TRPV1 Suppressed NLRP3 Through Regulating Autophagy in Microglia After Ischemia-Reperfusion Injury.

The microglia-mediated inflammatory response is one of the main causes of brain tissue damage after stroke. In recent years, it has been reported that autophagy in microglia played an important role in inflammatory response after stroke. Transient receptor potential vanilloid 1 (TRPV1) has been shown to regulate autophagy and inflammatory in microglia; however, the detailed mechanisms remain unclear. This study aimed to investigate whether autophagy regulates inflammatory is associated with TRPV1. Model of oxygen and glucose deprivation/reoxygenation (OGD/R) was established in vitro to induce cerebral ischemia-reperfusion injury (I/R). siRNA of Atg5, inhibitors, and agonists of both autophagy and TRPV1 were involved in our study. Autophagy was assayed by immunofluorescence staining LC-3 and autophagosome was observed using transmission electron microscopy (TEM). Autophagy/inflammation-related markers as Atg5, LC-3II/LC-3I, Beclin-1, NLRP3, IL-1ß, and Caspase-1 were also measured in the present study. Results indicated that I/R injury-induced inflammatory injury may be impeded by inhibition of autophagy, and TRPV1 could suppress OGD/R-induced autophagy of microglia. However, the effect of TRPV1's inhibitor on inflammatory response was attenuated when the autophagy was blocked. These findings suggested that TRPV1 exhibits an anti-inflammatory effect on OGD/R-induced microglia, which was at least correlated with the anti-autophagy action of TRPV1 partially.

The Synergistic Effect of TRPV1 on Oxidative Stress-Induced Autophagy and Apoptosis in Microglia.

Stroke mostly including ischemic stroke is the second leading mortality and disability worldwide. Oxidative stress injury occurred during ischemic stroke treatment generally. A high amount of reactive oxygen species (ROS) is involved in oxidative stress induction. Transient receptor potential vanilloid 1 (TRPV1) has been shown to regulate oxidative stress and apoptosis in microglia; however, the detailed mechanisms remain unclear. We aimed to explore whether autophagy-regulated oxidative stress and apoptosis are associated with TRPV1. The model of oxygen and glucose deprivation (OGD/R) in microglia was established. The siRNA of Atg5 and inhibitors and agonists of both autophagy and TRPV1 were involved in our study. Autophagy-related markers Atg5, LC3II/LC3I, and Beclin-1 were measured, and the autophagosome was observed under a transmission electron microscope (TEM). Caspase 3 was detected using ELISA. ROS and JC-1 were detected using flow cytometry. Apoptosis was observed by TUNEL. The results indicated that oxidative stress-induced injury and apoptosis may be impeded by the increasing autophagy, and TRPV1 inhibition could suppress the OGD/R-induced autophagy of microglia. However, the effect of TRPV1's inhibitor on oxidative stress and apoptosis was not obvious when the autophagy was blocked. These findings suggested that TRPV1 may exhibit antioxidative and antiapoptosis effect on OGD/R-induced microglia. However, the experimental results do not fully demonstrate that the TRPV1-mediated antioxidative and antiapoptosis effect is through the affecting autophagy entirely.

Treadmill exercise enhances synaptic plasticity in the ischemic penumbra of MCAO mice by inducing the expression of Camk2a via CYFIP1 upregulation.

AIMS: Physical exercise is beneficial to the recovery of patients with ischemic stroke. However, the underlying mechanism by which exercise promotes dendritic remodeling and synaptic plasticity is still obscure. This study explored the mechanism by which treadmill exercise enhances synaptic plasticity and dendritic remodeling in the ischemic penumbra. MAIN METHODS: A middle cerebral artery occlusion (MCAO) model was generated in C57BL/6 mice, and lentivirus-mediated cytoplasmic FMRP-associated protein 1 (CYFIP1) shRNA expression was utilized to confirm the role of CYFIP1 in the exercise-induced increase in synaptic plasticity and dendritic remodeling. Neurological deficits were measured using the Zea Longa scale. Hematoxylin-eosin (H&E) staining and Nissl staining were performed to assess cerebral ischemic injury. Golgi-Cox staining was used to observe changes in dendritic remodeling and synaptic plasticity. Transmission electron microscopy (TEM) was performed to observe the synaptic ultrastructure. Molecular mechanisms were explored using immunofluorescence staining and western blotting. KEY FINDINGS: Treadmill training enhanced synaptic plasticity in the penumbra. Additionally, we observed significant increases in the expression of CYFIP1 and calcium/calmodulin-dependent kinase 2a (Camk2a); enhanced neurological recovery and a decreased infarct volume. However, the injection of a lentivirus containing CYFIP1 shRNA into the lateral ventricle exerted negative effects on synaptic plasticity. Moreover, the exercise-induced neuroprotective effects were abolished by lentivirus-mediated CYFIP1 shRNA expression, consistent with the downregulation of Camk2a expression and the deterioration of neurological function. SIGNIFICANCE: Treadmill training enhances synaptic plasticity and dendritic remodeling in the ischemic penumbra by inducing the expression of Camk2a via upregulation of CYFIP1.

Intensive treadmill training promotes cognitive recovery after cerebral ischemia-reperfusion in juvenile rats.

Rehabilitation training is routine for children who experience stroke, but its protective mechanism remains unclear. To study the effect of treadmill training intensity on hippocampal synaptic plasticity after cerebral ischemia, a model of middle cerebral artery occlusion (MCAO)/reperfusion was established in young rats to simulate childhood ischemic stroke. The rats were randomly allocated into five groups: sham operation, MCAO, low-intensity exercise and MCAO (5 m/min), medium-intensity exercise and MCAO (10 m/min), and high-intensity exercise and MCAO (15 m/min). Intervention was continued for 14 days, and a series of experimental tests were conducted. After MCAO, the juvenile rats exhibited a series of morphological and functional alterations, including changes in their neurobehavior and cerebral infarct volumes. Compared with control rats, MCAO rats had a longer escape latency and crossed fewer platforms in the water maze test and exhibited decreased hippocampal neuron density and Synapsin I and PSD95 expression. Furthermore, MCAO rats exhibited synapse morphology changes and abnormal serum levels of lactic acid and corticosterone. Treadmill training effectively reduced the neurobehavioral scores and cerebral infarction volumes, with medium-intensity training showing the best effect. Treadmill training shortened the escape latency, increased the number of platform crossings, and improved the spatial cognitive abilities of the rats, with the medium intensity training having the best effect on spatial learning/memory efficiency. Treadmill training increased the neuron density in the hippocampus, with the medium-intensity training resulting in the highest density. Treadmill training had a positive effect on the expression of Synapsin I and PSD95, with the medium-intensity training showing the strongest effect. Treadmill training improved the sub-microstructure synapse morphology, with the medium-intensity training demonstrating the best effect. Treadmill training increased the plasma levels of lactic acid and corticosterone, with the high-intensity training having the most obvious effect. Treadmill training can provide neuroprotection by promoting hippocampal synaptic plasticity, with medium-intensity training showing the most optimal effects.

Effects of Treadmill Exercise on Motor and Cognitive Function Recovery of MCAO Mice Through the Caveolin-1/VEGF Signaling Pathway in Ischemic Penumbra.

Exercise has been regarded as an effective rehabilitation strategy to facilitate motor and cognitive functional recovery after stroke, even though the complex effects associated with exercise-induced repair of cerebral ischemic injury are not fully elucidated. The enhancement of angiogenesis and neurogenesis, and the improvement of synaptic plasticity following moderate exercise are conducive to functional recovery after ischemic damage. Our previous studies have confirmed the angiogenesis and neurogenesis through the caveolin-1/VEGF pathway in MCAO rats. As an essential neurotrophic factor, BDNF has multiple effects on ischemic injury. In this study, we attempted to determine an additional mechanism of treadmill exercise-mediated motor and cognitive functional recovery through the caveolin-1/VEGF pathway associated with BDNF in the ischemic penumbra of MCAO mice. We found that mice exposed to treadmill exercise after the MCAO operation showed a significant up-regulation in expression of caveolin-1, VEGF, BDNF, synapsin I and CYFIP1 proteins, numbers of cells positive for BrdU/CD34, BDNF, BrdU/NeuN, BrdU/Synapsin I and CYFIP1 expression were increased, which support the reduction in neurological deficit and infarction volume, as well as improved synaptic morphology and spatial learning abilities, compared with the non-exercise mice. However, the caveolin-1 inhibitor, daidzein, resulted in increase in neurological deficit and infarction volume. The selective VEGFR2 inhibitor, PD173074, significantly induced larger infarction volume and neurological injury, and decreased the expression of BDNF in the ischemic penumbra. These findings indicate that exercise improves angiogenesis, neurogenesis and synaptic plasticity to ameliorate motor and cognitive impairment after stroke partially through the caveolin-1/VEGF pathway, which is associated with the coregulator factor, BDNF.

Basic Fibroblast Growth Factor Protects Astrocytes Against Ischemia/Reperfusion Injury by Upregulating the Caveolin-1/VEGF Signaling Pathway.

A previous in vivo study demonstrated that intracerebroventricular injection of basic fibroblast growth factor (bFGF) in middle cerebral artery occlusion rats increased the expression of caveolin-1 (cav-1) and vascular endothelial growth factor (VEGF) in cerebral ischemia penumbra. Because astrocytes are the largest population in the brain, the aim of this in vitro study was to investigate the influence of bFGF on cav-1 and VEGF expression in rat astrocytes following oxygen glucose deprivation/reoxygenation (OGD/R). For this, an ischemic model in vitro of oxygen glucose deprivation lasting for 6 h, followed by 24 h of reoxygenation was used. Primary astrocytes from newborn rats were pre-treated with siRNA targeting bFGF before OGD/R. Cell viability was measured by a CCK-8 assay. The protein and mRNA expressions of bFGF, cav-1, and VEGF were evaluated by western blotting, immunofluorescence staining, and reverse transcription-quantitative polymerase chain reaction. The results showed that OGD/R reduced cell viability, which was decreased further following bFGF knockdown; however, restoring bFGF improved cell survival. A cav-1 inhibitor abrogated the effect of bFGF on cell viability. The expression levels of bFGF mRNA, bFGF protein, cav-1 mRNA, cav-1 protein, and VEGF protein were higher in OGD/R astrocytes. bFGF knockdown markedly decreased the expression levels of cav-1 mRNA, cav-1 protein, and VEGF protein, which were effectively reversed by exogenous bFGF treatment. Moreover, exogenous bFGF treatment significantly increased the expression levels of cav-1 mRNA, cav-1 protein, and VEGF protein in OGD/R astrocytes; however, a cav-1 inhibitor abolished the effect of bFGF on VEGF protein expression. These results suggested that bFGF may protect astrocytes against ischemia/reperfusion injury by upregulating caveolin-1/VEGF signaling pathway.

Apigenin Protects the Brain against Ischemia/Reperfusion Injury via Caveolin-1/VEGF In Vitro and In Vivo.

Apigenin is a natural flavonoid found in several dietary plant foods as vegetables and fruits. To investigate potential anti-ischemia/reperfusion injury properties of apigenin in vitro, cell proliferation assay, tube formation, cell migration, apoptosis, and autophagy were performed in human brain microvascular endothelial cells (HBMVECs) after oxygen-glucose deprivation/reoxygenation (OGD/R). The effect of apigenin was also explored in rats after middle cerebral artery occlusion/reperfusion (MCAO/R) via neurobehavioral scores, pathological examination, and measurement of markers involved in ischemia/reperfusion injury. Data in vitro indicated that apigenin could prompt cell proliferation, tube formation, and cell migration while inhibiting apoptosis and autophagy by affecting Caveolin-1/VEGF, Bcl-2, Caspase-3, Beclin-1, and mTOR expression. Results in vivo showed that apigenin significantly reduced neurobehavioral scores and volume of cerebral infarction while prompting vascular endothelial cell proliferation by upregulating VEGFR2/CD34 double-labeling endothelial progenitor cell (EPC) number and affecting Caveolin-1, VEGF, and eNOS expression in brain tissue of MCAO/R rats. All the data suggested that apigenin may be protective for the brain against ischemia/reperfusion injury by alleviating apoptosis and autophagy, promoting cell proliferation in HBMVECs of OGD/R, and attenuating brain damage and improved neurological function in rats of MCAO/R through the Caveolin-1/VEGF pathway.

Angiogenic effects of apigenin on endothelial cells after hypoxia-reoxygenation via the caveolin-1 pathway.

In the present study, we aimed to elucidate whether apigenin contributes to the induction of angiogenesis and the related mechanisms in cell hypoxia-reoxygenation injury. The role of apigenin was examined in human umbilical vein endothelial cell (HUVEC) viability, migration and tube formation in vitro. To investigate the related mechanisms, we used caveolin-1 short interfering RNA. The viability of HUVECs was measured using Cell Counting Kit-8 assays, HUVEC migration was analyzed by crystal violet staining, and a tube formation assay was performed using the branch point method. Expression of caveolin-1, vascular endothelial growth factor (VEGF), and endothelial nitric oxide synthase (eNOS) in HUVECs was examined by polymerase chain reaction and western blotting. Our data revealed that apigenin induced angiogenesis in vitro by increasing the tube formation ability of HUVECs, which was counteracted by caveolin-1 silencing. Compared to the NC group, Caveolin-1 and eNOS expression was upregulated by apigenin, whereas compared to the NC group, eNOS expression was increased upon caveolin-1 silencing. The expression of VEGF was increased by treatment with apigenin; however, compared to the NC group, caveolin-1 silencing did not affect VEGF expression, and apigenin did not increase VEGF expression in HUVECs after caveolin-1 silencing. These data suggest that apigenin may be a candidate therapeutic target for stroke recovery by promoting angiogenesis via the caveolin-1 signaling pathway.

Apigenin Ameliorates Post-Stroke Cognitive Deficits in Rats Through Histone Acetylation-Mediated Neurochemical Alterations.

BACKGROUND To identify the effect of apigenin on cognitive deficits of rats after cerebral ischemia and reperfusion injury, and to investigate the potential molecular mechanisms. MATERIAL AND METHODS The rats were given sodium butyrate (NaB) or apigenin (20 or 40 mg/kg) for 28 days. Cognition was investigated by the Morris water maze (MWM) test. On day 28, the rats were euthanized and their hippocampal brain regions were used to identify biochemical and neurochemical alterations. The content of histone deacetylase (HDAC) was measured by enzyme-linked immunosorbent assay (ELISA). Western blot analysis was performed to determine the levels of BDNF, phosphorylated cAMP response element-binding protein (pCREB), acetylated H3, and acetylated H4. The mRNA expressions of brain-derived neurotrophic factor (BDNF) and synapsin-I (Syn-I) were examined by polymerase chain reaction (PCR). RESULTS The rats with chronic administration of apigenin (20 and 40 mg/kg) showed better performance in the MWM task than the model rats; there was no significant difference between the apigenin-treated and NaB-treated rats. At the higher apigenin dose of 40 mg/kg, the HDAC content was decreased, the BDNF level was markedly increased, and acetylated H3 and acetylated H4 expressions and Syn-I expressions in the hippocampus was upregulated compared with the model group. Apigenin at 20 mg/kg did not show reversal of the neurochemical alterations. CONCLUSIONS The improvement effect of apigenin on cognitive impairments after cerebral ischemia and reperfusion injury may involve multiple mechanisms, such as the inhibition of HDAC, induction of BDNF and Syn-I expression, and regulation of histone acetylation.

Role of caveolin-1/vascular endothelial growth factor pathway in basic fibroblast growth factor-induced angiogenesis and neurogenesis after treadmill training following focal cerebral ischemia in rats.

Exercise is known to aid functional recovery following ischemia, though the mechanisms responsible for the beneficial effects of exercise on recovery from ischemic stroke are not fully understood. Basic fibroblast growth factor (bFGF) contributes to angiogenesis and promotes neurologic functional recovery after stroke. The present study aimed to investigate the possible mechanisms whereby treadmill exercise ameliorated impaired angiogenesis and neurogenesis following transient cerebral ischemia in middle cerebral artery occlusion (MCAO) rats. Treadmill exercise was started 2days after ischemia-reperfusion in MCAO rats and continued until 7 or 28days after MCAO, after which the animals were sacrificed. Changes in neurological deficit, infarction volume, neuronal morphology, expression levels of bFGF, caveolin-1, and vascular endothelial growth factor (VEGF), and angiogenesis and neurogenesis in the ischemic penumbra were examined by reverse transcription-polymerase chain reaction, western blots, and/or double immunofluorescence. The results suggested that treadmill exercise promoted the expression of bFGF, improved neurological recovery, and reduced infarct volume compared with non-exercised rats, and also enhanced the expression of caveolin-1, VEGF, VEGF receptor 2(FIK-1)/CD34, and Brdu/nestin staining. Small interfering RNA targeting bFGF blocked the protective effects of bFGF. In addition, 4weeks of post-stroke recovery still ameliorated ischemia-induced damage without bFGF shRNA. These findings suggest a novel mechanism underlying the beneficial effects of bFGF following stroke, and indicate that treadmill exercise may aid stroke recovery by regulating the caveolin-1/VEGF pathway in the ischemic zone.

Treadmill Exercise Promotes Neurogenesis in Ischemic Rat Brains via Caveolin-1/VEGF Signaling Pathways.

Using a model of middle cerebral artery occlusion (MCAO), we have previously demonstrated that treadmill exercise promotes angiogenesis in the ischemic penumbra through caveolin-1/VEGF signaling pathways. However, the function of caveolin-1/VEGF signaling in neurogenesis after MCAO has not been determined. In this study, we aimed to investigate the potential of treadmill exercise to promote neurogenesis after MCAO and whether caveolin-1/VEGF signaling pathways are involved. After MCAO, rats were subjected to a program of treadmill exercise. Daidzein (a specific inhibitor of caveolin-1 protein expression, 0.4 mg/kg) was used to confirm the effect of caveolin-1/VEGF signaling on exercise-mediated neurogenesis. We found that the total protein expression of both caveolin-1 and VEGF was increased by exercise and consistent with the improved neurological recovery, decreased infarct volumes and increased 5-bromo-2'-deoxyuridine (BrdU) in the ipsilateral Subventricular zone (SVZ), as well as increased numbers of BrdU/DCX and BrdU/Neun-positive cells in the peri-infarct region. Furthermore, we observed that the treadmill exercise-induced increased VEGF expression, improved neurological recovery, decreased infarct volumes, increased BrdU/DCX and BrdU/Neun-positive cells were significantly inhibited by the caveolin-1 inhibitor. Our results indicate that treadmill exercise improves neurological recovery in ischemic rats, possibly by enhancement of SVZ-derived neural stem cell (NSC) proliferation, migration and differentiation in the penumbra. Moreover, caveolin-1/VEGF signaling is involved in exercise-mediated NSC migration and neuronal differentiation.

Treadmill exercise promotes angiogenesis in the ischemic penumbra of rat brains through caveolin-1/VEGF signaling pathways.

The purpose of this study was to investigate the role of caveolin-1 in treadmill-exercise-induced angiogenesis in the ischemic penumbra of rat brains, and whether caveolin-1 changes correlated with reduced brain injury induced by treadmill exercise, in rats after cerebral ischemia. Rats were randomized into five groups: sham-operated (S, n=7), model (M, n=36), exercise and model (EM, n=36), inhibitor and model (IM, n=36), and inhibitor, exercise, and model (IEM, n=36). Rats in the model groups underwent middle cerebral artery occlusion (MCAO). Rats in the inhibitor groups received an IP injection of the caveolin-1 inhibitor, daidzein (0.4 mg/kg), every 24 h following reperfusion. Rats were killed at 7 or 28 days after the operation. The exercise group showed better neurological recovery and smaller infarction volumes compared with the non-exercise group. Correspondingly, significant increases of caveolin-1 and vascular endothelial growth factor (VEGF) protein expression were observed compared with the non-exercise group. Additionally, the number of Flk-1/CD34 double-positive cells towards the ischemic penumbra was increased in the exercise group. Furthermore, the induction of VEGF protein, microvessel density, decrease of infarct volumes and neurological recovery was significantly inhibited by daidzein. This study indicates that treadmill exercise reduces brain injury in stroke. Our findings suggest that the caveolin-1 pathway is involved in the regulation of VEGF in association with promoted angiogenesis in the ischemic penumbra of rat brains after treadmill exercise. The caveolin-1/VEGF signaling pathway may be a potential target for therapeutic intervention in rats following MCAO.

[Neuroprotective effects of apigenin on acute transient focal cerebral ischemia-reperfusion injury in rats].

OBJECTIVE: To evaluate the neuroprotective effects of apigenin on acute transient focal cerebral ischemia-reperfusion injury in rats. METHODS: The acute transient focal cerebral ischemia-reperfusion model was established with modified method of insertion of thread fish nylon into and staying for two hours and then withdrawing from middle cerebral artery in rats. In experiment groups the neurological behavior scores, TTC stain of brain slices, neurocyte morphology were observed, and brain water content and Evans blue (EB) content were measured. RESULTS: Abnormal neurological behavior scores were existed in apigenin-treated group and model group. Typical cortical infarct lesions in model group were found by TTC stain. The neurocyte morphology in model group 4 hours was found in swollen glia and obvious edema near capillary and within nervous process, and karyopycnosis in neuron of ischemic cortex and hippocampus CA1 under electric microscope. However lesion was alleviated in apigenin-treated group. The brain water content and EB content in apigenin-treated group were lower than model group. CONCLUSION: Apigenin may play an important neuroprotective role in acute transient focal cerebral ischemia-reperfusion injury in rats.