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Effects of Txk‑mediated activation of NF‑κB signaling pathway on neurological deficit and oxidative stress after ischemia‑reperfusion in rats.

ABSTRACT: Ischemic stroke is an extremely mortal cerebrovascular disease, and neuroinflammation and oxidative stress emerge as important traits of ischemic stroke. However, as an inflammation‑associated factor, Txk tyrosine kinases (Txk) has been poorly studied in neuroscience research. The aim of the present study was to investigate the role of Txk after ischemia‑reperfusion (I/R) in vivo and in vitro, observe the association between Txk knockdown and neurological deficit and oxidative stress, and to explore whether the process was mediated by the activation of nuclear factor (NF)‑κB signaling pathway. Middle cerebral artery occlusion (MCAO), oxygen and glucose deprivation/reperfusion (OGD/R) model and western blotting have been used to simulate the I/R injury to analyze the expression, and to approximate the localization of Txk, respectively. Brain infarct volume, neurological score, brain water content, apoptosis and oxidative stress assays in vivo and apoptosis, cellular viability, the LDH release and oxidative stress assays in vitro were observed using a Txk‑knockdown lentivirus. Finally, NF‑κB overexpression lentivirus was applied to discuss whether the role of Txk following I/R was regulated by the NF‑κB signaling pathway. The results show that the Txk expression peaked at 24 h after MCAO and 6 h after OGD/R, respectively. Txk molecules gradually entered the nucleus after MCAO and OGD/R. The Txk‑knockdown lentivirus resulted in decreased brain infarct volume, neurological score, brain water content, apoptosis and oxidative stress after MCAO in vivo. Besides, Txk knockdown decreased apoptosis, LDH release, oxidative stress, and increased cellular viability, after ODG in vitro. Finally, NF‑κB overexpression reversed the process of neurological deficit and oxidative stress after Txk regulation in vivo and vitro. Overall, the present study suggests that Txk potentially regulates apoptosis, neurological deficit, and oxidative stress after I/R, by entering the nucleus. NF‑κB maybe the downstream target factor of Txk.

SUBMITTER: Xu QL

PROVIDER: S-EPMC8160475 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:Ischemic stroke-induced neuroinflammation is mainly mediated by microglial cells. The nuclear factor kappa B (NF-κB) pathway is the key transcriptional pathway that initiates inflammatory responses following cerebral ischemia. OTULIN, a critical negative regulator of the NF-κΒ signaling pathway, exerts robust effects on peripheral immune cell-mediated inflammation and is regarded as an essential mediator for repressing inflammation in vivo. The effect of OTULIN on inflammatory responses in the central nervous system (CNS) was previously unstudied. This current study investigated the anti-inflammatory effect of OTULIN both in vitro and in vivo in ischemic stroke models. METHODS:Sprague-Dawley (SD) rats were subjected to transient middle cerebral artery occlusion (tMCAO) or an intraperitoneal injection of lipopolysaccharide (LPS). Overexpression of the OTULIN gene was utilized to observe the effect of OTULIN on ischemic stroke outcomes. The effect of OTULIN overexpression on microglia-mediated neuroinflammation was examined in rat primary microglia (PM) and in the microglial cell line N9 after induction by oxygen-glucose deprivation (OGD)-treated neuronal medium. The activation and inflammatory responses of microglia were detected using immunofluorescence, ELISA, and qRT-PCR. The details of molecular mechanism were assessed using Western blotting. RESULTS:In the tMCAO rats, the focal cerebral ischemia/reperfusion injury induced a continuous increase in OTULIN expression within 72 h, and OTULIN expression was increased in activated microglial cells. OTULIN overexpression obviously decreased the cerebral infarct volume, improved the neurological function deficits, and reduced neuronal loss at 72 h after reperfusion, and it also inhibited the activation of microglia and attenuated the release of TNF-α, IL-1β, and IL-6 by suppressing the NF-κB pathway at 24 h after tMCAO. In vitro, OTULIN overexpression inhibited the microglia-mediated neuroinflammation by reducing the production of TNF-α, IL-1β, and IL-6 via depressing the NF-κB pathway in both PM and N9 cells. CONCLUSIONS:OTULIN provides a potential therapeutic target for ischemic brain injury by ameliorating the excessive activation of microglial cells and neuroinflammation through repressing the NF-κB signaling pathway.

Project description:Liver ischemia-reperfusion injury (IRI) is a common clinical event with high morbidity in patients undergoing complex liver surgery or having abdominal trauma. Inflammatory and oxidative stress responses are the main contributing factors in liver IRI. The iridoid glucoside aucubin (AU) has good anti-inflammatory and antioxidative effects; however, there are no relevant reports on the protective effect of glucosides on hepatic IRI. The purpose of this study was to determine whether AU pretreatment could prevent liver IRI and to explore the mechanism. Sprague-Dawley rats were randomly divided into five groups. The sham operation and IRI control groups were given intraperitoneal injections of normal saline, while the AU low-dose (AU-L) group, AU medium-dose (AU-M) group, and AU high-dose (AU-H) group were given intraperitoneal injections of AU at doses of 1, 5, and 10 mg/kg/day, respectively. After 10 d, liver IRI (70% liver ischemia for 1 h, reperfusion for 6 h) was surgically established in all groups except the sham group. Our results confirmed that liver injury was significantly aggravated after hepatic ischemia-reperfusion. AU alleviated the increase of transaminase and pathological changes induced by ischemia-reperfusion and improved liver damage. AU could also ameliorate the inflammatory and oxidative stress responses induced by ischemia-reperfusion and reduced expression of high mobility group protein (HMG)B1, receptor for advanced glycation end-products (RAGE), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and reactive oxygen species (ROS). Moreover, AU reduced ischemia-reperfusion-induced mitochondrial dysfunction and cells apoptosis, increased peroxisome proliferator-activated receptor γ coactivator (PGC)-1α and uncoupling (UCP)2 protein expression, and reduced caspase-3, cleaved caspase-3, and Cytochrome P450 proteins (CYP) expression. To determine expression levels of the Toll-like receptor (TLR)-4/nuclear factor-κB (NF-κB) pathway-related proteins in vitro and in vivo, we also measured TLR-4, myeloid differentiation factor88 (MyD88), NF-κB P65, p-P65, I-kappa-B-alpha (IκB-α), and p-IκB-α levels. The results showed that AU effectively inhibited activation of the TLR-4/NF-κB signaling pathway. In conclusion, we showed for the first time a hepatoprotective effect for AU in liver IRI, which acted by inhibiting the HMGB1/TLR-4/NF-κB signaling pathway, oxidative stress, and apoptosis. Pretreatment with AU may be a promising strategy for preventing liver IRI.

Project description:BackgroundThe NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome is an important mediator of neuroinflammatory responses that regulates inflammatory injury following cerebral ischemia and may be a potential target. Salidroside (Sal) has good anti-inflammatory effects; however, it remains unclear whether Sal can regulate NLRP3 inflammasome activation through the Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway after cerebral ischemia to alleviate inflammatory injury.MethodsWe established an oxygen-glucose deprivation and reoxygenation (OGD/R) model of BV2 cells and a middle cerebral artery occlusion/reperfusion (MCAO/R) rat model. Cell Counting Kit-8 (CCK-8), flow cytometry and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay were used to detect the viability and apoptosis of BV2 cells. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of inflammatory factors. 2,3,5-triphenyltetrazolium chloride (TTC) staining and modified Neurological Severity Score (mNSS) were used to detect cerebral infarction volume and neurological deficit in rats. Western blot, immunohistochemistry and immunofluorescence staining were used to detect the protein expression levels.ResultsOur results showed that Sal increased viability, inhibited lactate dehydrogenase (LDH) release, and reduced apoptosis in OGD/R-induced BV2 cells. Sal reduced the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-8. Following induction by OGD/R, BV2 cells exhibited NLRP3 inflammasome activation and increased protein levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, IL-1β, and IL-18. Protein levels of key TLR4 signaling pathway elements, such as TLR4, myeloid differentiation primary response 88 (MyD88), and phosphorylated nuclear factor kappa B p65 (p-NF-κB p65)/NF-κB p65 were upregulated. Interestingly, it was revealed that Sal could reverse these changes. In addition, TAK242, a specific inhibitor of TLR4, had the same effect as Sal treatment on BV2 cells following induction by OGD/R. In the MCAO/R rat model, Sal was also observed to inhibit NLRP3 inflammasome activation in microglia, reduce cerebral infarction volume, and inhibit apoptosis.ConclusionsIn summary, we found that Sal inhibited NLRP3 inflammasome activation and apoptosis in microglia induced by cerebral ischemia/reperfusion injury by inhibiting the TLR4/NF-κB signaling pathway, thus playing a protective role. Therefore, Sal may be a promising drug for the clinical treatment of ischemic stroke.

Project description:BackgroundCerebral ischemia (CI) can lead to ischemic stroke. The most effective therapy for cerebral ischemic stroke is the early restoration of blood reperfusion. However, reperfusion after CI can result in cerebral ischemia reperfusion (CI/R) injury. This study aimed to detect the effect of eriocitrin on cerebral I/R injury and investigate the underlying mechanism.MethodsSeventy male Sprague-Dawley (SD) rats were randomly divided into 5 groups: the control group, the cerebral I/R group, the I/R + eriocitrin 8 mg/kg group, the I/R + eriocitrin 16 mg/kg group, and the I/R + eriocitrin 32 mg/kg group. Different doses of eriocitrin or 0.5% carboxymethyl cellulose sodium were administrated to the rats once daily for 7 days before middle cerebral artery occlusion (MCAO). PCR staining was performed to observe cerebral infarction. Hematoxylin and eosin (H&E) staining was carried out to observe the damage to the brain tissue. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to detect apoptosis. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the relative mRNA levels of related molecules. Western blot was used to detect the expression of related proteins. The detection kits were used to detect superoxide dismutase (SOD) and lactic dehydrogenase (LDH) activity, and malondialdehyde (MDA) content respectively. Enzyme-linked immunosorbent assay (ELISA) was used to detect TNF-radiation, interleukin-6 (IL-6), and interleukin-10 (IL-10).ResultsThe results showed that Eriocitrin significantly reduced the cerebral infarct volume, cerebral water content, and cerebral indexes. Eriocitrin treatment alleviated pathological injury, promoted cell proliferation, and inhibited cell apoptosis. Eriocitrin upregulated SOD activity and downregulated MDA and LDH content. Eriocitrin also effectively decreased the levels of IL-6 and tumor necrosis factor-α (TNF-α), but increased the content of IL-10 in serum and brain tissues. Furthermore, Eriocitrin increased the phosphorylation of nuclear factor erythroid 2-related factor (Nrf2), as well as the expressions of heme-oxygenase-1 (HO-1) and quinine oxidoreductase 1 (NQO1). Moreover, Eriocitrin decreased the phosphorylation of nuclear factor-κB (NF-κB) p65.ConclusionsOur results indicated that Eriocitrin attenuated oxidative injury and inflammatory response in rats with CI/R via the Nrf2/HO-1/NQO1/NF-κB signaling pathway.

Project description:Ischemic stroke is a devastating disease with long-term disability. However, the pathogenesis is unclear and treatments are limited. Meisoindigo, a second-generation derivative of indirubin, has general water solubility and is well-tolerated. Previous studies have shown that meisoindigo reduces inflammation by inhibiting leukocyte chemotaxis and migration. In the present study, we investigated the hypothesis that meisoindigo was also protective against ischemic stroke, then evaluated its underlying mechanisms. In vivo, adult male C57BL/6J wild-type mice were used to produce a middle cerebral artery occlusion (MCAO) stroke model. On day three after reperfusion, obvious improvement in neurological scores, infarct volume reduction and cerebral edema amelioration were observed in meisoindigo treatment. Moreover, immunofluorescence staining and western-blot showed that the expression of NLRP3 inflammasome and its associated proteins in neurons and microglia was inhibited by meisoindigo. The effects of Meisoindigo on NLRP3 inflammasome inactivation and increased the M2 phenotype of microglia/macrophage through shifting from a M1 phenotype, which was possibly mediated by inhibition of TLR4/NF-κB. Furthermore, we verified the inhibitory effect of meisoindigo on TLR4/NF-κB signaling pathway, and found that meisoindigo treatment could significantly suppressed the expression of TLR4/NF-κB pathway-associated proteins in a dose-dependent manner, meanwhile, which resulted in downregulation of HMGB1 and IL-1β. Next, we established an in vitro oxygen glucose deprivation/Reperfusion (OGD/R) model in HT-22 and BV2 cells to simulate ischemic conditions. Cytotoxicity assay showed that meisoindigo substantially improved relative cell vitality and in HT-22 and BV2 cells following OGD/R in vitro. After suffering OGD/R, the TLR4/NF-κB pathway was activated, the expression of NLRP3 inflammasome-associated proteins and M1 microglia/macrophage were increased, but meisoindigo could inhibit above changes in both HT-22 and BV2 cells. Additionally, though lipopolysaccharide stimulated the activation of TLR4 signaling in OGD/R models, meisoindigo co-treatment markedly reversed the upregulation of TLR4 and following activation of NLRP3 inflammasome and polarization of M1 microglia/macrophages mediated by TLR4. Overall, we demonstrate for the first time that meisoindigo post-treatment alleviates brain damage induced by ischemic stroke in vivo and in vitro experiments through blocking activation of the NLRP3 inflammasome and regulating the polarization of microglia/macrophages via inhibition of the TLR4/NF-κB signaling pathway.

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Effects of Txk‑mediated activation of NF‑κB signaling pathway on neurological deficit and oxidative stress after ischemia‑reperfusion in rats.

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