Reduced Lactate Dehydrogenase Release Research Articles

Protective role of lidocaine against cerebral ischemia-reperfusion injury: An in vitro study.

Lidocaine, a local anesthetic, is a valuable agent for the treatment of neuronal ischemia/reperfusion (I/R) injury. The aim of the present study was to investigate the role of lidocaine in oxygen-glucose deprivation/reperfusion (OGD/R)-induced cortical neurons and explore the related molecular mechanisms. Cerebral cortical neurons were isolated from Sprague-Dawley rat embryos and stimulated with OGD/R to establish an in vitro I/R injury model. Subsequently, neuronal cell viability, cytotoxicity and apoptosis were evaluated by performing the MTT assay, lactate dehydrogenase (LDH) assay and flow cytometry, respectively. The results suggested that OGD/R exposure significantly decreased cerebral cortical neuron cell viability, accelerated LDH release and induced cell apoptosis compared with control neurons, indicating that cerebral I/R injury was stimulated by OGD/R treatment. Further investigation indicated that 10 µM lidocaine significantly enhanced neuronal cell viability, and reduced LDH release and neuronal cell apoptosis in OGD/R-exposed cells compared with the OGD/R + saline group, which indicated that lidocaine displayed neuroprotective effects against I/R damage. In addition, the findings of the present study suggested that OGD/R exposure significantly decreased Bcl-2 and Bcl-xl protein expression levels, but increased Bax protein expression levels, the Bax/Bcl-2 ratio and caspase-3 activity compared with control neurons. However, lidocaine reversed OGD/R-mediated alterations to apoptosis-related protein expression. Furthermore, the results of the present study indicated that lidocaine increased Wnt3a, β-catenin and cyclin D1 expression levels in OGD/R-exposed cells compared with the OGD/R + saline group, thus activating the Wnt/β-catenin signaling pathway. The findings of the present study suggested that lidocaine served a protective role in OGD/R-triggered neuronal damage by activating the Wnt/β-catenin signaling pathway; therefore, lidocaine may serve as a potential candidate for the treatment of cerebral I/R injury.

Regulatory role of miR-129 and miR-384-5p on apoptosis induced by oxygen and glucose deprivation in PC12 cell.

This study aimed to establish the role of miR-129 and miR-384-5p in cerebral ischemia-induced apoptosis. Using PC12 cells transfected with miR-129 or miR-384-5p mimics or inhibitors, oxygen glucose deprivation (OGD) conditions were applied for 4h to simulate transient cerebral ischemia. Apoptotic phenotypes were assessed via lactate dehydrogenase (LDH) assay, MTT cell metabolism assay, and fluorescence-activatedcell sorting(FACS). The effect of miR overexpression and inhibition was evaluated by protein and mRNA detection of bcl-2 and caspase-3, critical apoptosis factors. Finally, the direct relationship of miR-129 and bcl-2 and miR-384-5p and caspase-3 was measured by luciferase reporter assay. The overexpression of miR-384-5p and miR-129 deficiency significantly enhanced cell viability, reduced LDH release, and inhibited apoptosis. By contrast, overexpression of miR-129 and miR-384-5p deficiency aggravated hypoxia-induced apoptosis and cell injury. miR-129 overexpression significantly reduced mRNA and protein levels of bcl-2 and miR-129 inhibition significantly increased mRNA and protein levels of bcl-2 in hypoxic cells.miR-384-5p overexpression significantly reduced protein levels of caspase-3 while miR-384-5p deficiency significantly increased protein levels of caspase-3. However, no changes were observed in caspase-3 mRNA in either transfection paradigm. Finally, luciferase reporter assay confirmed caspase-3 to be a direct target of miR-384-5p; however, no binding activity was detected between bcl-2 and miR-129.Transient cerebral ischemia induces differential expression of miR-129 and miR-384-5p which influences apoptosis by regulating apoptotic factors caspase-3 and bcl-2, thereby participating in the pathological mechanism of cerebral ischemia, and becoming potential targets for the treatment of ischemic cerebral injury in the future.

Knockdown of long noncoding RNA growth arrest-specific transcript 5 regulates forkhead box O3 to inhibit lipopolysaccharide-induced human bronchial epithelial cell pyroptosis.

Pyroptosis is a novel proinflammatory programmed cell death process. This study was designed to investigate the functional mechanisms of long noncoding RNA growth arrest-specific transcript 5 (lncRNA GAS5) on lipopolysaccharide (LPS)-induced human bronchial epithelial cell (HBEC) pyroptosis. LPS was used to induce pyroptosis in HBECs, followed by the detection of the expression of GAS5, forkhead box O3 (FOXO3), and nuclear factor E2-related factor 2/heme oxygenase 1 (Nrf2/HO-1) signaling pathway-related factors. Cell viability was evaluated using CCK-8 assay, lactate dehydrogenase (LDH) release was assessed by LDH assay kit and caspase-1 activity by flow cytometry. Furthermore, expression of NOD-like receptor family pyrin domain containing 3 and pyroptosis-related proteins was evaluated using Western blot analysis, while enzyme-linked immunosorbent assay was used to determine the levels of inflammatory factors. The interaction between GAS5 and FOXO3 was confirmed using bioinformatic prediction, RNA immunoprecipitation assay, RNA pull-down, and dual-luciferase reporter gene assay. Treatment of HBECs with LPS upregulated the expression of GAS5 and FOXO3, resulting in the inactivation of the Nrf2/HO-1 signaling pathway. On the other hand, inhibition of both GAS5 and FOXO3 promoted cell viability, reduced LDH release, pyroptosis, and inflammatory response in LPS-induced HBECs. Furthermore, FOXO3 could interact with GAS5, while FOXO3 overexpression reversed the inhibitory effect of GAS5 knockdown on cell pyroptosis. Thus, mechanistically, inhibition of FOXO3 activates the Nrf2/HO-1 pathway to suppress LPS-induced pyroptosis in HBECs. This study revealed that GAS5 knockdown attenuates FOXO3 expression thereby activating the Nrf2/HO-1 pathway to inhibit LPS-induced pyroptosis in HBECs. These findings may contribute to identifying novel targets that inhibit pyroptosis in HBECs.

Albumin Might Attenuate Bacteria-Induced Damage on Kupffer Cells for Patients with Chronic Liver Disease.

Chronic liver diseases (CLDs) are complex diseases that cause long-term inflammation and infection, which in turn accelerate their development. The usage of albumin in patients with CLDs has been debated for years. Human serum albumin (HSA) plays a key role in immunomodulation during the process of CLDs. The correlation between albumin and C-reactive protein (CRP) in CLD patients was analyzed by linear regression with the Pearson statistic. The damage of THP-1 and primary cells was evaluated by measuring the lactate dehydrogenase (LDH) in the supernatant. Immunofluorescence staining was performed to determine underlying pathways in Kupffer cells (KCs). Albumin negatively correlated with infection in patients with CLDs. In vitro experiments with THP-1 cells and KCs showed that albumin reduced LDH release after stimulation with bacterial products, while no differences in hepatic stellate cells (HSCs) and sinusoidal endothelial cells (SECs) were detected. Moreover, immunofluorescence staining revealed an increase of p-ERK and p-NF-kB p65 density after albumin treatment of KCs stimulated by bacterial products. In conclusion, albumin could assist CLD patients in alleviating inflammation caused by bacterial products and might be beneficial to patients with CLDs by securing KCs from bacteria-induced damage, providing a compelling rationale for albumin therapy in patients with CLDs.

Polypeptide Globular Adiponectin Ameliorates Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury by Inhibiting Both Apoptosis and Necroptosis.

Adiponectin is a small peptide secreted and a key component of the endocrine system and immune system. Although globular adiponectin protects myocardial ischemia/reperfusion-induced cardiomyocyte injury, the protective mechanisms remain largely unresolved. Using a neonatal rat ventricular myocyte hypoxia/reoxygenation model, we investigated the role of its potential mechanisms of necroptosis in globular adiponectin-mediated protection in hypoxia/reoxygenation-induced cardiomyocyte injury as compared to apoptosis. We found that globular adiponectin treatment attenuated cardiomyocyte injury as indicated by increased cell viability and reduced lactate dehydrogenase release following hypoxia/reoxygenation. Immunofluorescence staining and Western blotting demonstrated that both necroptosis and apoptosis were triggered by hypoxia/reoxygenation and diminished by globular adiponectin. Necrostatin-1 (RIP1-specific inhibitor) and Z-VAD-FMK (pan-caspase inhibitor) only mimicked the inhibition of necroptosis and apoptosis, respectively, by globular adiponectin in hypoxia/reoxygenation-treated cardiomyocytes. Globular adiponectin attenuated reactive oxygen species production, oxidative damage, and p38MAPK and NF-κB signaling, all important for necroptosis and apoptosis. Collectively, our study suggests that globular adiponectin inhibits hypoxia/reoxygenation-induced necroptosis and apoptosis in cardiomyocytes probably by reducing oxidative stress and interrupting p38MAPK signaling.

Toxicogenicity and mechanistic pathways of aflatoxin B1 induced renal injury.

The study investigated the toxicogenic effects, molecular mechanisms and proteomic assessment of aflatoxin B1 (AFB1 ) on human renal cells. Hek293 cells were exposed to AFB1 (0-100 μM) for 24 h. The effect on cell viability was assessed using the methylthiazol tetrazolium (MTT) assay, which also produced the half maximal inhibitory concentration (IC50 ) used in subsequent assays. Free radical production was evaluated by quantifying malondialdehyde (MDA) and nitrate concentration, while DNA fragmentation was determined using the single cell gel electrophoresis (SCGE) assay and DNA gel electrophoresis. Damage to cell membranes was ascertained using the lactate dehydrogenase (LDH) assay. The concentration of ATP, reduced glutathione (GSH), necrosis, annexin V and caspase activity was measured by luminometry. Western blotting and quantitative PCR was used to assess the expression of proteins and genes associated with apoptosis and oxidative stress. The MTT assay revealed a reduction in cell viability of Hek293 cells as the AFB1 concentration was increased, with a half maximum inhibitory concentration (IC50 ) of 32.60 μM. The decreased viability corresponded to decreased ATP concentration. The upregulation of Hsp70 indicated that oxidative stress was induced in the AFB1 -treated cells. While this implies an increased production of free radicals, the accompanying upregulation of the antioxidant system indicates the activation of defense mechanisms to prevent cellular damage. Thus, membrane damage associated with increased radical formation was prevented as indicated by the reduced LDH release and necrosis. In addition, cytotoxic effects were evident as AFB1 activated the intrinsic pathway of apoptosis with corresponding increased DNA fragmentation, p53 and Bax upregulation and increased caspase activity, but externalization of phosphatidylserine (PS), a major hallmark of apoptosis, did not occur in AFB1 treated renal cells. The results suggest that AFB1 induced oxidative stress leading to cell death by the intrinsic pathway of apoptosis in renal cells.

Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes.

Dysregulation of long non-coding RNA (IncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is associated with the risk of myocardial infarction (MI). Therefore, the present study aimed to determine the mechanisms underlying this association, which is currently poorly understood, to the best of our knowledge. The current study used an in vitro myocardial ischemia and reperfusion (MI/R) model, in which H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R), which demonstrated that ANRIL expression was downregulated and that ANRIL positively regulated sirtuin 1 (SIRT1) expression following H/R injury. Subsequently, it was demonstrated that ANRIL upregulated SIRT1 expression by sponging microRNA-181a (miR-181a). In addition, ANRIL overexpression reduced lactate dehydrogenase release and apoptosis of H9c2 cardiomyocytes exposed to H/R, indicating that ANRIL prevented H/R-induced cardiomyocyte injury. Moreover, both miR-181a overexpression and SIRT1 knockdown significantly decreased the protective effects of ANRIL on H/R-induced cardiomyocyte injury, thus demonstrating that SIRT1 upregulation via sponging miR-181a is a critical mechanism that mediates the function of ANRIL. These results provided a novel mechanistic insight into the role of ANRIL in H/R-injured cardiomyocytes and suggested that the ANRIL/miR-181a/SIRT1 axis may be a therapeutic target for reducing MI/R injury.

Luteolin has a significant protective effect against cadmium-induced injury in lung epithelial Beas-2B cells

To investigate the protective effect of luteolin against cadmium (Cd)-induced injury in human lung epithelial Beas-2B cells. Beas-2B cells were treated with different concentrations of luteolin (0-160 μmol/L) or Cd (0-40 μmol/L) for 24 h, and the cell viability was examined using MTT assay. After treatment with luteolin (0.25, 0.5 and 0.75 μmol/L) with or without Cd (5 μmol/L) for 24 h, the cells were examined for viability, lactate dehydrogenase (LDH) activity and morphological changes of the cell nuclei using Hoechst fluorescent staining. The levels of ROS, SOD, GSH and MDA in the treated cells were detected, and the expression levels of Akt, p-Akt and nuclear factor E2-related factor 2 (Nrf2) proteins were determined using Western blotting. Luteolin within the concentration range of 0-80 μmol/L did not significantly affect the survival rate of Beas-2B cells (P>0.05), but Cd at 5 μmol/L significantly decreased the cell viability (P < 0.05) with an IC50 of 24.6 μmol/L. In Cd-treated cells, treatment with luteolin significantly mitigated the decrease of cell viability, reduced LDH release and cell apoptosis, enhanced SOD activity and GSH content, and inhibited the production of MDA and ROS (all P < 0.05). Luteolin also significantly up-regulated the expression levels of p-Akt and Nrf2 protein in Cd-treated Beas-2B cells (P < 0.05). Luteolin has a significant protective effect against Cd-induced injury in Beas-2B cells, and the effects are probably mediated, at least in part, by promoting the activation of Akt and Nrf2.

MiR-195-5p Ameliorates Cerebral Ischemia-Reperfusion Injury by Regulating the PTEN-AKT Signaling Pathway

BackgroundMiR-195-5p has been shown to play crucial roles in tumor inhibition, but its biological functions in cerebral ischemia-reperfusion (I/R) injury are unclear.MethodsTo mimic cerebral I/R injury, mice were induced by transient middle cerebral artery occlusion (MCAO). Human brain microvascular endothelial cells (HBMVECs) were treated with oxygen-glucose deprivation (OGD) to mimic I/R injury in vitro. The expression of miR-195-5p and PTEN was detected by qRT-PCR or Western blot. Cell viability was evaluated by CCK-8 assay. Cell apoptosis was detected by flow cytometer. Cell death was detected using specific lactate dehydrogenase (LDH) cytotoxicity kit. Infarct volume in mice brains was evaluated by TTC staining. Histopathological analysis was performed by HE staining and TUNEL staining. The interaction between miR-195-5p and PTEN was determined by TargetScan and luciferase reporter assay.ResultsMiR-195-5p was significantly downregulated and PTEN was upregulated during cerebral I/R injury both in vitro and in vivo. Overexpression of miR-195-5p efficiently enhanced cell viability, while reduced LDH release and apoptotic rate of OGD-treated HBMVECs in vitro. MiR-195-5p could negatively regulate the expression of PTEN by directly binding to its 3′-UTR. Overexpression of PTEN obviously attenuated the protective effect of miR-195-5p mimics on cell viability, LDH release and apoptosis in OGD-treated HBMVECs. Meanwhile, overexpression of miR-195-5p increased the expression levels of p-AKT in OGD-treated HBMVECs, while this effect was reversed by overexpression of PTEN. Moreover, overexpression of miR-195-5p efficiently ameliorated brain injury of mice after MCAO treatment in vivo.ConclusionOverexpression of miR-195-5p ameliorated cerebral I/R injury by regulating the PTEN-AKT signaling pathway, providing a potential therapeutic target for cerebral I/R injury.

MESENCHYMAL STROMAL CELLS PROTECT AGAINST VASCULAR DAMAGE AND DEPRESSION-LIKE BEHAVIOR IN MICE SURVIVING CEREBRAL MALARIA

<h3>Introduction</h3> Cerebral malaria (CM) is a severe systemic inflammatory disease, that leads to behavioral impairments, such as depression. Currently, there are no therapies to treat the neurological sequelae of CM, and mesenchymal stromal cells (MSC) may be an alternative. <h3>Objectives</h3> To investigate the effects of MSC therapy on the blood-brain barrier (BBB), leukocyte rolling and adherence in the brain, and depression like-behavior in experimental CM. <h3>Methods</h3> Male C57BL/6 mice were infected with Plasmodium berghei ANKA (PbA, 1 × 106, intraperitoneally). At day 6, PbA-infected animals received chloroquine (25 mg/kg orally for seven days) and were then randomized to receive MSC (105 cells) or saline intravenously. All animal experiments were approved by the ethics committee of the Oswaldo Cruz Institute (L-012/2015), and had a number of 3 to 12 animals per group. In vitro, endothelial cells were used to evaluate the effects of conditioned media derived from MSC (CMMSC) on cell viability by lactate dehydrogenase (LDH) release. We used a one-way analysis of variance with post-hoc Tukey test to assess the differences between more than two groups and Student‘s t test when data from only two groups were analyzed. <h3>Results</h3> MSC therapy improved the vascular damage at 7 days after infection (dpi). Evans blue assay and intravital microscopy showed that infected mice, who received only saline, had an increase in BBB permeability (n=7; 0.0084±0.0016) and adherent leukocytes (n=3; 3.76±0.23). PbA+MSC mice had reduced BBB permeability (n=4; 0.0063±0.0012) and leukocyte adhesion in the brain microvasculature (n=4; 2.18±0.37). PbA-infected mice presented reduced levels of BDNF 7 dpi (n=4; 0.025±0.0015), and depression-like behavior 15 dpi evaluated by the tail suspension test (TST, n=5; 44.38±4) and the forced swim test (FST, n=5; 45.5±4.8). MSC therapy restored BDNF levels (n=11; 0.029±0.0008) and mitigated behavioral alterations (n=5; TST:18±2.7; FST:18.2±2.8). CMMSC reduced LDH release in endothelial cells stimulated with heme (group heme: 1,89±0,11; group CMMSC: 0,96±0,06), suggesting a paracrine mechanism of action. <h3>Conclusion</h3> A single dose of MSC as an adjuvant therapy protected against vascular damage and improved depression-like behavior in mice that survived experimental CM.

Protection of the neurovascular unit from calcium-related ischemic injury by linalyl acetate.

Calcium-related ischemic injury (CRII) can damage cells of the neurovascular unit (NVU). Here, we investigate the protective effects of linalyl acetate (LA) against CRII-induced NVU damage and evaluate the underlying mechanisms. The protective effects of LA in cell lines representative of NVU components (BEND, SH-SY5Y, BV2, and U373 cells) were evaluated following exposure to oxygen-glucose deprivation/reoxygenation alone (OGD/R-only) or OGD/R in the presence of 5 mM extracellular calcium ([Ca2+]o) to mimic CRII. LA reversed damage under OGD/R-only conditions by blocking p47phox/NADPH oxidase (NOX) 2 expression, reactive oxygen species (ROS) production, nitric oxide (NO) abnormality, and lactate dehydrogenase (LDH) release only in the BEND cells. However, under CRII-mimicking conditions, LA reversed NO abnormality and matrix metalloproteinase (MMP)-9 activation in the BEND murine brain endothelial cells; inhibited p47phox expression in the human SH-SY5Y neural-like cells; decreased NOX2 expression and ROS generation in the BV2 murine microglial cells; and reduced p47phox expression in the U373 human astrocyte-like cells. Importantly, LA protected against impairment of the neural cells, astrocytes, and microglia, all of which are cellular components of the NVU induced by exposure to CRII-mimicking conditions, by reducing LDH release. We found that LA exerted a protective effect in the BEND cells that may differ from its protective effects in other NVU cell types, following OGD/R-induced damage in the context of elevated [Ca2+]o.

MiR-129-5p Protects H9c2 Cardiac Myoblasts From Hypoxia/Reoxygenation Injury by Targeting TRPM7 and Inhibiting NLRP3 Inflammasome Activation.

As a biomarker for heart failure, miR-129-5p is abnormally expressed during myocardial I/R, but its specific functions and mechanisms remain largely unclear. Thus, this study explored the roles and possible mechanisms of miR-129-5p in hypoxia/reoxygenation (H/R)-insulted H9c2 cardiac myoblasts. After H/R insult, miR-129-5p expression levels were decreased, along with reduced cell viability and enhanced lactate dehydrogenase release in H9c2 cells. Overexpression of miR-129-5p through transfection of miR-129-5p mimics effectively improved cell viability and reduced lactate dehydrogenase release in H9c2 cells exposed to H/R, along with decreased apoptosis and caspase-3 activities. Moreover, miR-129-5p mimics inhibited reactive oxygen species production and upsurged superoxide dismutase activity in H9c2 cells exposed to H/R, and suppressed H/R-caused massive release of proinflammatory cytokines TNF-α and IL-1β. TRPM7 was identified as the target of miR-129-5p and was negatively regulated by miR-129-5p. TRPM7 overexpression counteracted the antagonistic effect of miR-129-5p on H/R-induced increase in intracellular calcium levels. TRPM7 overexpression also abolished miR-129-5p-induced elevation on cell viability and reduction on apoptosis as well as attenuated miR-129-5p-induced inhibition on reactive oxygen species and IL-1β production. Besides, H/R-induced NLRP3 inflammasome activation was inhibited by miR-129-5p mimic but reactivated by TRPM7. In conclusion, miR-129-5p alleviates H/R injury of H9c2 cardiomyocytes by targeting TRPM7 and inhibiting NLRP3 inflammasome activation, suggesting that miR-129-5p and TRPM7 may be potential therapeutic targets for myocardial I/R injury.

SCO-spondin-derived Peptide Protects Neurons from Glutamate-induced Excitotoxicity

Subcommissural organ (SCO)-spondin is a brain-specific glycoprotein produced during embryogenesis, that strongly contributes to neuronal development. The SCO becomes atrophic in adults, halting SCO-spondin production and its neuroprotective functions. Using rat and human neuronal cultures, we evaluated the neuroprotective effect of an innovative peptide derived from SCO-spondin against glutamate excitotoxicity. Primary neurons were exposed to glutamate and treated with the linear (NX210) and cyclic (NX210c) forms of the peptide. Neuronal survival and neurite networks were assessed using immunohistochemistry or biochemistry. The mechanism of action of both peptide forms was investigated by exposing neurons to inhibitors targeting receptors and intracellular mediators that trigger apoptosis, neuronal survival, or neurite growth. NX210c promoted neuronal survival and prevented neurite network retraction in rat cortical and hippocampal neurons, whereas NX210 was efficient only in neuronal survival (cortical neurons) or neurite networks (hippocampal neurons). They triggered neuroprotection via integrin receptors and γ-secretase substrate(s), activation of the PI3K/mTOR pathway and disruption of the apoptotic cascade. The neuroprotective effect of NX210c was confirmed in human cortical neurons via the reduction of lactate dehydrogenase release and recovery of normal basal levels of apoptotic cells. Together, these results show that NX210 and NX210c protect against glutamate neurotoxicity through common and distinct mechanisms of action and that, most often, NX210c is more efficient than NX210. Proof of concept in central nervous system animal models are under investigation to evaluate the neuroprotective action of SCO-spondin-derived peptide.

ADENOSINE A3 RECEPTOR INTERACTS WITH GASDERMIN D TO MODULATE INTESTINAL EPITHELIAL CELL PYROPTOSIS IN ULCERATIVE COLITIS

Abstract Background Adenosine A3 receptor (A3AR) plays a role in intestinal inflammation, but little is known about its mechnisms in intestinal inflammation such as ulcerative colitis (UC). Pyroptosis, characterized by Gasdermin D (GSDMD) activation, is implicated in the pathogenesis of UC. We investigated the role of A3AR in GSDMD-mediated pyroptosis in UC and its underlying molecular mechanisms. Methods The expression of A3AR in colonic mucosa of patients with UC were examined. A3AR agonist was used to study the role of A3AR in ex vivo colonic explants of UC patients. In addition, human intestinal epithelial cells Caco-2 were used to further verify the effect of A3AR on pyroptosis induced by LPS+ATP. RT-qPCR and western blotting were used to detect the expression levels of pyroptosis-associated factors including NLRP3, caspase-1, gasdermin-D N-terminal domain (GSDMD-NT), IL-1β and IL-18 in colonic tissues and Caco-2 cells. Immunofluorescence was used to detect the protein expression in tissues and cells. Enzyme-linked immunosorbent assay was used to determine the levels of IL-1β and IL-18 in tissue and cell culture supernatants. Lactate dehydrogenase (LDH) release assay and propidium iodide (PI) staining were used to measure cell pyroptosis. Molecular interactions beween A3AR and GSDMD were investigated using Co-IP and GST pull-down assays. Results A3AR expression was significantly reduced in colonic mucosa of patients with active UC, and colonic epithelial cells were the main cell subpopulation with down-regulated A3AR expression. Pyroptosis-associated factors, including Caspase-1, NLRP3, and GSDMD-NT, were upregulated in UC colonic tissues. The expression of A3AR and GSDMD-NT was negatively correlated. A3AR agonist reduced the production of cytokines (IL-1β and IL-18) and attenuates the expression levels of NLRP3 and GSDMD-NT in the colonic tissues of patients with UC. Furthermore, A3AR overexpression alleviated pyroptosis with reduced LDH release, PI-stained cell number and decreased expressions of GSDMD-NT, NLRP3, caspase-1, IL-1β, and IL-18 in the LPS+ATP-stimulated Caco-2 cells, whereas the opposite occurred in cells treated with small interfeing RNA (siRNA) targeting A3AR. Knockdown of GSDMD in Caco-2 cells significantly blocked the effects of A3AR overexpression or down-regulation on pyroptosis, suggesting that A3AR acts through the GSDMD-mediated pyroptosis pathway. Co-IP and GST pull-down assays showed that A3AR interacted with GSDMD. Conclusion A3AR modulates intestinal inflammation in UC through GSDMD-mediated intestinal epithelial cell pyroptosis. We propose a novel mechanism by which A3AR-GSDMD interaction affects UC through pyroptosis, suggesting that A3AR is a potential target for the treatment of UC.

Ac-FLTD-CMK inhibits pyroptosis and exerts neuroprotective effect in a mice model of traumatic brain injury.

Pyroptosis has been reported to contribute to the traumatic brain injury (TBI) process. Ac-FLTD-CMK is a newly synthesized pyroptosis inhibitor. However, whether Ac-FLTD-CMK inhibits pyroptosis and plays a neuroprotective role after TBI is unknown. The present study aimed to determine the effects of Ac-FLTD-CMK on TBI in a mouse model. Male C57BL/6 mice were randomly divided into sham, TBI + vehicle, and TBI + Ac-FLTD-CMK groups. TBI was induced using a weight-drop apparatus. Intraventricular injection of Ac-FLTD-CMK was performed 30 min after TBI. Caspase-1, caspase-11, gasdermin-D (GSDMD), and caspase-3 expression in the peri-contusional cortex were assessed by western blotting. Interleukin-1β (IL-1β) and interleukin-18 (IL-18) expression in the peri-contusional cortex were measured using ELISA. Behavioral experiments, brain water content, Evans blue extravasation, lactate dehydrogenase (LDH) release, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling staining were also performed. The results showed that Ac-FLTD-CMK administration significantly downregulated caspase-1 p20, caspase-11 p20, GSDMD N-terminal, IL-1β, and IL-18 expression; reduced LDH release; alleviated neuronal death; attenuated brain edema and blood-brain barrier damage; and improved neurobehavioral function. These findings indicate that Ac-FLTD-CMK treatment suppresses pyroptosis and protects mice against TBI.

Cardioprotective Effect of Echinatin Against Ischemia/Reperfusion Injury: Involvement of Hippo/Yes-Associated Protein Signaling.

Background: Echinatin (Ech) has been reported to exert antioxidant and anti-inflammatory activities. In this study, we aimed to characterize the functional role of Ech in myocardial ischemic/reperfusion (MI/R) injury and elucidate its underlying mechanism of action. Method: We established in vivo and in vitro models of MI/R injury to determine the effect of Ech on MI/R injury. Gene expression was examined using quantitative real-time polymerase chain reaction and western blotting. Myocardial infarction was assessed using tetrazolium chloride staining and the degree of myocardial injury was evaluated by measuring lactate dehydrogenase (LDH) and creatine kinase-myocardial band (CK-MB) levels. Cell apoptosis was detected using the terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling (TUNEL) assay. The viability of H9c2 cells was determined using Cell Counting Kit-8 assay. Results: MI/R induced myocardial infarction, which was mitigated by Ech treatment. Moreover, Ech treatment resulted in a marked decline of LDH and CK-MB levels in the serum and myocardium of MI/R rats. Ech treatment also restrained cardiomyocyte apoptosis in vivo and in vitro, as evidenced by reduction in LDH release, the number of TUNEL-positive cells, and caspase-3 activity. Furthermore, Ech administration inhibited MI/R-induced activation of Hippo/Yes-associated protein signaling in vivo and in vitro, as indicated by inhibition of mammalian sterile 20-like protein kinase 1, large tumor suppressor one, and YAP phosphorylation and promotion of YAP nuclear translocation. However, silencing of YAP counteracted the protective effect of Ech on hypoxia/reoxygenation-induced myocardial injury in vitro. Conclusion: Ech exerted its protective effect against MI/R injury at least partially by suppressing the Hippo/YAP signaling pathway, providing novel insights into the remission of MI/R injury.

Norwogonin attenuates hypoxia-induced oxidative stress and apoptosis in PC12 cells

BackgroundNorwogonin is a natural flavone with three phenolic hydroxyl groups in skeletal structure and has excellent antioxidant activity. However, the neuroprotective effect of norwogonin remains unclear. Here, we investigated the protective capacity of norwogonin against oxidative damage elicited by hypoxia in PC12 cells.MethodsThe cell viability and apoptosis were examined by MTT assay and Annexin V-FITC/PI staining, respectively. Reactive oxygen species (ROS) content was measured using DCFH-DA assay. Lactate dehydrogenase (LDH), malondialdehyde (MDA) and antioxidant enzyme levels were determined using commercial kits. The expression of related genes and proteins was measured by real-time quantitative PCR and Western blotting, respectively.ResultsWe found that norwogonin alleviated hypoxia-induced injury in PC12 cells by increasing the cell viability, reducing LDH release, and ameliorating the changes of cell morphology. Norwogonin also acted as an antioxidant by scavenging ROS, reducing MDA production, maintaining the activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx), and decreasing the expression levels of HIF-1α and VEGF. In addition, norwogonin prevented cell apoptosis via inhibiting the expression levels of caspase-3, cytochrome c and Bax, while increasing the expression levels of Bcl-2 and the ratio of Bcl-2/Bax.ConclusionsNorwogonin attenuates hypoxia-induced injury in PC12 cells by quenching ROS, maintaining the activities of antioxidant enzymes, and inhibiting mitochondrial apoptosis pathway.

Physiological Oxygen Tension Enhances Competence and Functional Properties of Murine Cardiac Mesenchymal Cells.

Cardiac mesenchymal cells (CMCs), a newly-discovered and promising type of progenitor cells, are effective in improving cardiac function in rodents after myocardial infarction. Stem/progenitor cells are usually cultured at atmospheric O2 tension (21%); however, the physiologic O2 tension in the heart is ~5%, raising the concern that 21% O2 may cause toxicity due to oxidative stress. Thus, we compared mouse CMCs cultured at 21% or 5% O2 beginning at passage 2. At passage 5, CMCs underwent severe hypoxic stress (1% O2 for 24 h). Compared with CMCs cultured at 21% O2, culture at 5% O2 consistently improved cell morphology throughout 5 passages, markedly decreased cell size, increased cell number, shortened cell doubling time, and dramatically reduced lactate dehydrogenase release from CMCs into culture media after hypoxic stress. Furthermore, culture at 5% O2 increased telomerase activity and telomere length, implying that 21% O2 tension impairs telomerase activity, resulting in telomere shortening and decreased cell proliferation. Thus far, almost all preclinical and clinical studies of cell therapy for the heart disease have used atmospheric (21%) O2 to culture cells. Our data challenge this paradigm. Our results demonstrate that, compared with 21% O2, 5% O2 tension greatly enhances the competence and functional properties of CMCs. The increased proliferation rate at 5% O2 means that target numbers of CMCs can be achieved with much less time and cost. Furthermore, since this increased proliferation may continue in vivo after CMC transplantation, and since cells grown at 5% O2 are markedly resistant to severe hypoxic stress, and thus may be better able to survive after transplantation into scarred regions of the heart where O2 is very low, culture at 5% O2 may enhance the reparative properties of CMCs (and possibly other cell types). In conclusion, our data support a change in the methods used to culture CMCs and possibly other progenitor cells.

Sestrin2 is involved in the Nrf2-regulated antioxidative signaling pathway in luteolin-induced prevention of the diabetic rat heart from ischemia/reperfusion injury.

Luteolin attenuates myocardial ischemia/reperfusion (I/R) injury in diabetes through activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidative response. Though sestrin2, a highly conserved stress-inducible protein, is regarded as a modulator of Nrf2 and reduces I/R injury, the effect of sestrin2 on luteolin-induced prevention of the diabetic heart from I/R injury remains unclear. We hypothesized that luteolin could relieve myocardial I/R injury in diabetes by activating the sestrin2-modulated Nrf2 antioxidative response. Diabetes was induced in rats using a single dose of streptozotocin (65 mg kg-1, i.p.) for 6 weeks, and then luteolin (100 mg kg-1 d-1, i.g.), Nrf2 inhibitor brusatol, or sestrin2 blocker leucine was administered for 2 consecutive weeks. After that, the hearts were isolated and exposed to global I/R (30 min/120 min). Luteolin markedly improved cardiac function, myocardial viability and expressions of Nrf2-regulated antioxidative genes, and reduced lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R hearts. Ca2+-induced mitochondrial permeability transition and membrane potential disruption were markedly inhibited in luteolin-treated diabetic ventricular myocytes. All these effects of luteolin were significantly reversed by Nrf2 inhibitor brusatol or sestrin2 inhibitor leucine. Luteolin-induced diminished Keap1 and augmented nuclear translocation and ARE binding activity of Nrf2 were hampered by leucine in the diabetic I/R heart. In addition, luteolin-induced augmented transcription of sestrin2 was markedly blocked by brusatol in the diabetic I/R heart. These data suggest that sestrin2 and Nrf2 positively interact to promote antioxidative actions and attenuate mitochondrial damage, by which luteolin relieves diabetic myocardial I/R injury.

Protective effect of iridoid glycosides of radix scrophulariae on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model

To investigate the regulatory effect of iridoid glycoside of radix scrophulariae (IGRS) on endoplasmic reticulum stress induced by oxygen-glucose deprivation and reperfusion in vitro model. Rat pheochromocytoma PC12 cells were pretreated with IGRS (50, 100, 200 μg/mL) for 24h, and the in vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was applied. The cell viability was determined by MTT and lactate dehydrogenase (LDH) assay. The apoptotic rate was detected by flow cytometry. The expression of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), C/EBP homologous protein (CHOP), caspase-12 protein, and glucose-regulated protein-78(GRP78)were detected by Western blotting. The mRNA expression levels of sarco/endoplasmic reticulum Ca2+-ATPase2 (SERCA2), 1, 4, 5-triphosphate inositol receptor 1 (IP3R1), and ryanodine receptor 2 (RyR2)were detected by real-time RT-PCR. Free Ca2+ concentration [Ca2+]i was determined by using laser scanning confocal microscopy. The damage caused by OGD/R to PC12 cells was significantly reduced by IGRS, with significant effect on increasing survival rate and reducing LDH release (all P<0.01). The expression of GRP78, CHOP, Bax, and caspase-12 were down-regulated (all P<0.01), and the expression of Bcl-2 and Bcl-2/Bax ratio was up-regulated (all P<0.01); IGRS increased the expression of SERCA2 mRNA in PC12 cells after OGD/R injury (P<0.01), decreased [Ca2+]i and down-regulated the expression of RyR2 mRNA and IP3R1 mRNA. IGRS has neuroprotective effect, which may alleviate cerebral ischemia-reperfusion injury by regulating SERCA2, maintaining calcium balance, and inhibiting endoplasmic reticulum stress-mediated apoptosis.