Icariin alleviates cellular injury induced by cardiac ischemia-reperfusion injury by inhibiting IRE1/JNK-induced ferroptosis.

BACKGROUND: Ischemia-induced cellular damage and stress responses significantly impact cellular viability and function. Icariin (ICA), known for its protective effects, has been studied to understand its role in mitigating oxygen-glucose deprivation/reperfusion (OGD/R)-induced endoplasmic reticulum (ER) stress and ferroptosis in H9C2 cardiomyoblast cells. METHODS: We employed an in vitro OGD/R model using H9C2 cells. ICA's effects were analyzed across multiple concentrations. Key indicators of ER stress, autophagy, and ferroptosis-including markers like Bip, PERK, IRE1, ATF6, P62, FTH1, LC3II/LC3I, and NCOA4-were assessed using Western blotting, electron microscopy, and biochemical assays. Additionally, the role of the IRE1/JNK pathway in mitochondrial dynamics and its influence on mitochondrial dynamics protein was explored through specific inhibition and activation experiments. RESULTS: ICA significantly reduced the activation of UPR pathways, decreased autophagic vacuole formation, and maintained cell viability in response to OGD/R and Erastin-induced ferroptosis. These protective effects were associated with modulated autophagic processes, reduced lipid peroxidation, and decreased ferrous ion accumulation. Inhibition of the IRE1/JNK pathway and subsequent Drp1 activity demonstrated reduced mitochondrial recruitment and mitophagy, correlating with decreased ferroptosis markers and improved cell survival. CONCLUSION: Our findings highlight ICA's potential in modulating IRE1/JNK pathway, autophagy, providing a therapeutic avenue for mitigating ferroptosis in myocardial ischemia-reperfusion injury (MIRI).

Construction of HEK293T cell line stably expressing TRPM2 channel based on PiggyBac transposition system and its application in drug screening for cerebral ischemia and other diseases. / 基于PiggyBac转座子优化稳定表达细胞系的新型瞬时受体电位M2通道抑制剂筛选.

OBJECTIVES: To establish a cell line stably expressing the TRPM2 channel for screening TRPM2 inhibitors based on PiggyBac transposition system. METHODS: A pPB-hTRPM2 eukaryotic expression vector was constructed using PiggyBac transposition system. The constructed plasmid and helper plasmid were contransfected into HEK293T cells to express TRPM2, which was identified by fluorescence and patch-clamp assay. The high throughput screening was assessed with the Z ´ factor. Calcium imaging and patch clamp techniques were employed to assess the initial activity of the eleven compound molecules, confirming the inhibitory effects of the primary molecule on TRPM2. The protective impact of screened compounds on damaged cells was validated using the oxygen-glucose deprivation reperfusion (OGD/R) model and CCK-8 kit. The level of cellular reactive oxygen species (ROS) was detected by flow cytometry. The neuroprotective effects of the compounds were evaluated using a transient middle cerebral artery occlusion (tMCAO) mouse model. RESULTS: The HEK293T cells transfected with pPB-hTRPM2-EGFP showed high TRPM2 expression. Puromycin-resistant cells, selected through screening, exhibited robust fluorescence. Whole-cell patch results revealed that induced cells displayed classical TRPM2 current characteristics comparable to the control group, showing no significant differences (P>0.05). With a Z ´ factor of 0.5416 in calcium imaging (Z ´>0.5), the model demonstrated suitability for high-throughput screening of TRPM2 inhibitors. Calcium imaging and electrophysiological experiments indicated that compound 6 significantly inhibited the TRPM2 channel. Further experiments showed that 1 µmol/L of compound 6 enhanced the cell viability (P<0.05) and reduced the level of ROS (P<0.05) of SH-SY5Y under OGD/R-induced injury, 0.3 and 1 mg/kg of compound 6 reduced the cerebral infarction volume in tMCAO mice (both P<0.05). CONCLUSIONS: A stably TRPM2 gene expressing cell line has been successfully established using PiggyBac gene editing in this study. TRPM2 channel inhibitors were screened through calcium imaging and patch clamp techniques, an inhibitor compound 6 has been identified, which can alleviate cell damage after OGD/R by reducing cellular ROS levels, and has a protective effect against cerebral ischemia-reperfusion injury in mice.

Formononetin inhibits neuroinflammation in BV2 microglia induced by glucose and oxygen deprivation reperfusion through TLR4/NF-κB signaling pathway.

Ischemic stroke, caused by diminished or interrupted cerebral blood flow, triggers the activation of microglial cells and subsequent inflammatory responses. Formononetin (FMN) has been observed to inhibit BV2 microglial cell activation and alleviate ensuing neuroinflammatory reactions. Despite extensive research, the precise underlying mechanism remains unclear. To investigate the neuroinflammatory response following FMN-mediated inhibition of BV2 microglial activation, we employed an in vitro oxygen-glucose deprivation/reperfusion (OGD/R) model. BV2 microglial cells were categorized into four groups: control, FMN, OGD/R, and OGD/R+FMN. Cell viability was assessed using the CCK-8 assay, while flow cytometry assessed M1 and M2 cell populations within BV2 cells. Immunofluorescence was utilized to detect the expression levels of apoptosis-inducing factor (AIF), p53, Toll-like receptor 4 (TLR4), and NF-κB p65. Western blotting (WB) was conducted to quantify p65/p-p65, IκB-α/p-IκB-α, and TLR4 protein levels in each group. Additionally, ELISA was employed to measure IL-1ß and TNF-α levels in cell supernatants from each group. The results revealed a significant increase in the proportion of iNOS/CD206-positive M1/M2 cells in the OGD/R group compared to the control group (p < 0.05). There was also a notable increase in nuclear translocation of NF-κB p65 and elevated expression of inflammatory factors IL-1ß and TNF-α in cell supernatants. Moreover, levels of p-p65, p-IκB-α, and TLR4 proteins were significantly elevated in the OGD/R group (p < 0.05). However, the addition of FMN reversed these effects. Specifically, FMN administration notably attenuated cell death and inflammation in BV2 microglia induced by OGD/R through modulation of the TLR4/NF-κB signaling pathway.These findings suggest that FMN may serve as a potential therapeutic agent against neuroinflammation associated with ischemic stroke by targeting microglial activation pathways.

Neuroprotective effects of GPR68 against cerebral ischemia-reperfusion injury via the NF-κB/Hif-1α pathway.

BACKGROUND: G protein-coupled receptor 68 (GPR68), an orphan receptor, has emerged as a promising therapeutic target for mitigating neuronal inflammation and oxidative damage. This study explores the protective mechanisms of GPR68 in cerebral ischemia-reperfusion injury (CIRI). METHODS: An in vivo middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model was established. Mice received intraperitoneal injections of Ogerin, a selective GPR68 agonist. In vitro, GPR68 was overexpressed in SH-SY5Y and HMC3 cells, and the effects of oxygen-glucose deprivation/reperfusion (OGD/R) on cell viability were assessed using real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and flow cytometry. RESULTS: The expression of GPR68 was suppressed in cells subjected to OGD/R treatment, whereas its upregulation conferred protection to SH-SY5Y and HMC3 cells. In vivo, levels of GPR68 were reduced in brain tissues affected by MCAO/R, correlating with oxidative stress, inflammation, and neurological damage. Treatment with a GPR68 agonist decreased brain infarction, apoptosis, and dysregulated gene expression induced by MCAO/R. Mechanistically, GPR68 agonist treatment may inhibit the activation of the NF-κB/Hif-1α pathway, thereby reducing oxidative and inflammatory responses and enhancing protection against CIRI. CONCLUSIONS: This study confirms that the GPR68/NF-κB/Hif-1α axis modulates apoptosis, inflammation, and oxidative stress in CIRI, indicating that GPR68 is a potential therapeutic target for CIRI.

Transcriptomic Hallmarks of Hypoxic-Ischemic Brain Injury: Insights from an in Vitro Model.

BACKGROUND: Hypoxic-ischemic injury of neurons is a pathological process observed in several neurological conditions, including ischemic stroke and neonatal hypoxic-ischemic brain injury (HIBI). An optimal treatment strategy for these conditions remains elusive. The present study delved deeper into the molecular alterations occurring during the injury process in order to identify potential therapeutic targets. METHODS: Oxygen-glucose deprivation/reperfusion (OGD/R) serves as an established in vitro model for the simulation of HIBI. This study utilized RNA sequencing to analyze rat primary hippocampal neurons that were subjected to either 0.5 or 2 h of OGD, followed by 0, 9, or 18 h of reperfusion. Differential expression analysis was conducted to identify genes dysregulated during OGD/R. Time-series analysis was used to identify genes exhibiting similar expression patterns over time. Additionally, functional enrichment analysis was conducted to explore their biological functions, and protein-protein interaction (PPI) network analyses were performed to identify hub genes. Quantitative real-time polymerase chain reaction (qRT-PCR) was used for validation of hub-gene expression. RESULTS: The study included a total of 24 samples. Analysis revealed distinct transcriptomic alterations after OGD/R processes, with significant dysregulation of genes such as Txnip, Btg2, Egr1 and Egr2. In the OGD process, 76 genes, in two identified clusters, showed a consistent increase in expression; functional analysis showed involvement of inflammatory responses and signaling pathways like tumor necrosis factor (TNF), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and interleukin 17 (IL-17). PPI network analysis suggested that Ccl2, Jun, Cxcl1, Ptprc, and Atf3 were potential hub genes. In the reperfusion process, 274 genes, in three clusters, showed initial upregulation followed by downregulation; functional analysis suggested association with apoptotic processes and neuronal death regulation. PPI network analysis identified Esr1, Igf-1, Edn1, Hmox1, Serpine1, and Spp1 as key hub genes. qRT-PCR validated these trends. CONCLUSIONS: The present study provides a comprehensive transcriptomic profile of an in vitro OGD/R process. Key hub genes and pathways were identified, offering potential targets for neuroprotection after hypoxic ischemia.

Cytisine-N-methylene-(5,7,4'-trihydroxy)- isoflavone ameliorates ischemic stroke-induced brain injury in mouse by regulating the oxidative stress and BDNF-Trkb/Akt pathway.

BACKGROUND: A novel compound Cytisine-N-methylene-(5,7,4'-trihydroxy)- isoflavone (LY01) found in the Sophora alopecuroides L is a neuroprotective agent. However, the effect and potential mechanism of LY01 treatment for ischemic stroke (IS) have not been fully elucidated. AIM OF THE STUDY: The aim of this study is to demonstrate whether LY01 can rescue ischemic stroke-induced brain injury and oxygen-glucose deprivation/reperfusion (OGD/R). RESULTS: Our results show that intragastric administration of LY01 improves ischemic stroke behaviors in mice, as demonstrated by neurological score, infarct volume, cerebral water content, rotarod test for activity. Compared with the model group, the ginkgo biloba extract (EGb) and LY01 reversed the neurological score, infarct volume, cerebral water content, rotarod test in model mice. Further analysis showed that the LY01 rescued oxidative stress in the model mice, which was reflected in the increased levels of catalase, superoxide dismutase, total antioxidant capacity and decreased levels of malondialdehyde in the serum of the model mice. Moreover, the expression of the brain-derived neurotrophic factor brain-derived neurotrophic factor (BDNF), phosphorylated protein kinase B (p-Akt), Bax, Bcl-2, (p)-tropomysin related kinase B (p-Trkb) was restored and the expression of Bax, glial fibrillary acidic protein (GFAP) in the brains of the model mice was inhibited through LY01 treatment. In the polymerase chain reaction (PCR) data, after giving LY01, the expression in the brains of model mice was that, IL-10 increased and IL-1ß, Bax, Bcl-2 decreased. Furthermore, the results indicated that LY01 improved cell viability, reactive oxygen species content, and mitochondrial membrane potential dissipation induced by OGD/R in primary culture of rat cortical neurons. Bax and caspase-3 activity was upregulated compared to the before after treatment with LY01. CONCLUSIONS: Our study suggests that LY01 reversed ischemic stroke by reducing oxidative stress and activating the BDNF-TrkB/Akt pathway and exerted a neuroprotective action against OGD/R injury via attenuation, a novel approach was suggested to treat ischemic stroke. Our observations justify the traditional use of LY01 for a treatment of IS in nervous system.

Hyperchloremia and Hypernatremia Decreased Microglial and Neuronal Survival during Oxygen-Glucose Deprivation/Reperfusion.

Hyperchloremia and hypernatremia are associated with higher mortality in ischemic stroke, but it remains unclear whether their influence directly contributes to ischemic injury. We investigated the impact of 0.9% sodium chloride (154 mM NaCl), 0.9% sodium acetate (167 mM CH3COONa), and their different combinations (3:1, 2:1, and 1:1) on microglial (HMC-3) and neuronal (differentiated SH-SY5Y) survival during oxygen-glucose deprivation/reperfusion (OGD/R). Further, we assessed the effect of hyperchloremia and hypernatremia-treated and OGD/R-induced HMC-3-conditioned media on differentiated SH-SY5Y cells under OGD/R conditions. We performed cell viability, cell toxicity, and nitric oxide (NO) release assays and studied the alteration in expression of caspase-1 and caspase-3 in different cell lines when exposed to hyperchloremia and hypernatremia. Cell survival was decreased in 0.9% NaCl, 0.9% CH3COONa, combinations of HMC-3 and differentiated SH-SY5Y, and differentiated SH-SY5Y cells challenged with HMC-3-conditioned media under normal and OGD/R conditions. Under OGD/R conditions, differentiated SH-SY5Y cells were less likely to survive exposure to 0.9% NaCl. Expression of caspase-1 and caspase-3 in HMC-3 and differentiated SH-SY5Y cells was altered when exposed to 0.9% NaCl, 0.9% CH3COONa, and their combinations. A total of 0.9% NaCl and 0.9% CH3COONa and their combinations decreased the NO production in HMC-3 cells under normal and OGD/R conditions. Both hypernatremia and hyperchloremia reduced the survival of HMC-3 and differentiated SH-SY5Y cells under OGD/R conditions. Based on the OGD/R in vitro model that mimics human ischemic stroke conditions, it possibly provides a link for the increased death associated with hyperchloremia or hypernatremia in stroke patients.

Zuogui Pill Promotes Neurite Outgrowth by Regulating OPN/ IGF-1R/PTEN and Downstream mTOR Signaling Pathway.

AIM AND OBJECTIVE: Zuogui pill (ZGP) is the traditional Chinese medicine for tonifying kidney yin. Clinical and animal studies have shown that ZGP effectively enhances neurologic impairment after ischemic stroke, which may be related to promoting neurite outgrowth. This investigation aimed to prove the pro-neurite outgrowth impact of ZGP and define the underlying molecular pathway in vitro. MATERIALS AND METHODS: The major biochemical components in the ZGP were investigated using UPLC-QTOF-MS. All-trans retinoic acid (ATRA) was employed to stimulate SH-SY5Y cells to develop into mature neurons, followed by oxygen-glucose deprivation and reoxygenation damage (OGD/R). Then the cells were supplemented with different concentrations of ZGP, and cell viability was identified by CCK-8. The neurites' outgrowth abilities were detected by wound healing test, while immunofluorescence staining of ß-III-tubulin was used to label neurites and measure their length. Western blot was employed to discover the changes in protein levels. RESULTS: ZGP improved the cell viability of differentiated SH-SY5Y cells following OGD/R damage, according to the CCK-8 assay. Concurrently, ZGP promoted neurite outgrowth and improved neurite crossing and migration ability. Protein expression analysis showed that ZGP upregulated the expression of GAP43, OPN, p-IGF-1R, mTOR, and p-S6 proteins but downregulated the expression of PTEN protein. Blocking assay with IGF-1R specific inhibitor Linstinib suggested IGF-1R mediated mTOR signaling pathway was involved in the pro-neurite outgrowth effect of ZGP. CONCLUSION: This work illustrated the molecular mechanism underpinning ZGP's action and offered more proof of its ability to promote neurite outgrowth and regeneration following ischemic stroke.

Hydroxysafflor Yellow A Inhibits Pyroptosis and Protecting HUVECs from OGD/R via NLRP3/Caspase-1/GSDMD Pathway.

OBJECTIVE: To observe the protective effect and mechanism of hydroxyl safflower yellow A (HSYA) from myocardial ischemia-reperfusion injury on human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were treated with oxygen-glucose deprivation reperfusion (OGD/R) to simulate the ischemia reperfusion model, and cell counting kit-8 was used to detect the protective effect of different concentrations (1.25-160 µ mol/L) of HSYA on HUVECs after OGD/R. HSYA 80 µ mol/L was used for follow-up experiments. The contents of inflammatory cytokines interleukin (IL)-18, IL-1 ß, monocyte chemotactic protein 1 (MCP-1), tumor necrosis factor α (TNF-α) and IL-6 before and after administration were measured by enzyme-linked immunosorbent assay. The protein expressions of toll-like receptor, NOD-like receptor containing pyrin domain 3 (NLRP3), gasdermin D (GSDMD) and GSDMD-N-terminal domain (GSDMD-N) before and after administration were detected by Western blot. NLRP3 inflammasome inhibitor cytokine release inhibitory drug 3 sodium salt (CRID3 sodium salt, also known as MCC950) and agonist were added, and the changes of NLRP3, cysteine-aspartic acid protease 1 (Caspase-1), GSDMD and GSDMD-N protein expressions were detected by Western blot. RESULTS: HSYA inhibited OGD/R-induced inflammation and significantly decreased the contents of inflammatory cytokines IL-18, IL-1 ß, MCP-1, TNF-α and IL-6 (P<0.01 or P<0.05). At the same time, by inhibiting NLRP3/Caspase-1/GSDMD pathway, HSYA can reduce the occurrence of pyroptosis after OGD/R and reduce the expression of NLRP3, Caspase-1, GSDMD and GSDMD-N proteins (P<0.01). CONCLUSIONS: The protective effect of HSYA on HUVECs after OGD/R is related to down-regulating the expression of NLRP3 inflammasome and inhibiting pyroptosis.

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.

Ampelopsin alleviates OGD/R induced neuronal damage by regulating JAK2/STAT3 signaling pathway / 国际检验医学杂志

Objective To investigate the impact of ampelopsin(AMP)on oxygen glucose deprivation/reperfusion(OGD/R)induced neuronal damage and its mechanism,and to lay a foundation for the study of neonatal hypoxic-ischemic brain damage.Methods Neurons of newborn SD rats were isolated and cultured in vitro,and they were divided into 5 groups:control group(AMP 0 μmol/L),OGD/R group,low dose AMP group(OGD/R+AMP 20 μmol/L),high dose AMP group(OGD/R+AMP 30 μmol/L)and JAK2/STAT3 activator group(OGD/R+AMP 30 μmol/L+Coumermycin A1 10 μmol/L).CCK-8 method was used to de-tect the cell viability of different treatment groups,the lactate dehydrogenase(LDH)kit was used to detect the cell activity of LDH in the medium,flow cytometry was used to detect the apoptosis rate,enzyme-linked immunosorbent assay was used to detect the levels of interleukin-6(IL-6),interleukin-10(IL-10)and tumor necrosis factor α(TNF-α),the kit was used to detect the levels of reactive oxygen species(ROS),malondial-dehyde(MDA)and superoxide dismutase(SOD),and Western blotting was used to detect the expression of apoptosis related proteins B-cell lymphoma-2(Bcl-2),Bcl-2 associated X protein(Bax),enzymatic cleavage of cysteine containing aspartate protein hydrolase-3(C-caspase-3),tyrosine kinase 2(J AK2),phosphorylated JAK2(p-JAK2),signal transduction and transcription activating factor 3(STAT3)and phosphorylated STAT3(p-STAT3).Results Compared with the concentration of AMP of 0 μmol/L,the cell viability in con-centration of AMP of 5-30 μmol/L was not obvious different(P＞0.05),when the concentration of AMP was 40 μmol/L,the cell viability decreased obviously(P＜0.05).Compared with the control group,the cell viability,the levels of SOD fluorescence intensity,IL-10 and Bcl-2 in OGD/R group decreased obviously,the LDH activity,cell apoptosis rate,the levels of ROS,MDA,IL-6,TNF-α,Bax,C-caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 increased obviously(P＜0.05).Compared with OGD/R group,the cell viability,the levels of SOD fluorescence intensity,IL-10 and Bcl-2 in low and high dose AMP groups increased,the LDH activity,cell apoptosis rate,the levels of ROS,MDA,IL-6,TNF-α,Bax,C-caspase-3,p-JAK2/JAK2,and p-STAT3/STAT3 decreased(P＜0.05),and JAK2/STAT3 activator was able to reverse the protective effect of AMP on OGD/R induced neuronal.Conclusion AMP attenuates OGD/R induced neuronal by reducing oxidative stress and inflammatory response,and its mechanism may be related to inhibition of JAK2/STAT3 signal pathway phosphorylation.

Overexpression of circular RNA HIPK3 prevents activation of rat microglia / 安徽医科大学学报

Objective @#To investigate the relationship between circular RNA homeodomain interacting protein kinase 3 (circHIPK3) and the activation of rat microglia (RM) cells.@*Methods @#In vitro , RM cells were cultured and randomized into normal and oxygen⁃glucose deprivation/reperfusion (OGD/R) groups , and the expression level of circHIPK3 in each group was detected by RT⁃qPCR. The circHIPK3 lentiviral vector with puromycin resistance was constructed , and the overexpression (OE) group and negative control (NC) group were set up. The optimal multiplicity of infection (MOI) for RM cells was determined based on fluorescence expression , and puromycin was used to screen RM cells stably expressing circHIPK3. The cells of OE and NC groups were treated with OGD/R , and the expression levels of ionized calcium binding adaptor molecule 1 (Iba ⁃1) and eukaryotic tumor necrosis factor receptor superfamily (CD40) were detected by Western blot. The circHIPK3 translational protein potential was analyzed by the circRNAdb database , while the potential binding microRNAs on circHIPK3 were predicted by circBank and Starbase databases.@*Results @#OGD/R down⁃regulated circHIPK3 in RM cells ( P < 0. 000 1) . The sequencing results were accurate and the lentiviral vector of circHIPK3 was constructed successfully. The optimal MOI of RM cells was 80 , puromycin at a concentration of 2 μg/ml was used to screen RM cell lines stably expressing circHIPK3. RT⁃qPCR results showed that the expression level of circHIPK3 was significantly higher in the OE group compared with the NC group (P < 0. 01) . Western blot results revealed that the expression levels of Iba and CD40 in the OE group were markedly lower than those in the NC group (P < 0. 05) . Protein translation analysis showed that circHIPK3 encoded a polypeptide of 404 amino acids with two internal ribosome entry sites (IRES) and an open reading frame (ORF) . Database analysis uncovered that circHIPK3 could target eight specific miRNAs , namely hsa⁃miR⁃3529⁃5p , hsa⁃miR⁃379⁃5p , hsa⁃miR⁃506⁃3p , hsa⁃miR⁃33 , hsa⁃miR⁃450b⁃5p , hsa⁃miR551b⁃3p , hsa⁃miR⁃193 , and hsa⁃miR⁃508 ⁃3p.@*Conclusion @#The overexpression of circHIPK3 effectively suppresses OGD/R⁃induced activation of RM cells. It has the potential to encode peptides and may act as a miRNA sponge. These findings provide a foundation for further study of circHIPK3 functions.

Progesterone improved the behavior of PC12 cells under OGD/R by reducing FABP5 expression and inhibiting TLR4/NF-κB signaling pathway.

Herein, PC12 cells were applied to detect the impact of progesterone under oxygen glucose deprivation/reperfusion (OGD/R) stimulation. The cell proliferation of PC12 cells was evaluated by cell counting kit-8 assay, and the concentrations of MDA, ROS and SOD were examined by their corresponding Enzyme Linked Immunosorbent Assay kits. The invasion and migration properties of PC12 cells were evaluated by transwell and wound healing assays, respectively. The expression patterns of related genes were evaluated by western blot and qPCR. Under OGD/R stimulation, progesterone treatment could elevate the viability of PC12 cells, reduce the levels of MDA and ROS, and elevate the concentration of SOD. Moreover, progesterone treatment could strengthen the invasion and migration abilities of PC12 cells under OGD/R condition, as well as decrease the apoptosis and inflammation. FABP5 expression was significantly increased in PC12 cells under OGD/R stimulation, which was reversed after progesterone stimulation. Under OGD/R stimulation, the protective effects of progesterone on PC12 cells were strengthened after si-FABP5 treatment. The protein levels of TLR4, p-P65 NF-κB, and P65 NF-κB in OGD/R-induced PC12 cells were increased, which were inhibited after progesterone treatment. Progesterone exerted protective effects on PC12 cells by targeting FABP5 under OGD/R stimulation.

Puerarin reduces cell damage from cerebral ischemia-reperfusion by inhibiting ferroptosis.

This study explores the protective effects of Puerarin, a compound derived from the traditional Chinese herb Pueraria, against cellular damage induced by Oxygen-Glucose Deprivation/Reoxygenation (OGD/R) in PC12 cells. The research focuses on understanding how Puerarin influences the mechanisms of ferroptosis and oxidative stress, key factors in ischemia-reperfusion injury relevant to neurodegenerative diseases. In our in vitro model, we identified the optimal OGD duration to induce significant cell stress and confirmed the non-toxicity of Puerarin up to 100uM. The results reveal that Puerarin substantially mitigates the detrimental effects of OGD/R, including improvements in cell viability, mitochondrial integrity, and reductions in oxidative stress markers like ROS and lipid peroxidation. Notably, Puerarin modulates key proteins in the autophagy process and the Nrf2 pathway, crucial in cellular stress responses. Further, the use of 3-Methyladenine, an autophagy inhibitor, underscores the significance of autophagy in managing OGD/R-induced stress. These findings suggest Puerarin's potential as a therapeutic agent for conditions characterized by ischemic cellular damage, highlighting the need for further clinical exploration.

Smurf2-Mediated Ubiquitination of FOXO4 Regulates Oxygen-glucose Deprivation/Reperfusion-induced Pyroptosis of Cortical Neurons.

BACKGROUND: Smad ubiquitination regulatory factor 2 (Smurf2) has been observed to alleviate ischemia-reperfusion injury. This study sought to explore the molecular mechanism of Smurf2-mediated forkhead box O4 (FOXO4) ubiquitination in oxygen-glucose deprivation/ reperfusion (OGD/R)-induced pyroptosis of cortical neurons. METHODS: Human cortical neurons (HCN-2) were subjected to OGD/R to establish a cell model of cerebral stroke. Smurf2, FOXO4, and doublecortin domain containing 2 (DCDC2) expressions were determined by RT-qPCR and Western blot. LDH release, pyroptosis-related proteins NLRP3, GSDMD-N, and cleaved-caspase-3, as well as inflammatory factors IL-1ß and IL-18, were assessed by LDH assay kit, Western blot, and ELISA. The ubiquitination level of FOXO4 was determined by ubiquitination assay. The bindings of Smurf2 to FOXO4 and FOXO4 to DCDC2 were testified by Co-IP, ChIP, and dual-luciferase assays. Rescue experiments were designed to validate the role of FOXO4/DCDC2 in the pyroptosis of HCN-2 cells. RESULTS: Smurf2 was weakly expressed, while FOXO4 and DCDC2 were prominently expressed in OGD/R-treated HCN-2 cells. Smurf2 overexpression promoted LDH release, reduced NLRP3, GSDMD-N, and cleaved-caspase-3 proteins, and decreased IL-1ß and IL-18 concentrations. Sumrf2 improved the ubiquitination level of FOXO4 to downregulate its protein level. FOXO4 is bound to the DCDC2 promoter to facilitate its transcription. Overexpression of FOXO4 or DCDC2 reversed the inhibition of Smurf2 overexpression on pyroptosis of OGD/Rtreated HCN-2 cells. CONCLUSION: Smurf2 overexpression facilitated the ubiquitination of FOXO4 to reduce its protein level, thereby suppressing DCDC2 transcription and restricting OGD/R-induced pyroptosis of cortical neurons.

ErbB4 processing is involved in OGD/R induced neuron injury.

OBJECTIVE: Our previous study found that ErbB4 gene expression was changed after oxygen-glucose deprivation/reperfusion (OGD/R). However, the exact role and mechanism of ErbB4 in brain ischemia are largely unknown. In this study, we explored the protective effects of ErbB4 and its possible mechanism after OGD/R. METHODS: Cerebral ischemia/reperfusion (I/R) injury model was established in vitro and in vivo. Cell viability, apoptosis, and ROS production were measured by MTT, TUNEL, and fluorescent probe 2', 7'-dichlorofluorescein diacetate (DCFH-DA). Infarct size was evaluated by TTC. We performed bioinformatics analyses to screen for novel key genes involved in ErbB4 changes. RNA-Seq was used to transcriptome analysis. RNA and protein expression were detected by quantitative RT‒PCR and western bloting. RESULTS: The expression of 80-kDa ErbB4 decreased after cerebral I/R injury in vitro and in vivo. Co-expression network analysis revealed that ErbB4 expression was correlated with the changes in Adrb1, Adrb2, Ldlr, and Dab2. Quantitative RT‒PCR revealed that the mRNA expression levels of Adrb1, Adrb2, and Dab2 were upregulated, and that of Ldlr was decreased after OGD/R. Activation of ErbB4 expression by neuregulin 1 (NRG1) significantly promoted cell survival, attenuated hippocampal apoptosis, and decreased ROS production after OGD/R. Furthermore, the elimination of ErbB4 using a specific siRNA reversed these beneficial effects. CONCLUSION: Our data revealed the neuroprotective effects of ErbB4 against OGD/R injury, and the action could be related to changes in the ErbB4 membrane-associated fragment and the expression of Adrb1, Adrb2, Ldlr, and Dab2.

Pachymic acid protects hepatic cells against oxygen-glucose deprivation/reperfusion injury by activating sirtuin 1 to inhibit HMGB1 acetylation and inflammatory signaling.

Ischemia-reperfusion injury is an important cause of liver injury occurring during liver transplantation. It is usually caused by inflammatory response and oxidative stress-induced oxidative damage. Pachymic acid (PA) has various biological activities such as anti-inflammatory, antioxidant and anti-cancer. However, the action mechanism of PA in hepatic ischemia-reperfusion injury is currently unknown. In this study, liver cells were subjected to oxygen-glucose deprivation/reperfusion (OGD/R) to simulate a hepatic ischemia-reperfusion injury model. The binding relationship between PA and sirtuin 1 (SIRT1) was analyzed by molecular docking. Cell viability was detected by Cell Counting Kit-8. Expression levels of SIRT1 and high mobility group box 1 (HMGB1) were detected by western blot. Subsequent levels of inflammatory factors were detected by related kits and western blot. Meanwhile, related kits were used to examine levels of oxidative stress markers including reactive oxygen species, malondialdehyde, superoxide dismutase and cytotoxicity-associated lactate dehydrogenase. Finally, cell apoptosis was detected by flow cytometry and western blot. The results showed that PA significantly ameliorated OGD/R-induced decrease in SIRT1 expression, increase in HMGB1 acetylation and HMGB1 translocation. Moreover, the elevated levels of inflammatory factors, oxidative stress indexes and cell apoptosis upon exposure to OGD/R were reversed by PA treatment. Moreover, the addition of SIRT1 agonist and inhibitor further demonstrated that PA exerted the aforementioned effects in OGD/R-exposed cells by targeting SIRT1. Thus, the present study revealed the mechanism by which PA ameliorated OGD/R-induced hepatic injury via SIRT1. These results might provide a clearer theoretical basis for the targeted treatment of OGD/R-induced hepatic injury with PA.

Ultrashort wave alleviates oxygen-glucose deprivation/reoxygenation injury via up-regulation of SPCA1 expression in N2a cells. / è¶ çŸ­æ³¢é€šè¿‡ä¸Šè°ƒSPCA1的表达减轻N2a细胞氧糖剥夺复氧后的损伤.

OBJECTIVES: Application of ultrashort wave (USW) to rats with cerebral ischemia and reperfusion injury could inhibit the decrease of expression of secretory pathway Ca2+-ATPase 1 (SPCA1), an important participant in Golgi stress, reduce the damage of Golgi apparatus and the apoptosis of neuronal cells, thereby alleviating cerebral ischemia-reperfusion injury. This study aims to investigate the effect of USW on oxygen-glucose deprivation/reperfusion (OGD/R) injury and the expression of SPCA1 at the cellular level. METHODS: N2a cells were randomly divided into a control (Con) group, an OGD/R group, and an USW group. The cells in the Con group were cultured without exposure to OGD. The cells in the OGD/R group were treated with OGD/R. The cells in the USW group were treated with USW after OGD/R. Cell morphology was observed under the inverted phase-contrast optical microscope, cell activity was detected by cell counting kit-8 (CCK-8), apoptosis was detected by flow cytometry, and SPCA1 expression was detected by Western blotting. RESULTS: Most of the cells in the Con group showed spindle shape with a clear outline and good adhesion. In the OGD/R group, cells were wrinkled, with blurred outline, poor adhesion, and lots of suspended dead cells appeared; compared with the OGD/R group, the cell morphology and adherence were improved, with clearer outlines and fewer dead cells in the USW group. Compared with the Con group, the OGD/R group showed decreased cell activity, increased apoptotic rate, and down-regulating SPCA1 expression with significant differences (all P<0.001); compared with the OGD/R group, the USW group showed increased cell activity, decreased apoptotic rate, and up-regulating SPCA1 expression with significant differences (P<0.01 or P<0.001). CONCLUSIONS: USW alleviates the injury of cellular OGD/R, and its protective effect may be related to its up-regulation of SPCA1 expression.

[Naoluo Xintong Decoction activates caspase-1/Gasdermin D pathway to promote angiogenesis of rat brain microvascular endothelial cells after oxygen glucose deprivation/reperfusion injury].

OBJECTIVE: To investigate the effects of Naoluo Xintong Decoction (NLXTD) on pyroptosis and angiogenesis of brain microvascular endothelial cells (BMECs) and explore the possible mechanisms in rats with oxygen-glucose deprivation/ reperfusion (OGD/R). METHODS: Rat BMECs with or without caspase-1 siRNA transfection were cultured in the presence of 10% medicated serum from NLXTD-treated rats (or blank serum) and exposed to OGD/R. CCK-8 assay, Transwell chamber assay, and tube formation assay were used to assess proliferation, migration, and tube-forming abilities of the cells. The activity of lactate dehydrogenase (LDH) in the culture supernatant was determined using a commercial assay kit, and the levels of inflammatory factors IL-1ß and IL-18 were detected with ELISA. The cellular expressions of pro-caspase-1, caspase-1, NLRP3, Gasdermin D, and angiogenesis-related proteins VEGF and VEGFR2 were detected using Western blotting. RESULTS: The BMECs showed obvious injuries after OGD/R exposure. Compared with the blank serum, the medicated serum significantly improved the cell viability, migration ability, and lumen-forming ability (P < 0.01) and lowered the levels of IL-1ß and IL-18 and the LDH release (P < 0.01) of the cells with OGD/R exposure. Western blotting showed that in the BMECs exposed to OGD/R, the medicated serum strongly upregulated the expression of VEGF and VEGFR2 proteins (P < 0.01) and reduced the protein expressions of pro-caspase-1, caspase-1, NLRP3, and Gasdermin D (P < 0.01), and transfection of the cells with caspase-1 siRNA further promoted the expressions of VEGFR2 protein in the cells (P < 0.01). CONCLUSION: NLXTD can improve the proliferation, migration, and tube- forming ability and promote angiogenesis of BMECs with OGD/R injury probably by inhibiting the caspase-1/Gasdermin D pathway in pyroptosis, alleviating cell injury, and upregulating the expressions of VEGF and VEGFR2.

The protective role of sulforaphane and Homer1a in retinal ischemia-reperfusion injury: Unraveling the neuroprotective interplay.

AIMS: Retinal ischemia/reperfusion (I/R) injury is a common pathological basis for various ophthalmic diseases. This study aimed to investigate the potential of sulforaphane (SFN) and Homer1a in regulating cell apoptosis induced by retinal I/R injury and to explore the underlying regulatory mechanism between them. MATERIALS AND METHODS: In in vivo experiments, C57BL/6J mice and Homer1flox/-/Homer1a+/-/Nestin-Cre+/- mice were used to construct retinal I/R injury models. In vitro experiments utilized the oxygen-glucose deprivation-reperfusion (OGD/R) injury model with primary retinal ganglion cells (RGCs). The effects of Homer1a and SFN on cell apoptosis were observed through pathological analyses, flow cytometry, and visual electrophysiological assessments. KEY FINDINGS: We discovered that after OGD/R injury, apoptosis of RGCs and intracellular Ca2+ activity significantly increased. However, these changes were reversed upon the addition of SFN, and similar observations were reproduced in in vivo studies. Furthermore, both in vivo and in vitro studies confirmed the upregulation of Homer1a after I/R, which could be further enhanced by the administration of SFN. Moreover, upregulation of Homer1a resulted in a reduction in cell apoptosis and pro-apoptotic proteins, while downregulation of Homer1a had the opposite effect. Flash visual evoked potential, oscillatory potentials, and escape latency measurements in mice supported these findings. Furthermore, the addition of SFN strengthened the neuroprotective effects in the OGD/R + H+ group but weakened them in Homer1flox/-/Homer1a+/-/Nestin-Cre+/- mice. SIGNIFICANCE: These results indicate that Homer1a plays a significant role in the therapeutic potential of sulforaphane for retinal I/R injury, thereby providing a theoretical basis for clinical treatment.