Elevated plasma homocysteine (Hcy) levels have been indicated as a strong and modifiable risk factor of ischemic stroke; the previous studies have shown that exposure to Hcy activates cultured microglia. However, whether neurotoxicity of Hcy involves microglia activation following brain ischemia and the underlying mechanisms remains incompletely understood. The cerebral damage was evaluated by staining with 2,3,5-triphenyltetrazolium chloride, hematoxylin-eosin, and Fluoro Jade B. The activation state of microglia was assessed via immunoreaction using the microglial markers Iba1 and OX-42. Then, the inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were examined by Western blot analysis and fluorescence immunohistochemistry. Elevated Hcy level augmented brain damage and neural cell toxicity in the brain cortex and the dentate gyrus region of the hippocampus after cerebral ischemia/reperfusion. Meanwhile, Hcy activated microglia and induced the expression of the inflammatory factors such as TNF-α and IL-6. Moreover, Hcy caused an increase in pSTAT3 expression which occurs in microglial cells. AG490, a JAK2-STAT3 inhibitor, effectively inhibited the phosphorylation of STAT3, microglial cell activation and the secretion of IL-6, TNF-α raised by Hcy treatment. STAT3 signaling pathway located in microglia plays a critical role in mediating Hcy-induced activation of microglia and neuroinflammation in rat MCAO model. This suggests the feasibility of targeting the JAK2/STAT3 pathway as an effective therapeutic strategy to alleviate the progression of Hcy-associated ischemia stroke.

The elevated level of the amino acid metabolite homocysteine (Hcy) is known as a risk factor for ischemic stroke. The molecular mechanisms responsible for neurotoxicity of Hcy remain largely unknown in ischemic brains. The previous studies have shown that Hcy decreases the proliferation and viability of neural stem cells (NSCs) in vivo and in vitro. Autophagy is required for the maintenance of NSCs homeostasis. In the current study, we hypothesized that the toxic effect of Hcy on NSCs may involve the changes in autophagy level following cerebral ischemia/reperfusion injury. The results showed that Hcy reduced cell viability, increased LDH release, and induced nonapoptotic cell death in primary NSCs exposed to oxygen–glucose deprivation)/reoxygenation (OGD/R). Treatment with autophagy inhibitor 3-methyladenine (3MA) partly reversed the decrease in the viability and prevented LDH release triggered by Hcy combined with OGD/R. Increased punctate LC3 dots co-localizing with Nestin-stained NSCs were also observed in the subventricular zone of Hcy-treated MCAO animals, which were partially blocked by 3MA. In vitro studies further revealed that Hcy induced the formation of autophagosomes, markedly increased the expression of the autophagic markers and decreased p-ERK, p-PI3K, p-AKT, and p-mTOR levels. In addition, MHY1485, an activator of mTOR, reduced Hcy-induced increase in LC3 and Beclin 1 protein levels, meanwhile ERK and PI3K activators (TPA, curcumin for ERK and IGF-1 for PI3K, respectively) enhanced Hcy-triggered mTOR inhibition in OGD/R NSCs. Our findings suggest that Hcy may cause excessive autophagy by downregulation of both PI3K-AKT- and ERK- dependent mTOR signaling, thereby facilitates the toxicity of Hcy on NSCs in ischemic brains.

Homocysteine (Hcy) has been shown to have a neurotoxic effect on ischemic brain cells; however, the underlying mechanisms remain incompletely understood. Here, we examined whether Hcy treatment influences mitochondria injury, oxidative stress, and mitochondrial STAT3 (mitoStat3) expression in rat ischemic brain. Our results demonstrated that Hcy treatment aggravated the damage of mitochondrial ultrastructure in the brain cortex and the dentate gyrus region of the hippocampus after focal cerebral ischemia. An elevated Hcy level was also accompanied by the significant inhibition of mitochondrial complex I–III enzymatic activities in addition to an increase in cytochrome c release. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) content and mitoStat3 protein phosphorylation level were increased in Hcy-treated animals, whereas AG490, a Jak2 inhibitor, inhibited mitoStat3 phosphorylation as well as 8-OHdG levels induced by Hcy. In vitro studies revealed that Hcy also markedly increased reactive oxygen species (ROS) and mitoStat3 levels. In addition, the inhibition of pSTAT3 reduced Hcy-mediated increase in ROS levels, whereas quenching ROS using the ROS inhibitor glutathione ethyl ester inhibited Hcy-mediated pSTAT3 overactivation in Neuro2a cells. These findings suggest that the development of therapies that interfere with the ROS/pSTAT3 pathway may be helpful for treating cerebral infarction-related diseases associated with Hcy.

Elevated homocysteine (Hcy) levels have been reported to be involved in neurotoxicity after ischemic stroke. However, the underlying mechanisms remain incompletely understood to date. In the current study, we hypothesized that neuronal autophagy activation may be involved in the toxic effect of Hcy on cortical neurons following cerebral ischemia. Brain cell injury was determined by hematoxylin-eosin (HE) staining and TdT-mediated dUTP Nick-End Labeling (TUNEL) staining. The level and localization of autophagy were detected by transmission electron microscopy, western blot and immunofluorescence double labeling. The oxidative DNA damage was revealed by immunofluorescence of 8-Hydroxy-2′-deoxyguanosine (8-OHdG). Hcy treatment aggravated neuronal cell death, significantly increased the formation of autophagosomes and the expression of LC3B and Beclin-1 in the brain cortex after middle cerebral artery occlusion-reperfusion (MCAO). Immunofluorescence analysis of LC3B and Beclin-1 distribution indicated that their expression occurred mainly in neurons (NeuN-positive) and hardly in astrocytes (GFAP-positive). 8-OHdG expression was also increased in the ischemic cortex of Hcy-treated animals. Conversely, LC3B and Beclin-1 overexpression and autophagosome accumulation caused by Hcy were partially blocked by the autophagy inhibitor 3-methyladenine (3-MA). Hcy administration enhanced neuronal autophagy, which contributes to cell death following cerebral ischemia. The oxidative damage-mediated autophagy may be a molecular mechanism underlying neuronal cell toxicity of elevated Hcy level.

Recent studies revealed that folic acid deficiency (FD) increased the likelihood of stroke and aggravated brain injury after focal cerebral ischaemia. The microglia‐mediated inflammatory response plays a crucial role in the complicated pathologies that lead to ischaemic brain injury. However, whether FD is involved in the activation of microglia and the neuroinflammation after experimental stroke and the underlying mechanism is still unclear. The aim of the present study was to assess whether FD modulates the Notch1/nuclear factor kappa B (NF‐κB) pathway and enhances microglial immune response in a rat middle cerebral artery occlusion‐reperfusion (MCAO) model and oxygen‐glucose deprivation (OGD)‐treated BV‐2 cells. Our results exhibited that FD worsened neuronal cell death and exaggerated microglia activation in the hippocampal CA1, CA3 and Dentate gyrus (DG) subregions after cerebral ischaemia/reperfusion. The hippocampal CA1 region was more sensitive to ischaemic injury and FD treatment. The protein expressions of proinflammatory cytokines such as tumour necrosis factor‐α, interleukin‐1β and interleukin‐6 were also augmented by FD treatment in microglial cells of the post‐ischaemic hippocampus and in vitro OGD‐stressed microglia model. Moreover, FD not only dramatically enhanced the protein expression levels of Notch1 and NF‐κB p65 but also promoted the phosphorylation of pIkBα and the nuclear translocation of NF‐κB p65. Blocking of Notch1 with N‐[N‐(3, 5‐difluorophenacetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester partly attenuated the nuclear translocation of NF‐κB p65 and the protein expression of neuroinflammatory cytokines in FD‐treated hypoxic BV‐2 microglia. These results suggested that Notch1/NF‐κB p65 pathway‐mediated microglial immune response may be a molecular mechanism underlying cerebral ischaemia‐reperfusion injury worsened by FD treatment.