of new neuronal cells in the injured brain, correlated to neurological outcome, is a key component of post-stroke brain remodeling processes. Neural progenitor cells(NPCs) are multipotent stem cells that are capable of self-renewal in specific brain regions. If endogenous stroke-induced neurogenesis occurs in the adult brain, the two most admitted areas where it may happen are subventricular zone(SVZ) and dentate gyrus of the hippocampus(SGZ). Several stimuli such as ischemia and trauma promotes neurogenesis in the SVZ and newly generated neuroblasts in the SVZ migrate to the ischemic penumbra to replace damaged neurons which contribute to functional recovery. Several neuroregulatory pathways such as transforming growth factor-β, Notch, and Wnt/β-catenin are connected with embryonic development and as well as postnatal neurogenesis by regulating cell proliferation, migration and differentiation of NSCs. Wnt proteins/ligands are important extracellular factors. Canonical Wnt/ β-catenin signaling is activated after the occurance of stroke. The interaction of a Wnt protein with cell-surface receptors the Frizzled(Fz) family initiate the activation of the Wnt signaling pathway. Then, a complex signaling cascade involving the transcription factors results in increase of beta-catenin( β-catenin)and decrease of the unactive form(p-beta-catenin) in cell nucleus.Several growth and neurotrophic factors facilitate neurogenesis in adults brain. Brain derived neurotrophic factor(BDNF) which is a trophic factor that plays a central role in neuroprotection appears to promote the survival of newborn neurons originating in the subventricular zone. However, little is known about the relationship between the Wnt3a/ β-catenin signaling pathways and the expressions of BDNF under stroke.The present study demonstrated whether delayed treatment with Kaemps could enhance neurogenesis and promote long-term functional restoration after cerebral infarction, and whether the Wnt3a/ β-catenin pathway is responsible for the beneficial effects.Methods:Unilateral cortical ischemia with small area was induced in adult male CD1 mice by permanent occlusion of the distal cortical branches middle cerebral artery(MCAO) and ipsilateral common carotid artery. Mice subjected to MCAO were delayed treated with kaempferol. Keamp was intragastric administration once a day for 14 days initiated 24 hours after stroke. Neurologic function was evaluated by the rotarod-test at 7 days, 14 days and 28 days after duplicating the mold. Immunofluorescence staining were employed to measure the proliferation, migration and differentiation of NPCs. Protein and gene expressions of Wnt3a/β-catenin signaling pathway components and BDNF were measured after stroke 7 th,14 th and 28 th days by western blot and RT-PCR analyses respectively.Results:1 Neurological deficit was evaluated and scored by the rotarod test at 7 days,14 days and 28 days after stroke. In the ischemic mice, all dosage treatment of Kaemps showed significant functional improvement at 7 days after stroke onset compared with vehicle-treated mice(P<0.05). However, mice with middle dose are the only group whose beneficial effect can maintain to the 28 days after stroke(P>0.05).2 Kaemps promoted neuronal proliferation and differentiation in the ipsilateral SVZ after stroke.To investigate the effects of Kaemps on proliferation of NSCs, we counted BrdU positive cells in SVZ. BrdU is a synthetic thymidine analogue, which incorporates into DNA during cell replication. The adult male CD1 mouse received a daily single injection of BrdU(50 mg/kg body weight) for five consecutive days from postnatal day after stroke and were sacrificed 4 h after the last BrdU injection. Treatment with Kaemps(30 mg/kg) significantly increased the number of BrdU+ cells in ipsilateral vSVZ at 14 days when the treatment was initiated at 24 hours after the occurance of stroke(P<0.05) indicating that Kaemps treatment can increase neurogenesis in SVZ after stroke.We observed that newly born, immature neurons are present in large numbers in peri-infarct cortex, showing that neural progenitor cells in the SVZ could migrate to the ischemic penumbra to replace damaged neurons. Moreover, Kaemps significantly increased the number of BrdU/NeuN and BrdU/GFAP co-labeled cells as compared to the vehicle control, which suggested that Kaemps induces long-term survival and influence the phenotypic fate of newborn neurons after stroke.3 The pro-neuorgenesis effect of Kaemps may be through the Wnt3a/β-catenin signaling pathway mediated the expression of BDNF after ischemic stroke.It showed us that the protein expression of Wnt3 a,β-catenin/p-β-catenin at 3,7 and 14 days after stroke were up-regulated. They had significant differences compared with MCAO group(P<0.05). And RT-qPCR result revealed that treatment with Kaemps significantly upregulated the gene expression of and Wnt3 a, β-catenin, at 3,7 and 14 days after stroke in ipsilateral cortex(P<0.05). The protein and mRNA level of BDNF was increased at 3, 7 and 14 days after stroke. And the results of protein levels are statistically significant at 3 and 7days after stroke. And the increase of mRNA level 3rd day after stroke had significant differences compared with MCAO group(P<0.05)Conclusion: Delayed continuous treatment with Kaemps in mice markedly improved functional recovery after MCAO. Concurrently, Kaemps treatment promote post-ischemic neurogenesis by facilitating neuronal proliferation, migration and differentiation in the SVZ of infarct side, and activated Wnt3a/β-catenin signaling pathway. In addition, Kaemps strongly potentiated both the protein and mRNA levels of BDNF in the ipsilateral cortex on days 3,7 and 14 post-MCAO suggesting that the effects of Kaemps likely involve activation of Wnt3a/ β-catenin signaling pathway and upregulation of trophic factors BDNF. |
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