The role of LCN2 in exacerbating ferroptosis levels in acute ischemic stroke injury.

Due to the complex pathogenesis of acute ischemic stroke (AIS), further investigation into its underlying mechanisms is necessary. Presently, existing literature indicates a close association between ferroptosis and AIS injury; however, the precise mechanism and molecular target of ferroptosis in AIS injury remain elusive. By RNA sequencing, we found a significant increase in LCN2 expression in the ischemic cortex. In order to investigate the potential role of LCN2 in modulating AIS injury through the regulation of ferroptosis, we utilized RNA interference (RNAi) knockdown and gene overexpression experiments. The findings from experiments conducted both in vitro and in vivo revealed a marked increase in ferroptosis levels within the AIS model group. Suppression of the LCN2 gene resulted in a significant reduction in ferroptosis levels in OGD/R cells. Conversely, upregulation of LCN2 exacerbated ferroptosis levels in OGD/R cells. The results suggest that elevated levels of ferroptosis may result from heightened expression of LCN2, thereby exacerbating ischemia/reperfusion injury. This study indicates the involvement of ferroptosis in the pathogenesis of AIS and highlights LCN2 as a regulator of ferroptosis in AIS-induced injury, suggesting a potential therapeutic target for ischemic stroke.

The anti-apoptotic role of Ginkgolide B via mitochondrial permeability transition pore inhibition in retinal ischemia-reperfusion.

This research delves into the effectiveness of Ginkgolide B (GB), a compound from Ginkgo biloba, in combating cell death caused by glaucoma, with a focus on mitochondrial impairment and the mitochondrial permeability transition pore (mPTP). Utilizing models of high intraocular pressure and in vitro glaucoma simulations, the study investigates GB's impact on retinal progenitor cells (RPCs) under oxygen-glucose deprivation/reperfusion (OGD/R) and in a rat glaucoma model. The study methodologies included apoptosis assessment, apoptotic marker analysis via Western blot, and mitochondrial structure and function evaluation. The findings reveal that GB notably decreases apoptosis in RPCs exposed to OGD/R in vitro, and reduces ischemia-reperfusion damage in vivo. GB's protective role is attributed to its ability to preserve mitochondrial integrity, maintain membrane potential, regulate calcium levels, and inhibit mPTP opening. These results underscore GB's potential as a therapeutic agent for acute primary angle-closure glaucoma, highlighting its capability to alleviate mitochondrial damage and apoptosis in RPCs and retinal nerve fiber layer cells.

Regulating NCOA4-Mediated Ferritinophagy for Therapeutic Intervention in Cerebral Ischemia-Reperfusion Injury.

Ischemic stroke presents a global health challenge, necessitating an in-depth comprehension of its pathophysiology and therapeutic strategies. While reperfusion therapy salvages brain tissue, it also triggers detrimental cerebral ischemia-reperfusion injury (CIRI). In our investigation, we observed the activation of nuclear receptor coactivator 4 (NCOA4)-mediated ferritinophagy in an oxygen-glucose deprivation/reoxygenation (OGD/R) model using HT22 cells (P < 0.05). This activation contributed to oxidative stress (P < 0.05), enhanced autophagy (P < 0.05) and cell death (P < 0.05) during CIRI. Silencing NCOA4 effectively mitigated OGD/R-induced damage (P < 0.05). These findings suggested that targeting NCOA4-mediated ferritinophagy held promise for preventing and treating CIRI. Subsequently, we substantiated the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway effectively regulated the NCOA4-mediated ferritinophagy, by applying the cGAS inhibitor RU.521 and performing NCOA4 overexpression (P < 0.05). Suppressing the cGAS-STING pathway efficiently curtailed ferritinophagy (P < 0.05), oxidative stress (P < 0.05), and cell damage (P < 0.05) of CIRI, while NCOA4 overexpression could alleviate this effect (P < 0.05). Finally, we elucidated the specific molecular mechanism underlying the protective effect of the iron chelator deferoxamine (DFO) on CIRI. Our findings revealed that DFO alleviated hypoxia-reoxygenation injury in HT22 cells through inhibiting NCOA4-mediated ferritinophagy and reducing ferrous ion levels (P < 0.05). However, the protective effects of DFO were counteracted by cGAS overexpression (P < 0.05). In summary, our results indicated that the activation of the cGAS-STING pathway intensified cerebral damage during CIRI by inducing NCOA4-mediated ferritinophagy. Administering the iron chelator DFO effectively attenuated NCOA4-induced ferritinophagy, thereby alleviating CIRI. Nevertheless, the role of the cGAS-STING pathway in CIRI regulation likely involves intricate mechanisms, necessitating further validation in subsequent investigations.

Panax quinquefolium saponins attenuates microglia activation following acute cerebral ischemia-reperfusion injury via Nrf2/miR-103-3p/TANK pathway.

Ischemic stroke is one of the leading causes of death and disability among adults worldwide. Intravenous thrombolysis is the only approved pharmacological treatment for acute ischemic stroke. However, reperfusion by thrombolysis will lead to the rapid activation of microglia cells which induces interferon-inflammatory response in the ischemic brain tissues. Panax quinquefolium saponins (PQS) has been proven to be effective in acute ischemic stroke, but there is no unified understanding about its specific mechanism. Here, we will report for the first time that PQS can significantly inhibit the activation of microglia cells in cerebral of MCAO rats via activation of Nrf2/miR-103-3p/TANK axis. Our results showed that PQS can directly bind to Nrf2 protein and inhibit its ubiquitination, which result in the indirectly enhancing the expression of TANK protein via transcriptional regulation on miR-103-3p, and finally to suppress the nuclear factor kappa-B dominated rapid activation of microglial cells induced by oxygen-glucose deprivation/reoxygenation  vitro and cerebral ischemia-reperfusion injury in vivo. In conclusion, our study not only revealed the new mechanism of PQS in protecting against the inflammatory activation of microglia cells caused by cerebral ischemia-reperfusion injury, but also suggested that Nrf2 is a potential target for development of new drugs of ischemic stroke. More importantly, our study also reminded that miR-103-3p might be used as a prognostic biomarker for patients with ischemic stroke.

Piracetam reduces oxidative stress and mitochondrial function impairment in an in vitro model of vascular dementia.

Vascular dementia (VaD) is the most common cause of dementia in older adults. Due to the lack of effective treatment options, there is an urgent need to find an effective pharmaceutical compound to combat VaD. Piracetam has been reported to improve impaired cognitive function in a variety of conditions in both human and animal models. However, the role and mechanism of Piracetam in VaD remain unclear. Therefore this study aimed to elucidate the effect of Piracetam on a cellular model of VaD in vitro. We found that Piracetam enhanced the growth of OGD-stimulated SH-SY5Y cells. In addition, Piracetam inhibited the oxidative stress of OGD-stimulated SH-SY5Y cells. Further, Piracetam improved mitochondrial function of OGD-stimulated SH-SY5Y cells. Mechanistically, Piracetam inhibited the PI3K/Akt/mTOR pathway in OGD-stimulated SH-SY5Y cells. Collectively, Piracetam improved oxidative stress and mitochondrial dysfunction of OGD-stimulated SH-SY5Y cells through PI3K/Akt/mTOR axis. Hence, Piracetam has the potential to serve as a promising drug of VaD.

NDRG2 regulates glucose metabolism and ferroptosis of OGD/R-treated astrocytes by the Wnt/ß-catenin signaling.

Ischemic stroke is one main type of cerebrovascular disorders with leading cause of death and disability worldwide. Astrocytes are the only nerve cell type storing glycogen in the brain, which regulate the glucose metabolism and handle the energy supply and survive of neurons. Astrocyte ferroptosis contributes to neuron injury in brain disorders. N-myc downstream-regulated gene 2 (NDRG2) has been implicated in the progression of brain diseases, including ischemic stroke. However, whether NDRG2 could affect the glucose metabolism and ferroptosis of astrocytes during ischemic stroke remains largely unknown. Mouse astrocytes were treated with oxygen-glucose deprivation/reoxygenation (OGD/R) to establish the in vitro model. Glial fibrillary acidic protein, NDRG2, Wnt3a and ß-catenin expression levels were detected by immunofluorescence staining and western blot analyses. Glucose metabolism was investigated by glucose uptake, lactate production, nicotinamide adenine dinucleotide phosphate hydrogen/nicotinamide adenine dinucleotide phosphate (NADPH/NADP+), ATP and glycolysis enzymes (HK2, PKM2 and lactate dehydrogenase A [LDHA]) levels. Ferroptosis was assessed via reactive oxygen species (ROS), glutathione (GSH), iron and ferroptosis-related markers (GPX4 and PTGS2) contents. Glycolysis enzymes and ferroptosis-related markers levels were measured via western blot. NDRG2 expression was elevated in OGD/R-induced astrocytes. NDRG2 overexpression aggravated OGD/R-induced loss of glucose metabolism through reducing glucose uptake, lactate production, NADPH/NADP+ and ATP levels. NDRG2 upregulation exacerbated OGD/R-caused reduction of glycolysis enzymes (HK2, PKM2 and LDHA) levels. NDRG2 promoted OGD/R-induced ferroptosis of astrocytes by increasing ROS, iron and PTGS2 levels and decreasing GSH and GPX4 levels. NDRG2 overexpression enhanced OGD/R-induced decrease of Wnt/ß-catenin signaling activation by reducing Wnt3a and ß-catenin expression. NDRG2 silencing played an opposite effect. Inhibition of Wnt/ß-catenin signaling activation by IWR-1 attenuated the influences of NDRG2 knockdown on glucose metabolism, glycolysis enzymes levels and ferroptosis. These findings demonstrated that NDRG2 contributes to OGD/R-induced inhibition of glucose metabolism and promotion of ferroptosis in astrocytes through inhibiting Wnt/ß-catenin signaling activation, which might be associated with ischemic stroke progression.

Ginsenoside Rg1 attenuates cerebral ischemia-reperfusion injury through inhibiting the inflammatory activation of microglia.

It is recognized that the cerebral ischemia/reperfusion (I/R) injury triggers inflammatory activation of microglia and supports microglia-driven neuronal damage. Our previous studies have shown that ginsenoside Rg1 had a significant protective effect on focal cerebral I/R injury in middle cerebral artery occlusion (MCAO) rats. However, the mechanism still needs further clarification. Here, we firstly reported that ginsenoside Rg1 effectively suppressed the inflammatory activation of brain microglia cells under I/R conditions depending on the inhibition of Toll-likereceptor4 (TLR4) proteins. In vivo experiments showed that the ginsenoside Rg1 administration could significantly improve the cognitive function of MCAO rats, and in vitro experimental data showed that ginsenoside Rg1 significantly alleviated neuronal damage via inhibiting the inflammatory response in microglia cells co-cultured under oxygen and glucose deprivation/reoxygenation (OGD/R) condition in gradient dependent. The mechanism study showed that the effect of ginsenoside Rg1 depends on the suppression of TLR4/MyD88/NF-κB and TLR4/TRIF/IRF-3 pathways in microglia cells. In a word, our research shows that ginsenoside Rg1 has great application potential in attenuating the cerebral I/R injury by targeting TLR4 protein in the microglia cells.

CTRP 9 mitigates the apoptosis and unfolded protein response of OGD/R-induced retinal ganglion cells by regulating the AMPK pathway.

C1q/TNF-related protein-9 (CTRP9) has been reported to play roles in several types of retinal diseases. However, the role and the potential mechanism of CTRP9 in glaucoma are still incompletely understood. The expression of CTRP9 in OGD/R-induced retinal ganglion cells (RGCs) was detected by quantitative real-time polymerase chain reaction and western blot assay. Cell proliferation was identified by cell counting Kit-8 assay. Flow cytometry, enzyme-linked immunosorbent assay and western blot assay were performed to assess cell apoptosis. Unfolded protein response (UPR), endoplasmic reticulum (ER) stress and the AMPK pathway were evaluated by western blot assay. The data showed that the expression of CTRP9 was significantly downregulated in OGD/R-induced 661W cells. OGD/R treatment reduced cell viability, promoted cell apoptosis and activated the UPR and ER stress. The overexpression of CTRP9 reversed the effects of OGD/R on 661W cell viability, apoptosis, the UPR and ER stress, as well as the AMPK pathway. However, Compound C, an inhibitor of AMPK signaling, reversed the protection of CTRP9 overexpression against injury from OGD/R in 661W cells. In summary, the results revealed that CTRP9 abated the apoptosis and UPR of OGD/R-induced RGCs by regulating the AMPK pathway, which may provide a promising target for the treatment of glaucoma.

2,3-Diphosphoglyceric Acid Alleviating Hypoxic-Ischemic Brain Damage through p38 MAPK Modulation.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a critical condition characterized by significant brain damage due to insufficient blood flow and oxygen delivery at birth, leading to high rates of neonatal mortality and long-term neurological deficits worldwide. 2,3-Diphosphoglyceric acid (2,3-DPG), a small molecule metabolite prevalent in erythrocytes, plays an important role in regulating oxygen delivery, but its potential neuroprotective role in hypoxic-ischemic brain damage (HIBD) has yet to be fully elucidated. Our research reveals that the administration of 2,3-DPG effectively reduces neuron damage caused by hypoxia-ischemia (HI) both in vitro and in vivo. We observed a notable decrease in HI-induced neuronal cell apoptosis, attributed to the downregulation of Bax and cleaved-caspase 3, alongside an upregulation of Bcl-2 expression. Furthermore, 2,3-DPG significantly alleviates oxidative stress and mitochondrial damage induced by oxygen-glucose deprivation/reperfusion (OGD/R). The administration of 2,3-DPG in rats subjected to HIBD resulted in a marked reduction in brain edema and infarct volume, achieved through the suppression of neuronal apoptosis and neuroinflammation. Using RNA-seq analysis, we validated that 2,3-DPG offers protection against neuronal apoptosis under HI conditions by modulating the p38 MAPK pathway. These insights indicated that 2,3-DPG might act as a promising novel therapeutic candidate for HIE.

Velvet antler polypeptide (VAP) protects against cerebral ischemic injury through NF-κB signaling pathway in vitro.

OBJECTIVE: Velvet antler polypeptide (VAP) has been shown to play important roles in the immune and nervous systems. The purpose of this study was to investigate the protective effects of VAP on cerebral ischemic injury with the involvement of NF-κB signaling pathway in vitro. MATERIALS AND METHODS: PC-12 cells stimulated by oxygen-glucose deprivation/reperfusion (OGD/R) was used to mimic cerebral ischemic injury in vitro. The levels of ROS, SOD, and intracellular concentrations of Ca2+ were measured by the relevant kits. Meanwhile, the expressions of inflammatory cytokines (IL-6, IL-1ß, and TNF-α) were determined by ELISA kit assay. In addition, MTT, EdU, and flow cytometry assays were used to measure the cell proliferation and apoptosis. Besides which, the related proteins of NF-κB signaling pathway were measured by western blotting assay. RESULTS: VAP alleviated cerebral ischemic injury by reducing OGD/R-induced oxidative stress, inflammation, and apoptosis in PC-12 cells in a time dependent manner. Mechanistically, VAP inhibited the levels of p-p65 and p-IkB-α in a time dependent manner, which was induced by OGD/R operation. Moreover, NF-κB agonist diprovocim overturned the suppression effects of VAP on OGD/R-induced oxidative stress, inflammation, and apoptosis in PC-12 cells. CONCLUSIONS: The results demonstrate that VAP may alleviate cerebral ischemic injury by suppressing the activation of NF-κB signaling pathway.

LncRNA SNHG12 regulated by WTAP aggravated the oxygen-glucose deprivation/reperfusion-induced injury in bEnd.3 cell.

OBJECTIVES: Previous studies have identified abnormal expression of lncRNA SNHG12 in ischemic stroke, but the underlying molecular mechanism remains unclear. MATERIALS AND METHODS: Through database predictions, m6A methylation sites were found on SNHG12, suggesting post-transcriptional modification. To further elucidate the role of SNHG12 and m6A methyltransferase WTAP in oxygen-glucose deprivation/reperfusion (OGD/R)-induced damage in cerebral microvascular endothelial cells, we conducted investigations. Additionally, we examined the impact of m6A methyltransferase WTAP on SNHG12 expression. RESULTS: Overexpressing SNHG12 in bEnd.3 cells was found to inhibit cell proliferation and promote apoptosis, as well as activate the production of reactive oxygen species and inflammatory cytokines (E-selectin, IL-6 and MCP-1), along with angiogenic proteins (VEGFA and FGFb). Conversely, SNHG12 knockdown alleviated OGD/R-induced damage to BEnd.3 cells, resulting in improved cell proliferation, reduced apoptosis, decreased ROS and LDH production, as well as diminished expression of inflammatory cytokines (E-selectin, IL-6 and MCP-1) and angiogenic proteins (VEGFA and FGFb). Furthermore, WTAP was found to positively regulate SNHG12 expression, and WTAP knockdown in bEnd.3 cells under the OGD/R conditions inhibited cell proliferation, promoted apoptosis, and increased ROS and LDH production. CONCLUSION: These findings suggest that WTAP may play a crucial role in SNHG12-mediated OGD/R-induced damage in bEnd.3 cells. More molecular experiments are needed to further analyze its mechanism. Overall, our study helps to enrich our understanding of the dysregulation of SNHG12 in ischemic stroke.

[Ginsenoside Rg_1 ameliorates OGD/R-induced PC12 cell injury by inhibiting autography via IRE1-JNK-CHOP pathway].

This study investigated the protective effect of ginsenoside Rg_1(GRg_1) on oxygen and glucose deprivation/reoxygenation(OGD/R)-injured rat adrenal pheochromocytoma(PC12) cells and whether the underlying mechanism was related to the regulation of inositol-requiring enzyme 1(IRE1)-c-Jun N-terminal kinase(JNK)-C/EBP homologous protein(CHOP) signaling pathway. An OGD/R model was established in PC12 cells, and PC12 cells were randomly classified into control, model, OGD/R+GRg_1(0.1, 1, 10 µmol·L~(-1)), OGD/R+GRg_1+rapamycin(autophagy agonist), OGD/R+GRg_1+3-methyladenine(3-MA,autophagy inhibitor), OGD/R+GRg_1+tunicamycin(endoplasmic reticulum stress agonist), OGD/R+GRg_1+4-phenylbutyric acid(4-PBA, endoplasmic reticulum stress inhibitor), and OGD/R+GRg_1+3,5-dibromosalicylaldehyde(DBSA, IRE1 inhibitor) groups. Except the control group, the other groups were subjected to OGD/R treatment, i.e., oxygen and glucose deprivation for 6 h followed by reoxygenation for 6 h. Cell viability was detected by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide(MTT) assay. Apoptosis was detected by Hoechst 33342 staining, and the fluorescence intensity of autophagosomes by the monodansylcadaverine(MDC) assay. Western blot was employed to determine the expression of autophagy-related proteins(Beclin1, LC3-Ⅱ, and p62) and the pathway-related proteins [IRE1, p-IRE1, JNK, p-JNK, glucose-regulated protein 78(GRP78), and CHOP]. The results showed that GRg_1 dose-dependently increased the viability of PC12 cells and down-regulated the expression of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, compared with the model group. Furthermore, GRg_1 decreased the apoptosis rate and MDC fluorescence intensity and up-regulated the expression of p62 protein. Compared with the OGD/R+GRg_1(10 µmol·L~(-1)) group, OGD/R+GRg_1+rapamycin and OGD/R+GRg_1+tunicamycin groups showed increased apoptosis rate and MDC fluorescence intensity, up-regulated protein levels of Beclin1, LC3-Ⅱ, p-IRE1, p-JNK, GRP78, and CHOP, decreased relative cell survival rate, and down-regulated protein level of p62. The 3-MA, 4-PBA, and DBSA groups exerted the opposite effects. Taken together, GRg_1 may ameliorate OGD/R-induced PC12 cell injury by inhibiting autophagy via the IRE1-JNK-CHOP pathway.

Role of NKCC1 and KCC2 during hypoxia-induced neuronal swelling in the neonatal neocortex.

Neonatal hypoxia causes cytotoxic neuronal swelling by the entry of ions and water. Multiple water pathways have been implicated in neurons because these cells lack water channels, and their membrane has a low water permeability. NKCC1 and KCC2 are cation-chloride cotransporters (CCCs) involved in water movement in various cell types. However, the role of CCCs in water movement in neonatal neurons during hypoxia is unknown. We studied the effects of modulating CCCs pharmacologically on neuronal swelling in the neocortex (layer IV/V) of neonatal mice (post-natal day 8-13) during prolonged and brief hypoxia. We used acute brain slices from Clomeleon mice which express a ratiometric fluorophore sensitive to Cl- and exposed them to oxygen-glucose deprivation (OGD) while imaging neuronal size and [Cl-]i by multiphoton microscopy. Neurons were identified using a convolutional neural network algorithm, and changes in the somatic area and [Cl-]i were evaluated using a linear mixed model for repeated measures. We found that (1) neuronal swelling and Cl- accumulation began after OGD, worsened during 20 min of OGD, or returned to baseline during reoxygenation if the exposure to OGD was brief (10 min). (2) Neuronal swelling did not occur when the extracellular Cl- concentration was low. (3) Enhancing KCC2 activity did not alter OGD-induced neuronal swelling but prevented Cl- accumulation; (4) blocking KCC2 led to an increase in Cl- accumulation during prolonged OGD and aggravated neuronal swelling during reoxygenation; (5) blocking NKCC1 reduced neuronal swelling during early but not prolonged OGD and aggravated Cl- accumulation during prolonged OGD; and (6) treatment with the "broad" CCC blocker furosemide reduced both swelling and Cl- accumulation during prolonged and brief OGD, whereas simultaneous NKCC1 and KCC2 inhibition using specific pharmacological blockers aggravated neuronal swelling during prolonged OGD. We conclude that CCCs, and other non-CCCs, contribute to water movement in neocortical neurons during OGD in the neonatal period.

Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12 cells.

The brain ischemia/reperfusion (I/R) injury has a great impact on human life and property safety. As far as we know, mild hypothermia (MH) is an effective measure to reduce neuronal injury after I/R. However, the precise mechanism is not extremely clear. The purpose of this study was to investigate whether mild therapeutic hypothermia can play a protective role in nerve cells dealing with brain I/R injury and explore its specific mechanism in vitro. A flow cytometer, cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay were performed to detect apoptotic rate of cells, cell viability and cytotoxicity, respectively, reactive oxygen species (ROS) assay kit, JC-1 fluorescent methods, immunofluorescence and western blot were used to explore ROS, mitochondrial transmembrane potential (Δψm), mitochondrial permeability transition pore (MPTP) and protein expression, respectively. The result indicated that the cell activity was decreased, while the cytotoxicity and apoptosis rate were increased after treating with oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 cells. However, MH could antagonize this phenomenon. Interestingly, treating with OGD/R increased the release of ROS and the transfer of Cytochrome C (Cyt-C) from mitochondria to cytoplasm. In addition, it up-regulated the expression of γH2AX, Bax and Clv-caspase3, down-regulated the expression of PCNA, Rad51 and Bcl-2, and inhibited the function of mitochondria in PC12 cells. Excitingly, the opposite trend was observed after MH treatment. Therefore, our results suggest that MH protects PC12 cells against OGD/R-induced injury with the mechanism of inhibiting cell apoptosis by reducing ROS production, improving mitochondrial function, reducing DNA damage, and enhancing DNA repair.

Nrf2 attenuates oxidative stress to mediate the protective effect of ciprofol against cerebral ischemia-reperfusion injury.

Neuroinflammation and oxidative stress damage are involved in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). Ferroptosis emerged as a new player in the regulation of lipid peroxidation processes. This study aimed at exploring the potential involvement of ciprofol on ferroptosis-associated CIRI and subsequent neurological deficits in the mouse model of transient cerebral ischemia and reperfusion. Cerebral ischemia was built in male C57BL/6 J wild-type (WT) and Nrf2-knockout (Nrf2 KO) mice in the manner of middle cerebral artery occlusion (MCAO) followed by reperfusion. Ciprofol improved autonomic behavior, alleviated reactive oxygen species output and ferroptosis-induced neuronal death by nucleus transportation of NFE2 like BZIP transcription factor 2 (Nrf2) and the promotion of heme oxygenase 1 (Ho-1), solute carrier family 7 member 11 (SLC7A11/xCT), and glutathione peroxidase 4 (GPX4). Additionally, ciprofol improved neurological scores and reduced infarct volume, brain water content, and necrotic neurons. Cerebral blood flow in MCAO-treated mice was also improved. Furthermore, absence of Nrf2 abrogated the neuroprotective actions of ciprofol on antioxidant capacity and sensitized neurons to oxidative stress damage. In vitro, the primary-cultured cortical neurons from mice were pre-treated with oxygen-glucose deprivation/reperfusion (OGD/R), followed by ciprofol administration. Ciprofol effectively reversed OGD/R-induced ferroptosis and accelerated transcription of GPX4 and xCT. In conclusion, we investigated the ciprofol-induced inhibition effect of ferroptosis-sheltered neurons from lipid preoxidation in the pathogenesis of CIRI via Nrf2-xCT-GPX4 signaling pathway.

Oxygen-Glucose Deprivation Decreases the Motility and Length of Axonal Mitochondria in Cultured Dorsal Root Ganglion Cells of Rats.

Controlling axonal mitochondria is important for maintaining normal function of the neural network. Oxygen-glucose deprivation (OGD), a model used for mimicking ischemia, eventually induces neuronal cell death similar to axonal degeneration. Axonal mitochondria are disrupted during OGD-induced neural degeneration; however, the mechanism underlying mitochondrial dysfunction has not been completely understood. We focused on the dynamics of mitochondria in axons exposed to OGD; we observed that the number of motile mitochondria significantly reduced in 1 h following OGD exposure. In our observation, the decreased length of stationary mitochondria was affected by the following factors: first, the halt of motile mitochondria; second, the fission of longer stationary mitochondria; and third, a transformation from tubular to spherical shape in OGD-exposed axons. Motile mitochondria reduction preceded stationary mitochondria fragmentation in OGD exposure; these conditions induced the decrease of stationary mitochondria in three different ways. Our results suggest that mitochondrial morphological changes precede the axonal degeneration while ischemia-induced neurodegeneration.

Inhibition of ERK downregulates autophagy via mitigating mitochondrial fragmentation to protect SH-SY5Y cells from OGD/R injury.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is the main cause leading to high mortality and neurological disability in patients with cardiac arrest/cardiopulmonary resuscitation (CA/CPR). Our previous study found that extracellular signal-regulated kinase (ERK) activation, dynamin-related protein1 (Drp1)/Mitofusin2 (Mfn2)-dependent mitochondrial dynamics imbalance, and excessive autophagy were involved in the mechanism of nerve injury after CA/CPR. However, the specific pathological signaling pathway is still unknown. This study aimed to explore the molecular function changes of ERK-Drp1/Mfn2-autophagy signaling pathway in SH-SY5Y cell oxygen-glucose deprivation/reoxygenation (OGD/R) model, to further clarify the pathophysiological mechanism of CIRI, and to provide a new strategy for cerebral protection after CIRI. METHODS: SH-SY5Y cells were pretreated with drugs 24 h before OGD/R. The Drp1 and Mfn2 knockdown were adopted small interfering RNAs. The overexpression of p-Drp1S616 and Mfn2 were used recombinant plasmids. The expression levels of mitochondrial dynamics proteins (p-Drp1, Drp1, Mfn2, Mfn1 and Opa1) and autophagy markers (LC3, Beclin1 and p62) were measured with the Western blotting. The mRNA levels after transfection were determined by PCR. Cell injury and viability were evaluated with released LDH activity and CCK8 assay kits. Mitochondria morphology and autophagosome were observed under transmission electron microscopy. Mitochondrial function was detected by the mitochondrial permeability transition pore assay kit. The co-expression of p-ERK, p-Drp1 and LC3 was assessed with multiple immunofluorescences. One-way analysis of variance followed by least significance difference post hoc analysis (for equal homogeneity) or Dunnett's T3 test (for unequal homogeneity) were used for statistical tests. RESULTS: ERK inhibitor-PD98059 (PD) protects SH-SY5Y cells from OGD/R-induced injury; while ERK activator-TPA had the opposite effect. Similar to autophagy inhibitor 3-MA, PD downregulated autophagy to improve cell viability; while autophagy activator-rapamycin further aggravated cell death. PD and Drp1-knockdown synergistically attenuated OGD/R-induced Drp1 activation, mPTP opening and cell injury; overexpression of Drp1S616E or ablating Mfn2 partly abolished the protective effects of PD. Multiple immunofluorescences showed that p-ERK, p-Drp1 and LC3 were co-expressed. CONCLUSION: Inhibition of ERK downregulates autophagy via reducing Drp1/Mfn2-dependent mitochondrial fragmentation to antagonize mitochondrial dysfunction and promotes cell survival in the SH-SY5Y cells OGD/R model. Video Abstract.

cPKCγ Inhibits Caspase-9-Initiated Neuronal Apoptosis in an Ischemia Reperfusion Model In Vitro Through p38 MAPK-p90RSK-Bad Pathway.

Strokes are one of the leading causes of death and disability in the world. Previously we have found that conventional protein kinase Cγ (cPKCγ) plays neuroprotective role in ischemic strokes. Further, we found that cPKCγ knockdown increased the level of cleaved (cl)-Caspase-3. However, the precise mechanisms underlying cPKCγ-mediated neuronal death remain unclear. To this end, a model incorporating 1 h oxygen-glucose deprivation/24 h reoxygenation (1 h OGD/24 h R) was established in cortical neurons. We found that cPKCγ knockdown remarkably increased neuronal death after OGD. We also found that cPKCγ knockdown increased the level of cl-Caspase-3 through the upstream initiators Capsases-9 (not Caspase-8/12) in OGD-treated neurons. Overexpression of cPKCγ could decrease neuronal death and cl-Caspase-3 and -9 levels. Moreover, cPKCγ knockdown further reduced the phosphorylation levels of p38 MAPK, p90RSK, and Bad. In addition, the protein levels of Bcl-2 and Bcl-xl were decreased after cPKCγ knockdown, whereas that of Bax was increased. In conclusion, our results suggest that cPKCγ partly alleviates ischemic injury through activating the p38 MAPK-p90RSK-Bad pathway and inhibiting Caspase-9 initiated apoptosis. This may have potential as a therapeutic target for ischemic stroke.

Sodium Tanshinone IIA Sulfonate Ameliorates Oxygen-glucose Deprivation/Reoxygenation-induced Neuronal Injury via Protection of Mitochondria and Promotion of Autophagy.

Sodium tanshinone IIA sulfonate (STS) has shown significant clinical therapeutic effects in cerebral ischemic stroke (CIS), but the molecular mechanisms of neuroprotection remain partially known. The purpose of this study was to explore whether STS plays a protective role in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal injury by regulating microglia autophagy and inflammatory activity. Co-cultured microglia and neurons were subjected to OGD/R injury, an in vitro model of ischemia/reperfusion (I/R) injury with or without STS treatment. Expression of protein phosphatase 2 A (PP2A) and autophagy-associated proteins Beclin 1, autophagy related 5 (ATG5), and p62 in microglia was determined by Western blotting. Autophagic flux in microglia was observed with confocal laser scanning microscopy. Neuronal apoptosis was measured by flow cytometric and TUNEL assays. Neuronal mitochondrial function was determined via assessments of reactive oxygen species generation and mitochondrial membrane potential integrity. STS treatment markedly induced PP2A expression in microglia. Forced overexpression of PP2A increased levels of Beclin 1 and ATG5, decreased the p62 protein level, and induced autophagic flux. Silencing of PP2A or administration of 3-methyladenine inhibited autophagy and decreased the production of anti-inflammatory factors (IL-10, TGF-ß and BDNF) and induced the release of proinflammatory cytokines (IL-1ß, IL-2 and TNF-α) by STS-treated microglia, thereby inducing mitochondrial dysfunction and apoptosis of STS-treated neurons. STS exerts protection against neuron injury, and the PP2A gene plays a crucial role in improving mitochondrial function and inhibiting neuronal apoptosis by regulating autophagy and inflammation in microglia.

Upregulation of glutamate transporter 1 by mTOR/Akt pathway in astrocyte culture during oxygen-glucose deprivation and reoxygenation.

Astrocyte-specific glutamate transporter subtype 1 (GLT-1) plays an important role in influencing glutamate excitatory toxicity and preventing the death of excitatory toxic neurons. Although the mammalian target of rapamycin (mTOR)/protein kinase B(Akt)/nuclear factor kappa B signaling cascade is involved in the upregulation of astrocytic GLT-1 in oxygen-glucose deprivation (OGD), it is unclear whether the mTOR/Akt pathway is involved in astrocytic GLT-1 upregulation in OGD and reoxygenation (OGD/R). In this study, we found that the treatment of cultured astrocytes with rapamycin and triciribine led to the decreased astrocytes' protrusions, smaller nuclei, and an increased apoptotic rate. The inhibitors of mTOR complex 1 significantly increased the expression levels of phosphorylated Akt-Ser473 (p-Akt), phosphorylated Akt-Thr308(p-Akt), and GLT-1, while Akt-specific inhibitors blocked GLT-1 expression, suggesting that the mTOR/Akt pathway is involved in GLT-1 upregulation. We further demonstrated that astrocytes under OGD/R adapted to environmental changes through the mTOR/Akt pathway, mainly by altering cell morphology and apoptosis and upregulating the expression levels of p-Akt and GLT-1. Our results suggested that astrocytes may adapt to short-term ischemic-reperfusion injury by regulating cell morphology, apoptosis and GLT-1 upregulation.