Recombinant Human Ciliary Neurotrophic Factor Protects MCAO/R Rat Brain against Neuronal Degeneration and Apoptosis by Regulating NOS Expression

To research the effects and mechanisms of recombinant human ciliary neurotrophic factor (rhCNTF) on ischemia/reperfusion in vivo and in vitro, rhCNTF was biosynthesized, and ischemia/reperfusion-like models were used. Protection by rhCNTF was studied at the in vivo level using a model of middle cerebral artery occlusion and reperfusion (MCAO/R) in rats. RhCNTF was administrated just before reperfusion. RhCNTF markedly increased animal viability, decreased infarct volumes and neurological deficit scores. Primary cortical neuronal cultures were subjected to oxygen-glucose deprivation/reoxygenation, and treated with rhCNTF prophylactically. Results indicated that neuronal survival rates were increased, LDH release was decreased and lose of neurite length were alleviated in rhCNTF group, and this protection was associated with nerotrophic effect, nitric oxide and neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS). The data suggest that rhCNTF may be a good therapeutic reagent to reduce cerebral ischemia/reperfusion injury, and may act by NOS regulation.

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LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽

10.1007/s13577-021-00527-x ◽

2021 ◽

Author(s):

Jiaying Zhu ◽

Zhu Zhu ◽

Yipin Ren ◽

Yukang Dong ◽

Yaqi Li ◽

...

Keyword(s):

Cerebral Ischemia ◽

Nuclear Translocation ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Mice Model ◽

Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

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miR-19a/b-3p promotes inflammation during cerebral ischemia/reperfusion injury via SIRT1/FoxO3/SPHK1 pathway

Journal of Neuroinflammation ◽

10.1186/s12974-021-02172-5 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Feng Zhou ◽

Yu-Kai Wang ◽

Cheng-Guo Zhang ◽

Bing-Yi Wu

Keyword(s):

Inflammatory Responses ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Luciferase Assay ◽

In Vivo Model ◽

Tunel Staining

Abstract Background Stroke affects 3–4% of adults and kills numerous people each year. Recovering blood flow with minimal reperfusion-induced injury is crucial. However, the mechanisms underlying reperfusion-induced injury, particularly inflammation, are not well understood. Here, we investigated the function of miR-19a/b-3p/SIRT1/FoxO3/SPHK1 axis in ischemia/reperfusion (I/R). Methods MCAO (middle cerebral artery occlusion) reperfusion rat model was used as the in vivo model of I/R. Cultured neuronal cells subjected to OGD/R (oxygen glucose deprivation/reperfusion) were used as the in vitro model of I/R. MTT assay was used to assess cell viability and TUNEL staining was used to measure cell apoptosis. H&E staining was employed to examine cell morphology. qRT-PCR and western blot were performed to determine levels of miR-19a/b-3p, SIRT1, FoxO3, SPHK1, NF-κB p65, and cytokines like TNF-α, IL-6, and IL-1β. EMSA and ChIP were performed to validate the interaction of FoxO3 with SPHK1 promoter. Dual luciferase assay and RIP were used to verify the binding of miR-19a/b-3p with SIRT1 mRNA. Results miR-19a/b-3p, FoxO3, SPHK1, NF-κB p65, and cytokines were elevated while SIRT1 was reduced in brain tissues following MCAO/reperfusion or in cells upon OGD/R. Knockdown of SPHK1 or FoxO3 suppressed I/R-induced inflammation and cell death. Furthermore, knockdown of FoxO3 reversed the effects of SIRT1 knockdown. Inhibition of the miR-19a/b-3p suppressed inflammation and this suppression was blocked by SIRT1 knockdown. FoxO3 bound SPHK1 promoter and activated its transcription. miR-19a/b-3p directly targeted SIRT1 mRNA. Conclusion miR-19a/b-3p promotes inflammatory responses during I/R via targeting SIRT1/FoxO3/SPHK1 axis.

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Nitric oxide-induced regulation of renal organic cation transport after renal ischemia-reperfusion injury

AJP Renal Physiology ◽

10.1152/ajprenal.00264.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. F997-F1004 ◽

Cited By ~ 11

Author(s):

R. Schneider ◽

M. Meusel ◽

B. Betz ◽

M. Kersten ◽

K. Möller-Ehrlich ◽

...

Keyword(s):

Nitric Oxide ◽

Organic Cation ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Fractional Excretion ◽

Renal Ischemia ◽

Organic Cation Transporters ◽

Cation Transporters

Renal organic cation transporters are downregulated by nitric oxide (NO) in rat endotoxemia. NO generated by inducible NO synthase (iNOS) is substantially increased in the renal cortex after renal ischemia-reperfusion (I/R) injury. Therefore, we investigated the effects of iNOS-specific NO inhibition on the expression of the organic cation transporters rOct1 and rOct2 (Slc22a1 and Slc22a2, respectively) after I/R injury both in vivo and in vitro. In vivo, N6-(1-iminoethyl)-l-lysine (l-NIL) completely inhibited NO generation after I/R injury. Moreover, l-NIL abolished the ischemia-induced downregulation of rOct1 and rOct2 as determined by qPCR and Western blotting. Functional evidence was obtained by measuring the fractional excretion (FE) of the endogenous organic cation serotonin. Concordant with the expression of the rate-limiting organic cation transporter, the FE of serotonin decreased after I/R injury and was totally abolished by l-NIL. In vitro, ischemia downregulated both rOct1 and rOct2, which were also abolished by l-NIL; the same was true for the uptake of the organic cation MPP. We showed that renal I/R injury downregulates rOct1 and rOct2, which is most probably mediated via NO. In principle, this may be an autocrine effect of proximal tubular epithelial cells. We conclude that rOct1, or rOct1 and rOct2 limit the rate of the renal excretion of serotonin.

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Remifentanil Preconditioning Reduces Hepatic Ischemia–Reperfusion Injury in Rats via Inducible Nitric Oxide Synthase Expression

Anesthesiology ◽

10.1097/aln.0b013e3182104956 ◽

2011 ◽

Vol 114 (5) ◽

pp. 1036-1047 ◽

Cited By ~ 43

Author(s):

Li-Qun Yang ◽

Kun-Ming Tao ◽

Yan-Tao Liu ◽

Chi-Wai Cheung ◽

Michael G. Irwin ◽

...

Keyword(s):

Nitric Oxide ◽

Methyl Ester ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Hepatic Ischemia ◽

Hepatic Ischemia Reperfusion ◽

Inducible Nos

Background Opioid preconditioning against ischemia reperfusion injury has been well studied in myocardial and neuronal tissues. The objective of this study was to determine whether remifentanil could attenuate hepatic injury and to investigate the mechanisms. Methods A rat model of hepatic ischemia reperfusion injury and a hepatocyte hypoxia reoxygenation (HR) injury model were used, respectively, in two series of experiments. Remifentanil was administered before ischemia or hypoxia and the experiments were repeated with previous administration of naloxone, L-arginine and N-ω-nitro-L-arginine methyl ester, a nonselective opioid receptor antagonist, a nitric oxide donor, and nitric oxide synthase (NOS) inhibitor, respectively. Serum aminotransferase, cytokines, and hepatic lipid peroxidation were measured. Histopathology examination and apoptotic cell detection were assessed. For the in vitro study, cell viability, intracellular nitric oxide, apoptosis, and NOS expression were evaluated. Results Remifentanil and L-arginine pretreatment reduced concentrations of serum aminotransferases and cytokines, decreased the concentrations of hepatic malondialdehyde and myeloperoxidase activity, and increased superoxide dismutase, nitric oxide, and inducible NOS expression in vivo. Decreased histologic damage and apoptosis were also seen in these two groups. These changes were prevented by previous administration of N-ω-nitro-L-arginine methyl ester but not naloxone. There was an increase in inducible NOS protein expression but not endogenous NOS in remifentanil and L-arginine pretreated groups compared with control, naloxone, and N-ω-nitro-L-arginine methyl ester groups. Conclusion Pretreatment with remifentanil can attenuate liver injury both in vivo and in vitro. Inducible NOS but not opioid receptors partly mediate this effect by exhausting reactive oxygen species and attenuating the inflammatory response.

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VX765, a Specific Caspase-1 Inhibitor, Alleviates Lung Ischemia Reperfusion Injury by Suppressing Endothelial Pyroptosis and Barrier Dysfunction

BioMed Research International ◽

10.1155/2021/4525988 ◽

2021 ◽

Vol 2021 ◽

pp. 1-16

Author(s):

Siyi Wu ◽

Zhao Li ◽

Mengling Ye ◽

Chunxia Liu ◽

Hao Liu ◽

...

Keyword(s):

Protective Effect ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

High Morbidity ◽

Barrier Dysfunction ◽

Caspase 1

Lung ischemia reperfusion injury (LIRI) is a complex pathophysiological process with high morbidity and mortality. An important pathophysiological characteristic of LIRI is endothelial barrier dysfunction, although the mechanism involved in this process remains unclear. VX765, a specific caspase-1 inhibitor, has been shown to have a protective effect against several diseases including sepsis, atherosclerosis, and glial inflammatory disease. The objective of this study was to determine whether VX765 had a protective effect in LIRI. The results showed that lung ischemia/reperfusion (I/R) and oxygen/glucose deprivation and reoxygenation (OGD/R) induced endothelial pyroptosis and barrier dysfunction characterized by an inflammatory response. Treatment with VX765 successfully alleviated I/R- and OGD/R-induced endothelial pyroptosis and barrier dysfunction by inhibiting caspase-1 in vivo and in vitro. In conclusion, these findings showed that VX765 provided effective protection against lung I/R-induced endothelial pyroptosis and barrier dysfunction.

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Xingnaojing Injection Protects against Cerebral Ischemia Reperfusion Injury via PI3K/Akt-Mediated eNOS Phosphorylation

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2018/2361046 ◽

2018 ◽

Vol 2018 ◽

pp. 1-13 ◽

Cited By ~ 5

Author(s):

Yue-Ming Zhang ◽

Xiao-Yu Qu ◽

Jing-Hui Zhai ◽

Li-Na Tao ◽

Huan Gao ◽

...

Keyword(s):

Cerebral Ischemia ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Underlying Mechanism ◽

Dapi Staining ◽

Pi3k Inhibitor Ly294002 ◽

Cerebral Ischemic

Xingnaojing (XNJ) injection, derived from traditional Chinese medicine formulation, has a protective effect against stroke, but the underlying mechanism is unclear, which severely limited its clinical application. This research aims to elucidate the role and mechanism of XNJ in reducing cerebral ischemic reperfusion (I/R) injury. Rats received 2 h cerebral ischemia followed by reperfusion of 24 h and were intraperitoneally given 5, 10, or 15 ml/kg XNJ 24 h before ischemia and at the onset of reperfusion, respectively. TTC staining, HE staining, and neurological score were implied to evaluate the effectiveness of XNJ. The protein expressions of PI3K/Akt and eNOS signaling were measured. Experiments were further performed in human brain microvascular endothelial cells (HBMECs) to investigate the protective mechanisms of XNJ. HBMECs were subjected to 3 h oxygen and glucose deprivation following 24 h of reoxygenation (OGD) to mimic cerebral I/R in vitro. PI3K inhibitor LY294002 was added with or without the preconditioning of XNJ. Multiple methods including western blot, immunofluorescence, DAPI staining, JC-1, and flow cytometry were carried out to evaluate the effect of XNJ on HBMECs. XNJ could improve rat cerebral ischemic injury and OGD induced HBMECs apoptosis. In vivo and in vitro researches indicated that the mechanism might be relevant to the activation of PI3K/Akt/eNOS signaling.

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Microglia-derived FGF21 as a modulator of astrocytic phenotype and cerebral ischemia injury

10.21203/rs.3.rs-15781/v1 ◽

2020 ◽

Author(s):

Fei Liu ◽

Dongxue Wang ◽

Liyun Zhu ◽

Jingting Du ◽

Ping Lin ◽

...

Keyword(s):

Cerebral Ischemia ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Cell Counting ◽

Fibroblast Growth Factor 21 ◽

Protective Effects ◽

Triphenyltetrazolium Chloride

Abstract Background: Fibroblast growth factor 21 (FGF21) is an important neuroprotective factor in the central nervous system (CNS), and it has been reported that FGF21 can protect against cerebral ischemia during the acute phase. However, the possible effects of FGF21 on ischemic brains and the interactions between FGF21 and nonneuronal cells have not been examined. Thus, the aim of this study was to elucidate the protective effects of endogenous FGF21 in ischemic brains.Methods: In this study, in vivo ischemia/reperfusion injury mouse model established by transient middle cerebral artery occlusion (MCAO)/reperfusion and in vitro cell models of oxygen/glucose deprivation (OGD)/reoxygenation (R) were used. Western blot analysis, RT-PCR, double immunofluorescence staining, immunohistochemistry, 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, neurobehavioral tests, cell counting kit-8 (CCK-8) assay and high-throughput gene sequencing were employed to explore the mechanism by which FGF21 unleash neuroprotective effort of astrocyte phenotype shifts in ischemic stroke.Results: We found that cortical FGF21 expression significantly increased after MCAO/reperfusion, peaking at 7 d. Ischemia-activated microglia were the main sources of endogenous FGF21 in brain tissue. However, FGF21 deficiency aggravated brain injury and slowed neurological functional recovery in FGF21 knockout mice. The in vitro and vivo studies revealed that FGF21 could activate astrocytes and mediate astrocytic phenotype. FGF21-activated astrocytes contributed to neuronal survival and synaptic protein upregulation after ischemia.Conclusion: Collectively, our data indicate that FGF21 plays vital roles in alleviating ischemic brain by mediating the manifestation of potentially pro-recovery astrocytic phenotypes. Therefore, modulation of FGF21 is a potential target strategy for stroke.

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Sphk1-induced Autophagy in Microglia Promotes Neuronal Injury Following Cerebral Ischemia-Reperfusion

10.21203/rs.3.rs-91557/v1 ◽

2020 ◽

Author(s):

Manhua Lv ◽

Yongjia Jiang ◽

Dayong Zhang ◽

Dan Yao ◽

Yuefeng Cheng ◽

...

Keyword(s):

Cerebral Ischemia ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Neuronal Injury ◽

Glucose Deprivation ◽

Enzyme Linked Immunosorbent Assay ◽

Cerebral Ischemia Reperfusion ◽

The Impact

Abstract Background: Microglial hyperactivation driven by SphK1/S1P signaling and consequent inflammatory mediator production is a key driver of cerebral ischemia-reperfusion injury (CIRI). While SphK1 reportedly controls autophagy and microglial activation, it remains uncertain as to whether it is similarly able to regulate damage mediated by CIRI-activated microglia. Methods: In the present study, we utilized both an in vitro oxygen-glucose deprivation reperfusion (OGDR) model and an in vivo rat model of focal CIRI to test whether Sphk1 and autophagy is expressed in microglia. Western blot analysis was used to estimate the autophagy protein level (LC3 and SQSTM ) at different time points after OGDR. To detect cytokine secretion in microglial supernatants in response to OGDR, we measured the concentration of IL-1β, IL-6 and TNF-α in the culture supernatants using an enzyme-linked immunosorbent assay (ELISA). To evaluate whether microglia subjected to OGDR exhibited neuronal injury, we used a commercially available terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) kit and flow cytometry to detect apoptotic neurons.Results: We determined that in the context of CIRI, microglia upregulated SphK1 and induced autophagy, while inhibiting these changes by lentivirus targeting SphK1 significantly decreased expression of autophagy . Moreover, we determined that autophagic body formation was enhanced in cerebral tissues following I/R. We also explored the impact of SphK1-induced autophagy on microglial inflammatory cytokine production and associated neuronal apoptosis using an in vitro OGDR model system. At a mechanistic level, we found that SphK1 promotes autophagy via the tumor necrosis factor receptor-associated factor 2 (TRAF2) pathway. Conclusion: These results reveal a novel mechanism whereby SphK1-induced autophagy in microglia can contribute to the pathogenesis of CIRI, potentially highlighting novel avenues for future therapeutic intervention in IS patients.

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Neurons can upregulate Cav-1 to increase intake of endothelial cells-derived extracellular vesicles that attenuate apoptosis via miR-1290

Cell Death and Disease ◽

10.1038/s41419-019-2100-5 ◽

2019 ◽

Vol 10 (12) ◽

Cited By ~ 8

Author(s):

Kang-Yi Yue ◽

Pei-Ran Zhang ◽

Min-Hua Zheng ◽

Xiu-Li Cao ◽

Yuan Cao ◽

...

Keyword(s):

Endothelial Cells ◽

Extracellular Vesicles ◽

Neuronal Apoptosis ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Cell Communication ◽

Glucose Deprivation ◽

Neuronal Protection

AbstractExtracellular vesicles (EVs) including exosomes can serve as mediators of cell–cell communication under physiological and pathological conditions. However, cargo molecules carried by EVs to exert their functions, as well as mechanisms for their regulated release and intake, have been poorly understood. In this study, we examined the effects of endothelial cells-derived EVs on neurons suffering from oxygen-glucose deprivation (OGD), which mimics neuronal ischemia-reperfusion injury in human diseases. In a human umbilical endothelial cell (HUVEC)–neuron coculture assay, we found that HUVECs reduced apoptosis of neurons under OGD, and this effect was compromised by GW4869, a blocker of exosome release. Purified EVs could be internalized by neurons and alleviate neuronal apoptosis under OGD. A miRNA, miR-1290, was highly enriched in HUVECs-derived EVs and was responsible for EV-mediated neuronal protection under OGD. Interestingly, we found that OGD enhanced intake of EVs by neurons cultured in vitro. We examined the expression of several potential receptors for EV intake and found that caveolin-1 (Cav-1) was upregulated in OGD-treated neurons and mice suffering from middle cerebral artery occlusion (MCAO). Knock-down of Cav-1 in neurons reduced EV intake, and canceled EV-mediated neuronal protection under OGD. HUVEC-derived EVs alleviated MCAO-induced neuronal apoptosis in vivo. These findings suggested that ischemia likely upregulates Cav-1 expression in neurons to increase EV intake, which protects neurons by attenuating apoptosis via miR-1290.

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RTN1-C mediates cerebral ischemia/reperfusion injury via modulating autophagy

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa162 ◽

2020 ◽

Author(s):

Jun Ling ◽

Haijian Cai ◽

Muya Lin ◽

Shunli Qi ◽

Jian Du ◽

...

Keyword(s):

Cerebral Ischemia ◽

Ischemia Reperfusion Injury ◽

Infarct Volume ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Neurological Deficits ◽

Cerebral Ischemia Reperfusion

Abstract It has been widely accepted that autophagic cell death exacerbates the progression of cerebral ischemia/reperfusion (I/R). Our previous study revealed that overexpression of reticulon protein 1-C (RTN1-C) is involved in cerebral I/R injury. However, the underlying mechanisms have not been studied intensively. This study was designed to evaluate the effect of RTN1-C on autophagy under cerebral I/R. Using an in vitro oxygen-glucose deprivation followed by reoxygenation and a transient middle cerebral artery occlusion model in rats, we found that the expression of RTN1-C protein was significantly upregulated. We also revealed that RTN1-C knockdown suppressed overactivated autophagy both in vivo and in vitro, as indicated by decreased expressions of autophagic proteins. The number of Beclin-1/propidium iodide-positive cells was significantly less in the LV-shRTN1-C group than in the LV-shNC group. In addition, rapamycin, an activator of autophagy, aggravated cerebral I/R injury. RTN1-C knockdown reduced brain infarct volume, improved neurological deficits, and attenuated cell vulnerability to cerebral I/R injury after rapamycin treatment. Taken together, our findings demonstrated that the modulation of autophagy from RTN1-C may play vital roles in cerebral I/R injury, providing a potential therapeutic treatment for ischemic brain injury.

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